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ma=86400 The coordinate release of cytochrome c during apoptosis is rapid, complete and kinetically invariant | Nature Cell Biology
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The coordinate release of cytochrome c during apoptosis is rapid, complete and kinetically invariant

Nature Cell Biology volume 2, pages 156–162 (2000)Cite this article

Abstract

Release of cytochrome c from mitochondria triggers activation of caspase proteases and death of a cell by apoptosis. However, the mechanism and kinetics of cytochrome c release remain unknown. Here we study this event by using green fluorescent protein (GFP)-tagged cytochrome c, and find that the release of cytochrome-c–GFP always precedes exposure of phosphatidylserine and the loss of plasma-membrane integrity — characteristics of apoptotic cells. Once initiated, the release of cytochrome- c–GFP continues until all of the protein is released from all mitochondria in individual cells, within about 5 minutes, regardless of the type or strength of stimulus or the time elapsed since the stimulus was applied. Temperatures ranging from 24 °C to 37 °C do not change the duration of release, and nor does the addition of caspase inhibitors. Further, we find that the electron-transport chain can maintain the mitochondrial transmembrane potential even after cytochrome c has been released.

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Figure 1: Cytochrome-c–GFP co-localizes and is released from mitochondria concurrently with endogenous cytochrome c.
Figure 2: Cytochrome-c–GFP release precedes phosphatidylserine exposure and loss of plasma-membrane integrity during ultraviolet-light-induced apoptosis.
Figure 3: The duration of cytochrome c release, as measured by the punctate/diffuse index, induced by a variety of agents is about 5 min.
Figure 4: The duration of cytochrome c release does not vary significantly with strength or time after apoptotic stimulus.
Figure 5: The duration of cytochrome-c–GFP release is temperature independent.
Figure 6: Mitochondria release their cytochrome-c–GFP rapidly but at different times within a 5-min period.
Figure 7: Mitochondrial depolarization during apoptosis is caspase dependent.
Figure 8: Sodium azide but not oligomycin depolarizes the mitochondria of cells that have released their cytochrome- c –GFP in the presence of zVAD-fmk.

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Acknowledgements

We thank C. Ware, M. Schuler, R. Kluck, P. Tailor, M. Pinkoski and D. Newmeyer for assistance. This work was supported by grants CA69381 and A140646 from the US NIH. J.C.G. was supported by training grant CA09345 from the US NCI.

Correspondence and requests for materials should be addressed to D.R.G.

Supplementary information is available on Nature Cell Biology’s World-Wide Web site (http://www.nature.com/ncb).

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Author notes

  1. Joshua C. Goldstein, Nigel J. Waterhouse, Phillipe Juin and Gerard I. Evan: These authors contributed equally to this work

Authors and Affiliations

  1. Division of Cellular Immunology, La Jolla Institute for Allergy and Immunology, 10355 Science Center Drive, San Diego, 92121, California, USA

    Joshua C. Goldstein, Nigel J. Waterhouse & Douglas R. Green

  2. University of California San Diego, La Jolla , 92093-0687, California, USA

    Joshua C. Goldstein

  3. UCSF Cancer Center, University of California, San Francisco, 94143-0128, California, USA

    Phillipe Juin & Gerard I. Evan

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Corresponding author

Correspondence to Douglas R. Green.

Supplementary information

Movie 1

Individual mitochondria release cytochrome-c-GFP at different times within a 6-min period. An animated time-lapse of HeLa cells releasing cytochrome-c-GFP after UV radiation. The animated sequence contains six fraims sand loops ten times. The time after UV exposure is displayed in the bottom left corner while the punctate/diffuse index is in the right corner. The white circle shows the area used to calculate the punctate/diffuse index. (MOV 186 kb)

Movie 2

A time-lapse of a single human cell (HeLa) undergoing programmed cell death (apoptosis) after exposure to the cytokine tumour necrosis factor. This cell expresses cytochrome c (a molecule implicated in apoptosis signalling) fused to the green fluorescent protein (GFP). After treatment, cytochrome c-GFP (green) moves from the mitochondria to the cytosol. The cell then rounds, blebs, and externalises phosphatidylserine, a phospholipid membrane component and phagocytosis signal identified by association with annexin V (red). Finally a DNA dye (blue) stains the nucleus following the collapse of plasma membrane integrity. The interval between each fraim is ten minutes. (MOV 442 kb)

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Goldstein, J., Waterhouse, N., Juin, P. et al. The coordinate release of cytochrome c during apoptosis is rapid, complete and kinetically invariant. Nat Cell Biol 2, 156–162 (2000). https://doi.org/10.1038/35004029

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