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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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Cytochrome c concentration limits respiration in mitochondria that have undergone outer membrane permeabilization. (A) Permeabilized cells stained with TMRE (50 nM), either untreated or treated with tBid, were incubated at 37°C for 20 min with the concentrations of horse heart cytochrome c indicated. ΔΨm was measured by flow cytometry. FCCP (10 μM) was used as a control for depolarized mitochondria. KCN (1 mM) was used to block the involvement of cytochrome c in the electron transport chain. (B) Cc-GFP-HeLa cells were treated for 12 h with actinomycin D (1 μM) and stained with TMRE (50 nM). The cells were permeabilized with digitonin and incubated for 20 min with horse heart cytochrome c. ΔΨm and cytochrome c–GFP were measured by flow cytometry. The cells were gated for cytochrome c–GFP release, and the relative fluorescence of TMRE was compared with that of cells that had not released cytochrome c–GFP. Error bars indicate SD.
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Figure 2: Cytochrome c concentration limits respiration in mitochondria that have undergone outer membrane permeabilization. (A) Permeabilized cells stained with TMRE (50 nM), either untreated or treated with tBid, were incubated at 37°C for 20 min with the concentrations of horse heart cytochrome c indicated. ΔΨm was measured by flow cytometry. FCCP (10 μM) was used as a control for depolarized mitochondria. KCN (1 mM) was used to block the involvement of cytochrome c in the electron transport chain. (B) Cc-GFP-HeLa cells were treated for 12 h with actinomycin D (1 μM) and stained with TMRE (50 nM). The cells were permeabilized with digitonin and incubated for 20 min with horse heart cytochrome c. ΔΨm and cytochrome c–GFP were measured by flow cytometry. The cells were gated for cytochrome c–GFP release, and the relative fluorescence of TMRE was compared with that of cells that had not released cytochrome c–GFP. Error bars indicate SD.

Mentions: Cc-GFP-HeLa cells (2 × 106) were incubated for 20 min at 37°C in media containing TMRE (50 nM). Buffers in all subsequent steps contained TMRE (50 nM). The cells were trypsinized and incubated for 5 min on ice in 1 ml of ice-cold mitochondria isolation buffer (MIB) (200 mM mannitol, 50 mM sucrose, 10 mM Hepes, 10 mM succinate, 70 mM KCl, 1 mM DTT) containing 50 μg/ml digitonin. When >95% of cells were permeable to Trypan blue, the cells were washed twice in ice-cold MIB containing 0.1% BSA. For Fig. 2 A, the cells were incubated for 30 min at 37°C in 1 ml of MIB containing truncated Bid (tBid) (20 μg/ml) and diluted in 1:10 in MIB containing BSA (0.1%) ATP (1 mM), creatin-phosphate (5 mM), creatin kinase (0.1 mg/ml), oligomycin (10 μg/ml), and the concentrations of cytochrome c indicated. Carbonyl cyanide p-(trifluoromethoxy) phenylhydrazone (FCCP) (10 μM) and KCN (1 mM) were added as indicated. The cells were analyzed by flow cytometry after 20 min, measuring cytochrome c–GFP fluorescence in FL-1 and TMRE fluorescence in FL-2. Analysis confirmed that >95% of cells treated with tBid had released cytochrome c–GFP. For Fig. 2 B, the permeabilized apoptotic cells were directly resuspended in the MIB containing cytochrome c, and the cells were analyzed by flow cytometry after 20 min.


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)

Cytochrome c concentration limits respiration in mitochondria that have undergone outer membrane permeabilization. (A) Permeabilized cells stained with TMRE (50 nM), either untreated or treated with tBid, were incubated at 37°C for 20 min with the concentrations of horse heart cytochrome c indicated. ΔΨm was measured by flow cytometry. FCCP (10 μM) was used as a control for depolarized mitochondria. KCN (1 mM) was used to block the involvement of cytochrome c in the electron transport chain. (B) Cc-GFP-HeLa cells were treated for 12 h with actinomycin D (1 μM) and stained with TMRE (50 nM). The cells were permeabilized with digitonin and incubated for 20 min with horse heart cytochrome c. ΔΨm and cytochrome c–GFP were measured by flow cytometry. The cells were gated for cytochrome c–GFP release, and the relative fluorescence of TMRE was compared with that of cells that had not released cytochrome c–GFP. Error bars indicate SD.
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Related In: Results  -  Collection

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Figure 2: Cytochrome c concentration limits respiration in mitochondria that have undergone outer membrane permeabilization. (A) Permeabilized cells stained with TMRE (50 nM), either untreated or treated with tBid, were incubated at 37°C for 20 min with the concentrations of horse heart cytochrome c indicated. ΔΨm was measured by flow cytometry. FCCP (10 μM) was used as a control for depolarized mitochondria. KCN (1 mM) was used to block the involvement of cytochrome c in the electron transport chain. (B) Cc-GFP-HeLa cells were treated for 12 h with actinomycin D (1 μM) and stained with TMRE (50 nM). The cells were permeabilized with digitonin and incubated for 20 min with horse heart cytochrome c. ΔΨm and cytochrome c–GFP were measured by flow cytometry. The cells were gated for cytochrome c–GFP release, and the relative fluorescence of TMRE was compared with that of cells that had not released cytochrome c–GFP. Error bars indicate SD.
Mentions: Cc-GFP-HeLa cells (2 × 106) were incubated for 20 min at 37°C in media containing TMRE (50 nM). Buffers in all subsequent steps contained TMRE (50 nM). The cells were trypsinized and incubated for 5 min on ice in 1 ml of ice-cold mitochondria isolation buffer (MIB) (200 mM mannitol, 50 mM sucrose, 10 mM Hepes, 10 mM succinate, 70 mM KCl, 1 mM DTT) containing 50 μg/ml digitonin. When >95% of cells were permeable to Trypan blue, the cells were washed twice in ice-cold MIB containing 0.1% BSA. For Fig. 2 A, the cells were incubated for 30 min at 37°C in 1 ml of MIB containing truncated Bid (tBid) (20 μg/ml) and diluted in 1:10 in MIB containing BSA (0.1%) ATP (1 mM), creatin-phosphate (5 mM), creatin kinase (0.1 mg/ml), oligomycin (10 μg/ml), and the concentrations of cytochrome c indicated. Carbonyl cyanide p-(trifluoromethoxy) phenylhydrazone (FCCP) (10 μM) and KCN (1 mM) were added as indicated. The cells were analyzed by flow cytometry after 20 min, measuring cytochrome c–GFP fluorescence in FL-1 and TMRE fluorescence in FL-2. Analysis confirmed that >95% of cells treated with tBid had released cytochrome c–GFP. For Fig. 2 B, the permeabilized apoptotic cells were directly resuspended in the MIB containing cytochrome c, and the cells were analyzed by flow cytometry after 20 min.

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