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. 1997 Dec 1;139(5):1281-92.
doi: 10.1083/jcb.139.5.1281.

Movement of Bax from the cytosol to mitochondria during apoptosis

K G Wolter  1 , Y T Hsu, C L Smith, A Nechushtan, X G Xi, R J Youle
Affiliations

Movement of Bax from the cytosol to mitochondria during apoptosis

K G Wolter et al. J Cell Biol. .

Abstract

Bax, a member of the Bcl-2 protein family, accelerates apoptosis by an unknown mechanism. Bax has been recently reported to be an integral membrane protein associated with organelles or bound to organelles by Bcl-2 or a soluble protein found in the cytosol. To explore Bcl-2 family member localization in living cells, the green fluorescent protein (GFP) was fused to the NH2 termini of Bax, Bcl-2, and Bcl-XL. Confocal microscopy performed on living Cos-7 kidney epithelial cells and L929 fibroblasts revealed that GFP-Bcl-2 and GFP-Bcl-XL had a punctate distribution and colocalized with a mitochondrial marker, whereas GFP-Bax was found diffusely throughout the cytosol. Photobleaching analysis confirmed that GFP-Bax is a soluble protein, in contrast to organelle-bound GFP-Bcl-2. The diffuse localization of GFP-Bax did not change with coexpression of high levels of Bcl-2 or Bcl-XL. However, upon induction of apoptosis, GFP-Bax moved intracellularly to a punctate distribution that partially colocalized with mitochondria. Once initiated, this Bax movement was complete within 30 min, before cellular shrinkage or nuclear condensation. Removal of a COOH-terminal hydrophobic domain from GFP-Bax inhibited redistribution during apoptosis and inhibited the death-promoting activity of both Bax and GFP-Bax. These results demonstrate that in cells undergoing apoptosis, an early, dramatic change occurs in the intracellular localization of Bax, and this redistribution of soluble Bax to organelles appears important for Bax to promote cell death.

