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. 2001 Apr 16;153(2):319-28.
doi: 10.1083/jcb.153.2.319.

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

N J Waterhouse  1 , J C Goldstein, O von Ahsen, M Schuler, D D Newmeyer, D R Green
Affiliations

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

N J Waterhouse et al. J Cell Biol. .

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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Figures

Figure 1
Figure 1
Loss of ΔΨm is caspase dependent during apoptosis. (A) Jurkat cells treated with etoposide (40 μM) or Cc-GFP-HeLa cells were treated with actinomycin D (1 μM) in the presence or absence of zVADfmk (100 μM), harvested at the times indicated, stained with TMRE (50 nM), and analyzed by flow cytometry. Low fluorescence indicates a loss of ΔΨm. (B) A representation of A, in which untreated cells (thin lines) are overlaid directly on cells treated with the apoptosis inducer in the presence of zVADfmk (zVAD) (100 μM). In the Cc-GFP-Hela cells, 57% of cells had released cytochrome c–GFP by this point.
Figure 2
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.
Figure 5
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.
Figure 3
Figure 3
Time course analysis of ΔΨm during apoptosis. Cc-GFP-HeLa cells stained with TMRE and treated with actinomycin D (1 μM) in the presence (+zVAD) or absence (−zVAD) of 100 μM zVADfmk as indicated, and were followed by time-lapse confocal microscopy. Images were taken every 2 min and the relative brightness of TMRE fluorescence and punctate–diffuse index of cytochrome c–GFP was calculated and plotted over time. (A) Two cells, which commenced cytochrome c–GFP release at 534 and 474 min show a subsequent drop in ΔΨm, commencing at 536 and 478 min, respectively. (B) Similar cells in the absence or presence of zVADfmk (100 μM) were followed by time-lapse confocal microscopy, and the relative brightness of TMRE fluorescence was calculated and plotted over time. For comparative purposes, cytochrome c–GFP release (indicated by arrow) was set at 4 h in each case. (C) The mean brightness of TMRE in individual cells aligned for cytochrome c release at 4 h. n represents the number of cells averaged. Error bars indicate SD.
Figure 4
Figure 4
Dissipation and regeneration of ΔΨm after cytochrome c release in the absence of caspases. (A) Time lapse of the relative brightness of TMRE and the punctate–diffuse index of cytochrome c–GFP in one Cc-GFP-HeLa cell treated with actinomycin D (1 μM) in the presence of zVADfmk showing a drop in ΔΨm, followed by a slow regeneration of ΔΨm after cytochrome c–GFP release. Cytochrome c–GFP release was set at 120 min. (B) Pictographic representation of ΔΨm and cytochrome c–GFP in the cell depicted in A shows that ΔΨm is regenerated, whereas cytochrome c–GFP remains diffuse throughout the cell. More cells can be seen in Quicktime movie format at http://www.jcb.org/cgi/content/full/153/2/319/DC1. In the movie, green (left) indicates cytochrome c–GFP, whereas red (right) indicates TMRE staining. (C and D) Time-lapse of the relative brightness of TMRE fluorescence of one Apaf-1–deficient murine embryonic fibroblast (apaf-deficient MEF) treated with 1 μM actinomycin D (C), one Cc-GFP-HeLa cell treated with 1 μM staurosporine in the presence of 100 μM zVADfmk (Di), or one Cc-GFP-HeLa cell treated with 1 μM staurosporine in the presence of 100 μM zVADfmk and 10 μg/ml of oligomycin (Dii). Bars, 10 μm.
Figure 6
Figure 6
