Figure 8.

Disruption of cytoskeleton-dependent mitochondrial translocation to the IS rescues the Ca²⁺ microdomain-dependent modulation of PMCA activity. (A) Brightfield images (before stimulation) and TIRFM images of single representative MitoTracker^® Green/AM-loaded Jurkat T cells that were pre-treated with nocodazole (2 μM) latrunculin A (10 μg/ml), latrunculin B (10 μg/ml) or cytochalsin D (10 μM) as indicated in order to block the cytoskeleton-dependent mitochondrial translocation to the IS. Cells were settled on anti-CD3 antibody-coated coverslips (IS) for 5–7 min in a Ca²⁺-free solution to induce the maximal depletion of intracellular Ca²⁺ stores. Three minutes after starting the acquisition, cells were exposed to 20 mM Ca²⁺ for the next 22 min. The brightest point in the TIRFM picture belongs to a fluorescent bead (Invitrogen) used as control of the focus (see Materials and methods). (B) Statistical analysis of the normalized MitoTracker^® fluorescence from 40 (Control, red trace), 10 (Noco, purple trace), 20 (Lat A, blue trace), 20 (Lat B, green trace) and 16 (Cyto D, black trace) cells analysed as the ones shown in (A) (Noco, Lat A, Lat B and Cyto D versus control at 20 min; P=0.079, P=0.041, P=0.029, P=0.028). (C, F) Average [Ca²⁺]_i responses and Ca²⁺ clearance rates from control iso-cells induced by 1 or 20 mM extracellular Ca²⁺ solutions in the presence of nocodazole (C), latrunculin A (D), latrunculin B (E) and cytochalasin D (F) following IS formation. Average Ca²⁺ clearance rates following [Ca²⁺]_i elevation (indicated by the thick lines) were calculated by the dRatio (340/380)/dt slopes over 10 s periods following external Ca²⁺ removal. (G) Statistical analysis of Ca²⁺ clearance rates from the cells shown in (C, F) compared with controls cells from Figure 6A (1 versus 20; control: n=60, P=0.76; Noco: n=14, P<0.0001; Lat A: n=20, P<0.0001; Lat B: n=15, P<0.001; Cyto D: n=12, P<0.0001). (H) Normalized Fluo-5F fluorescence from 15 (IS, red Δ) and 23 (No IS, blue □) cells, respectively (at 20 min, P=0.6). Inset shows brightfield images (before stimulation) and TIRFM pictures of Fluo-5F/AM-loaded Jurkat T cells. Cells were settled on either anti-CD3 antibody-coated coverslips (IS) or poly-L-ornithine-coated coverslips (No IS) in the absence of extracellular Ca²⁺ solution containing 1 μM TG for 5–7 min. Cells were pre-treated with latrunculin B (Lat B) as in (A). Three minutes after starting the acquisition, cells were exposed to 20 mM Ca²⁺ for the next 22 min. Errors bars indicate s.e.m. Scale bars are 5 μm.