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Essential role of voltage-dependent anion channel in various forms of apoptosis in mammalian cells.

Shimizu S, Matsuoka Y, Shinohara Y, Yoneda Y, Tsujimoto Y - J. Cell Biol. (2001)

Bottom Line: Through direct interaction with the voltage-dependent anion channel (VDAC), proapoptotic members of the Bcl-2 family such as Bax and Bak induce apoptogenic cytochrome c release in isolated mitochondria, whereas BH3-only proteins such as Bid and Bik do not directly target the VDAC to induce cytochrome c release.When microinjected into cells, these anti-VDAC antibodies, but not control antibodies, also prevented Bax-induced cytochrome c release and apoptosis, whereas the antibodies did not prevent Bid-induced apoptosis, indicating that the VDAC is essential for Bax-induced, but not Bid-induced, apoptogenic mitochondrial changes and apoptotic cell death.Taken together, our data provide evidence that the VDAC plays an essential role in apoptogenic cytochrome c release and apoptosis in mammalian cells.

View Article: PubMed Central - PubMed

Affiliation: Osaka University Graduate School of Medicine, Biomedical Research Center, Department of Medical Genetics, Osaka 565-0871, Japan.

ABSTRACT
Through direct interaction with the voltage-dependent anion channel (VDAC), proapoptotic members of the Bcl-2 family such as Bax and Bak induce apoptogenic cytochrome c release in isolated mitochondria, whereas BH3-only proteins such as Bid and Bik do not directly target the VDAC to induce cytochrome c release. To investigate the biological significance of the VDAC for apoptosis in mammalian cells, we produced two kinds of anti-VDAC antibodies that inhibited VDAC activity. In isolated mitochondria, these antibodies prevented Bax-induced cytochrome c release and loss of the mitochondrial membrane potential (Deltapsi), but not Bid-induced cytochrome c release. When microinjected into cells, these anti-VDAC antibodies, but not control antibodies, also prevented Bax-induced cytochrome c release and apoptosis, whereas the antibodies did not prevent Bid-induced apoptosis, indicating that the VDAC is essential for Bax-induced, but not Bid-induced, apoptogenic mitochondrial changes and apoptotic cell death. In addition, microinjection of these anti-VDAC antibodies significantly inhibited etoposide-, paclitaxel-, and staurosporine-induced apoptosis. Furthermore, we used these antibodies to show that Bax- and Bak-induced lysis of red blood cells was also mediated by the VDAC on plasma membrane. Taken together, our data provide evidence that the VDAC plays an essential role in apoptogenic cytochrome c release and apoptosis in mammalian cells.

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Inhibition of rBax-induced cytochrome c release by anti-VDAC antibodies. HeLa cells were microinjected with 12 μg/μl of NRI or Ab#25 plus 0.4 μg/μl of Cy5-mouse IgG (to identify the injected cells), and then 1 μg/μl of rBax was injected. After 12 h, the intracellular distribution of cytochrome c and that of F1 ATPase (as a mitochondrial marker) was assessed by immunostaining. Fluorescence images of cytochrome c and F1 ATPase are merged in the right panels (overlay).
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Figure 5: Inhibition of rBax-induced cytochrome c release by anti-VDAC antibodies. HeLa cells were microinjected with 12 μg/μl of NRI or Ab#25 plus 0.4 μg/μl of Cy5-mouse IgG (to identify the injected cells), and then 1 μg/μl of rBax was injected. After 12 h, the intracellular distribution of cytochrome c and that of F1 ATPase (as a mitochondrial marker) was assessed by immunostaining. Fluorescence images of cytochrome c and F1 ATPase are merged in the right panels (overlay).

Mentions: To test whether Ab#20 and Ab#25 inhibited rBax-induced cytochrome c release from mitochondria in cells, Cy5-IgG was coinjected together with Ab#20 or Ab#25 to identify injected cells, followed by injection of rBax, and the distribution of cytochrome c was examined in comparison with that of mitochondrial F1 ATPase. In cells injected with NRI and rBax, cytochrome c was diffusely distributed at 12 h, and in some cells, it was mainly localized in the nucleus (Fig. 5), and mitochondria tended to come together in the cells, as defined by F1 ATPase staining. Thus the distribution of cytochrome c was quite different from that of F1 ATPase as seen better in a merged photograph (Fig. 5, top right), indicating that cytochrome c had been released from the mitochondria. On the other hand, in cells injected with Ab#25 and rBax, the distribution of cytochrome c and F1 ATPase was almost completely concordant (Fig. 5), as is the case in healthy cells (data not shown), indicating that Bax-induced cytochrome c release was prevented by Ab#25. Virtually identical results were obtained with Ab#20 (data not shown). These results indicated that the VDAC is essential for Bax-induced cytochrome c release in mammalian cells.


