Figure 4.
Drp1 controls IS organization. (A) Distribution of Drp1 (red) and CD3 (green) in conjugates between control or Drp1-silenced J77 T cells and SEE-pulsed Raji B cells (CMAC loaded, blue). Scale bar, 10 μm (B) Quantification of CD3 accumulation at the IS, calculated from maximal confocal Z-projections in 30 cells from two independent experiments (P<0.0001, Mann–Whitney test) (C) 3D reconstructions of the cell–cell contact area in control and Drp1-silenced J77-APC conjugates; note the dispersed organization of the CD3/TCR in Drp1-silenced cells. Scale bar, 5 μm (D) Profile plot of the staining intensities of CD3 (green) and mitochondria (red) in the structures depicted in (C). (E, F) CD3 diameter (P<0.001 in Student's t-test; 58 conjugates) and CD3 cluster number (P<0.001, Mann–Whitney test; 50 conjugates) calculated from 3D reconstructions of T cell–APC contacts. Data are means±s.d. from three independent experiments. (G) Confocal analysis of IS structures formed in mitotracker-loaded control or Drp1-silenced J77 cells (red) adhered for 20 min to lipid bilayers containing anti-CD3 (green) and GPI-ICAM-1. A representative cell from one experiment of three is shown. Scale bar, 5 μm. (H) Profile plots of fluorescence intensities for CD3 and mitochondria along the lines indicated in (G).