Preparation of asymmetric phospholipid vesicles for use as cell membrane models

Doktorova, Milka; Heberle, Frederick A.; Eicher, Barbara; Standaert, Robert F.; Katsaras, John; London, Erwin; Pabst, Georg; Marquardt, Drew

doi:10.1038/s41596-018-0033-6

Title: Preparation of asymmetric phospholipid vesicles for use as cell membrane models

Journal Article · Thu Sep 06 00:00:00 EDT 2018 · Nature Protocols

DOI:https://doi.org/10.1038/s41596-018-0033-6· OSTI ID:1561603

^[1]; Heberle, Frederick A. ^[2]; Eicher, Barbara ^[3]; Standaert, Robert F. ^[4];

^[5];

^[6]; Pabst, Georg ^[3]; Marquardt, Drew ^[7]

Weill Cornell Medical College, New York, NY (United States)
Oak Ridge National Lab. (ORNL), Oak Ridge, TN (United States); Univ. of Tennessee, Knoxville, TN (United States)
Univ. of Graz (Austria)
Oak Ridge National Lab. (ORNL), Oak Ridge, TN (United States); East Tennessee State Univ., Johnson City, TN (United States)
Oak Ridge National Lab. (ORNL), Oak Ridge, TN (United States)
Stony Brook Univ., NY (United States)
Univ. of Windsor, ON (Canada)

Freely suspended liposomes are widely used as model membranes for studying lipid–lipid and protein–lipid interactions. Liposomes prepared by conventional methods have chemically identical bilayer leaflets. By contrast, living cells actively maintain different lipid compositions in the two leaflets of the plasma membrane, resulting in asymmetric membrane properties that are critical for normal cell function. Here, we present a protocol for the preparation of unilamellar asymmetric phospholipid vesicles that better mimic biological membranes. Asymmetry is generated by methyl-β-cyclodextrin-catalyzed exchange of the outer leaflet lipids between vesicle pools of differing lipid composition. Lipid destined for the outer leaflet of the asymmetric vesicles is provided by heavy-donor multilamellar vesicles containing a dense sucrose core. Donor lipid is exchanged into extruded unilamellar acceptor vesicles that lack the sucrose core, facilitating the post-exchange separation of the donor and acceptor pools by centrifugation because of differences in vesicle size and density. We present two complementary assays allowing quantification of each leaflet’s lipid composition: the overall lipid composition is determined by gas chromatography–mass spectrometry, whereas the lipid distribution between the two leaflets is determined by NMR, using the lanthanide shift reagent Pr³⁺. The preparation protocol and the chromatographic assay can be applied to any type of phospholipid bilayer, whereas the NMR assay is specific to lipids with choline-containing headgroups, such as phosphatidylcholine and sphingomyelin. In ~12 h, the protocol can produce a large yield of asymmetric vesicles (up to 20 mg) suitable for a wide range of biophysical studies.

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Research Organization:: Oak Ridge National Laboratory (ORNL), Oak Ridge, TN (United States)

Sponsoring Organization:: USDOE Office of Science (SC), Basic Energy Sciences (BES); National Science Foundation (NSF)

Grant/Contract Number:: AC05-00OR22725

OSTI ID:: 1561603

Journal Information:: Nature Protocols, Vol. 13, Issue 9; ISSN 1754-2189

Publisher:: Nature Publishing GroupCopyright Statement

Country of Publication:: United States

Language:: English

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Cited by: 87 works

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References (39)