Essential role of voltage-dependent anion channel in various forms of apoptosis in mammalian cells.

Shimizu S, Matsuoka Y, Shinohara Y, Yoneda Y, Tsujimoto Y - J. Cell Biol. (2001)

Inhibition of rBax-induced cytochrome c release by anti-VDAC antibodies. HeLa cells were microinjected with 12 μg/μl of NRI or Ab#25 plus 0.4 μg/μl of Cy5-mouse IgG (to identify the injected cells), and then 1 μg/μl of rBax was injected. After 12 h, the intracellular distribution of cytochrome c and that of F1 ATPase (as a mitochondrial marker) was assessed by immunostaining. Fluorescence images of cytochrome c and F1 ATPase are merged in the right panels (overlay).
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2199613&req=5

Figure 5: Inhibition of rBax-induced cytochrome c release by anti-VDAC antibodies. HeLa cells were microinjected with 12 μg/μl of NRI or Ab#25 plus 0.4 μg/μl of Cy5-mouse IgG (to identify the injected cells), and then 1 μg/μl of rBax was injected. After 12 h, the intracellular distribution of cytochrome c and that of F1 ATPase (as a mitochondrial marker) was assessed by immunostaining. Fluorescence images of cytochrome c and F1 ATPase are merged in the right panels (overlay).
Mentions: To test whether Ab#20 and Ab#25 inhibited rBax-induced cytochrome c release from mitochondria in cells, Cy5-IgG was coinjected together with Ab#20 or Ab#25 to identify injected cells, followed by injection of rBax, and the distribution of cytochrome c was examined in comparison with that of mitochondrial F1 ATPase. In cells injected with NRI and rBax, cytochrome c was diffusely distributed at 12 h, and in some cells, it was mainly localized in the nucleus (Fig. 5), and mitochondria tended to come together in the cells, as defined by F1 ATPase staining. Thus the distribution of cytochrome c was quite different from that of F1 ATPase as seen better in a merged photograph (Fig. 5, top right), indicating that cytochrome c had been released from the mitochondria. On the other hand, in cells injected with Ab#25 and rBax, the distribution of cytochrome c and F1 ATPase was almost completely concordant (Fig. 5), as is the case in healthy cells (data not shown), indicating that Bax-induced cytochrome c release was prevented by Ab#25. Virtually identical results were obtained with Ab#20 (data not shown). These results indicated that the VDAC is essential for Bax-induced cytochrome c release in mammalian cells.

Bottom Line: Through direct interaction with the voltage-dependent anion channel (VDAC), proapoptotic members of the Bcl-2 family such as Bax and Bak induce apoptogenic cytochrome c release in isolated mitochondria, whereas BH3-only proteins such as Bid and Bik do not directly target the VDAC to induce cytochrome c release.When microinjected into cells, these anti-VDAC antibodies, but not control antibodies, also prevented Bax-induced cytochrome c release and apoptosis, whereas the antibodies did not prevent Bid-induced apoptosis, indicating that the VDAC is essential for Bax-induced, but not Bid-induced, apoptogenic mitochondrial changes and apoptotic cell death.Taken together, our data provide evidence that the VDAC plays an essential role in apoptogenic cytochrome c release and apoptosis in mammalian cells.

View Article: PubMed Central - PubMed

Affiliation: Osaka University Graduate School of Medicine, Biomedical Research Center, Department of Medical Genetics, Osaka 565-0871, Japan.

ABSTRACT
Through direct interaction with the voltage-dependent anion channel (VDAC), proapoptotic members of the Bcl-2 family such as Bax and Bak induce apoptogenic cytochrome c release in isolated mitochondria, whereas BH3-only proteins such as Bid and Bik do not directly target the VDAC to induce cytochrome c release. To investigate the biological significance of the VDAC for apoptosis in mammalian cells, we produced two kinds of anti-VDAC antibodies that inhibited VDAC activity. In isolated mitochondria, these antibodies prevented Bax-induced cytochrome c release and loss of the mitochondrial membrane potential (Deltapsi), but not Bid-induced cytochrome c release. When microinjected into cells, these anti-VDAC antibodies, but not control antibodies, also prevented Bax-induced cytochrome c release and apoptosis, whereas the antibodies did not prevent Bid-induced apoptosis, indicating that the VDAC is essential for Bax-induced, but not Bid-induced, apoptogenic mitochondrial changes and apoptotic cell death. In addition, microinjection of these anti-VDAC antibodies significantly inhibited etoposide-, paclitaxel-, and staurosporine-induced apoptosis. Furthermore, we used these antibodies to show that Bax- and Bak-induced lysis of red blood cells was also mediated by the VDAC on plasma membrane. Taken together, our data provide evidence that the VDAC plays an essential role in apoptogenic cytochrome c release and apoptosis in mammalian cells.

Show MeSH
Related in: MedlinePlus