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Fig. 3. CCR precedes Bax redistribution and propagates within cells. (A) Simultaneous single-cell monitoring of EGFP-Bax and C3/7 activity with the mom-C3/7 probe (Schembri et al., 2007). Frames are numbered in minutes. Ellipse: nuclear region used to detect late EGFP-Bax relocalization (drop in green fluorescence) and t-HcRed released by C3/7 from mitochondria (rise in red fluorescence) as shown in the graph. Late EGFP-Bax relocalization slowly develops after C3/7 activity has already plateaued. (B) Second updated model of Bax relocalization: the late relocalization phase occurs secondary to C3/7 activation, which itself precedes CCR (supplementary material Fig. S3). (C) Simultaneous single-cell monitoring of t-HcRed–Bax motion and of cytochrome-c–EGFP release. Frames are numbered in minutes. Enclosed region shows the group of mitochondria that was used to quantify early and late t-HcRed–Bax recruitment (red-fluorescence increase) and cytochrome-c–EGFP release (green-fluorescence decrease) as shown in the graphs. (D) Simultaneous single-cell monitoring of t-HcRed–Bax motion and of cytochrome-c–EGFP release shows a polarized and propagating CCR. The experiment and conclusions are identical to those in C, with CCR and Bax motion quantified this time using a punctuate/diffuse index (Goldstein et al., 2000). In the first five frames, polarized CCR is observable (from left to right). (E, left) Subcellular quantification in mitochondrial regions 1 to 8 (R1-R8) shows a propagating wavefront of CCR. Left panel: in a HeLa cell, R1-R8 indicate a wave propagating perpendicularly to the cell main axis. (Center) Duration of elemental CCR in R1-R8 (blue bars) is fairly constant. (Right) The CCR wave elicited by STS in a HCT116 Bax–/– cell.
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