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First published online 7 October 2008
doi: 10.1242/jcs.029587

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An intracellular wave of cytochrome c propagates and precedes Bax redistribution during apoptosis

Lydia Lartigue^1,*, Chantal Medina^1,*, Laura Schembri¹, Paul Chabert¹, Marion Zanese^1,2, Flora Tomasello¹, Renée Dalibart¹, Didier Thoraval³, Marc Crouzet³, François Ichas^1,2, and Francesca De Giorgi^1,2

¹ INSERM U916, Université Bordeaux 2, Institut Bergonié, 229 cours de l'Argonne, 33000 Bordeaux, France
² FLUOFARMA, 2 rue Robert Escarpit, 33600 Pessac, France
³ CNRS UMR 5095, Université Bordeaux 2, 146 rue Léo Saignat, 33076 Bordeaux Cedex, France

View larger version (55K): [in this window] [in a new window]   Fig. 1. The two phases of Bax relocalization. (A) HCT116 Bax–/– cells expressing EGFP-Bax after transfection: basal EGFP-Bax distribution is cytosolic and nuclear (representative of >80% of the cells after 12 hours of expression); early EGFP-Bax redistribution is partial and delineates mitochondria (representative of >50% of the cells after 48 hours of expression); late EGFP-Bax redistribution is total and forms clusters scattered at the mitochondrial surface (De Giorgi et al., 2002; Nechushtan et al., 2001) (representative of >90% cells after 72 hours of expression). Scale bars: 10 µm. (B) iFRAP analysis (van Drogen and Peter, 2004) of the mobility of EGFP-Bax by recording the drop in EGFP-Bax fluorescence (right diagram) that is associated with a small group of mitochondria (left panels, circle) and induced by bleaching over 80% of EGFP-Bax located outside the recording zone (left panels, dotted perimeter). (Right) Early relocalized EGFP-Bax is not anchored to the MOM, whereas late relocalized EGFP-Bax is anchored to the MOM, similar to the reference tail-anchored construct EGFP-Cb5TMDRR (Schembri et al., 2007). (C) FRET analysis of HCT116 Bax–/– cells expressing ECFP-Bax and EYFP-Bax (De Giorgi et al., 2002) shows that Bax multimerizes only during the late relocalization phase, concomitant with its anchorage and the formation of clusters. **Statistically significant P<0.001 (n=5), t-test. Scale bars: 1 µm. (D) Two possible models for Bax (blue) relocalization: (1) the early and late phases are connected in a continuum (upper model); (2) the early and late phases are independent and chronologically separated (lower model). Irrespective of the model, the phase of cytochrome c (green) release is unknown.

View larger version (59K): [in this window] [in a new window]   Fig. 2. Differential involvement of 9 reveals that the early and late phases of Bax motion are distinct. (A) Coexpression of EYFP-BaxC and ECFP-Bax shows that 9 deletion prevents the early redistribution phase detected at the level of a single mitochondrion. Arrow points to a single mitochondrion. Scale bars: 1 µm. (B) Enforced dimerization of CopGFP-Bax provokes early mitochondrial relocalization without directly causing CCR. Deleting 9 (CopGreen-BaxC) prevents this relocalization. Note the appearance of inclusion bodies that are clearly distinguishable from mitochondria. Cc, cytochrome c. Scale bars: 10 µm. (C) 9 constitutively targets EGFP to the MOM in live yeast cells (EGFP-9). Mitochondria are labeled with DiOC6. Scale bars: 1 µm. (D) Effect of 9 deletion or 9 immobilization by cyclization (cycloBax) (supplementary material Fig. S1) on the proapoptotic potency of Bax. Loss of mitochondrial membrane potential (low) was used as a late apoptosis reporter (Goldstein et al., 2000). **Statistically significant P<0.001 (n=5), t-test. (E) After STS challenge (1 µM, 5 hours), EGFP-BaxC bypasses the early relocalization phase and directly undergoes the late one (compare with t-HcRed–Bax in the two upper rows). Direct late relocalization of EGFP-BaxC is observed in the absence of full-length Bax (HCT116 Bax–/– cells, lower row); cycloBax (revealed by anti-myc) (supplementary material Fig. S1) behaves similarly to EGFP-BaxC. Scale bars: 10 µm. (F) First updated model of Bax relocalization: the early and late relocalization phases are disconnected; CCR is not associated with the early phase.

View larger version (53K): [in this window] [in a new window]   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.

View larger version (56K): [in this window] [in a new window]   Fig. 4. Giant syncytia as a tool for characterizing the CCR wave. (A) Syncytium were created by fusing cytochrome-c–EGFP-expressing HeLa cells (Goldstein et al., 2000), which were then challenged with FasL and imaged over time. The CCR wave propagates over a distance >100 µm, at 16.1±5.1 µm.second–1 (broken line shows the wavefront). Time is shown in minutes (top right). Signal intensity is coded in pseudocolor. See corresponding supplementary material Movie 2. (B) CCR recorded in regions organized along the wave-propagation axis in the syncytium shown in A and supplementary material Movie 2. Note the rise in basal cytochrome-c-EGFP levels as the wave proceeds. (C) Immunodetection of cytochrome c (Cc) and of endogenous activated Bax (N20 antibody) in a HeLa syncytium. The CCR wave precedes Bax conformational activation. Compare the positions of wavefronts (broken lines) and intensity recordings (plot) in regions aligned along the CCR-wave main axis (R1-R17). (D) Diffuse treatment with 20 µM BH3I-1 triggers a polarized CCR wave in a single HeLa cell expressing cytochrome-c–EGFP. (E) Bax/Bak double-knockout mouse embryonic fibroblasts incorporated into a chimeric syncytium with cytochrome-c–EGFP-expressing HeLa cells neither prevent nor slow down the propagation of a CCR wave elicited by FasL. The position of the mouse fibroblasts within the live syncytium was determined by the nuclear stain Hoechst (distinctive chromatin clumps; left panels, and red-hatched discs in the cytochrome-c–EGFP channel). Velocity of the CCR wave recorded between regions 1 and 2 (HeLa field), and 2 and 3 (fibroblasts field) (lower right panel) is unchanged (upper right panel). Arrows point to the mouse fibroblasts. (F) Cytosolic Ca2+ buffering with BAPTA-AM does not affect CCR induced by STS in HeLa cells expressing cytochrome-c–EGFP (left panel). CCR propagates in a Ca2+-depleted cell (right panels) that was pre-treated for 30 minutes with the combination thapsigargin (1 µM), 2APB (50 µM) and BAPTA-AM (2 µM). Note the delayed movement of cytochrome-c–EGFP back to mitochondria (arrow). Scale bars: 20 µm.

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