Distribution of cholesterol in asymmetric membranes driven by composition and differential stress

Published:August 03, 2022DOI:


      Many lipid membranes of eukaryotic cells are asymmetric, which means the two leaflets differ in at least one physical property, such as lipid composition or lateral stress. Maintaining this asymmetry is helped by the fact that ordinary phospholipids rarely transition between leaflets, but cholesterol is an exception: its flip-flop times are in the microsecond range, so that its distribution between leaflets is determined by a chemical equilibrium. In particular, preferential partitioning can draw cholesterol into a more saturated leaflet, and phospholipid number asymmetry can force it out of a compressed leaflet. Combining highly coarse-grained membrane simulations with theoretical modeling, we investigate how these two driving forces play against each other until cholesterol’s chemical potential is equilibrated. The theory includes two coupled elastic sheets and a Flory-Huggins mixing free energy with a χ parameter. We obtain a relationship between χ and the interaction strength between cholesterol and lipids in either of the two leaflets, and we find that it depends, albeit weakly, on lipid number asymmetry. The differential stress measurements under various asymmetry conditions agree with our theoretical predictions. Using the two kinds of asymmetries in combination, we find that it is possible to counteract the phospholipid number bias, and the resultant stress in the membrane, via the control of cholesterol mixing in the leaflets.
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        • Alberts B.
        • Johnson A.
        • Walter P.
        • et al.
        Molecular Biology of the Cell.
        Garland Science, 2002
        • Karp G.
        Cell and Molecular Biology: Concepts and Experiments.
        John Wiley & Sons, 2009
        • Bretscher M.S.
        Asymmetrical lipid bilayer structure for biological membranes.
        Nat. New Biol. 1972; 236: 11-12
        • Verkleij A.J.
        • Zwaal R.F.
        • van Deenen L.L.
        • et al.
        The asymmetric distribution of phospholipids in the human red cell membrane. A combined study using phospholipases and freeze-etch electron microscopy.
        Biochim. Biophys. Acta. 1973; 323: 178-193
        • Sandra A.
        • Pagano R.E.
        Phospholipid asymmetry in LM cell plasma membrane derivatives: polar head group and acyl chain distributions.
        Biochemistry. 1978; 17: 332-338
        • Devaux P.F.
        Static and dynamic lipid asymmetry in cell membranes.
        Biochemistry. 1991; 30: 1163-1173
        • Lorent J.H.
        • Levental K.R.
        • Levental I.
        • et al.
        Plasma membranes are asymmetric in lipid unsaturation, packing and protein shape.
        Nat. Chem. Biol. 2020; 16: 644-652
        • Doktorova M.
        • Symons J.L.
        • Levental I.
        Structural and functional consequences of reversible lipid asymmetry in living membranes.
        Nat. Chem. Biol. 2020; 16: 1321-1330
        • Pautot S.
        • Frisken B.J.
        • Weitz D.A.
        Engineering asymmetric vesicles.
        Proc. Natl. Acad. Sci. USA. 2003; 100: 10718-10721
        • Hamada T.
        • Miura Y.
        • Takagi M.
        • et al.
        Construction of asymmetric cell-sized lipid vesicles from lipid-coated water-in-oil microdroplets.
        J. Phys. Chem. B. 2008; 112: 14678-14681
        • Matosevic S.
        • Paegel B.M.
        Layer-by-layer cell membrane assembly.
        Nat. Chem. 2013; 5: 958-963
        • Elani Y.
        • Purushothaman S.
        • Ces O.
        • et al.
        Measurements of the effect of membrane asymmetry on the mechanical properties of lipid bilayers.
        Chem. Commun. 2015; 51: 6976-6979
        • Karamdad K.
        • Law R.V.
        • Ces O.
        • et al.
        Studying the effects of asymmetry on the bending rigidity of lipid membranes formed by microfluidics.
        Chem. Commun. 2016; 52: 5277-5280
        • Cheng H.-T.
        • London E.
        • London E.
        Preparation and properties of asymmetric vesicles that mimic cell membranes.
        J. Biol. Chem. 2009; 284: 6079-6092
        • Cheng H.-T.
        • London E.
        Preparation and properties of asymmetric large unilamellar vesicles: interleaflet coupling in asymmetric vesicles is dependent on temperature but not curvature.
