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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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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.
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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.

Mentions: Supplemental video of Fig. 4 shows loss and regeneration of ΔΨm after cytochrome c release. Cc-GFP-HeLa cells were treated with actinomycin D (1 μM) in the presence of N-benzoylcarbonyl-Val-Ala-Asp-fluoromethylketone (zVADfmk) (100 μM), and confocal images were taken every 2 min. The cytochrome c–GFP (green, left) shows the coordinate release of cytochrome c in the individual cells (the staining goes from punctate to diffuse upon release). TMRE fluorescence in the same cells (red, right) shows the loss and recovery of ΔΨm. The red and green images are of the same cells taken at the same time. The frames are separate rather than overlaid for clarity, and a mathematical representation of loss and regeneration of ΔΨm in a similarly treated cell is shown in Fig. 4 A. Video is available at http://www.jcb.org/cgi/content/full/153/2/319/DC1.


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)

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.
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Related In: Results  -  Collection

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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.
Mentions: Supplemental video of Fig. 4 shows loss and regeneration of ΔΨm after cytochrome c release. Cc-GFP-HeLa cells were treated with actinomycin D (1 μM) in the presence of N-benzoylcarbonyl-Val-Ala-Asp-fluoromethylketone (zVADfmk) (100 μM), and confocal images were taken every 2 min. The cytochrome c–GFP (green, left) shows the coordinate release of cytochrome c in the individual cells (the staining goes from punctate to diffuse upon release). TMRE fluorescence in the same cells (red, right) shows the loss and recovery of ΔΨm. The red and green images are of the same cells taken at the same time. The frames are separate rather than overlaid for clarity, and a mathematical representation of loss and regeneration of ΔΨm in a similarly treated cell is shown in Fig. 4 A. Video is available at http://www.jcb.org/cgi/content/full/153/2/319/DC1.

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