Mitochondria are crucial compartments of eukaryotic cells because they function as the cellular power plant and play a central role in the early stages of programmed cell death (apoptosis). To avoid undesired cell death, this apoptotic pathway is tightly regulated by members of the Bcl-2 protein family, which interact on the external surface of the mitochondria, i.e., the mitochondrial outer membrane (MOM), and modulate its permeability to apoptotic factors, controlling their release into the cytosol. A growing body of evidence suggests that the MOM lipids play active roles in this permeabilization process. In particular, oxidized phospholipids (OxPls) formed under intracellular stress seem to directly induce apoptotic activity at the MOM. Here we show that the process of MOM pore formation is sensitive to the type of OxPls species that are generated. We created MOM-mimicking liposome systems, which resemble the cellular situation before apoptosis and upon triggering of oxidative stress conditions. These vesicles were studied using 31P solid-state magic-angle-spinning nuclear magnetic resonance spectroscopy and differential scanning calorimetry, together with dye leakage assays. Direct polarization and cross-polarization nuclear magnetic resonance experiments enabled us to probe the heterogeneity of these membranes and their associated molecular dynamics. The addition of apoptotic Bax protein to OxPls-containing vesicles drastically changed the membranes' dynamic behavior, almost completely negating the previously observed effect of temperature on the lipids' molecular dynamics and inducing an ordering effect that led to more cooperative membrane melting. Our results support the hypothesis that the mitochondrion-specific lipid cardiolipin functions as a first contact site for Bax during its translocation to the MOM in the onset of apoptosis. In addition, dye leakage assays revealed that different OxPls species in the MOM-mimicking vesicles can have opposing effects on Bax pore formation.

Department of Chemistry Umeå University Umeå Sweden

J Heyrovský Institute of Physical Chemistry Academy of Sciences of the Czech Republic Prague Czech Republic

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Fulda S., Galluzzi L., Kroemer G. Targeting mitochondria for cancer therapy. Nat. Drug. Discov. 2010;9:447–464. PubMed

Kerr J.F., Wyllie A.H., Currie A.R. Apoptosis: a basic biological phenomenon with wide-ranging implications in tissue kinetics. Br. J. Cancer. 1972;26:239–257. PubMed PMC

Ulukaya E., Acilan C., Yilmaz Y. Apoptosis: why and how does it occur in biology? Cell Biochem. Funct. 2011;29:468–480. PubMed

Adams J.M., Cory S. Bcl-2-regulated apoptosis: mechanism and therapeutic potential. Curr. Opin. Immunol. 2007;19:488–496. PubMed PMC

Youle R.J., Strasser A. The BCL-2 protein family: opposing activities that mediate cell death. Nat. Rev. Mol. Cell Biol. 2008;9:47–59. PubMed

Czabotar P.E., Lessene G., Adams J.M. Control of apoptosis by the BCL-2 protein family: implications for physiology and therapy. Nat. Rev. Mol. Cell Biol. 2014;15:49–63. PubMed

Leber B., Lin J., Andrews D.W. Still embedded together binding to membranes regulates Bcl-2 protein interactions. Oncogene. 2010;29:5221–5230. PubMed PMC

Martinou J.C., Youle R.J. Mitochondria in apoptosis: Bcl-2 family members and mitochondrial dynamics. Dev. Cell. 2011;21:92–101. PubMed PMC

Renault T.T., Floros K.V., Chipuk J.E. Mitochondrial shape governs BAX-induced membrane permeabilization and apoptosis. Mol. Cell. 2015;57:69–82. PubMed PMC

Lucken-Ardjomande S., Montessuit S., Martinou J.C. Contributions to Bax insertion and oligomerization of lipids of the mitochondrial outer membrane. Cell Death Differ. 2008;15:929–937. PubMed

Schellenberg B., Wang P., Gilmore A.P. Bax exists in a dynamic equilibrium between the cytosol and mitochondria to control apoptotic priming. Mol. Cell. 2013;49:959–971. PubMed PMC

Wallgren M., Lidman M., Gröbner G. The oxidized phospholipid PazePC modulates interactions between Bax and mitochondrial membranes. Biochim. Biophys. Acta. 2012;1818:2718–2724. PubMed

Westphal D., Dewson G., Kluck R.M. Apoptotic pore formation is associated with in-plane insertion of Bak or Bax central helices into the mitochondrial outer membrane. Proc. Natl. Acad. Sci. USA. 2014;111:E4076–E4085. PubMed PMC

Luckey M. Cambridge University Press; New York: 2008. Membrane Structural Biology.