        Biophys. J. 2011; 100: 2671-2678
        • Chiantia S.
        • Schwille P.
        • London E.
        • et al.
        Asymmetric GUVs prepared by MβCD-mediated lipid exchange: an FCS study.
        Biophys. J. 2011; 100: L1-L3
        • Chiantia S.
        • London E.
        Acyl chain length and saturation modulate interleaflet coupling in asymmetric bilayers: effects on dynamics and structural order.
        Biophys. J. 2012; 103: 2311-2319
        • Doktorova M.
        • Heberle F.A.
        • Marquardt D.
        • et al.
        Preparation of asymmetric phospholipid vesicles for use as cell membrane models.
        Nat. Protoc. 2018; 13: 2086-2101
        • Enoki T.A.
        • Feigenson G.W.
        Asymmetric bilayers by hemifusion: method and leaflet behaviors.
        Biophys. J. 2019; 117: 1037-1050
        • Lu L.
        • Doak W.J.
        • Chiarot P.R.
        • et al.
        Membrane mechanical properties of synthetic asymmetric phospholipid vesicles.
        Soft Matter. 2016; 12: 7521-7528
        • Eicher B.
        • Marquardt D.
        • Pabst G.
        • et al.
        Intrinsic curvature-mediated transbilayer coupling in asymmetric lipid vesicles.
        Biophys. J. 2018; 114: 146-157
        • Hossein A.
        • Deserno M.
        Spontaneous curvature, differential stress, and bending modulus of asymmetric lipid membranes.
        Biophys. J. 2020; 118: 624-642
        • Hossein A.
        • Deserno M.
        Stiffening transition in asymmetric lipid bilayers: the role of highly ordered domains and the effect of temperature and size.
        J. Chem. Phys. 2021; 154: 014704
        • Mohideen N.
        • Weiner M.D.
        • Feigenson G.W.
        Bilayer compositional asymmetry influences the nanoscopic to macroscopic phase domain size transition.
        Chem. Phys. Lipids. 2020; 232: 104972
        • Doktorova M.
        • Weinstein H.
        Accurate in silico modeling of asymmetric bilayers based on biophysical principles.
        Biophys. J. 2018; 115: 1638-1643
        • Miettinen M.S.
        • Lipowsky R.
        Bilayer membranes with frequent flip-flops have tensionless leaflets.
        Nano Lett. 2019; 19: 5011-5016
        • Park S.
        • Im W.
        • Pastor R.W.
        Developing initial conditions for simulations of asymmetric membranes: a practical recommendation.
        Biophys. J. 2021; 120: 5041-5059
        • Daleke D.L.
        Regulation of transbilayer plasma membrane phospholipid asymmetry.
        J. Lipid Res. 2003; 44: 233-242
        • Kobayashi T.
        • Menon A.K.
        Transbilayer lipid asymmetry.
        Curr. Biol. 2018; 28: R386-R391
        • Kornberg R.D.
        • McConnell H.M.
        Insideoutside transitions of phospholipids in vesicle membranes.
        Biochemistry. 1971; 10: 1111-1120
        • Zachowski A.
        • Devaux P.F.
        Transmembrane movements of lipids.
        Experientia. 1990; 46: 644-656
        • Hamilton J.A.
        Fast flip-flop of cholesterol and fatty acids in membranes: implications for membrane transport proteins.
        Curr. Opin. Lipidol. 2003; 14: 263-271
        • Leventis R.
        • Silvius J.R.
        Use of cyclodextrins to monitor transbilayer movement and differential lipid affinities of cholesterol.
        Biophys. J. 2001; 81: 2257-2267
        • Bennett W.F.D.
        • MacCallum J.L.
        • Tieleman D.P.
        • et al.
        Molecular view of cholesterol flip-flop and chemical potential in different membrane environments.
        J. Am. Chem. Soc. 2009; 131: 12714-12720
        • Gu R.-X.
        • Baoukina S.
        • Tieleman D.P.
        Cholesterol flip-flop in heterogeneous membranes.
        J. Chem. Theory Comput. 2019; 15: 2064-2070
        • Florence Trentacosti Presti
        The role of cholesterol in regulating membrane fluidity.
