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.2002 Dec 23;159(6):931-8.
doi: 10.1083/jcb.200209124. Epub 2002 Dec 23.

Spatial and temporal association of Bax with mitochondrial fission sites, Drp1, and Mfn2 during apoptosis

Affiliations

Spatial and temporal association of Bax with mitochondrial fission sites, Drp1, and Mfn2 during apoptosis

Mariusz Karbowski et al. J Cell Biol..

Abstract

We find that Bax, a proapoptotic member of the Bcl-2 family, translocates to discrete foci on mitochondria during the initial stages of apoptosis, which subsequently become mitochondrial scission sites. A dominant negative mutant of Drp1, Drp1K38A, inhibits apoptotic scission of mitochondria, but does not inhibit Bax translocation or coalescence into foci. However, Drp1K38A causes the accumulation of mitochondrial fission intermediates that are associated with clusters of Bax. Surprisingly, Drp1 and Mfn2, but not other proteins implicated in the regulation of mitochondrial morphology, colocalize with Bax in these foci. We suggest that Bax participates in apoptotic fragmentation of mitochondria.

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Figures

Figure 1.
Figure 1.
Localization of Bax clusters on mitochondria in apoptotic Cos-7 cells. (A) Cells transfected with mito-DsRed2 (green) were treated with 1 μM STS for 6 h, fixed, stained with anti-Bax 6A7 mAb (red), and analyzed by confocal microscopy. (B) Cells were cotransfected with CFP-Bax and mito-YFP. At 12 h after transfection, cells were treated with 1 μM STS for 90 min and examined by confocal microscopy. Each field illustrates one of the typical morphologies of mitochondria (green) and Bax foci (red) in apoptotic Cos-7 cells. Bax associates with mitochondrial constriction sites (arrowheads) or at the tips of tubular elongated mitochondria and appositions of mitochondria. (C and D) Cos-7 cells were treated with 1 μM STS and analyzed by electron microscopy after staining endogenous Bax (C) or CFP-Bax (D) with anti-Bax 6A7 mAb and anti-GFP mAb, respectively, and processing by silver-enhancement of immunogold labeling. In dying cells, endogenous Bax localized to mitochondrial tips and constriction sites. Bars, 0.5 μm.
Figure 2.
Figure 2.
Apoptotic Bax clusters colocalize with sites of mitochondrial fragmentation. HeLa (A and B) and Cos-7 (C) cells were cotransfected with CFP-Bax and mito-YFP. At 12 h after transfection cells were treated with 1 μM STS and analyzed by confocal microscopy over time. Fragmentation and vesiculation of mitochondria began at the same time (between 75 and 80 min in A) that Bax (red) coalesced at mitochondrial scission sites (arrowheads). Fragmented mitochondria often remained connected by Bax-containing structures (A and B). Complete separation of fragmented mitochondria was also detected (C).
Figure 3.
Figure 3.
Drp1K38A cause accumulation of Bax-associated fission intermediates. (A) Induction of WT Drp1 and Drp1K38A. T-Rex- Drp1, T-Rex- Drp1K38A, and T-Rex-V cells were treated with 1 μg/ml of tetracycline for 24 h and analyzed for expression of WT Drp1, Drp1K38A by Western Blotting. (B) T-Rex- Drp1, T-Rex- Drp1K38A, and T-Rex-V cells were treated with 1 μg/ml of tetracycline for 24 h and then treated with 1 μM STS for 5 h, and analyzed for the translocation of Bax to the membranes by Western Blotting. (C) Viability T-Rex- Drp1, T-Rex- Drp1K38A, and T-Rex-V was assayed as described in Materials and methods. Data represent the mean ± SD of two experiments with the starting number of 100 cells per field. Two fields per sample were counted at each time