Figure 7.
Drp1 regulates TCR signal strength. (A, B) Confocal 3D stack reconstructions showing the distribution of phosphotyrosine (pY) (A) and phosphorylated ERK1/2 (B) at the contact site between mitotracker-loaded control or Drp1-silenced J77 T cells and SEE-pulsed Raji B cells (asterisks). (C, D) Time course of the phosphorylation status of PLC-γ1 and ERK 1/2 in control and Drp1-silenced human primary T lymphoblasts (C) or J77 T cells (D) activated with SEE-pulsed Raji B cells. Tubulin or total protein expression are shown as loading controls. Immunoblots are representative of three independent experiments. (E) Confocal microscopy analysis of intracellular Ca2+ flux in control and Drp1-silenced J77 T cells preloaded with Fluo4 AM and conjugated with SEE-pulsed Raji B cells (CMAC, blue). The time profile shows the evolution of Fluo4 AM fluorescence in control (56 conjugates) and siDrp1 (86 conjugates) conjugates. Data are means±s.d. from three independent experiments (P<0.0001, two-tailed t-test). (F) IL-2 secretion from conjugates formed between SEE-pulsed Raji cells and control or Drp1-silenced J77 cells. IL-2 was measured by ELISA 16 h after initial conjugate formation. Data are means±s.d. of six independent experiments (*P<0.05).