Dynamic membrane protein topological switching upon changes in phospholipid environment Vitrac, Heidi; MacLean, David M.; Jayaraman, Vasanthi Proceedings of the National Academy of Sciences, Vol. 112, Issue 45 https://doi.org/10.1073/pnas.1512994112	journal	October 2015
Engineering asymmetric vesicles Pautot, S.; Frisken, B. J.; Weitz, D. A. Proceedings of the National Academy of Sciences, Vol. 100, Issue 19 https://doi.org/10.1073/pnas.1931005100	journal	September 2003
Asymmetry and transposition rate of phosphatidylcholine in rat erythrocyte ghosts Bloj, Bernabe; Zilversmit, D. B. Biochemistry, Vol. 15, Issue 6 https://doi.org/10.1021/bi00651a017	journal	March 1976
Transbilayer diffusion of phospholipids: dependence on headgroup structure and acyl chain length Homan, Reynold; Pownall, Henry J. Biochimica et Biophysica Acta (BBA) - Biomembranes, Vol. 938, Issue 2 https://doi.org/10.1016/0005-2736(88)90155-1	journal	February 1988
Formation of asymmetric vesicles via phospholipase D-mediated transphosphatidylation Takaoka, Rina; Kurosaki, Haruko; Nakao, Hiroyuki Biochimica et Biophysica Acta (BBA) - Biomembranes, Vol. 1860, Issue 2 https://doi.org/10.1016/j.bbamem.2017.10.011	journal	February 2018
Preparation and Properties of Asymmetric Large Unilamellar Vesicles: Interleaflet Coupling in Asymmetric Vesicles Is Dependent on Temperature but Not Curvature Cheng, Hui-Ting; London, Erwin Biophysical Journal, Vol. 100, Issue 11 https://doi.org/10.1016/j.bpj.2011.04.048	journal	June 2011
Engineering Asymmetric Lipid Vesicles: Accurate and Convenient Control of the Outer Leaflet Lipid Composition Markones, Marie; Drechsler, Carina; Kaiser, Michael Langmuir, Vol. 34, Issue 5 https://doi.org/10.1021/acs.langmuir.7b03189	journal	January 2018
Dynamic Tension Spectroscopy and Strength of Biomembranes Evans, Evan; Heinrich, Volkmar; Ludwig, Florian Biophysical Journal, Vol. 85, Issue 4 https://doi.org/10.1016/S0006-3495(03)74658-X	journal	October 2003
Joint small-angle X-ray and neutron scattering data analysis of asymmetric lipid vesicles Eicher, Barbara; Heberle, Frederick A.; Marquardt, Drew Journal of Applied Crystallography, Vol. 50, Issue 2 https://doi.org/10.1107/S1600576717000656	journal	February 2017
Phospholipid asymmetry in large unilamellar vesicles induced by transmembrane pH gradients Hope, Michael J.; Redelmeier, Tom E.; Wong, Kim F. Biochemistry, Vol. 28, Issue 10 https://doi.org/10.1021/bi00436a009	journal	May 1989
Cholesterol Promotes Protein Binding by Affecting Membrane Electrostatics and Solvation Properties Doktorova, Milka; Heberle, Frederick A.; Kingston, Richard L. Biophysical Journal, Vol. 113, Issue 9 https://doi.org/10.1016/j.bpj.2017.08.055	journal	November 2017
Curvature Effect on the Structure of Phospholipid Bilayers Kučerka, Norbert; Pencer, Jeremy; Sachs, Jonathan N. Langmuir, Vol. 23, Issue 3 https://doi.org/10.1021/la062455t	journal	January 2007
The dependence of lipid asymmetry upon polar headgroup structure Son, Mijin; London, Erwin Journal of Lipid Research, Vol. 54, Issue 12 https://doi.org/10.1194/jlr.M041749	journal	October 2013
Preparation of Artificial Plasma Membrane Mimicking Vesicles with Lipid Asymmetry Lin, Qingqing; London, Erwin PLoS ONE, Vol. 9, Issue 1 https://doi.org/10.1371/journal.pone.0087903	journal	January 2014
Lipid Asymmetry in Membranes Op den Kamp, J. A. F. Annual Review of Biochemistry, Vol. 48, Issue 1 https://doi.org/10.1146/annurev.bi.48.070179.000403	journal	June 1979
Intrinsic Curvature-Mediated Transbilayer Coupling in Asymmetric Lipid Vesicles Eicher, Barbara; Marquardt, Drew; Heberle, Frederick A. Deutsches Elektronen-Synchrotron, DESY, Hamburg https://doi.org/10.3204/pubdb-2018-05680	text	January 2018
Protein-mediated phospholipid translocation in the endoplasmic reticulum with a low lipid specificity Herrmann, Andreas; Zachowski, Alain; Devaux, Philippe F. Biochemistry, Vol. 29, Issue 8 https://doi.org/10.1021/bi00460a010	journal	February 1990
The dependence of lipid asymmetry upon phosphatidylcholine acyl chain structure Son, Mijin; London, Erwin Journal of Lipid Research, Vol. 54, Issue 1 https://doi.org/10.1194/jlr.M032722	journal	October 2012