point. (D) Cos-7 cells were cotransfected with pcDNA 3.1-Drp1 and pcDNA 3.1-Bax or pcDNA3.1- Drp1K38A and pcDNA 3.1-Bax (ratios 3:1), treated with 1 μM STS for 2 h and, after cell fractionation, heavy membrane fractions and cytosols were analyzed by Western Blotting. (E and F) Cells were transfected with CFP-Bax, YFP-20, and pcDNA3.1-Drp1 (E), or CFP-Bax, YFP-20, and pcDNA3.1-Drp1K38A (F) (ratio 2:0.5:6), treated with 1 μM STS for 90 min and analyzed with confocal microscopy. Arrowheads indicate invaginations of OMM; possibly representing fission intermediates localized with CFP-Bax clusters. Bars, 20 μm. (G) High magnification of some mitochondria from F (G1 and G2, control; G3–6, STS-treated cells). Arrowheads show the sites where Bax clusters localize, which may represent early fission sites. (H and I) Cells were transfected with CFP-Bax and pcDNA3.1-Drp1 (H) or CFP-Bax and pcDNA3.1-Drp1K38A (I), treated with 1 μM STS for 90 min and analyzed for submitochondrial localization of Bax clusters by electron microscopy. Arrow in I1 points to membrane vesiculations associated with Bax clusters. Arrowheads in I3 point to constriction sites associated with Bax clusters. Bars: (H, I1, I3 insert, I4, I5) 0.2 μm, (I2) 0.1 μm, (I3) 1 μm.
Figure 3.
Figure 3.
Drp1K38A cause accumulation of Bax-associated fission intermediates. (A) Induction of WT Drp1 and Drp1K38A. T-Rex- Drp1, T-Rex- Drp1K38A, and T-Rex-V cells were treated with 1 μg/ml of tetracycline for 24 h and analyzed for expression of WT Drp1, Drp1K38A by Western Blotting. (B) T-Rex- Drp1, T-Rex- Drp1K38A, and T-Rex-V cells were treated with 1 μg/ml of tetracycline for 24 h and then treated with 1 μM STS for 5 h, and analyzed for the translocation of Bax to the membranes by Western Blotting. (C) Viability T-Rex- Drp1, T-Rex- Drp1K38A, and T-Rex-V was assayed as described in Materials and methods. Data represent the mean ± SD of two experiments with the starting number of 100 cells per field. Two fields per sample were counted at each time point. (D) Cos-7 cells were cotransfected with pcDNA 3.1-Drp1 and pcDNA 3.1-Bax or pcDNA3.1- Drp1K38A and pcDNA 3.1-Bax (ratios 3:1), treated with 1 μM STS for 2 h and, after cell fractionation, heavy membrane fractions and cytosols were analyzed by Western Blotting. (E and F) Cells were transfected with CFP-Bax, YFP-20, and pcDNA3.1-Drp1 (E), or CFP-Bax, YFP-20, and pcDNA3.1-Drp1K38A (F) (ratio 2:0.5:6), treated with 1 μM STS for 90 min and analyzed with confocal microscopy. Arrowheads indicate invaginations of OMM; possibly representing fission intermediates localized with CFP-Bax clusters. Bars, 20 μm. (G) High magnification of some mitochondria from F (G1 and G2, control; G3–6, STS-treated cells). Arrowheads show the sites where Bax clusters localize, which may represent early fission sites. (H and I) Cells were transfected with CFP-Bax and pcDNA3.1-Drp1 (H) or CFP-Bax and pcDNA3.1-Drp1K38A (I), treated with 1 μM STS for 90 min and analyzed for submitochondrial localization of Bax clusters by electron microscopy. Arrow in I1 points to membrane vesiculations associated with Bax clusters. Arrowheads in I3 point to constriction sites associated with Bax clusters. Bars: (H, I1, I3 insert, I4, I5) 0.2 μm, (I2) 0.1 μm, (I3) 1 μm.
Figure 3.
Figure 3.
