Abstract
Mitochondrial outer membrane permeabilisation (MOMP) during apoptosis is triggered by the activation and oligomerisation of Bax and Bak, but a quantification of these processes in individual cells has not yet been performed. Single-cell imaging of Bax translocation and oligomerisation in Bax-deficient DU-145 cells expressing CFP-Bax and YFP-Bax revealed that both processes started only minutes before or concomitantly with MOMP, with the majority of Bax translocation and oligomerisation occurring downstream of MOMP. Quantification of YFP-Bax concentrations at mitochondria revealed an increase of only 1.8±1.5% at MOMP onset. This was increased to 11.2±3.6% in bak-silenced cells. These data suggested that Bax activation exceeded by far the quantities required for MOMP induction, and that minimal Bax or Bak activation may be sufficient to trigger rapid pore formation. In a cellular automaton modelling approach that incorporated the quantities and movement probabilities of Bax and its inhibitors, activators and enablers in the mitochondrial membrane, we could re-model rapid pore formation kinetics at submaximal Bax activation.
Similar content being viewed by others
Log in or create a free account to read this content
Gain free access to this article, as well as selected content from this journal and more on nature.com
or
Abbreviations
- AOTF:
-
acousto optical tunable filter
- CA:
-
cellular automaton
- CFP:
-
cyan fluorescent protein
- CHX:
-
cycloheximide
- Cyt-C:
-
cytochrome c
- FRET:
-
fluorescence resonance energy transfer
- FWHM:
-
full-width half maximum
- GFP:
-
green fluorescent protein
- ΔΨM:
-
mitochondrial membrane potential
- MOMP:
-
mitochondrial outer membrane permeabilisation
- STS:
-
staurosporine
- tBid:
-
truncated Bid
- TRAIL:
-
tumour necrosis factor-related apoptosis inducing ligand
- YFP:
-
yellow fluorescent protein
References
Green DR, Kroemer G . The pathophysiology of mitochondrial cell death. Science 2004; 305: 626–629.
Wei MC, Zong W-X, Cheng EHY, Lindsten T, Panoutsakopoulou V, Ross AJ et al. Proapoptotic BAX and BAK: a requisite gateway to mitochondrial dysfunction and death. Science 2001; 292: 727–730.
Wolter KG, Hsu Y-T, Smith CL, Nechushtan A, Xi X-G, Youle RJ . Movement of Bax from the Cytosol to mitochondria during apoptosis. J Cell Biol 1997; 139: 1281–1292.
Desagher S, Osen-Sand A, Nichols A, Eskes R, Montessuit S, Lauper S et al. Bid-induced conformational change of Bax is responsible for mitochondrial cytochrome c release during apoptosis. J Cell Biol 1999; 144: 891–901.
Kuwana T, Mackey MR, Perkins G, Ellisman MH, Latterich M, Schneiter R et al. Bid, Bax, and lipids cooperate to form supramolecular openings in the outer mitochondrial membrane. Cell 2002; 111: 331.
Nechushtan A, Smith CL, Lamensdorf I, Yoon SH, Youle RJ . Bax and Bak coalesce into novel mitochondria-associated clusters during apoptosis. J Cell Biol 2001; 153: 1265–1276.
Lovell JF, Billen LP, Bindner S, Shamas-Din A, Fradin C, Leber B et al. Membrane binding by tBid initiates an ordered series of events culminating in membrane permeabilization by Bax. Cell 2008; 135: 1074–1084.
Annis MG, Soucie EL, Dlugosz PJ, Cruz-Aguado JA, Penn LZ, Leber B et al. Bax forms multispanning monomers that oligomerize to permeabilize membranes during apoptosis. EMBO J 2005; 24: 2096–2103.
Dussmann H, Rehm M, Kogel D, Prehn JHM . Outer mitochondrial membrane permeabilization during apoptosis triggers caspase-independent mitochondrial and caspase-dependent plasma membrane potential depolarization: a single-cell analysis. J Cell Sci 2003; 116: 525–536.
Goldstein JC, Waterhouse NJ, Juin P, Evan GI, Green DR . The coordinate release of cytochrome c during apoptosis is rapid, complete and kinetically invariant. Nat Cell Biol 2000; 2: 156–162.
Munoz-Pinedo C, Guio-Carrion A, Goldstein JC, Fitzgerald P, Newmeyer DD, Green DR . Different mitochondrial intermembrane space proteins are released during apoptosis in a manner that is coordinately initiated but can vary in duration. PNAS 2006; 103: 11573–11578.
