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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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References

  1. Wolf, B. B. & Green, D. R. Suicidal tendencies: apoptotic cell death by caspase family proteinases. J. Biol. Chem. 274, 20049–20052 (1999).

    Article  CAS  Google Scholar 

  2. Slee, E. A. et al. Ordering the cytochrome c-initiated caspase cascade: hierarchical activation of caspases-2, -3, -6, -7, -8, and -10 in a caspase-9-dependent manner. J. Cell Biol. 144, 281– 292 (1999).

    Article  CAS  Google Scholar 

  3. Nicholson, D. W. & Thornberry, N. A. Caspases: killer proteases. Trends Biochem. Sci. 22, 299–306 (1997).

    Article  CAS  Google Scholar 

  4. Li, P. et al. Cytochrome c and dATP-dependent formation of Apaf-1/caspase-9 complex initiates an apoptotic protease cascade. Cell 91, 479–489 (1997).

    Article  CAS  Google Scholar 

  5. Zou, H., Li, Y., Liu, X. & Wang, X. An APAF-1/cytochrome c multimeric complex is a functional apoptosome that activates procaspase-9 . J. Biol. Chem. 274, 11549– 11556 (1999).

    Article  CAS  Google Scholar 

  6. Zou, H., Henzel, W. J., Liu, X., Lutschg, A. & Wang, X. Apaf-1, a human protein homologous to C. elegans CED-4, participates in cytochrome c-dependent activation of caspase-3 . Cell 90, 405–413 (1997).

    Article  CAS  Google Scholar 

  7. Cecconi, F., Alvarez-Bolado, G., Meyer, B. I., Roth, K. A. & Gruss, P. Apaf 1 (CED-4 homolog) regulates programmed cell death in mammalian development. Cell 94, 727–737 (1998).

    Article  CAS  Google Scholar 

  8. Hu, Y., Benedict, M. A., Ding, L. & Nunez, G. Role of cytochrome c and dATP/ATP hydrolysis in Apaf-1-mediated caspase- 9 activation and apoptosis. EMBO J. 18, 3586–3595 (1999).

    Article  CAS  Google Scholar 

  9. Srinivasula, S. M., Ahmad, M., Fernandes-Alnemri, T. & Alnemri, E. S. Autoactivation of procaspase-9 by Apaf-1-mediated oligomerization. Mol. Cell 1, 949–957 ( 1998).

    Article  CAS  Google Scholar 

  10. Kennedy, S. G., Kandel, E. S., Cross, T. K. & Hay, N. Akt/Protein kinase B inhibits cell death by preventing the release of cytochrome c from mitochondria. Mol. Cell. Biol. 19, 5800–5810 (1999).

    Article  CAS  Google Scholar 

  11. Narita, M. et al. Bax interacts with the permeability transition pore to induce permeability transition and cytochrome c release in isolated mitochondria . Proc. Natl Acad. Sci. USA 95, 14681– 14686 (1998).

    Article  CAS  Google Scholar 

  12. Wolter, K. G. et al. Movement of Bax from the cytosol to mitochondria during apoptosis . J. Cell Biol. 139, 1281– 1292 (1997).

    Article  CAS  Google Scholar 

  13. Finucane, D. M., Bossy-Wetzel, E., Waterhouse, N. J., Cotter, T. G. & Green, D. R. Bax-induced caspase activation and apoptosis via cytochrome c release from mitochondria is inhibitable by Bcl-xL. J. Biol. Chem. 274, 2225– 2233 (1999).

    Article  CAS  Google Scholar 

  14. Eskes, R. et al. Bax-induced cytochrome c release from mitochondria is independent of the permeability transition pore but highly dependent on Mg2+ ions. J. Cell Biol. 143, 217– 224 (1998).

    Article  CAS  Google Scholar 

  15. Luo, X., Budihardjo, I., Zou, H., Slaughter, C. & Wang, X. Bid, a Bcl2 interacting protein, mediates cytochrome c release from mitochondria in response to activation of cell surface death receptors. Cell 94, 481– 490 (1998).

    Article  CAS  Google Scholar 

  16. Desagher, S. et al. Bid-induced conformational change of Bax is responsible for mitochondrial cytochrome c release during apoptosis. J. Cell Biol. 144, 891–901 (1999).

    Article  CAS  Google Scholar 

  17. Basanez, G. et al. Bax, but not Bcl-xL, decreases the lifetime of planar phospholipid bilayer membranes at subnanomolar concentrations. Proc. Natl Acad. Sci. USA 96, 5492–5497 ( 1999).

    Article  CAS  Google Scholar 

  18. Marchetti, P. et al. Mitochondrial permeability transition is a central coordinating event of apoptosis. J. Exp. Med. 184, 1155 –1160 (1996).

