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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Abbreviations
- Act D:
-
actinomycin D
- CHX:
-
cycloheximide
- cyt. c-GFP:
-
cytochrome c-GFP
- ΔΨm:
-
mitochondrial membrane potential
- EDT:
-
1,2-ethanedithiol
- GFP:
-
green fluorescent protein
- MOMP:
-
mitochondrial outer membrane permeabilization
- STS:
-
staurosporine
- TMRE:
-
tetramethylrhodamine ethyl ester
- TNF:
-
tumor necrosis factor-α
- UV:
-
ultraviolet
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Acknowledgements
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.
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Goldstein, J., Muñoz-Pinedo, C., Ricci, JE. et al. Cytochrome c is released in a single step during apoptosis. Cell Death Differ 12, 453–462 (2005). https://doi.org/10.1038/sj.cdd.4401596
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DOI: https://doi.org/10.1038/sj.cdd.4401596
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