Rehm M, Dussmann H, Prehn JHM . Real-time single cell analysis of Smac/DIABLO release during apoptosis. J Cell Biol 2003; 162: 1031–1043.
Li H, Zhu H, Xu CJ, Yuan J . Cleavage of BID by caspase 8 mediates the mitochondrial damage in the Fas pathway of apoptosis. Cell 1998; 94: 491–501.
Kroemer G, Martin SJ . Caspase-independent cell death. Nat Med 2005; 11: 725–730.
Rehm M, Huber HJ, Hellwig CT, Anguissola S, Dussmann H, Prehn JH . Dynamics of outer mitochondrial membrane permeabilization during apoptosis. Cell Death Differ 2009; 16: 613–623.
von Haefen C, Wieder T, Gillissen B, Starck L, Graupner V, Dorken B et al. Ceramide induces mitochondrial activation and apoptosis via a Bax-dependent pathway in human carcinoma cells. Oncogene 2002; 21: 4009–4019.
Hsu YT, Youle RJ . Nonionic detergents induce dimerization among members of the Bcl-2 family. J Biol Chem 1997; 272: 13829–13834.
Peyerl FW, Dai S, Murphy GA, Crawford F, White J, Marrack P et al. Elucidation of some Bax conformational changes through crystallization of an antibody-peptide complex. Cell Death Differ 2006; 14: 447.
Heiskanen KM, Bhat MB, Wang HW, Ma J, Nieminen AL . Mitochondrial depolarization accompanies cytochrome c release during apoptosis in PC6 cells. J Biol Chem 1999; 274: 5654–5658.
Mootha VK, Wei MC, Buttle KF, Scorrano L, Panoutsakopoulou V, Mannella CA et al. A reversible component of mitochondrial respiratory dysfunction in apoptosis can be rescued by exogenous cytochrome c. EMBO J 2001; 20: 661–671.
Waterhouse NJ, Goldstein JC, von Ahsen O, Schuler M, Newmeyer DD, Green DR . Cytochrome c maintains mitochondrial transmembrane potential and ATP generation after outer mitochondrial membrane permeabilization during the apoptotic process. J Cell Biol 2001; 153: 319–328.
Chen C, Cui J, Lu H, Wang R, Zhang S, Shen P . Modeling of the role of a Bax-activation switch in the mitochondrial apoptosis decision. Biophys J 2007; 92: 4304–4315.
Siehs C, Oberbauer R, Mayer G, Lukas A, Mayer B . Discrete simulation of regulatory homo- and heterodimerization in the apoptosis effector phase. Bioinformatics 2002; 18: 67–76.
Luetjens CM, Kogel D, Reimertz C, Dussmann H, Renz A, Schulze-Osthoff K et al. Multiple kinetics of mitochondrial cytochrome c release in drug-induced apoptosis. Mol Pharmacol 2001; 60: 1008–1019.
Smaili SS, Hsu YT, Sanders KM, Russell JT, Youle RJ . Bax translocation to mitochondria subsequent to a rapid loss of mitochondrial membrane potential. Cell Death Differ 2001; 8: 909–920.
Albeck JG, Burke JM, Spencer SL, Lauffenburger DA, Sorger PK . Modeling a snap-action, variable-delay switch controlling extrinsic cell death. PLoS Biol 2008; 6: 2831–2852.
Lartigue L, Medina C, Schembri L, Chabert P, Zanese M, Tomasello F et al. An intracellular wave of cytochrome c propagates and precedes Bax redistribution during apoptosis. J Cell Sci 2008; 121 (Pt 21): 3515–3523.
Zhou L, Chang DC . Dynamics and structure of the Bax-Bak complex responsible for releasing mitochondrial proteins during apoptosis. J Cell Sci 2008; 121 (Pt 13): 2186–2196.
Ward MW, Rehm M, Duessmann H, Kacmar S, Concannon CG, Prehn JHM . Real time single cell analysis of BID cleavage and BID translocation during caspase-dependent and neuronal caspase-independent apoptosis. J Biol Chem 2005; 281: 5837–5844.
Gross A, Jockel J, Wei MC, Korsmeyer SJ . Enforced dimerization of BAX results in its translocation, mitochondrial dysfunction and apoptosis. EMBO J 1998; 17: 3878–3885.
Nechushtan A, Smith CL, Hsu YT, Youle RJ . Conformation of the Bax C-terminus regulates subcellular location and cell death. EMBO J 1999; 18: 2330–2341.
