Cytochrome c is released in a single step during apoptosis

J. C. Goldstein; C. Muñoz-Pinedo; J. E. Ricci; S. R. Adams; A. Kelekar; M. Schuler; R. Y. Tsien; Doug R. Green

doi:10.1038/sj.cdd.4401596

Cytochrome c is released in a single step during apoptosis

J. C. Goldstein
, C. Muñoz-Pinedo
, J. E. Ricci
, S. R. Adams
, A. Kelekar
, M. Schuler
, R. Y. Tsien
, Doug R. Green

Laboratory Medicine and Pathology

Research output: Contribution to journal › Article › peer-review

200 Scopus citations

Abstract

Release of cytochrome c from mitochondria is a central event in apoptotic signaling. In this study, we utilized a cytochrome c fusion that binds fluorescent biarsenical ligands (cytochrome c-4CYS (cyt. c-4CYS)) as well as cytochrome c-green fluorescent protein (cyt. c-GFP) to measure its release from mitochondria in different cell types during apoptosis. In single cells, the kinetics of cyt. c-4CYS release was indistinguishable from that of cyt. c-GFP in apoptotic cells expressing both molecules. Lowering the temperature by 7°C did not affect this corelease, but further separated cytochrome c release from the subsequent decrease in mitochondrial membrane potential (ΔΨ_m). Cyt. c-GFP rescued respiration in cells lacking endogenous cytochrome c, and the duration of cytochrome c release was approximately 5 min in a variety of cell types induced to die by various forms of cellular stress. In addition, we could observe no evidence of caspase-dependent amplification of cytochrome c release or changes in ΔΨ_m preceding the release of cyt. c-GFP. We conclude that there is a general mechanism responsible for cytochrome c release that proceeds in a single step that is independent of changes in ΔΨ_m.

Original language	English (US)
Pages (from-to)	453-462
Number of pages	10
Journal	Cell Death and Differentiation
Volume	12
Issue number	5
DOIs	https://doi.org/10.1038/sj.cdd.4401596
State	Published - May 2005

Bibliographical note

Funding Information:
This work was supported by NIH Grants AI40646, AI47891, GM52735, and CA69381. C Muñoz-Pinedo is a recipient of a fellowship from the Secretaría de Estado de Educación y Universidades of Spain. We thank G Gaietta, TJ Durnick, DL Price, and MH Ellisman for helpful imaging discussions, AL Nieminen for the gift of the rat cyt. c-GFP construct, T Kanda and GM Wahl for the gift of the H2B-GFP construct, P Fitzgerald and E Ehlert for cloning advice, and CJ Lena for cell sorting.

Keywords

Apoptosis
Cytochrome c
Mitochondria
Mitochondrial membrane potential
Video microscopy

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10.1038/sj.cdd.4401596

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title = "Cytochrome c is released in a single step during apoptosis",

abstract = "Release of cytochrome c from mitochondria is a central event in apoptotic signaling. In this study, we utilized a cytochrome c fusion that binds fluorescent biarsenical ligands (cytochrome c-4CYS (cyt. c-4CYS)) as well as cytochrome c-green fluorescent protein (cyt. c-GFP) to measure its release from mitochondria in different cell types during apoptosis. In single cells, the kinetics of cyt. c-4CYS release was indistinguishable from that of cyt. c-GFP in apoptotic cells expressing both molecules. Lowering the temperature by 7°C did not affect this corelease, but further separated cytochrome c release from the subsequent decrease in mitochondrial membrane potential (ΔΨm). Cyt. c-GFP rescued respiration in cells lacking endogenous cytochrome c, and the duration of cytochrome c release was approximately 5 min in a variety of cell types induced to die by various forms of cellular stress. In addition, we could observe no evidence of caspase-dependent amplification of cytochrome c release or changes in ΔΨm preceding the release of cyt. c-GFP. We conclude that there is a general mechanism responsible for cytochrome c release that proceeds in a single step that is independent of changes in ΔΨm.",

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note = "Funding Information: This work was supported by NIH Grants AI40646, AI47891, GM52735, and CA69381. C Mu{\~n}oz-Pinedo is a recipient of a fellowship from the Secretar{\'i}a de Estado de Educaci{\'o}n y Universidades of Spain. We thank G Gaietta, TJ Durnick, DL Price, and MH Ellisman for helpful imaging discussions, AL Nieminen for the gift of the rat cyt. c-GFP construct, T Kanda and GM Wahl for the gift of the H2B-GFP construct, P Fitzgerald and E Ehlert for cloning advice, and CJ Lena for cell sorting.",

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AU - Goldstein, J. C.