Drp1K38A cause accumulation of Bax-associated fission intermediates. (A) Induction of WT Drp1 and Drp1K38A. T-Rex- Drp1, T-Rex- Drp1K38A, and T-Rex-V cells were treated with 1 μg/ml of tetracycline for 24 h and analyzed for expression of WT Drp1, Drp1K38A by Western Blotting. (B) T-Rex- Drp1, T-Rex- Drp1K38A, and T-Rex-V cells were treated with 1 μg/ml of tetracycline for 24 h and then treated with 1 μM STS for 5 h, and analyzed for the translocation of Bax to the membranes by Western Blotting. (C) Viability T-Rex- Drp1, T-Rex- Drp1K38A, and T-Rex-V was assayed as described in Materials and methods. Data represent the mean ± SD of two experiments with the starting number of 100 cells per field. Two fields per sample were counted at each time point. (D) Cos-7 cells were cotransfected with pcDNA 3.1-Drp1 and pcDNA 3.1-Bax or pcDNA3.1- Drp1K38A and pcDNA 3.1-Bax (ratios 3:1), treated with 1 μM STS for 2 h and, after cell fractionation, heavy membrane fractions and cytosols were analyzed by Western Blotting. (E and F) Cells were transfected with CFP-Bax, YFP-20, and pcDNA3.1-Drp1 (E), or CFP-Bax, YFP-20, and pcDNA3.1-Drp1K38A (F) (ratio 2:0.5:6), treated with 1 μM STS for 90 min and analyzed with confocal microscopy. Arrowheads indicate invaginations of OMM; possibly representing fission intermediates localized with CFP-Bax clusters. Bars, 20 μm. (G) High magnification of some mitochondria from F (G1 and G2, control; G3–6, STS-treated cells). Arrowheads show the sites where Bax clusters localize, which may represent early fission sites. (H and I) Cells were transfected with CFP-Bax and pcDNA3.1-Drp1 (H) or CFP-Bax and pcDNA3.1-Drp1K38A (I), treated with 1 μM STS for 90 min and analyzed for submitochondrial localization of Bax clusters by electron microscopy. Arrow in I1 points to membrane vesiculations associated with Bax clusters. Arrowheads in I3 point to constriction sites associated with Bax clusters. Bars: (H, I1, I3 insert, I4, I5) 0.2 μm, (I2) 0.1 μm, (I3) 1 μm.
Figure 3.
Figure 3.
Drp1K38A cause accumulation of Bax-associated fission intermediates. (A) Induction of WT Drp1 and Drp1K38A. T-Rex- Drp1, T-Rex- Drp1K38A, and T-Rex-V cells were treated with 1 μg/ml of tetracycline for 24 h and analyzed for expression of WT Drp1, Drp1K38A by Western Blotting. (B) T-Rex- Drp1, T-Rex- Drp1K38A, and T-Rex-V cells were treated with 1 μg/ml of tetracycline for 24 h and then treated with 1 μM STS for 5 h, and analyzed for the translocation of Bax to the membranes by Western Blotting. (C) Viability T-Rex- Drp1, T-Rex- Drp1K38A, and T-Rex-V was assayed as described in Materials and methods. Data represent the mean ± SD of two experiments with the starting number of 100 cells per field. Two fields per sample were counted at each time point. (D) Cos-7 cells were cotransfected with pcDNA 3.1-Drp1 and pcDNA 3.1-Bax or pcDNA3.1- Drp1K38A and pcDNA 3.1-Bax (ratios 3:1), treated with 1 μM STS for 2 h and, after cell fractionation, heavy membrane fractions and cytosols were analyzed by Western Blotting. (E and F) Cells were transfected with CFP-Bax, YFP-20, and pcDNA3.1-Drp1 (E), or CFP-Bax, YFP-20, and pcDNA3.1-Drp1K38A (F) (ratio 2:0.5:6), treated with 1 μM STS for 90 min and analyzed with confocal microscopy. Arrowheads indicate invaginations of OMM; possibly representing fission intermediates localized with CFP-Bax clusters. Bars, 20 μm. (G) High magnification of some mitochondria from F (G1 and G2, control; G3–6, STS-treated cells). Arrowheads show the sites where Bax clusters localize, which may represent early fission sites. (H and I) Cells were transfected with CFP-Bax and pcDNA3.1-Drp1 (H) or CFP-Bax and pcDNA3.1-Drp1K38A (I), treated with 1 μM STS for 90 min and analyzed for submitochondrial localization of Bax clusters by electron microscopy. Arrow in I1 points to membrane vesiculations associated with Bax clusters. Arrowheads in I3 point to constriction sites associated with Bax clusters. Bars: (H, I1, I3 insert, I4, I5) 0.2 μm, (I2) 0.1 μm, (I3) 1 μm.
