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. 2006 Oct;26(20):7397-408.
doi: 10.1128/MCB.02282-05.

Inhibiting the mitochondrial fission machinery does not prevent Bax/Bak-dependent apoptosis

Philippe A Parone  1 , Dominic I James, Sandrine Da Cruz, Yves Mattenberger, Olivier Donzé, François Barja, Jean-Claude Martinou
Affiliations

Inhibiting the mitochondrial fission machinery does not prevent Bax/Bak-dependent apoptosis

Philippe A Parone et al. Mol Cell Biol. 2006 Oct.

Abstract

Apoptosis, induced by a number of death stimuli, is associated with a fragmentation of the mitochondrial network. These morphological changes in mitochondria have been shown to require proteins, such as Drp1 or hFis1, which are involved in regulating the fission of mitochondria. However, the precise role of mitochondrial fission during apoptosis remains elusive. Here we report that inhibiting the fission machinery in Bax/Bak-mediated apoptosis, by down-regulating of Drp1 or hFis1, prevents the fragmentation of the mitochondrial network and partially inhibits the release of cytochrome c from the mitochondria but fails to block the efflux of Smac/DIABLO. In addition, preventing mitochondrial fragmentation does not inhibit cell death induced by Bax/Bak-dependent death stimuli, in contrast to the effects of Bcl-xL or caspase inhibition. Therefore, the fission of mitochondria is a dispensable event in Bax/Bak-dependent apoptosis.

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Figures

FIG. 1.
FIG. 1.
Depleting cells of Drp1 by RNA interference inhibits mitochondrial fission and prevents fragmentation of the mitochondrial network during apoptosis. (A) HeLa cells were transiently transfected with the control (Ctrl), D1, or D2 shRNA expression constructs, selected with puromycin for 24 h, and collected for Western blot analysis using the indicated antibodies 72 h after transfection. M, molecular mass markers (in kilodaltons). (B) HeLa cells were transiently cotransfected with the control or D1 shRNA expression constructs together with pEYFPmito and selected with puromycin for 24 h. At 72 h after transfection the cells were UV irradiated in the presence of zVAD-fmk and fixed 4 or 14 h later, and the morphology of the mitochondrial network was visualized by fluorescent microscopy. The scale bar corresponds to 20 μm. (C) HeLa Drp1 shRNA-inducible cells cultured in the presence or absence of tetracycline (Tet) for 5 days were UV irradiated in the presence of zVAD-fmk, grown for 14 h in their respective growth media supplemented with zVAD-fmk, and fixed for ultrastructural analysis by electron microscopy. The scale bar corresponds to 1 μm.
FIG. 2.
FIG. 2.
Depleting cells of Drp1 partially inhibits the release of cytochrome c from the mitochondria but not that of Smac/DIABLO. (A) HeLa cells were transiently transfected with the control (Ctrl), D1, or D2 shRNA expression constructs and selected with puromycin for 24 h. At 72 h posttransfection the cells were UV irradiated in the presence of zVAD-fmk, grown for 4 h and 14 h in the same medium, and fixed to determine the localization of cytochrome c (Cyto c) and Smac/DIABLO by immunostaining. The scale bar corresponds to 20 μm. (B) Quantitative analysis of data from panel A at 14 h postirradiation. Data are expressed as the means + SEMs of the results of at least three independent experiments. ***, P < 0.0005; ns, not significant. (C) HeLa cells were transfected and selected as described for panel A. At 72 h posttransfection the cells were treated with 3 μM ActD in the presence of zVAD-fmk, grown for 14 h in the same medium, and fixed to determine the localization of cytochrome c and Smac/DIABLO by immunostaining. Quantitative analysis of the data is expressed as the means + SEMs of the results of at least three independent experiments. (D) HeLa cells were transfected and selected as described for panel A. At 72 h posttransfection the cells were UV irradiated in the absence of zVAD-fmk, grown for 14 h in the same medium, and fixed. The localization of cytochrome c and Smac/DIABLO was assessed by immunostaining. The cells were costained with Hoechst 33342 to observe changes in the nuclear morphology. The scale bar corresponds to 20 μm. (E) Quantitative analysis of the data presented in panel D is expressed as the means + SEMs of the results of at least three independent experiments. (F) HeLa cells were infected with the control, D1, or F1 shRNA retroviruses, grown for 96 h, and treated with 3 μM ActD for 8 h. The cells were then collected and fractionated, and the protein contents of the cytosolic fraction (CF) and heavy-membrane fractions (HMF) were analyzed by Western blotting with the indicated antibodies. (G) The protein content of the cytosolic fractions (CF) and heavy-membrane fractions (HMF), as well as that of the alkali-treated heavy-membrane fraction, was analyzed by Western blotting with the indicated antibodies.
FIG. 3.
FIG. 3.
Cytochrome c (Cyto C) and Smac/DIABLO are differentially released from mitochondria (Mito) isolated from Drp1-depleted cells treated with recombinant tBid. (A) Mitochondria isolated from HeLa cells infected with the control (Ctrl), D1, or F1 shRNA retroviruses were incubated in the presence or absence of tBid, and the pellets were alkali treated to determine the amount of Bax insertion. (B) Isolated mitochondria were treated as described for panel A, and the protein contents of the supernatant and pellet were assessed by Western blot analysis with the indicated antibodies.
FIG. 4.
FIG. 4.
Depleting cells of Drp1 or hFis1 does not inhibit apoptosis. (A) HeLa cells were infected with the control (Ctrl), D1, or F1 shRNA retroviruses, grown for 96 h, treated with ActD 3 μM for the indicated times, and stained with annexin V FITC for flow cytometric analysis. The histograms represent a quantitative analysis of the percentage of annexin V FITC-positive cells after induction of apoptosis by ActD in the absence (0 h, 4 h, 8 h, 14 h) or presence (14 h + zVAD-fmk) of the caspase inhibitor zVAD-fmk. The data are expressed as the means + SEMs of the results of three independent experiments. formula image, P < 0.05; formula imageformula image, P < 0.005; formula imageformula imageformula image, P < 0.0005; ns, not significant). Panel B presents data obtained as described for panel A except that apoptosis was triggered by UV irradiation. formula imageformula image, P < 0.005; ns, not significant. (C) Cos-7 cells were cotransfected with pEYFPmito and control, D1, or F1 shRNA constructs or pCiDrpK38A or pCiBcl-Xl; 72 h after transfection, the cells were treated with ActD (6 μM) for 14 h and just before collection the cells were incubated with propidium iodide and Hoechst 33342 for 15 min. The histogram represents a quantitative analysis of the percentage of the propidium iodide-negative Cos-7 cells expressing yellow fluorescent protein (YFP) that displayed apoptotic nuclei as observed by Hoechst 33342 staining. The data are expressed as means + SEMs of the results of three independent experiments.formula imageformula imageformula image, P < 0.0005; ns, not significant. (D) HeLa cells were treated as described for panel B, and the cleavage of caspase-3 was assessed by Western blotting at the indicated times.
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References

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