        Membrane fluidity in biology. 1985; 4: 97-146
        • Mouritsen O.G.
        • Jørgensen K.
        Dynamical order and disorder in lipid bilayers.
        Chem. Phys. Lipids. 1994; 73: 3-25
        • Chakraborty S.
        • Doktorova M.
        • Ashkar R.
        • et al.
        How cholesterol stiffens unsaturated lipid membranes.
        Proc. Natl. Acad. Sci. USA. 2020; 117: 21896-21905
        • Simons K.
        • Toomre D.
        Lipid rafts and signal transduction.
        Nat. Rev. Mol. Cell Biol. 2000; 1: 31-39
        • Silvius J.R.
        Role of cholesterol in lipid raft formation: lessons from lipid model systems.
        Biochim. Biophys. Acta. 2003; 1610: 174-183
        • Crane J.M.
        • Tamm L.K.
        Role of cholesterol in the formation and nature of lipid rafts in planar and spherical model membranes.
        Biophys. J. 2004; 86: 2965-2979
        • Levental I.
        • Levental K.R.
        • Heberle F.A.
        Lipid rafts: controversies resolved, mysteries remain.
        Trends Cell Biol. 2020; 30: 341-353
        • Liu S.-L.
        • Sheng R.
        • Cho W.
        • et al.
        Orthogonal lipid sensors identify transbilayer asymmetry of plasma membrane cholesterol.
        Nat. Chem. Biol. 2017; 13: 268-274
        • Mondal M.
        • Mesmin B.
        • Maxfield F.R.
        • et al.
        Sterols are mainly in the cytoplasmic leaflet of the plasma membrane and the endocytic recycling compartment in CHO cells.
        Mol. Biol. Cell. 2009; 20: 581-588
        • Courtney K.C.
        • Pezeshkian W.
        • Zha X.
        • et al.
        C24 sphingolipids govern the transbilayer asymmetry of cholesterol and lateral organization of model and live-cell plasma membranes.
        Cell Rep. 2018; 24: 1037-1049
        • Foley S.
        • Deserno M.
        Stabilizing leaflet asymmetry under differential stress in a highly coarse-grained lipid membrane model.
        J. Chem. Theory Comput. 2020; 16: 7195-7206
        • Cooke I.R.
        • Kremer K.
        • Deserno M.
        Tunable generic model for fluid bilayer membranes.
        Phys. Rev. E Stat. Nonlin. Soft Matter Phys. 2005; 72: 011506
        • Cooke I.R.
        • Deserno M.
        Solvent-free model for self-assembling fluid bilayer membranes: stabilization of the fluid phase based on broad attractive tail potentials.
        J. Chem. Phys. 2005; 123: 224710
        • Deserno M.
        Mesoscopic membrane physics: concepts, simulations, and selected applications.
        Macromol. Rapid Commun. 2009; 30: 752-771
        • Hill T.L.
        An Introduction to Statistical Thermodynamics.
        Dover, 1986
        • Richard Anthony Lewis Jones
        Soft Condensed Matter.
        Oxford University Press, 2002
        • Weik F.
        • Weeber R.
        • Holm C.
        • et al.
        ESPResSo 4.0–an extensible software package for simulating soft matter systems.
        Eur. Phys. J. Spec. Top. 2019; 227: 1789-1816
        • Marrink S.J.
        • Tieleman D.P.
        Perspective on the Martini model.
        Chem. Soc. Rev. 2013; 42: 6801-6822
        • Flyvbjerg H.
        • Petersen H.G.
        Error estimates on averages of correlated data.
        J. Chem. Phys. 1989; 91: 461-466
        • Irving J.H.
        • Kirkwood J.G.
        The statistical mechanical theory of transport processes. IV. The equations of hydrodynamics.
        J. Chem. Phys. 1950; 18: 817-829
        • Hardy R.J.
        Formulas for determining local properties in molecular-dynamics simulations: Shock waves.
        J. Chem. Phys. 1982; 76: 622-628
        • John Shipley Rowlinson and Benjamin Widom
        Molecular Theory of Capillarity.
        Courier Corporation, 2013
        • Allender D.W.
        • Sodt A.J.
        • Schick M.
