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Cytochrome c maintains mitochondrial transmembrane potential and ATP generation after outer mitochondrial membrane permeabilization during the apoptotic process.

Waterhouse NJ, Goldstein JC, von Ahsen O, Schuler M, Newmeyer DD, Green DR - J. Cell Biol. (2001)

Bottom Line: After outer membrane permeabilization, mitochondria can use cytoplasmic cytochrome c to maintain mitochondrial transmembrane potential and ATP production.Furthermore, both cytochrome c release and apoptosis proceed normally in cells in which mitochondria have been uncoupled.These studies demonstrate that cytochrome c release does not affect the integrity of the mitochondrial inner membrane and that, in the absence of caspase activation, mitochondrial functions can be maintained after the release of cytochrome c.

View Article: PubMed Central - PubMed

Affiliation: Division of Cellular Immunology, La Jolla Institute for Allergy and Immunology, San Diego, California 92121, USA.

ABSTRACT
During apoptosis, cytochrome c is released into the cytosol as the outer membrane of mitochondria becomes permeable, and this acts to trigger caspase activation. The consequences of this release for mitochondrial metabolism are unclear. Using single-cell analysis, we found that when caspase activity is inhibited, mitochondrial outer membrane permeabilization causes a rapid depolarization of mitochondrial transmembrane potential, which recovers to original levels over the next 30-60 min and is then maintained. After outer membrane permeabilization, mitochondria can use cytoplasmic cytochrome c to maintain mitochondrial transmembrane potential and ATP production. Furthermore, both cytochrome c release and apoptosis proceed normally in cells in which mitochondria have been uncoupled. These studies demonstrate that cytochrome c release does not affect the integrity of the mitochondrial inner membrane and that, in the absence of caspase activation, mitochondrial functions can be maintained after the release of cytochrome c.

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Mitochondria that maintain ΔΨm after cytochrome c release also produce ATP. (A) Cc-GFP-HeLa cells cultured in the absence of glucose for 12–15 h were treated with actinomycin D (1 μM) in the presence or absence of zVADfmk (100 μM). (Ai) After 12 h, the cells were stained with TMRE (50 nM) and analyzed by flow cytometry. Cells treated in the presence of zVADfmk, maintained ΔΨm. (Aii) Similarly treated cells were harvested at the times indicated and percentage of cells with polarized mitochondria, and the percentage of cells that had not released cytochrome c were determined by flow cytometry. (Aiii) Aliquots of cells in Aii were analyzed for total cellular ATP. (B) Cc-GFP-HeLa cells cultured in the absence of glucose for 12–15 h were treated with actinomycin D (1 μM) in the presence or absence of zVADfmk (100 μM). After 12 h, when ∼80% of the cells had released cytochrome c, oligomycin (10 μg/ml) was added to the sample indicated. All cells were harvested 1 h later, and total cellular ATP was measured. Error bars indicate SEM.
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Figure 5: Mitochondria that maintain ΔΨm after cytochrome c release also produce ATP. (A) Cc-GFP-HeLa cells cultured in the absence of glucose for 12–15 h were treated with actinomycin D (1 μM) in the presence or absence of zVADfmk (100 μM). (Ai) After 12 h, the cells were stained with TMRE (50 nM) and analyzed by flow cytometry. Cells treated in the presence of zVADfmk, maintained ΔΨm. (Aii) Similarly treated cells were harvested at the times indicated and percentage of cells with polarized mitochondria, and the percentage of cells that had not released cytochrome c were determined by flow cytometry. (Aiii) Aliquots of cells in Aii were analyzed for total cellular ATP. (B) Cc-GFP-HeLa cells cultured in the absence of glucose for 12–15 h were treated with actinomycin D (1 μM) in the presence or absence of zVADfmk (100 μM). After 12 h, when ∼80% of the cells had released cytochrome c, oligomycin (10 μg/ml) was added to the sample indicated. All cells were harvested 1 h later, and total cellular ATP was measured. Error bars indicate SEM.

Mentions: One of the major functions of the electron transport chain is to generate ATP via complex V. If, after the permeabilization of the outer membrane, the mitochondria can use the decreased amounts of cytochrome c to sustain electron transport, then ATP levels should be maintained. We therefore examined ATP levels in Cc-GFP-HeLa cells deprived of glucose and provided with pyruvate to ensure that the majority of ATP generation was dependent on mitochondrial electron transport and the function of complex V. Consistent with our hypothesis, we noted a decrease in ATP levels in the glucose-deprived cells upon treatment with oligomycin for 1 h (indicated by the dashed lines in Fig. 5 Aiii), and the majority of cells died within 12 h (not shown). In the presence of glucose, Cc-GFP-HeLa cells were resistant to oligomycin-induced death for >3 d (not shown).


