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Cytofluorometric quantitation of apoptosis-driven inner mitochondrial membrane permeabilization

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Abstract

The mitochondrial matrix can be specifically labeled by loading cells with calcein and simultaneous quenching of the non-mitochondrial calcein fluorescence with cobalt (Co2+). Positive staining of mitochondria thus requires that the inner mitochondrial membrane functions as a barrier separating calcein (within the matrix) from Co2+ (outside of the matrix). Upon induction of apoptosis, such calcein/Co2+-labeled cells, demonstrate a decrease in the overall calcein fluorescence resulting from inner mitochondrial membrane permeabilization. This decrease can be quantified by cytofluorometry and can be dissociated from other apoptosis-associated mitochondrial perturbations such as the loss of the mitochondrial transmembrane potential (ΔΨ m ), the local overproduction of reactive oxygen species, and the mitochondrial release of cytochrome c. In some paradigms of apoptosis the loss of calcein/Co2+ (CC) staining can be dissociated from the ΔΨ m loss, both of which may occur in a caspase-dependent or caspase-independent fashion, depending on the apoptosis inducer. Importantly, inner membrane permeabilization to CC may occur without a permanent ΔΨ m dissipation in apoptosis, suggesting that transient permeabilization events could participate at the apoptotic cascade. Altogether, our data demonstrate that inner mitochondrial membrane permeabilization constitutes an early event in the apoptotic cascade.

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References

  1. Kroemer G, Reed JC. Mitochondrial control of cell death. Nat Med 2000; 6: 513-519

    Google Scholar 

  2. Zamzami N, Kroemer G. Mitochondria in apoptosis. How Pandora's box opens Nat Rev Mol Cell Biol 2001; 2: 67-71

    Google Scholar 

  3. Martinou J-C, Green DR. Breaking the mitochondrial barrier. Nat Rev Mol Cell Biol 2001; 2: 63-67

    Google Scholar 

  4. Zamzami N, Kroemer G. Mitochondrial membrane permeabilization in apoptosis The (w)hole story? Curr Biol 2001; 13: R71-R73.

    Google Scholar 

  5. Kroemer G. The proto-oncogene Bcl-2 and its role in regulating apoptosis. Nat Med. 1997; 3: 614-620.

    Google Scholar 

  6. Zamzami N, Brenner C, Marzo I, Susin SA, Kroemer G. Subcellular and submitochondrial mechanisms of apoptosis inhibition by Bcl-2-related proteins. Oncogene 1998; 16: 2265- 2282.

    Google Scholar 

  7. Vander Heiden MG, Thompson CB. Bcl-2 proteins: Inhibitors of apoptosis or regulators of mitochondrial homeostasis? Nat Cell Biol 1999; 1: E209-E216.

    Google Scholar 

  8. Gross A, McDonnell JM, Korsmeyer SJ. Bcl-2 family members and the mitochondria in apoptosis. Genes Dev 1999; 13: 1988- 1911.

    Google Scholar 

  9. Patterson S, Spahr CS, Daugas E, et al. Mass spectrometric identification of proteins released from mitochondria undergoing permeability transition. Cell Death Differ 2000; 7: 137-144.

    Google Scholar 

  10. Kuwana T, Mackey MR, Perkins GA, et al. Bid Bax, lipids cooperate to form supramolecular openings in the outer mitochondrial membrane. Cell 2002; 111: 1-12.

    Google Scholar 

  11. Wang X. The expanding role of mitochondria in apoptosis. Genes Dev 2002; 15: 2922-2933.

    Google Scholar 

  12. Hüser J, Rechenmacher CE, Blatter LA. Imaging the permeability transition pore transition in single mitochondria. Biophys J 1998; 74: 2129-2137.

    Google Scholar 

  13. Petronilli V, Miotto G, Canton M, et al. Transient and longlasting openings of the mitochondrial permeability transition pore can be monitored directly in intact cells by changes in mitochondrial calcein fluorescence. Biophys J 1999; 76: 725- 734.

    Google Scholar 

  14. Pastorino JG, Tafani M, Rothman RJ, Macineviciute A, Hoek JB, Farber JL. Functional consequences of sustained or transient activation by Bax of the mitochondrial permeability transition pore. J Biol Chem 1999; 274: 31734-31739.

    Google Scholar 

  15. Bernardi P, Scorrano L, Colonna R, Petronilli V, Di Lisa F. Mitochondria and cell death-Mechanistic aspects and methodological issues. Eur J Biochem 1999; 264: 687-701.