Ardail D., Privat J.P., Louisot P. Mitochondrial contact sites. Lipid composition and dynamics. J. Biol. Chem. 1990;265:18797–18802. PubMed

Karbowski M., Youle R.J. Dynamics of mitochondrial morphology in healthy cells and during apoptosis. Cell Death Differ. 2003;10:870–880. PubMed

Korytowski W., Basova L.V., Girotti A.W. Permeabilization of the mitochondrial outer membrane by Bax/truncated Bid (tBid) proteins as sensitized by cardiolipin hydroperoxide translocation: mechanistic implications for the intrinsic pathway of oxidative apoptosis. J. Biol. Chem. 2011;286:26334–26343. PubMed PMC

Gonzalvez F., Gottlieb E. Cardiolipin: setting the beat of apoptosis. Apoptosis. 2007;12:877–885. PubMed

Sani M.A., Dufourc E.J., Gröbner G. How does the Bax-α1 targeting sequence interact with mitochondrial membranes? The role of cardiolipin. Biochim. Biophys. Acta. 2009;1788:623–631. PubMed

Subburaj Y., Cosentino K., García-Sáez A.J. Bax monomers form dimer units in the membrane that further self-assemble into multiple oligomeric species. Nat. Commun. 2015;6:8042. PubMed PMC

Gahl R.F., He Y., Tjandra N. Conformational rearrangements in the pro-apoptotic protein, Bax, as it inserts into mitochondria: a cellular death switch. J. Biol. Chem. 2014;289:32871–32882. PubMed PMC

Shamas-Din A., Bindner S., Fradin C. Distinct lipid effects on tBid and Bim activation of membrane permeabilization by pro-apoptotic Bax. Biochem. J. 2015;467:495–505. PubMed

Fruhwirth G.O., Hermetter A. Mediation of apoptosis by oxidized phospholipids. Subcell. Biochem. 2008;49:351–367. PubMed

Fruhwirth G.O., Loidl A., Hermetter A. Oxidized phospholipids: from molecular properties to disease. Biochim. Biophys. Acta. 2007;1772:718–736. PubMed

Volinsky R., Kinnunen P.K.J. Oxidized phosphatidylcholines in membrane-level cellular signaling: from biophysics to physiology and molecular pathology. FEBS J. 2013;280:2806–2816. PubMed

Khandelia H., Mouritsen O.G. Lipid gymnastics: evidence of complete acyl chain reversal in oxidized phospholipids from molecular simulations. Biophys. J. 2009;96:2734–2743. PubMed PMC

Wallgren M., Beranova L., Gröbner G. Impact of oxidized phospholipids on the structural and dynamic organization of phospholipid membranes: a combined DSC and solid state NMR study. Faraday Discuss. 2013;161:499–513. discussion 563–589. PubMed

Hermetter A., Kinnunen P., Spickett C. Oxidized phospholipids-their properties and interactions with proteins. Biochim. Biophys. Acta. 2012;1818:2373. PubMed

Lidman M., Pokorná Š., Gröbner G. The oxidized phospholipid PazePC promotes permeabilization of mitochondrial membranes by Bax. Biochim. Biophys. Acta. 2016;1858:1288–1297. PubMed

Wallgren M., Pham Q.D., Grobner G. Impact of oxidized phospholipids on membrane organization. Biophys. J. 2013;104:249a. PubMed

Lindström F., Williamson P.T., Gröbner G. Molecular insight into the electrostatic membrane surface potential by 14N/31P MAS NMR spectroscopy: nociceptin-lipid association. J. Am. Chem. Soc. 2005;127:6610–6616. PubMed

Bonev B.B., Chan W.C., Watts A. Interaction of the lantibiotic nisin with mixed lipid bilayers: a 31P and 2H NMR study. Biochemistry. 2000;39:11425–11433. PubMed

Cornell B.A., Hiller R.G., Morris C. Biological membranes are rich in low-frequency motion. Biochim. Biophys. Acta. 1983;732:473–478. PubMed

Suzuki M., Youle R.J., Tjandra N. Structure of Bax: coregulation of dimer formation and intracellular localization. Cell. 2000;103:645–654. PubMed

Banigan J.R., Gayen A., Traaseth N.J. Correlating lipid bilayer fluidity with sensitivity and resolution of polytopic membrane protein spectra by solid-state NMR spectroscopy. Biochim. Biophys. Acta. 2015;1848(1 Pt. B):334–341. PubMed PMC

Angelova M.I., Soleau S., Bothorel P. Trends in Colloid and Interface Science VI. Dr Dietrich Steinkopff Verlag; Berlin, Germany: 1992. Preparation of giant vesicles by external AC electric fields—kinetics and applications.