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Figures

Figure 7
Figure 7
GFP–Bax distribution in Cos-7 cells is not affected by coexpression of Bcl-2. Cells were transiently transfected with a 3:1 ratio of plasmid containing Bcl-2 and GFP–Bax, and examined 48 h later by confocal microscopy. (A) In Cos-7 cells cotransfected with Bcl-2 and GFP– Bax, the GFP–Bax distributes throughout the cell in a pattern that is indistinguishable from that of cells expressing GFP–Bax alone. (B) The coexpression of GFP–Bax and human Bcl-2 in Cos-7 cells was monitored by Western blotting with α human Bax 2D2 and α human Bcl-2 8C8 monoclonal antibodies. Lanes a are the Cos-7 cell lysates from transfection with the pcDNA 3 vector alone. Lanes b are the cell lysates from the pcDNA 3–Bcl-2 transfection, and lanes c are from the cotransfection of C3–EGFP-Bax and pcDNA 3–Bcl-2. Arrows a, b, and c represent the GFP–Bax, its NH2-terminal translational variant, and endogenous simian Bax, respectively. Bar, 20 μm.
Figure 7
Figure 7
GFP–Bax distribution in Cos-7 cells is not affected by coexpression of Bcl-2. Cells were transiently transfected with a 3:1 ratio of plasmid containing Bcl-2 and GFP–Bax, and examined 48 h later by confocal microscopy. (A) In Cos-7 cells cotransfected with Bcl-2 and GFP– Bax, the GFP–Bax distributes throughout the cell in a pattern that is indistinguishable from that of cells expressing GFP–Bax alone. (B) The coexpression of GFP–Bax and human Bcl-2 in Cos-7 cells was monitored by Western blotting with α human Bax 2D2 and α human Bcl-2 8C8 monoclonal antibodies. Lanes a are the Cos-7 cell lysates from transfection with the pcDNA 3 vector alone. Lanes b are the cell lysates from the pcDNA 3–Bcl-2 transfection, and lanes c are from the cotransfection of C3–EGFP-Bax and pcDNA 3–Bcl-2. Arrows a, b, and c represent the GFP–Bax, its NH2-terminal translational variant, and endogenous simian Bax, respectively. Bar, 20 μm.
Figure 1
Figure 1
Distribution of GFP-fusion proteins expressed in living Cos-7 cells, before and after STS treatment. At 48 h after transfection, cells were examined by confocal microscopy. Each field was visualized by laser fluorescence to detect GFP (a, c, e, g, i, k, m, and o), and by DIC to illustrate cell morphology (b, d, f, h, j, l, n, and p). Native GFP (a and b) distributes throughout untreated cells, that display a healthy morphology. In contrast, GFP–Bcl-2 (e and f) displays a punctate distribution, mostly perinuclear. GFP–Bcl-XL (i and j) appears to distribute in a mainly punctate pattern similar to GFP–Bcl-2, but also has a faint diffuse fluorescence that extends throughout cells. GFP-Bax (m and n) distributes freely throughout cells, in a pattern indistinguishable from that of GFP alone. After 6 h of treatment with 1 μM STS, Cos-7 cells assume a morphology indicative of apoptosis, including loss of cell volume, retraction of processes, and blebbing of cell membrane. Despite these changes, native GFP (c and d) still distributes throughout the cell. GFP–Bcl-2 (g and h) retains a punctate distribution after STS treatment. GFP–Bcl-XL (k and l) also does not appear to change distribution with STS treatment, remaining mainly punctate with a background of diffuse fluorescence. However, GFP–Bax (o and p) undergoes a striking change in distribution, becoming entirely punctate after STS treatment. Bar, 20 μm.
Figure 2
Figure 2
Distribution of GFP-fusion proteins expressed in living L929 cells, before and after STS treatment. At 48 h after transfection, cells were examined by confocal microscopy. Each field was visualized by laser fluorescence to detect GFP (a, c, e, g, i, k, m, and o), and by DIC to illustrate cell morphology (b, d, f, h, j, l, n, and p). Native GFP (a and b) distributes throughout healthy cells. GFP–Bcl-2 (e and f) displays a punctate perinuclear distribution. GFP–Bcl-XL (i and j) appears to have both punctate and diffuse fractions. GFP–Bax (m and n) distributes freely throughout cells. After 6 h of treatment with 1 μM STS, L929 cells undergo a loss of cell volume and retraction and blebbing of extended processes. Native GFP (c and d) is found throughout the cells after STS treatment, including blebs. GFP–Bcl-2 (g and h) retains a punctate distribution after STS treatment. After STS treatment, GFP–Bcl-XL (k and l) shows a largely punctate distribution with a background of diffuse fluorescence. As in Cos-7 cells, GFP–Bax (o and p) undergoes a change in distribution, becoming punctate after STS treament. Bar, 30 μm.
Figure 3
Figure 3
Expression of truncated and full-length GFP–Bax, GFP– Bcl-2, and GFP–Bcl-XL fusion proteins in COS cells. COOH-terminal truncated and full-length GFP fusion constructs of Bax (lanes a and b), Bcl-2 (lanes c and d) and Bcl-XL (lanes e and f) were transfected into COS cells. The expression of the fusion products was monitored by Western blotting of the cell lysates with α human Bax 2D2, α human Bcl-2 8C8, and α universal Bcl-XL 2H12 monoclonal antibodies for the detection of Bax, Bcl-2, and Bcl-XL, respectively. Arrows a are the expressed fusion products. Arrows b are likely the in-frame NH2-terminal translational variants. Arrows c represent the endogenous simian Bax, Bcl-2, or Bcl-XL that cross-react with the above specified antibodies.
Figure 4
Figure 4
Effect on cell viability of transient expression of wild-type and COOH-terminal truncated forms of Bcl-2 family members, with and without GFP fused at the NH2-terminus. Cells were either singly transfected with the GFP fusion constructs or cotransfected with GFP and various Bcl-2 family members. At 48 h after transfection, cells were treated with STS. The number of GFP-positive cells was counted within a given field at regular intervals, starting at the time of STS addition (∼500 GFP-positive cells at time zero was typical for each experiment). Changes in cell viability are displayed as number of glowing cells within a field and expressed as a percentage of time zero value for that field. (A) Bcl-2 constructs expressed in L929 cells. (n = 4). pcDNA3 vector control (⋄), Bcl-2 (○), GFP–Bcl-2 (•), Bcl-2 ΔCT (□), GFP–Bcl-2 ΔCT (▪), and Bax (▵) (B) Bcl-XL constructs expressed in L929 cells. (n = 4). pcDNA3 vector control (⋄), Bcl-XL (○), GFP–Bcl-XL (•), Bcl-XL ΔCT (□), GFP–Bcl-XL ΔCT (▪), and Bax (▵) (n = 4). (C) Bax constructs expressed in Cos-7 cells. (n = 3). pcDNA3 vector control (⋄), Bax (○), GFP–Bax (•), Bax ΔCT (▵), GFP–Bax ΔCT (▴).