    Google Scholar 

  16. Leach JK, Van Tuyle G, Lin PS, Schmidt-Ullrich R, Mikkelsen RB. Ionizing radiation-induced mitochondria-dependent generation of reactive oxygen/nitrogen. Cancer Res 2001; 61: 3894- 3901.

    Google Scholar 

  17. Green DR, Reed JC. Mitochondria and apoptosis. Science 1998; 281: 1309-1312.

    Google Scholar 

  18. Susin SA, Zamzami N, Kroemer G. Mitochondrial regulation of apoptosis Doubt no more. Biochim Biophys Acta (Bioenergetics) 1998; 1366: 151-165.

    Google Scholar 

  19. Chen LB. Mitochondrial membrane potential in living cells. Annu Rev Cell Biol 1988; 4: 155-181.

    Google Scholar 

  20. Rottenberg H, Wu SL. Quantitative assay by flow cytometry of the mitochondrial membrane potential in intact cells. Biochim Biophys Acta Mol Cell Res 1998; 1404: 393-404.

    Google Scholar 

  21. Metivier D, Dallaporta B, Zamzami N, et al. Cytofluorometric detection of mitochondrial alterations in early CD95/Fas/APO-1-triggered apoptosis of Jurkat T lymphoma cells Comparison of seven mitochondrion-specific fluorochromes. Immunol Lett 1998; 61: 157-164.

    Google Scholar 

  22. Castedo M, Ferri KF, Roumier T, Metivier D, Zamzami N, Kroemer G. Quantitation of mitochondrial alterations associated with apoptosis. J Immunol Methods 2002; 265: 39-47.

    Google Scholar 

  23. Ferri KF, Jacotot E, Blanco J, et al. Apoptosis control in syncytia induced by the HIV-1-envelope glycoprotein complex Role of mitochondria and caspases. J Exp Med 2000; 192: 1081- 1092.

    Google Scholar 

  24. Zoratti M, Szabò I. The mitochondrial permeability transition. Biochem Biophys Acta Rev Biomembranes 1995; 1241: 139-176.

    Google Scholar 

  25. Nieminen AL, Saylor AK, Tesfai SA, Herman B, Lemasters JJ. Contribution of the mitochondrial permeability transition to lethal injury after exposure of hepatocytes to t-butylhydroperoxide. Biochem J 1995; 307: 99-106.

    Google Scholar 

  26. Scorrano L, Petronilli V, Di Lisa F, Bernardi P. Commitment to apoptosis by GD3 ganglioside depends on opening of the mitochondrial permeability transition pore. J Biol Chem 1999; 274: 22581-22585.

    Google Scholar 

  27. Yang JH, Gross RL, Basinger SF, Wu, SM. Apoptotic cell death of cultured salamander photoreceptors induced by CCCP: A CsA-insensitive mitochondrial permeability transition. J Cell Sci 2001; 114: 1655-1664.

    Google Scholar 

  28. Chauvin C, De Oliveira F, Ronot X, Mousseau M, Leverve X, Fontaine E. Rotenone inhibits the mitochondrial permeability transition-induced cell death in U937 andKBcells. J Biol Chem 2001; 276: 41394-41398.

    Google Scholar 

  29. Gison EM, Henson ES, Villanueva J, Gibson SB. MEK Kinase 1 induces pitochondtial permeability transition leading to apoptosis independent of cytochrome c release. J Biol Chem 2001; 277: 10573-10580.

    Google Scholar 

  30. Goldmacher VS, Bartle LM, Skletskaya S, et al. A cytomegalovirus-encoded mitochondria-localized inhibitor of apoptosis structurally unrelated to Bcl-2. Proc Natl Acad Sci USA 1999; 96: 12536-12541.

    Google Scholar 

  31. Zhu W, Cowie A, Wasfy GW, Penn LZ, Leber B, Andrews DW. Bcl-2 mutants with restricted subcellular localization reveal spatially distinct pathways for apoptosis in different cell types. EMBO J 1996; 15: 4130-4141.

    Google Scholar 

  32. Daugas E, Susin SA, Zamzami N, et al. Mitochondrio-nuclear redistribution of AIF in apoptosis and necrosis. FASEB J 2000; 14: 729-739.