AU - Muñoz-Pinedo, C.

AU - Ricci, J. E.

AU - Adams, S. R.

AU - Kelekar, A.

AU - Schuler, M.

AU - Tsien, R. Y.

AU - Green, Doug R.

N1 - Funding Information: This work was supported by NIH Grants AI40646, AI47891, GM52735, and CA69381. C Muñoz-Pinedo is a recipient of a fellowship from the Secretaría de Estado de Educación y Universidades of Spain. We thank G Gaietta, TJ Durnick, DL Price, and MH Ellisman for helpful imaging discussions, AL Nieminen for the gift of the rat cyt. c-GFP construct, T Kanda and GM Wahl for the gift of the H2B-GFP construct, P Fitzgerald and E Ehlert for cloning advice, and CJ Lena for cell sorting.

PY - 2005/5

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N2 - Release of cytochrome c from mitochondria is a central event in apoptotic signaling. In this study, we utilized a cytochrome c fusion that binds fluorescent biarsenical ligands (cytochrome c-4CYS (cyt. c-4CYS)) as well as cytochrome c-green fluorescent protein (cyt. c-GFP) to measure its release from mitochondria in different cell types during apoptosis. In single cells, the kinetics of cyt. c-4CYS release was indistinguishable from that of cyt. c-GFP in apoptotic cells expressing both molecules. Lowering the temperature by 7°C did not affect this corelease, but further separated cytochrome c release from the subsequent decrease in mitochondrial membrane potential (ΔΨm). Cyt. c-GFP rescued respiration in cells lacking endogenous cytochrome c, and the duration of cytochrome c release was approximately 5 min in a variety of cell types induced to die by various forms of cellular stress. In addition, we could observe no evidence of caspase-dependent amplification of cytochrome c release or changes in ΔΨm preceding the release of cyt. c-GFP. We conclude that there is a general mechanism responsible for cytochrome c release that proceeds in a single step that is independent of changes in ΔΨm.

AB - Release of cytochrome c from mitochondria is a central event in apoptotic signaling. In this study, we utilized a cytochrome c fusion that binds fluorescent biarsenical ligands (cytochrome c-4CYS (cyt. c-4CYS)) as well as cytochrome c-green fluorescent protein (cyt. c-GFP) to measure its release from mitochondria in different cell types during apoptosis. In single cells, the kinetics of cyt. c-4CYS release was indistinguishable from that of cyt. c-GFP in apoptotic cells expressing both molecules. Lowering the temperature by 7°C did not affect this corelease, but further separated cytochrome c release from the subsequent decrease in mitochondrial membrane potential (ΔΨm). Cyt. c-GFP rescued respiration in cells lacking endogenous cytochrome c, and the duration of cytochrome c release was approximately 5 min in a variety of cell types induced to die by various forms of cellular stress. In addition, we could observe no evidence of caspase-dependent amplification of cytochrome c release or changes in ΔΨm preceding the release of cyt. c-GFP. We conclude that there is a general mechanism responsible for cytochrome c release that proceeds in a single step that is independent of changes in ΔΨm.

KW - Apoptosis

KW - Cytochrome c

KW - Mitochondria

KW - Mitochondrial membrane potential

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UR - https://www.scopus.com/pages/publications/18644362771#tab=citedBy

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DO - 10.1038/sj.cdd.4401596

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AN - SCOPUS:18644362771

SN - 1350-9047

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EP - 462

JO - Cell Death and Differentiation

JF - Cell Death and Differentiation

IS - 5

ER -

Cytochrome c is released in a single step during apoptosis

Abstract

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Keywords

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