Apoptosis and cytochrome c release proceed in the absence of hyperpolarization. (A) Jurkat cells were treated with etoposide (40 μM) or actinomycin D (500 nM), and Cc-GFP-HeLa cells were treated with UV (180 mJ/cm2) or actinomycin D (1 μM) in the presence or absence of FCCP (5 μM) for the times indicated. The cells were analyzed for phosphatidylserine exposure as a measure of apoptosis. (B) Similar cells to those assayed in A were analyzed for cytochrome c release (Bi) by western blotting (Jurkat) or cytochrome c–GFP release (Bii) by flow cytometry (Cc-GFP-HeLa). (C) Untreated Cc-GFP-HeLa cells or Cc-GFP-HeLa cells treated with UV (180 mJ/cm2) in the presence or absence of the concentrations of FCCP indicated were harvested at 6 h and assayed for phosphatidylserine exposure (annexin V-FITC binding) or cytochrome c–GFP release (CLAMI assay) by flow cytometry.
Figure 7
Figure 7
Bcl-2 family members maintain their proapoptotic and antiapoptotic functions in the presence of uncouplers of ΔΨm. (A) Confocal micrographs of immunocytochemistry of Bax (left) in Cc-GFP-HeLa cells treated with UV (180 mJ/cm2) for 6 h in the absence (top) or presence (bottom) of FCCP (5 μM). Cytochrome c–GFP fluorescence (right) in the same cells is also shown. (B) Permeabilized HeLa cells were treated with tBid (20 μg/ml) or Bax (10 μg/ml) in the absence (left) or presence (right) of FCCP (5 μM). Cytochrome c–GFP fluorescence was detected by flow cytometry in FL-1. Release of cytochrome c–GFP was observed as a drop in overall fluorescence in the cells. (C) Cc-GFP-HeLa cells, which overexpress Bcl-2, were treated with UV (180 mJ/cm2) in the presence or absence of FCCP (5 μM), CCCP (10 μM), or DNP (800 μM). Cells were harvested after 6 h and assayed for phosphatidylserine exposure or cytochrome c–GFP release. The extent of annexin V binding and cytochrome c–GFP release was compared with cells that did not express Bcl-2, which were treated with UV at the same time. Bars, 20 μm.
Figure 7
Figure 7
Bcl-2 family members maintain their proapoptotic and antiapoptotic functions in the presence of uncouplers of ΔΨm. (A) Confocal micrographs of immunocytochemistry of Bax (left) in Cc-GFP-HeLa cells treated with UV (180 mJ/cm2) for 6 h in the absence (top) or presence (bottom) of FCCP (5 μM). Cytochrome c–GFP fluorescence (right) in the same cells is also shown. (B) Permeabilized HeLa cells were treated with tBid (20 μg/ml) or Bax (10 μg/ml) in the absence (left) or presence (right) of FCCP (5 μM). Cytochrome c–GFP fluorescence was detected by flow cytometry in FL-1. Release of cytochrome c–GFP was observed as a drop in overall fluorescence in the cells. (C) Cc-GFP-HeLa cells, which overexpress Bcl-2, were treated with UV (180 mJ/cm2) in the presence or absence of FCCP (5 μM), CCCP (10 μM), or DNP (800 μM). Cells were harvested after 6 h and assayed for phosphatidylserine exposure or cytochrome c–GFP release. The extent of annexin V binding and cytochrome c–GFP release was compared with cells that did not express Bcl-2, which were treated with UV at the same time. Bars, 20 μm.
Figure 7
Figure 7
Bcl-2 family members maintain their proapoptotic and antiapoptotic functions in the presence of uncouplers of ΔΨm. (A) Confocal micrographs of immunocytochemistry of Bax (left) in Cc-GFP-HeLa cells treated with UV (180 mJ/cm2) for 6 h in the absence (top) or presence (bottom) of FCCP (5 μM). Cytochrome c–GFP fluorescence (right) in the same cells is also shown. (B) Permeabilized HeLa cells were treated with tBid (20 μg/ml) or Bax (10 μg/ml) in the absence (left) or presence (right) of FCCP (5 μM). Cytochrome c–GFP fluorescence was detected by flow cytometry in FL-1. Release of cytochrome c–GFP was observed as a drop in overall fluorescence in the cells. (C) Cc-GFP-HeLa cells, which overexpress Bcl-2, were treated with UV (180 mJ/cm2) in the presence or absence of FCCP (5 μM), CCCP (10 μM), or DNP (800 μM). Cells were harvested after 6 h and assayed for phosphatidylserine exposure or cytochrome c–GFP release. The extent of annexin V binding and cytochrome c–GFP release was compared with cells that did not express Bcl-2, which were treated with UV at the same time. Bars, 20 μm.
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