Cytochrome c maintains mitochondrial transmembrane potential and ATP generation after outer mitochondrial membrane permeabilization during the apoptotic process.

Waterhouse NJ, Goldstein JC, von Ahsen O, Schuler M, Newmeyer DD, Green DR - J. Cell Biol. (2001)

Mitochondria that maintain ΔΨm after cytochrome c release also produce ATP. (A) Cc-GFP-HeLa cells cultured in the absence of glucose for 12–15 h were treated with actinomycin D (1 μM) in the presence or absence of zVADfmk (100 μM). (Ai) After 12 h, the cells were stained with TMRE (50 nM) and analyzed by flow cytometry. Cells treated in the presence of zVADfmk, maintained ΔΨm. (Aii) Similarly treated cells were harvested at the times indicated and percentage of cells with polarized mitochondria, and the percentage of cells that had not released cytochrome c were determined by flow cytometry. (Aiii) Aliquots of cells in Aii were analyzed for total cellular ATP. (B) Cc-GFP-HeLa cells cultured in the absence of glucose for 12–15 h were treated with actinomycin D (1 μM) in the presence or absence of zVADfmk (100 μM). After 12 h, when ∼80% of the cells had released cytochrome c, oligomycin (10 μg/ml) was added to the sample indicated. All cells were harvested 1 h later, and total cellular ATP was measured. Error bars indicate SEM.
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Related In: Results  -  Collection

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Figure 5: Mitochondria that maintain ΔΨm after cytochrome c release also produce ATP. (A) Cc-GFP-HeLa cells cultured in the absence of glucose for 12–15 h were treated with actinomycin D (1 μM) in the presence or absence of zVADfmk (100 μM). (Ai) After 12 h, the cells were stained with TMRE (50 nM) and analyzed by flow cytometry. Cells treated in the presence of zVADfmk, maintained ΔΨm. (Aii) Similarly treated cells were harvested at the times indicated and percentage of cells with polarized mitochondria, and the percentage of cells that had not released cytochrome c were determined by flow cytometry. (Aiii) Aliquots of cells in Aii were analyzed for total cellular ATP. (B) Cc-GFP-HeLa cells cultured in the absence of glucose for 12–15 h were treated with actinomycin D (1 μM) in the presence or absence of zVADfmk (100 μM). After 12 h, when ∼80% of the cells had released cytochrome c, oligomycin (10 μg/ml) was added to the sample indicated. All cells were harvested 1 h later, and total cellular ATP was measured. Error bars indicate SEM.
Mentions: One of the major functions of the electron transport chain is to generate ATP via complex V. If, after the permeabilization of the outer membrane, the mitochondria can use the decreased amounts of cytochrome c to sustain electron transport, then ATP levels should be maintained. We therefore examined ATP levels in Cc-GFP-HeLa cells deprived of glucose and provided with pyruvate to ensure that the majority of ATP generation was dependent on mitochondrial electron transport and the function of complex V. Consistent with our hypothesis, we noted a decrease in ATP levels in the glucose-deprived cells upon treatment with oligomycin for 1 h (indicated by the dashed lines in Fig. 5 Aiii), and the majority of cells died within 12 h (not shown). In the presence of glucose, Cc-GFP-HeLa cells were resistant to oligomycin-induced death for >3 d (not shown).

Bottom Line: After outer membrane permeabilization, mitochondria can use cytoplasmic cytochrome c to maintain mitochondrial transmembrane potential and ATP production.Furthermore, both cytochrome c release and apoptosis proceed normally in cells in which mitochondria have been uncoupled.These studies demonstrate that cytochrome c release does not affect the integrity of the mitochondrial inner membrane and that, in the absence of caspase activation, mitochondrial functions can be maintained after the release of cytochrome c.

View Article: PubMed Central - PubMed

Affiliation: Division of Cellular Immunology, La Jolla Institute for Allergy and Immunology, San Diego, California 92121, USA.

ABSTRACT
During apoptosis, cytochrome c is released into the cytosol as the outer membrane of mitochondria becomes permeable, and this acts to trigger caspase activation. The consequences of this release for mitochondrial metabolism are unclear. Using single-cell analysis, we found that when caspase activity is inhibited, mitochondrial outer membrane permeabilization causes a rapid depolarization of mitochondrial transmembrane potential, which recovers to original levels over the next 30-60 min and is then maintained. After outer membrane permeabilization, mitochondria can use cytoplasmic cytochrome c to maintain mitochondrial transmembrane potential and ATP production. Furthermore, both cytochrome c release and apoptosis proceed normally in cells in which mitochondria have been uncoupled. These studies demonstrate that cytochrome c release does not affect the integrity of the mitochondrial inner membrane and that, in the absence of caspase activation, mitochondrial functions can be maintained after the release of cytochrome c.

Show MeSH
Related in: MedlinePlus