Koukalová A., Pokorná Š., Hof M. Membrane activity of the pentaene macrolide didehydroroflamycoin in model lipid bilayers. Biochim. Biophys. Acta. 2015;1848:444–452. PubMed

Sani M.A., Keech O., Gröbner G. Magic-angle phosphorus NMR of functional mitochondria: in situ monitoring of lipid response under apoptotic-like stress. FASEB J. 2009;23:2872–2878. PubMed

Stohrer J., Grobner G., Kothe G. Collective lipid motions in bilayer-membranes studied by transverse deuteron spin relaxation. J. Chem. Phys. 1991;95:672–678.

Satsoura D., Kučerka N., Fradin C. Interaction of the full-length Bax protein with biomimetic mitochondrial liposomes: a small-angle neutron scattering and fluorescence study. Biochim. Biophys. Acta. 2012;1818:384–401. PubMed

Bleicken S., Jeschke G., Bordignon E. Structural model of active Bax at the membrane. Mol. Cell. 2014;56:496–505. PubMed PMC

Mannock D.A., Lewis R.N., McElhaney R.N. A calorimetric and spectroscopic comparison of the effects of ergosterol and cholesterol on the thermotropic phase behavior and organization of dipalmitoylphosphatidylcholine bilayer membranes. Biochim. Biophys. Acta. 2010;1798:376–388. PubMed

Dufourc E.J., Mayer C., Kothe G. Dynamics of phosphate head groups in biomembranes. Comprehensive analysis using phosphorus-31 nuclear magnetic resonance lineshape and relaxation time measurements. Biophys. J. 1992;61:42–57. PubMed PMC

Weisz K., Gröbner G., Kothe G. Deuteron nuclear magnetic resonance study of the dynamic organization of phospholipid/cholesterol bilayer membranes: molecular properties and viscoelastic behavior. Biochemistry. 1992;31:1100–1112. PubMed

Baoukina S., Mendez-Villuendas E., Tieleman D.P. Computer simulations of the phase separation in model membranes. Faraday Discuss. 2013;161:63–75. discussion 113–150. PubMed

Leber B., Lin J., Andrews D.W. Embedded together: the life and death consequences of interaction of the Bcl-2 family with membranes. Apoptosis. 2007;12:897–911. PubMed PMC

Fuertes G., García-Sáez A.J., Salgado J. Pores formed by Baxα5 relax to a smaller size and keep at equilibrium. Biophys. J. 2010;99:2917–2925. PubMed PMC

Last N.B., Rhoades E., Miranker A.D. Islet amyloid polypeptide demonstrates a persistent capacity to disrupt membrane integrity. Proc. Natl. Acad. Sci. USA. 2011;108:9460–9465. PubMed PMC

Apellániz B., Nieva J.L., García-Sáez A.J. All-or-none versus graded: single-vesicle analysis reveals lipid composition effects on membrane permeabilization. Biophys. J. 2010;99:3619–3628. PubMed PMC

Duer M.J. Blackwell Science; Oxford, UK: 2002. Solid-State NMR Spectroscopy: Principles and Applications.

Benetis N.P., Kyrikou I., Mavromoustakos T. Static CP P-31 NMR multilamellar bilayer broadlines in the absence and presence of the bioactive dipeptide beta-Ala-Tyr or Glu. Chem. Phys. 2005;314:57–72.

Holland G.P., McIntyre S.K., Alam T.M. Distinguishing individual lipid headgroup mobility and phase transitions in raft-forming lipid mixtures with 31P MAS NMR. Biophys. J. 2006;90:4248–4260. PubMed PMC

Ivanova V.P., Makarov I.M., Heimburg T. Analyzing heat capacity profiles of peptide-containing membranes: cluster formation of gramicidin A. Biophys. J. 2003;84:2427–2439. PubMed PMC

Cornell B.A., Davenport J.B., Separovic F. Low-frequency motion in membranes. The effect of cholesterol and proteins. Biochim. Biophys. Acta. 1982;689:337–345. PubMed

Mendes Ferreira T., Sood R., Ollila O.H.S. Acyl chain disorder and azelaoyl orientation in lipid membranes containing oxidized lipids. Langmuir. 2016;32:6524–6533. PubMed

The minimal membrane requirements for BAX-induced pore opening upon exposure to oxidative stress