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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.

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