Figure 4.
Figure 4.
Mfn2 protein forms mitochondria-associated punctate foci that colocalize with Bax during apoptosis. Cos-7 cells were cotransfected with CFP-Bax (blue) and Mfn2-YFP (green), incubated without (A) and with (B) 1 μM STS for 90 min, and analyzed by confocal microscopy after staining with Mitotracker CMXRos (red). In untreated cells (A) Bax is mostly cytosolic and Mfn2 is concentrated in punctate mitochondrial foci. In apoptotic cells (B) Bax localizes to foci containing Mfn2. The line scans plot the intensity of CFP-Bax (blue) and Mfn2-YFP (green). Bars, 20 μm.
Figure 5.
Figure 5.
Upon induction of apoptosis Bax localizes to mitochondrial foci containing Drp1. HeLa (A) and Cos-7 (B) cells were triple-transfected with CFP-Bax (blue), YFP-Drp1 (green), and mito-DsRed2 (red). At 12 h after transfection, cells were treated with 1 μM STS to induce apoptosis and analyzed by confocal microscopy over time. The same field of the cell was analyzed before (−STS) and after (+STS) Bax translocation/clustering (A, top). The bottom panel in A shows an independent example of CFP-Bax and YFP-Drp1 localization. The line scans plot the intensity of CFP-Bax (blue) and YFP-Drp1 (green). Colocalization of Bax with Drp1 at the mitochondrial foci is apparent in apoptotic cells. (B) Localization of CFP-Bax (blue) and YFP-Drp1 (green) in healthy (−STS) and apoptotic Cos-7 cells (+STS). The mitochondrial matrix is marked with mito-DsRed2 (red). CFP-Bax and YFP-Drp1 colocalize in clusters on mitochondria (cyan in overlay). (C) Colocalization of endogenous Bax and Drp1 in apoptotic cells. Cos-7 cells were treated for 6 h with 1 μM STS in the presence of zVAD-fmk. Cells were stained with anti-Bax 6A7 mAb (red), and anti-Drp1 polyclonal antibodies (green). Arrowheads point to some of the foci where endogenous Bax and Drp1 colocalize. Arrows shows some of the Drp1 foci, which do not colocalize with Bax, and may represent the nonmitochondrial subpopulation of Drp1 (Yoon et al., 1998). Bar, 20 μm.
Figure 5.
Figure 5.
Upon induction of apoptosis Bax localizes to mitochondrial foci containing Drp1. HeLa (A) and Cos-7 (B) cells were triple-transfected with CFP-Bax (blue), YFP-Drp1 (green), and mito-DsRed2 (red). At 12 h after transfection, cells were treated with 1 μM STS to induce apoptosis and analyzed by confocal microscopy over time. The same field of the cell was analyzed before (−STS) and after (+STS) Bax translocation/clustering (A, top). The bottom panel in A shows an independent example of CFP-Bax and YFP-Drp1 localization. The line scans plot the intensity of CFP-Bax (blue) and YFP-Drp1 (green). Colocalization of Bax with Drp1 at the mitochondrial foci is apparent in apoptotic cells. (B) Localization of CFP-Bax (blue) and YFP-Drp1 (green) in healthy (−STS) and apoptotic Cos-7 cells (+STS). The mitochondrial matrix is marked with mito-DsRed2 (red). CFP-Bax and YFP-Drp1 colocalize in clusters on mitochondria (cyan in overlay). (C) Colocalization of endogenous Bax and Drp1 in apoptotic cells. Cos-7 cells were treated for 6 h with 1 μM STS in the presence of zVAD-fmk. Cells were stained with anti-Bax 6A7 mAb (red), and anti-Drp1 polyclonal antibodies (green). Arrowheads point to some of the foci where endogenous Bax and Drp1 colocalize. Arrows shows some of the Drp1 foci, which do not colocalize with Bax, and may represent the nonmitochondrial subpopulation of Drp1 (Yoon et al., 1998). Bar, 20 μm.
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