It is a fundamental question in cell biology and biophysics whether sphingomyelin (SM)- and cholesterol (Chol)- driven nanodomains exist in living cells and in model membranes. Biophysical studies on model membranes revealed SM and Chol driven micrometer-sized liquid-ordered domains. Although the existence of such microdomains has not been proven for the plasma membrane, such lipid mixtures have been often used as a model system for 'rafts'. On the other hand, recent super resolution and single molecule results indicate that the plasma membrane might organize into nanocompartments. However, due to the limited resolution of those techniques their unambiguous characterization is still missing. In this work, a novel combination of Förster resonance energy transfer and Monte Carlo simulations (MC-FRET) identifies directly 10 nm large nanodomains in liquid-disordered model membranes composed of lipid mixtures containing SM and Chol. Combining MC-FRET with solid-state wide-line and high resolution magic angle spinning NMR as well as with fluorescence correlation spectroscopy we demonstrate that these nanodomains containing hundreds of lipid molecules are fluid and disordered. In terms of their size, fluidity, order and lifetime these nanodomains may represent a relevant model system for cellular membranes and are closely related to nanocompartments suggested to exist in cellular membranes.

Centre for Biological Sciences Institute for Life Sciences University of Southampton Southampton SO17 1BJ United Kingdom

Department of Biophysical Chemistry J Heyrovský Institute of Physical Chemistry of the A S C R v v i Prague Czech Republic

Department of Chemistry University of Umeå SE 901 87 Umeå Sweden

Shemyakin Ovchinnikov Institute of Bioorganic Chemistry of the Russian Academy of Science Moscow GSP 7 Russian Federation

Zobrazit více v PubMed

Simons K, Ikonen E. Functional rafts in cell membranes. Nature. 1997;387:569–572. doi: 10.1038/42408. PubMed DOI

Brown DA, London E. Structure and origin of ordered lipid domains in biological membranes. J. Membr. Biol. 1998;164:103–114. doi: 10.1007/s002329900397. PubMed DOI

Baumgart T, et al. Large-scale fluid/fluid phase separation of proteins and lipids in giant plasma membrane vesicles. Proc. Natl. Acad. Sci. USA. 2007;104:3165–3170. doi: 10.1073/pnas.0611357104. PubMed DOI PMC

Lingwood D, Simons K. Lipid rafts as a membrane-organizing principle. Science. 2010;327:46–50. doi: 10.1126/science.1174621. PubMed DOI

Levental I, Grzybek M, Simons K. Raft domains of variable properties and compositions in plasma membrane vesicles. Proc. Natl. Acad. Sci. USA. 2011;108:11411–6. doi: 10.1073/pnas.1105996108. PubMed DOI PMC

Sezgin E, et al. Adaptive lipid packing and bioactivity in membrane domains. PLoS One. 2015;10:1–14. doi: 10.1371/journal.pone.0123930. PubMed DOI PMC

Eggeling C, et al. Direct observation of the nanoscale dynamics of membrane lipids in a living cell. Nature. 2009;457:1159–1162. doi: 10.1038/nature07596. PubMed DOI

Bernardino de la Serna J, Schütz GJ, Eggeling C, Cebecauer M. There Is No Simple Model of the Plasma Membrane Organization. Front. Cell Dev. Biol. 2016;4:1–17. doi: 10.3389/fcell.2016.00106. PubMed DOI PMC

Owen DM, Williamson DJ, Magenau A, Gaus K. Sub-resolution lipid domains exist in the plasma membrane and regulate protein diffusion and distribution. Nat. Commun. 2012;3:1256. doi: 10.1038/ncomms2273. PubMed DOI

Kreder R, et al. Solvatochromic Nile Red Probes with FRET Quencher Reveal Lipid Order Heterogeneity in Living and Apoptotic Cells. ACS Chem. Biol. 2015;10:1435–1442. doi: 10.1021/cb500922m. PubMed DOI

Sanchez SA, Tricerri MA, Gratton E. Laurdan generalized polarization fluctuations measures membrane packing micro-heterogeneity in vivo. Proc. Natl. Acad. Sci. USA. 2012;109:7314–9. doi: 10.1073/pnas.1118288109. PubMed DOI PMC

Ritchie K, Iino R, Fujiwara T, Murase K, Kusumi A. The fence and picket structure of the plasma membrane of live cells as revealed by single molecule techniques (Review) Mol. Membr. Biol. 2003;20:13–18. doi: 10.1080/0968768021000055698. PubMed DOI

Varma R, Mayor S. GPI-anchored proteins are organized in submicron domains at the cell surface. Nature. 1998;394:798–801. doi: 10.1038/29563. PubMed DOI

Smith AK, Freed JH. Determination of Tie-Line Fields for Coexisting Lipid Phases: An ESR Study. J. Phys. Chem. B. 2009;113:3957–3971. doi: 10.1021/jp808412x. PubMed DOI PMC

Farkas, E. R. & Webb, W. W. Precise and millidegree stable temperature control for fluorescence imaging: Application to phase transitions in lipid membranes. Rev. Sci. Instrum. 81 (2010). PubMed PMC