Figure 5
Figure 5
GFP–Bcl-2 and the punctate portion of GFP–Bcl-XL colocalize with mitochondria in Cos-7 cells. Transiently transfected Cos-7 cells were treated with 20 ng/ml Mitotracker Red CMXRos to stain mitochondria and then examined by laser fluorescence confocal microscopy. Each field was independently visualized with the appropriate wavelength for GFP (a, d, and g) and for Mitotracker Red CMXRos (b, e, and h) and then the two images were overlaid (c, f, and i). GFP–Bcl-2 localizes primarily to mitochondria (a–c). A majority GFP–Bcl-XL also localizes to mitochondria, although there is a faint, diffuse background which is not punctate (d–f). GFP–Bax does not localize to mitochondria in healthy cells (g–i). Bar, 20 μm.
Figure 6
Figure 6
FRAP analysis of GFP, and GFP–Bax, and GFP–Bcl-2 in healthy and cells treated with STS. (A) Fluorescence recovery visualized in individual treated cells. In a healthy Cos-7 cell expressing GFP–Bax (a–d), fluorescence recovers rapidly in a photobleached area. After treatment with STS, a Cos-7 cell with punctate GFP–Bax (e–h) no longer recovers after photobleaching. In a healthy Cos-7 cell expressing GFP–Bcl-2 (i–l), recovery does not occur in the photobleached area. (B) Quantitative FRAP analysis to determine protein mobilities in cells expressing GFP alone, GFP–Bax, and GFP–Bcl-2. Fluorescence intensities during recovery after photobleach are plotted versus time. Data was collected at 0.46-s intervals during recovery. GFP and GFP–Bax rapidly recover with a time course consistent with diffusion. GFP–Bcl-2 shows minimal recovery, indicating that it is largely immobile. All intensity values were normalized to prebleach intensity (I = 100) and to the intensity immediately after photobleach (I = 0). Bars, 20 μm.
Figure 6
Figure 6
FRAP analysis of GFP, and GFP–Bax, and GFP–Bcl-2 in healthy and cells treated with STS. (A) Fluorescence recovery visualized in individual treated cells. In a healthy Cos-7 cell expressing GFP–Bax (a–d), fluorescence recovers rapidly in a photobleached area. After treatment with STS, a Cos-7 cell with punctate GFP–Bax (e–h) no longer recovers after photobleaching. In a healthy Cos-7 cell expressing GFP–Bcl-2 (i–l), recovery does not occur in the photobleached area. (B) Quantitative FRAP analysis to determine protein mobilities in cells expressing GFP alone, GFP–Bax, and GFP–Bcl-2. Fluorescence intensities during recovery after photobleach are plotted versus time. Data was collected at 0.46-s intervals during recovery. GFP and GFP–Bax rapidly recover with a time course consistent with diffusion. GFP–Bcl-2 shows minimal recovery, indicating that it is largely immobile. All intensity values were normalized to prebleach intensity (I = 100) and to the intensity immediately after photobleach (I = 0). Bars, 20 μm.
Figure 8
Figure 8
GFP–Bax colocalizes with mitochondria in Cos-7 cells after STS treatment. Cos-7 cells transiently expressing GFP–Bax were treated with 1 μM STS to induce apoptosis and with 20 ng/ml Mitotracker Red CMXRos to stain for mitochondria and then examined after 4 h by laser fluorescence confocal microscopy. The field shown was independently visualized by laser fluorescence confocal microscopy at the appropriate wavelength for GFP (a) and for Mitotracker Red CMXRos (b), and the two images were then overlaid (c). Whereas a majority of the punctate GFP–Bax appears to localize to mitochondria, there are also areas where the GFP–Bax is punctate but which do not label with the mitochondrial dye (c, arrow). Bar, 20 μm.
Figure 9
Figure 9
GFP–Bax redistribution occurs before cell shrinkage associated with apoptosis. A field containing three living Cos-7 cells expressing GFP–Bax was followed over time after addition of 1 μM STS. At each timepoint, the field was visualized by laser fluorescence to detect GFP–Bax (a–e and k–o), and by DIC to illustrate cell morphology (f–j and p–t). Time elapsed after addition of STS is indicated in the corner of each of the laser fluorescence panels. Redistribution of the GFP–Bax was first detectable for the two cells towards the top of the field at 1 h, 24 min after STS addition (c). Changes in cell shape first became apparent 12–24 min later, as evidenced by a retraction of cell outlines (arrows, i and j). Notice that at these timepoints, the lowermost cell has not yet initiated either GFP–Bax redistribution or cell shrinkage. GFP–Bax redistribution is first detectable for the lowermost cell at 2 h, 36 min (n). Bar, 20 μm.
Figure 10
Figure 10
GFP–Bax redistribution precedes nuclear fragmentation. A field containing three living Cos-7 cells expressing GFP–Bax was followed over time after addition of 1 μM STS, and 100 ng/ml of the nuclear stain bis-benzamide (a–e). In each panel, laser fluorescence confocal microscopy was used at the appropriate wavelength to visualize GFP (green) and bis-benzamide (blue). Time elapsed after STS addition is indicated in each panel. After 15 h (f) cells show fragmented nuclei associated with apoptosis. Bar, 20 μm.
Figure 11
Figure 11
Distribution of GFP-fusion proteins lacking COOH-terminal hydrophobic domains before and after STS treatment. Cos-7 and L929 cells were transiently transfected with the appropriate construct, and examined 48 h later by confocal microscopy. Each field was visualized by laser fluorescence to detect GFP. In both healthy Cos-7 cells (a) and L929 cells (c), ΔCT GFP– Bcl-2 was diffusely distributed throughout the cytosol. Likewise, in healthy Cos-7 cells (e) and L929 cells (g), ΔCT GFP–Bcl-XL was diffusely distributed throughout the cytosol. Finally, in both healthy Cos-7 cells (i) and L929 cells (k), ΔCT GFP– Bax was diffusely distributed throughout the cytosol. Cells expressing truncated GFP-fusion proteins were treated with 1 μM STS, and examined after 6 h. In both Cos-7 cells (b, f, and j) and L929 cells (d, h, and l), none of the truncated proteins redistributed after STS treatment. Bar, 20 μm.
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