        Cholesteroldependent bending energy is important in cholesterol distribution of the plasma membrane.
        Biophys. J. 2019; 116: 2356-2366
        • van Dijck P.W.
        Negatively charged phospholipids and their position in the cholesterol affinity sequence.
        Biochim. Biophys. Acta. 1979; 555: 89-101
        • Yeagle P.L.
        • Young J.E.
        Factors contributing to the distribution of cholesterol among phospholipid vesicles.
        J. Biol. Chem. 1986; 261: 8175-8181
        • Niu S.-L.
        • Litman B.J.
        Determination of membrane cholesterol partition coefficient using a lipid vesicle–cyclodextrin binary system: effect of phospholipid acyl chain unsaturation and headgroup composition.
        Biophys. J. 2002; 83: 3408-3415
        • Tsamaloukas A.
        • Szadkowska H.
        • Heerklotz H.
        Thermodynamic comparison of the interactions of cholesterol with unsaturated phospholipid and sphingomyelins.
        Biophys. J. 2006; 90: 4479-4487
        • Bradley E.
        • Tibshirani R.J.
        An Introduction to the Bootstrap.
        CRC press, 1994
        • Doktorova M.
        • Symons J.L.
        • Levental I.
        • et al.
        Challenging the dogma-cell plasma membranes are asymmetric not only in phospholipid composition but also abundance.
        Biophys. J. 2021; 120: 147a
        • Symons J.L.
        • Doktorova M.
        • Levental I.
        • et al.
        Challenging old dogma with new tech: asymmetry of lipid abundances within the plasma membrane.
        Biophys. J. 2022; 121: 289a-290a
        • Rawicz W.
        • Olbrich K.C.
        • Evans E.
        • et al.
        Effect of chain length and unsaturation on elasticity of lipid bilayers.
        Biophys. J. 2000; 79: 328-339
        • Leeb F.
        • Maibaum L.
        Spatially resolving the condensing effect of cholesterol in lipid bilayers.
        Biophys. J. 2018; 115: 2179-2188
        • Huang J.
        • Buboltz J.T.
        • Feigenson G.W.
        Maximum solubility of cholesterol in phosphatidylcholine and phosphatidylethanolamine bilayers.
        Biochim. Biophys. Acta, Biomembr. 1999; 1417: 89-100
        • Veatch S.L.
        • Keller S.L.
        Separation of liquid phases in giant vesicles of ternary mixtures of phospholipids and cholesterol.
        Biophys. J. 2003; 85: 3074-3083
        • Zhao J.
        • Wu J.
        • Feigenson G.W.
        • et al.
        Phase studies of model biomembranes: complex behavior of DSPC/DOPC/cholesterol.
        Biochim. Biophys. Acta. 2007; 1768: 2764-2776
        • Veatch S.L.
        • Keller S.L.
        Seeing spots: complex phase behavior in simple membranes.
        Biochim. Biophys. Acta. 2005; 1746: 172-185
        • Veatch S.L.
        • Cicuta P.
        • Baird B.
        • et al.
        Critical fluctuations in plasma membrane vesicles.
        ACS Chem. Biol. 2008; 3: 287-293
        • Marsh D.
        Cholesterol-induced fluid membrane domains: a compendium of lipid-raft ternary phase diagrams.
        Biochim. Biophys. Acta. 2009; 1788: 2114-2123
        • Pike L.J.
        Rafts defined: a report on the Keystone Symposium on lipid rafts and cell function.
        J. Lipid Res. 2006; 47: 1597-1598
        • Ciana A.
        • Achilli C.
        • Minetti G.
        Membrane rafts of the human red blood cell.
        Mol. Membr. Biol. 2014; 31: 47-57
        • Sodt A.J.
        • Venable R.M.
        • Pastor R.W.
        • et al.
        Nonadditive compositional curvature energetics of lipid bilayers.
        Phys. Rev. Lett. 2016; 117: 138104
        • Shi Z.
        • Graber Z.T.
        • Cohen A.E.
        • et al.
        Cell membranes resist flow.
        Cell. 2018; 175: 1769-1779.e13
        • Cohen A.E.
        • Shi Z.
        Do cell membranes flow like honey or jiggle like jello?.
        Bioessays. 2020; 42: 1900142