Loura LMS, Fernandes F, Prieto M. Membrane microheterogeneity: Forster resonance energy transfer characterization of lateral membrane domains. Eur. Biophys. J. with Biophys. Lett. 2010;39:589–607. doi: 10.1007/s00249-009-0547-5. PubMed DOI

Loura LM, Fedorov A, Prieto M. Fluid-fluid membrane microheterogeneity: a fluorescence resonance energy transfer study. Biophys. J. 2001;80:776–788. doi: 10.1016/S0006-3495(01)76057-2. PubMed DOI PMC

Bader AN, et al. Homo-FRET imaging as a tool to quantify protein and lipid clustering. ChemPhysChem. 2011;12:475–483. doi: 10.1002/cphc.201000801. PubMed DOI

Benda A, et al. How to determine diffusion coefficients in planar phospholipid systems by confocal fluorescence correlation spectroscopy. Langmuir. 2003;19:4120–4126. doi: 10.1021/la0270136. DOI

Šachl R, Johansson LB-Å, Hof M. Förster resonance energy transfer (FRET) between heterogeneously distributed probes: Application to lipid nanodomains and pores. Int. J. Mol. Sci. 2012;13:16141–16156. doi: 10.3390/ijms131216141. PubMed DOI PMC

Šachl R, et al. Distribution of BODIPY-labelled phosphatidylethanolamines in lipid bilayers exhibiting different curvatures. Phys. Chem. Chem. Phys. 2011;13:11694–11701. doi: 10.1039/c1cp20608g. PubMed DOI

Štefl M, et al. Dynamics and size of cross-linking-induced lipid nanodomains in model membranes. Biophys. J. 2012;102:2104–2113. doi: 10.1016/j.bpj.2012.03.054. PubMed DOI PMC

Honigmann A, et al. Scanning STED-FCS reveals spatiotemporal heterogeneity of lipid interaction in the plasma membrane of living cells. Nat. Commun. 2014;5:5412. doi: 10.1038/ncomms6412. PubMed DOI

Šachl R, et al. On multivalent receptor activity of GM1 in cholesterol containing membranes. Biochim. Biophys. Acta. 2015;1853:850–7. doi: 10.1016/j.bbamcr.2014.07.016. PubMed DOI

Amaro M, et al. GM1 Ganglioside Inhibits b-amyloid Oligomerization Induced by Sphingomyelin. Angew. Chemie. 2016;55:9411–9415. doi: 10.1002/anie.201603178. PubMed DOI PMC

Marushchak D, Gretskaya N, Mikhalyov I, Johansson LB-Å. Self-aggregation - an intrinsic property of G(M1) in lipid bilayers. Mol. Membr. Biol. 2007;24:102–112. doi: 10.1080/09687860600995235. PubMed DOI

Nyholm TKM, Lindroos D, Westerlund B, Slotte JP. Construction of a DOPC/PSM/cholesterol phase diagram based on the fluorescence properties of trans-parinaric acid. Langmuir. 2011;27:8339–50. doi: 10.1021/la201427w. PubMed DOI

Veatch SL, Keller SL. Miscibility Phase Diagrams of Giant Vesicles Containing Sphingomyelin. Phys. Rev. Lett. 2005;94:148101. doi: 10.1103/PhysRevLett.94.148101. PubMed DOI

Rheinstädter MC, Mouritsen OG. Small-scale structure in fluid cholesterol-lipid bilayers. Curr. Opin. Colloid Interface Sci. 2013;18:440–447. doi: 10.1016/j.cocis.2013.07.001. DOI

Leidy C, Wolkers WF, Jørgensen K, Mouritsen OG, Crowe JH. Lateral Organization and Domain Formation in a Two-Component Lipid Membrane System. Biophys. J. 2001;80:1819–1828. doi: 10.1016/S0006-3495(01)76152-8. PubMed DOI PMC

Epand RM, et al. Novel properties of cholesterol-dioleoylphosphatidylcholine mixtures. Biochim. Biophys. Acta - Biomembr. 2003;1616:196–208. doi: 10.1016/j.bbamem.2003.08.006. PubMed DOI

Šachl, R., Bergstrand, J., Widengren, J. & Hof, M. Fluorescence correlation spectroscopy diffusion laws in the presence of moving nanodomains. J. Phys.D Appl. Phys. 49, 114002 (11pp) (2016).

Korlach J, Schwille P, Webb W, Feigenson GW. Characterization of Lipid Bilayer Phases By Confocal Microscopy and Fluorescence Correlation Spectroscopy. Proc. Natl. Acad. Sci. 1999;96:8461–8466. doi: 10.1073/pnas.96.15.8461. PubMed DOI PMC

Dufourc EJ, Mayer C, Stohrer J, Althoff G, 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. doi: 10.1016/S0006-3495(92)81814-3. PubMed DOI PMC

Holland GP, McIntyre SK, Alam TM. Distinguishing individual lipid headgroup mobility and phase transitions in raft-forming lipid mixtures with 31P MAS NMR. Biophys. J. 2006;90:4248–4260. doi: 10.1529/biophysj.105.077289. PubMed DOI PMC

Lindström F, Williamson PTF, Gröbner G. Molecular insight into the electrostatic membrane surface potential by 14N/31p MAS NMR spectroscopy: nociceptin-lipid association. J. Am. Chem. Soc. 2005;127:6610–6. doi: 10.1021/ja042325b. PubMed DOI

Cullis PR, De Kruijff B. Lipid polymorphism and the functional roles of lipids in biological membranes. Biochim. Biophys. Acta - Rev. Biomembr. 1979;559:399–420. doi: 10.1016/0304-4157(79)90012-1. PubMed DOI

Ernst, R. R., Bodenhausen, G. & Wokaun, A. Principles of Nuclear Resonance in One and Two Dimensions. (Oxford University Press, 1987).

