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.
Bibliographical noteFunding 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.
Copyright 2008 Elsevier B.V., All rights reserved.
- Cytochrome c
- Mitochondrial membrane potential
- Video microscopy