Rehm M, Dussmann H, Janicke RU, Tavare JM, Kogel D, Prehn JHM . Single-cell fluorescence resonance energy transfer analysis demonstrates that caspase activation during apoptosis is a rapid process. ROLE OF CASPASE-3. J Biol Chem 2002; 277: 24506–24514.
Rehm M, Huber HJ, Dussmann H, Prehn JHM . Systems analysis of effector caspase activation and its control by X-linked inhibitor of apoptosis protein. EMBO J 2006; 25: 4338–4349.
Saito M, Korsmeyer SJ, Schlesinger PH . BAX-dependent transport of cytochrome c reconstituted in pure liposomes. Nat Cell Biol 2000; 2: 553.
Pomerening JR . Uncovering mechanisms of bistability in biological systems. Curr Opin Biotechnol 2008; 19: 381–388.
Tan C, Dlugosz PJ, Peng J, Zhang Z, Lapolla SM, Plafker SM et al. Auto-activation of the apoptosis protein BAX increases mitochondrial membrane permeability and is inhibited by BCL-2. J Biol Chem 2006; 281: 14764–14775.
Korsmeyer SJ, Wei MC, Saito M, Weiler S, Oh KJ, Schlesinger PH . Pro-apoptotic cascade activates BID, which oligomerizes BAK or BAX into pores that result in the release of cytochrome c. Cell Death Differ 2000; 7: 1166–1173.
Letai AG . Diagnosing and exploiting cancer's addiction to blocks in apoptosis. Nat Rev Cancer 2008; 8: 121–132.
Kitano H . Biological robustness. Nat Rev Genet 2004; 5: 826–837.
Marcelli M, Marani M, Li X, Sturgis L, Haidacher SJ, Trial JA et al. Heterogeneous apoptotic responses of prostate cancer cell lines identify an association between sensitivity to staurosporine-induced apoptosis, expression of Bcl-2 family members, and caspase activation. Prostate 2000; 42: 260–273.
Chiu SM, Xue LY, Usuda J, Azizuddin K, Oleinick NL . Bax is essential for mitochondrion-mediated apoptosis but not for cell death caused by photodynamic therapy. Br J Cancer 89: 1590.
Li X, Marani M, Yu J, Nan B, Roth JA, Kagawa S et al. Adenovirus-mediated Bax overexpression for the induction of therapeutic apoptosis in prostate cancer. Cancer Res 2001; 61: 186–191.
Zal T, Gascoigne NRJ . Photobleaching-corrected FRET efficiency imaging of live cells. Biophys J 2004; 86: 3923–3939.
Acknowledgements
We thank Hannes Herzel for technical support and Dr. Peter Daniel (Charité, Humboldt University, Berlin) for DU-145 and DU-145-Bax cells. This research was supported by grants from Science Foundation Ireland (03/RP1/B344; 08/IN1/1949), the Irish Health Research Board (RP/2006/258), the National Biophotonics and Imaging Platform Ireland (Higher Education Authority PRTLI Cycle 4), the European Union (FP 7 Health–APO-SYS), and by Siemens Ireland.
Author information
Authors and Affiliations
Corresponding author
Additional information
Edited by D Vaux
Supplementary Information accompanies the paper on Cell Death and Differentiation website (http://www.nature.com/cdd)
Supplementary information
Rights and permissions
About this article
Cite this article
Düssmann, H., Rehm, M., Concannon, C. et al. Single-cell quantification of Bax activation and mathematical modelling suggest pore formation on minimal mitochondrial Bax accumulation. Cell Death Differ 17, 278–290 (2010). https://doi.org/10.1038/cdd.2009.123
Received:
Revised:
Accepted:
Published:
Issue date:
DOI: https://doi.org/10.1038/cdd.2009.123
Keywords
This article is cited by
-
Endogenous BAX and BAK form mosaic rings of variable size and composition on apoptotic mitochondria
Cell Death & Differentiation (2024)
-
Regorafenib induces Bim-mediated intrinsic apoptosis by blocking AKT-mediated FOXO3a nuclear export
Cell Death Discovery (2023)
-
Stoichiometry and regulation network of Bcl-2 family complexes quantified by live-cell FRET assay
Cellular and Molecular Life Sciences (2020)
-
High PGAM5 expression induces chemoresistance by enhancing Bcl-xL-mediated anti-apoptotic signaling and predicts poor prognosis in hepatocellular carcinoma patients
Cell Death & Disease (2018)
-
Bax retrotranslocation potentiates Bcl-xL’s antiapoptotic activity and is essential for switch-like transitions between MOMP competency and resistance
Cell Death & Disease (2018)
