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Figure 4


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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