    Article  CAS  Google Scholar 

  19. Heiskanen, K. M., Bhat, M. B., Wang, H. W., Ma, J. & Nieminen, A. L. Mitochondrial depolarization accompanies cytochrome c release during apoptosis in PC 6 cells. J. Biol. Chem. 274, 5654–5658 (1999).

    Article  CAS  Google Scholar 

  20. Heiden, M. G., Chandel, N. S., Schumacker, P. T. & Thompson, C. B. Bcl-xL prevents cell death following growth factor withdrawal by facilitating mitochondrial ATP/ADP exchange. Mol. Cell 3, 159–167 (1999).

    Article  Google Scholar 

  21. Susin, S. A. et al. Mitochondrial release of caspase-2 and -9 during the apoptotic process. J. Exp. Med. 189, 381– 394 (1999).

    Article  CAS  Google Scholar 

  22. Susin, S. A. et al. Molecular characterization of mitochondrial apoptosis-inducing factor. Nature 397, 441– 446 (1999).

    Article  CAS  Google Scholar 

  23. Green, D. R. & Reed, J. C. Mitochondria and apoptosis. Science 281, 1309–1312 ( 1998).

    Article  CAS  Google Scholar 

  24. Marzo, I. et al. The permeability transition pore complex: a target for apoptosis regulation by caspases and bcl-2-related proteins. J. Exp. Med. 187, 1261–1271 ( 1998).

    Article  CAS  Google Scholar 

  25. Bossy-Wetzel, E., Newmeyer, D. D. & Green, D. R. Mitochondrial cytochrome c release in apoptosis occurs upstream of DEVD-specific caspase activation and independently of mitochondrial transmembrane depolarization. EMBO J. 17, 37–49 (1998).

    Article  CAS  Google Scholar 

  26. Single, B., Leist, M. & Nicotera, P. Simultaneous release of adenylate kinase and cytochrome c in cell death. Cell Death Differ. 5, 1001–1003 (1998).

    Article  CAS  Google Scholar 

  27. Kohler, C. et al. Release of adenylate kinase 2 from the mitochondrial intermembrane space during apoptosis. FEBS Lett. 447, 10–12 (1999).

    Article  CAS  Google Scholar 

  28. Kluck, R. M. et al. The pro-apoptotic proteins, Bid and Bax, cause a limited permeabilization of the mitochondrial outer membrane that is enhanced by cytosol. J. Cell Biol. 147, 809–822 (1999).

    Article  CAS  Google Scholar 

  29. Martin, S. J. et al. Early redistribution of plasma membrane phosphatidylserine is a general feature of apoptosis regardless of the initiating stimulus: inhibition by overexpression of Bcl-2 and Abl. J. Exp. Med. 182 , 1545–1556 (1995).

    Article  CAS  Google Scholar 

  30. Vermes, I., Haanen, C., Steffens-Nakken, H. & Reutelingsperger, C. A novel assay for apoptosis. Flow cytometric detection of phosphatidylserine expression on early apoptotic cells using fluorescein labelled Annexin V. J. Immunol. Methods 184, 39–51 (1995).

    Article  CAS  Google Scholar 

  31. Srinivasan, A. et al. Bcl-xL functions downstream of caspase-8 to inhibit Fas- and tumor necrosis factor receptor 1-induced apoptosis of MCF7 breast carcinoma cells. J. Biol. Chem. 273, 4523– 4529 (1998).

    Article  CAS  Google Scholar 

  32. Matsuyama, S., Xu, Q., Velours, J. & Reed, J. C. The mitochondrial F0F1-ATPase proton pump is required for function of the proapoptotic protein Bax in yeast and mammalian cells. Mol. Cell 1, 327–336 ( 1998).

    Article  CAS  Google Scholar 

  33. Saleh, A., Srinivasula, S. M., Acharya, S., Fishel, R. & Alnemri, E. S. Cytochrome c and dATP-mediated oligomerization of Apaf-1 is a prerequisite for procaspase-9 activation. J. Biol. Chem. 274, 17941–17945 (1999).

    Article  CAS  Google Scholar 

  34. Eguchi, Y., Shimizu, S. & Tsujimoto, Y. Intracellular ATP levels determine cell death fate by apoptosis or necrosis. Cancer Res. 57, 1835 –1840 (1997).

    CAS  PubMed  Google Scholar 

  35. Leist, M., Single, B., Castoldi, A. F., Kuhnle, S. & Nicotera, P. Intracellular adenosine triphosphate (ATP) concentration: a switch in the decision between apoptosis and necrosis . J. Exp. Med. 185, 1481– 1486 (1997).

    Article  CAS  Google Scholar 

  36. Kroemer, G., Dallaporta, B. & Resche-Rigon, M. The mitochondrial death/life regulator in apoptosis and necrosis. Annu. Rev. Physiol. 60, 619 –642 (1998).

    Article  CAS  Google Scholar 

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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 frames 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 frame 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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