Bonev BB, Chan WC, Bycroft BW, Roberts GCK, Watts A. Interaction of the lantibiotic nisin with mixed lipid bilayers: A 31P and 2H NMR study. Biochemistry. 2000;39:11425–11433. doi: 10.1021/bi0001170. PubMed DOI

Armstrong CL, et al. The Observation of Highly Ordered Domains in Membranes with Cholesterol. PLoS One. 2013;8:1–10. PubMed PMC

Baoukina, S., Mendez-Villuendas, E., Bennett, W. F. D. & Tieleman, D. P. Computer simulations of the phase separation in model membranes. Faraday Discuss. 63–75, doi:10.1039/c2fd20117h (2013). PubMed

Honerkamp-Smith, A. R. R. Machta, B., Benjamin, B. & Keller, S. Experimental Observations of Dynamic Critical Phenomena in a Lipid Membrane. Phys. Rev. Lett. 108, 265702 (5pp) (2012). PubMed PMC

Ohvo-Rekilä H, Ramstedt B, Leppimäki P, Peter Slotte J. Cholesterol interactions with phospholipids in membranes. Prog. Lipid Res. 2002;41:66–97. doi: 10.1016/S0163-7827(01)00020-0. PubMed DOI

Silvius JR, Del Giudice D, Lafleur M. Cholesterol at different bilayer concentrations can promote or antagonize lateral segregation of phospholipids of differing acyl chain length. Biochemistry. 1996;35:15198–15208. doi: 10.1021/bi9615506. PubMed DOI

Slotte JP. Sphingomyelin-cholesterol interactions in biological and model membranes. Chem. Phys. Lipids. 1999;102:13–27. doi: 10.1016/S0009-3084(99)00071-7. PubMed DOI

Boggs JM. Lipid intermolecular hydrogen bonding: influence on structural organization and membrane function. Biochim. Biophys. Acta - Rev. Biomembr. 1987;906:353–404. doi: 10.1016/0304-4157(87)90017-7. PubMed DOI

Hyvonen MT, et al. Molecular dynamics simulation of sphingomyelin bilayer. J. Phys. Chem. B. 2003;107:9102–9108. doi: 10.1021/jp035319v. DOI

Huang J, Feigenson GW. Monte Carlo simulation of lipid mixtures: finding phase separation. Biophys. J. 1993;65:1788–1794. doi: 10.1016/S0006-3495(93)81234-7. PubMed DOI PMC

Sezgin E, et al. Elucidating membrane structure and protein behavior using giant plasma membrane vesicles. Nat. Protoc. 2012;7:1042–51. doi: 10.1038/nprot.2012.059. PubMed DOI

Angelova M, Soleau S, Meleard P. Preparation of Giant Vesicles by External AC Electric Fields - Kinetics and Applications. Trends Colloid Interface Sci. VI. 1992;89:127–131. doi: 10.1007/BFb0116295. DOI

Wallgren M, Lidman M, Pham QD, Cyprych K, Gröbner G. The oxidized phospholipid PazePC modulates interactions between Bax and mitochondrial membranes. Biochim. Biophys. Acta - Biomembr. 2012;1818:2718–2724. doi: 10.1016/j.bbamem.2012.06.005. PubMed DOI

Bennett AE, Rienstra CM, Auger M, Lakshmi KV, Griffin RG. Heteronuclear decoupling in rotating solids. J. Chem. Phys. 1995;103:6951. doi: 10.1063/1.470372. DOI

van Beek J. D. matNMR: A flexible toolbox for processing, analyzing and visualizing magnetic resonance data in Matlab? J. Magn. Reson. 2007;187:19–26. doi: 10.1016/j.jmr.2007.03.017. PubMed DOI

Valeur, B. Molecular Fluorescence Principles and Applications (2001).

Macháň R, Hof M. Recent Developments in Fluorescence Correlation Spectroscopy for Diffusion Measurements in Planar Lipid Membranes. Int. J. Mol. Sci. 2010;11:427–457. doi: 10.3390/ijms11020427. PubMed DOI PMC

Which Moiety Drives Gangliosides to Form Nanodomains?

Interleaflet organization of membrane nanodomains: What can(not) be resolved by FRET?

The impact of the glycan headgroup on the nanoscopic segregation of gangliosides

Interleaflet Coupling of Lipid Nanodomains - Insights From in vitro Systems

FSCS Reveals the Complexity of Lipid Domain Dynamics in the Plasma Membrane of Live Cells