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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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Effect of the microinjection of anti-VDAC antibodies. (A) Localization of injected Ab#25. HeLa cells were microinjected with 3 μg/μl of Ab#25. After fixing, the cells were incubated with anti-cytochrome c antibody for 12 h at 4°C, and then with anti–rabbit IgG-Alexa488 (which reacted with Ab#25) and anti–mouse IgG-Alexa568 (which reacted with anti-cytochrome c antibody), followed by observation under a fluorescence microscope. (B) Lack of inhibition of mitochondrial respiration by Ab#25. HeLa cells were microinjected with 12 μg/μl of Ab#25. Then noninjected and injected cells were incubated in glucose-containing (+ glc) or glucose-free (− glc) medium in the presence or absence of 10 μM oligomycin (oligo). After 24 h, cells were stained with JC-1 dye and observed under a fluorescence microscope. The dye gave an orange color to cells with a high Δψ and a green color to cells with a low Δψ.
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Figure 3: Effect of the microinjection of anti-VDAC antibodies. (A) Localization of injected Ab#25. HeLa cells were microinjected with 3 μg/μl of Ab#25. After fixing, the cells were incubated with anti-cytochrome c antibody for 12 h at 4°C, and then with anti–rabbit IgG-Alexa488 (which reacted with Ab#25) and anti–mouse IgG-Alexa568 (which reacted with anti-cytochrome c antibody), followed by observation under a fluorescence microscope. (B) Lack of inhibition of mitochondrial respiration by Ab#25. HeLa cells were microinjected with 12 μg/μl of Ab#25. Then noninjected and injected cells were incubated in glucose-containing (+ glc) or glucose-free (− glc) medium in the presence or absence of 10 μM oligomycin (oligo). After 24 h, cells were stained with JC-1 dye and observed under a fluorescence microscope. The dye gave an orange color to cells with a high Δψ and a green color to cells with a low Δψ.

Mentions: To determine whether the VDAC had an essential role in Bax-induced apoptosis in mammalian cells, we carried out an experiment in which Ab#20 and Ab#25 were microinjected into the cytoplasm of HeLa cells. Consistent with the predominant localization of VDAC in the mitochondria, Ab#25 was mainly observed in the mitochondria by immunostaining, when injected at a lower concentration (3 μg/μl) (Fig. 3 A), whereas it was observed in the mitochondria with a small amount throughout the cytoplasm when injected at a higher concentration of 12 μg/μl (data not shown), suggesting that a concentration of 3 μg/μl was less than the saturation level for mitochondrial VDAC. Therefore, microinjection experiments were mainly done with Ab#20 and Ab#25 at 12 μg/μl, which was close to the saturation level for mitochondrial VDAC. Injection of these antibodies showed little toxicity in HeLa cells (data not shown). To test the effect of Ab#20 and Ab#25 on mitochondrial respiration, cells were incubated in glucose-free medium for 24 h. Since glycolysis was halted under these conditions, inhibition of mitochondrial respiration (e.g., by the addition of oligomycin, an F1 ATPase inhibitor) led to mitochondrial Δψ loss (Fig. 3 B, second panel). However, when cells were microinjected with Ab#20 or Ab#25, Δψ remained high during incubation in glucose-free medium (Fig. 3 B, far right panel; data not shown), indicating that these antibodies did not significantly affect mitochondrial respiration, consistent with the results obtained using isolated mitochondria (Fig. 2 D).


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)

Effect of the microinjection of anti-VDAC antibodies. (A) Localization of injected Ab#25. HeLa cells were microinjected with 3 μg/μl of Ab#25. After fixing, the cells were incubated with anti-cytochrome c antibody for 12 h at 4°C, and then with anti–rabbit IgG-Alexa488 (which reacted with Ab#25) and anti–mouse IgG-Alexa568 (which reacted with anti-cytochrome c antibody), followed by observation under a fluorescence microscope. (B) Lack of inhibition of mitochondrial respiration by Ab#25. HeLa cells were microinjected with 12 μg/μl of Ab#25. Then noninjected and injected cells were incubated in glucose-containing (+ glc) or glucose-free (− glc) medium in the presence or absence of 10 μM oligomycin (oligo). After 24 h, cells were stained with JC-1 dye and observed under a fluorescence microscope. The dye gave an orange color to cells with a high Δψ and a green color to cells with a low Δψ.
© Copyright Policy
Related In: Results  -  Collection

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

Figure 3: Effect of the microinjection of anti-VDAC antibodies. (A) Localization of injected Ab#25. HeLa cells were microinjected with 3 μg/μl of Ab#25. After fixing, the cells were incubated with anti-cytochrome c antibody for 12 h at 4°C, and then with anti–rabbit IgG-Alexa488 (which reacted with Ab#25) and anti–mouse IgG-Alexa568 (which reacted with anti-cytochrome c antibody), followed by observation under a fluorescence microscope. (B) Lack of inhibition of mitochondrial respiration by Ab#25. HeLa cells were microinjected with 12 μg/μl of Ab#25. Then noninjected and injected cells were incubated in glucose-containing (+ glc) or glucose-free (− glc) medium in the presence or absence of 10 μM oligomycin (oligo). After 24 h, cells were stained with JC-1 dye and observed under a fluorescence microscope. The dye gave an orange color to cells with a high Δψ and a green color to cells with a low Δψ.
Mentions: To determine whether the VDAC had an essential role in Bax-induced apoptosis in mammalian cells, we carried out an experiment in which Ab#20 and Ab#25 were microinjected into the cytoplasm of HeLa cells. Consistent with the predominant localization of VDAC in the mitochondria, Ab#25 was mainly observed in the mitochondria by immunostaining, when injected at a lower concentration (3 μg/μl) (Fig. 3 A), whereas it was observed in the mitochondria with a small amount throughout the cytoplasm when injected at a higher concentration of 12 μg/μl (data not shown), suggesting that a concentration of 3 μg/μl was less than the saturation level for mitochondrial VDAC. Therefore, microinjection experiments were mainly done with Ab#20 and Ab#25 at 12 μg/μl, which was close to the saturation level for mitochondrial VDAC. Injection of these antibodies showed little toxicity in HeLa cells (data not shown). To test the effect of Ab#20 and Ab#25 on mitochondrial respiration, cells were incubated in glucose-free medium for 24 h. Since glycolysis was halted under these conditions, inhibition of mitochondrial respiration (e.g., by the addition of oligomycin, an F1 ATPase inhibitor) led to mitochondrial Δψ loss (Fig. 3 B, second panel). However, when cells were microinjected with Ab#20 or Ab#25, Δψ remained high during incubation in glucose-free medium (Fig. 3 B, far right panel; data not shown), indicating that these antibodies did not significantly affect mitochondrial respiration, consistent with the results obtained using isolated mitochondria (Fig. 2 D).

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