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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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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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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.

Mentions: The proapoptotic Bcl-2 family members tBid and Bax can translocate to the mitochondria during apoptosis and effectively and rapidly trigger mitochondrial outer membrane permeabilization (von Ahsen et al. 2000; Wei et al. 2000). Immunocytochemistry of Cc-GFP-HeLa cells treated with UV (Fig. 7 A) or actinomycin D (not shown) revealed that Bax had translocated to the mitochondria of all cells in which cytochrome c–GFP was in the cytoplasm. Furthermore, the addition of recombinant tBid or Bax to digitonin-permeabilized Cc-GFP-HeLa cells resulted in loss of green fluorescence, indicative of the release of cytochrome c–GFP from mitochondria (Fig. 7 B). Dissipation of the mitochondrial transmembrane potential with FCCP had no effect on the ability of these proapoptotic Bcl-2 family proteins to permeabilize the mitochondrial outer membrane (Fig. 7 B). Furthermore, Bax overexpressed in Cc-GFP-HeLa treated with FCCP induced cytochrome c–GFP release in a similar number of cells as compared with control cells (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)

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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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.
Mentions: The proapoptotic Bcl-2 family members tBid and Bax can translocate to the mitochondria during apoptosis and effectively and rapidly trigger mitochondrial outer membrane permeabilization (von Ahsen et al. 2000; Wei et al. 2000). Immunocytochemistry of Cc-GFP-HeLa cells treated with UV (Fig. 7 A) or actinomycin D (not shown) revealed that Bax had translocated to the mitochondria of all cells in which cytochrome c–GFP was in the cytoplasm. Furthermore, the addition of recombinant tBid or Bax to digitonin-permeabilized Cc-GFP-HeLa cells resulted in loss of green fluorescence, indicative of the release of cytochrome c–GFP from mitochondria (Fig. 7 B). Dissipation of the mitochondrial transmembrane potential with FCCP had no effect on the ability of these proapoptotic Bcl-2 family proteins to permeabilize the mitochondrial outer membrane (Fig. 7 B). Furthermore, Bax overexpressed in Cc-GFP-HeLa treated with FCCP induced cytochrome c–GFP release in a similar number of cells as compared with control cells (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