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.
References
Kroemer G, Reed JC. Mitochondrial control of cell death. Nat Med 2000; 6: 513-519
Zamzami N, Kroemer G. Mitochondria in apoptosis. How Pandora's box opens Nat Rev Mol Cell Biol 2001; 2: 67-71
Martinou J-C, Green DR. Breaking the mitochondrial barrier. Nat Rev Mol Cell Biol 2001; 2: 63-67
Zamzami N, Kroemer G. Mitochondrial membrane permeabilization in apoptosis The (w)hole story? Curr Biol 2001; 13: R71-R73.
Kroemer G. The proto-oncogene Bcl-2 and its role in regulating apoptosis. Nat Med. 1997; 3: 614-620.
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.
Vander Heiden MG, Thompson CB. Bcl-2 proteins: Inhibitors of apoptosis or regulators of mitochondrial homeostasis? Nat Cell Biol 1999; 1: E209-E216.
Gross A, McDonnell JM, Korsmeyer SJ. Bcl-2 family members and the mitochondria in apoptosis. Genes Dev 1999; 13: 1988- 1911.
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.
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.
Wang X. The expanding role of mitochondria in apoptosis. Genes Dev 2002; 15: 2922-2933.
Hüser J, Rechenmacher CE, Blatter LA. Imaging the permeability transition pore transition in single mitochondria. Biophys J 1998; 74: 2129-2137.
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.
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.
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.
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.
Green DR, Reed JC. Mitochondria and apoptosis. Science 1998; 281: 1309-1312.
Susin SA, Zamzami N, Kroemer G. Mitochondrial regulation of apoptosis Doubt no more. Biochim Biophys Acta (Bioenergetics) 1998; 1366: 151-165.
Chen LB. Mitochondrial membrane potential in living cells. Annu Rev Cell Biol 1988; 4: 155-181.
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.
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.
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.
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.
Zoratti M, Szabò I. The mitochondrial permeability transition. Biochem Biophys Acta Rev Biomembranes 1995; 1241: 139-176.
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.
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.
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.
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.
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.
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.
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.
Daugas E, Susin SA, Zamzami N, et al. Mitochondrio-nuclear redistribution of AIF in apoptosis and necrosis. FASEB J 2000; 14: 729-739.
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.
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.
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.
Lin DT, Lechleiter JD. Mitochondrial targeted cyclophilin D protects cells from Cell death by peptidyl prolyl isomerization. J Biol Chem 2002; 277: 31134-31141.
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.
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.
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.
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.
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.
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.
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.
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.
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.
Cai J, Jones DP. Superoxide in apoptosis Mitochondrial generation triggered by cytochrome c loss. J Biol Chem 1998; 273: 11401-11404.
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.
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.
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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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DOI: https://doi.org/10.1023/A:1025546525894