Original Paper

Cell Death and Differentiation (2005) 12, 453–462. doi:10.1038/sj.cdd.4401596

Cytochrome c is released in a single step during apoptosis

Edited by G Melino

J C Goldstein1,7, C Muñoz-Pinedo1, J-E Ricci1, S R Adams2, A Kelekar3, M Schuler1,4, R Y Tsien5,6 and D R Green1

  1. 1 La Jolla Institute for Allergy and Immunology, 10355 Science Center Dr., San Diego, CA 92121, USA
  2. 2 Department of Pharmacology, University of California, San Diego, La Jolla, CA 92093-0647, USA
  3. 3 Department of Laboratory Medicine and Pathology, University of Minnesota, Minneapolis, MN 55455, USA
  4. 4 Department of Medicine III, Johannes Gutenberg University, Mainz 55101, Germany
  5. 5 HHMI, Department of Chemistry and Biochemistry, and Department of Pharmacology, University of California, San Diego, La Jolla, CA 92093-0647, USA
  6. 6 Department of Pharmacology, University of California, San Diego, La Jolla, CA 92093-0647, USA
  7. 7 Current address: Lawrence Berkeley National Laboratory, MS 84-171, 1 Cyclotron Road, Berkeley, CA 94720, USA

Correspondence: DR Green, La Jolla Institute for Allergy and Immunology, 10355 Science Center Dr., San Diego, CA 92121, USA. Tel: +858-558-3543; Fax: +858-558-3594; E-mail: doug@liai.org

Received 13 December 2004; Accepted 19 January 2005.

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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 (DeltaPsim). 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 DeltaPsim 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 DeltaPsim.

Keywords:

apoptosis, cytochrome c , mitochondria, mitochondrial membrane potential, video microscopy

Abbreviations:

Act D, actinomycin D; CHX, cycloheximide; cyt. c-GFP, cytochrome c-GFP; DeltaPsim, 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-alpha; UV, ultraviolet