    Google Scholar 

  33. Ferri KF, Jacotot E, LeDuc P, Geuskens M, Ingber DE, Kroemer G. Apoptosis of syncytia induced by HIV-1-Envelope glycoprotein complex Influence of cell shape and size. Exp Cell Sci 2000; 261: 119-126.

    Google Scholar 

  34. Scarlett JL, Sheard PW, Hughes G, Legerwoo EC, Ku H, Murphy MP. Changes in mitochondrial membrane potential during staurosporin-induced apoptosis in Jurkat cells. FEBS Lett 2000; 475: 267-272.

    Google Scholar 

  35. Tafani M, Minchenko DA, Serroni A, Farber JL. Induction of the mitochondrial permeability transition mediates the killing of HeLa cells by staurosporine. Cancer Res 2001; 61: 2459- 2466.

    Google Scholar 

  36. Lin DT, Lechleiter JD. Mitochondrial targeted cyclophilin D protects cells from Cell death by peptidyl prolyl isomerization. J Biol Chem 2002; 277: 31134-31141.

    Google Scholar 

  37. Zamzami N, Marchetti P, Castedo M, et al. Sequential reduction of mitochondrial transmembrane potential and generation of reactive oxygen species in early programmed cell death. J Exp Med 1995; 182: 367-377.

    Google Scholar 

  38. Saxena K, Henry TR, Solem LE, Wallace KB. Enhanced induction of the mitochondrial permeability transition following acute menadione administration. Arch Biochem Biophys 1995; 317: 79-84.

    Google Scholar 

  39. Susin SA, Zamzami N, Castedo M, et al. Bcl-2 inhibits the mitochondrial release of an apoptogenic protease. J Exp Med 1996; 184: 1331-1342.

    Google Scholar 

  40. Kluck RM, Bossy-Wetzel E, Green DR, Newmeyer DD. The release of cytochrome c from mitochondria: A primary site for Bcl-2 regulation of apoptosis. Science 1997; 275: 1132-1136.

    Google Scholar 

  41. Yang J, Liu X, Bhalla K, et al. Prevention of apoptosis by Bcl-2: Release of cytochrome c from mitochondria blocked. Science 1997; 275: 1129-1132.

    Google Scholar 

  42. Decaudin D, Geley S, Hirsch T, et al. Bcl-2 and Bcl-XL antagonize the mitochondrial dysfunction preceding nuclear apoptosis induced by chemotherapeutic agents. Cancer Res 1997; 57: 62-67.

    Google Scholar 

  43. Vieira HL, Belzacq A-S, Haouzi D, et al. The adenine nucleotide translocator: A target of nitric oxide peroxynitrite and 4-hydroxynonenal. Oncogene 2001; 20: 4305-4316.

    Google Scholar 

  44. Annis MG, Zamzami N, Zhu W, et al. Endoplasmic reticulum localized Bcl-2 prevents apoptosis when redistribution of cytochrome c is a late event. Oncogene 2001; 20: 1939- 1952.

    Google Scholar 

  45. Boya P, Andreau K, Poncet D, et al. Lysosomal membrane permeabilization induces cell death in a mitochondrion-dependent fashion. J Exp Med 2003; 197: 1323-1334.

    Google Scholar 

  46. Cai J, Jones DP. Superoxide in apoptosis Mitochondrial generation triggered by cytochrome c loss. J Biol Chem 1998; 273: 11401-11404.

    Google Scholar 

  47. Mootha VK, Wei MC, Buttle KF, et al. A reversible component of mitochondrial respiratory dysfunction in apoptosis can be rescued by exogenous cytochrome c. EMBO J 2001; 20: 661- 671.

    Google Scholar 

  48. Ricci JE, Gottlieb RA, Green DR. Caspase-mediated loss of mitochondrial function and generation of reactive oxygen species during apoptosis.J Cell Biol 2003; 160: 65-75.

    Google Scholar 

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Authors and Affiliations

  1. Centre National de la Recherche Scientifique, UMR8125, Institut Gustave Roussy, 39 rue Camille-Desmoulins, F-94805, Villejuif, France

    D. Poncet, P. Boya, D. Métivier, N. Zamzami & G. Kroemer

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  1. D. Poncet
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  2. P. Boya
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  3. D. Métivier
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  4. N. Zamzami
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  5. G. Kroemer
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Poncet, D., Boya, P., Métivier, D. et al. Cytofluorometric quantitation of apoptosis-driven inner mitochondrial membrane permeabilization. Apoptosis 8, 521–530 (2003). https://doi.org/10.1023/A:1025546525894

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