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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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Related in: MedlinePlus

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.
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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.

Mentions: Previously, we have found that in CEM and HeLa cells treated with UV radiation in the presence of the caspase inhibitor zVADfmk, cytochrome c can be released without significantly affecting ΔΨm (Bossy-Wetzel et al. 1998). To confirm this finding in the cell lines used in the this study, we treated Jurkat or Cc-GFP-HeLa cells with actinomycin D, staurosporine, UV, or etoposide and examined ΔΨm by TMRE staining. As shown in Fig. 1, loss of ΔΨm in Jurkat cells treated with etoposide or Cc-GFP-HeLa cells treated with actinomycin D was prevented by addition of the caspase inhibitor. Similar results were obtained in both cell lines treated with staurosporine and in Cc-GFP-HeLa cells treated with UV (data not shown), and when other dyes including CMTM-ROS and DiOC(6)3 were used to measure ΔΨm (not shown). In every case, zVADfmk did not affect cytochrome c release in Cc-GFP-HeLa cells (Goldstein et al. 2000).


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)

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.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2169468&req=5

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.
Mentions: Previously, we have found that in CEM and HeLa cells treated with UV radiation in the presence of the caspase inhibitor zVADfmk, cytochrome c can be released without significantly affecting ΔΨm (Bossy-Wetzel et al. 1998). To confirm this finding in the cell lines used in the this study, we treated Jurkat or Cc-GFP-HeLa cells with actinomycin D, staurosporine, UV, or etoposide and examined ΔΨm by TMRE staining. As shown in Fig. 1, loss of ΔΨm in Jurkat cells treated with etoposide or Cc-GFP-HeLa cells treated with actinomycin D was prevented by addition of the caspase inhibitor. Similar results were obtained in both cell lines treated with staurosporine and in Cc-GFP-HeLa cells treated with UV (data not shown), and when other dyes including CMTM-ROS and DiOC(6)3 were used to measure ΔΨm (not shown). In every case, zVADfmk did not affect cytochrome c release in Cc-GFP-HeLa cells (Goldstein et al. 2000).

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