Lipid somersaults: Uncovering the mechanisms of protein-mediated lipid flipping Pomorski, Thomas Günther; Menon, Anant K. Progress in Lipid Research, Vol. 64 https://doi.org/10.1016/j.plipres.2016.08.003	journal	October 2016
Preparation and Properties of Asymmetric Vesicles That Mimic Cell Membranes Cheng, Hui-Ting; London, Erwin Journal of Biological Chemistry, Vol. 284, Issue 10 https://doi.org/10.1074/jbc.M806077200	journal	January 2009
Ordered Raft Domains Induced by Outer Leaflet Sphingomyelin in Cholesterol-Rich Asymmetric Vesicles Lin, Qingqing; London, Erwin Biophysical Journal, Vol. 108, Issue 9 https://doi.org/10.1016/j.bpj.2015.03.056	journal	May 2015
Lipid Rafts As a Membrane-Organizing Principle Lingwood, D.; Simons, K. Science, Vol. 327, Issue 5961 https://doi.org/10.1126/science.1174621	journal	December 2009
On the mechanism of transbilayer transport of phosphatidylglycerol in response to transmembrane pH gradients Redelmeier, T. E.; Hope, M. J.; Cullis, P. R. Biochemistry, Vol. 29, Issue 12 https://doi.org/10.1021/bi00464a022	journal	March 1990
¹ H NMR Shows Slow Phospholipid Flip-Flop in Gel and Fluid Bilayers Marquardt, Drew; Heberle, Frederick A.; Miti, Tatiana Langmuir, Vol. 33, Issue 15 https://doi.org/10.1021/acs.langmuir.6b04485	journal	February 2017
Osmotic properties of large unilamellar vesicles prepared by extrusion Mui, B. L.; Cullis, P. R.; Evans, E. A. Biophysical Journal, Vol. 64, Issue 2 https://doi.org/10.1016/S0006-3495(93)81385-7	journal	February 1993
Bilayer Asymmetry Influences Integrin Sequestering in Raft-Mimicking Lipid Mixtures Hussain, Noor F.; Siegel, Amanda P.; Ge, Yifan Biophysical Journal, Vol. 104, Issue 10 https://doi.org/10.1016/j.bpj.2013.04.020	journal	May 2013
Asymmetric GUVs Prepared by MβCD-Mediated Lipid Exchange: An FCS Study Chiantia, Salvatore; Schwille, Petra; Klymchenko, Andrey S. Biophysical Journal, Vol. 100, Issue 1 https://doi.org/10.1016/j.bpj.2010.11.051	journal	January 2011
Flip-Flop of Phospholipids in Vesicles: Kinetic Analysis with Time-Resolved Small-Angle Neutron Scattering Nakano, Minoru; Fukuda, Masakazu; Kudo, Takayuki The Journal of Physical Chemistry B, Vol. 113, Issue 19 https://doi.org/10.1021/jp900913w	journal	May 2009
Phosphatidylserine decarboxylase: generation of asymmetric vesicles and determination of the transbilayer distribution of fluorescent phosphatidylserine in model membrane systems Denkins, Yvonne M.; Schroit, Alan J. Biochimica et Biophysica Acta (BBA) - Biomembranes, Vol. 862, Issue 2 https://doi.org/10.1016/0005-2736(86)90237-3	journal	November 1986
Intrinsic Curvature-Mediated Transbilayer Coupling in Asymmetric Lipid Vesicles Eicher, Barbara; Marquardt, Drew; Heberle, Frederick A. Biophysical Journal, Vol. 114, Issue 1 https://doi.org/10.1016/j.bpj.2017.11.009	journal	January 2018
Thermodynamics of Methyl-β-cyclodextrin-Induced Lipid Vesicle Solubilization: Effect of Lipid Headgroup and Backbone Bozelli, José Carlos; Hou, Yu Heng; Epand, Richard M. Langmuir, Vol. 33, Issue 48 https://doi.org/10.1021/acs.langmuir.7b03447	journal	November 2017
The asymmetric distribution of phospholipids in the human red cell membrane. A combined study using phospholipases and freeze-etch electron microscopy Verkleij, A. J.; Zwaal, R. F. A.; Roelofsen, B. Biochimica et Biophysica Acta (BBA) - Biomembranes, Vol. 323, Issue 2 https://doi.org/10.1016/0005-2736(73)90143-0	journal	October 1973
Introduction and General Overview of Cyclodextrin Chemistry Szejtli, József Chemical Reviews, Vol. 98, Issue 5 https://doi.org/10.1021/cr970022c	journal	July 1998
Structural determinants and functional consequences of protein affinity for membrane rafts Lorent, Joseph H.; Diaz-Rohrer, Blanca; Lin, Xubo Nature Communications, Vol. 8, Issue 1 https://doi.org/10.1038/s41467-017-01328-3	journal	October 2017
Subnanometer Structure of an Asymmetric Model Membrane: Interleaflet Coupling Influences Domain Properties Heberle, Frederick A.; Marquardt, Drew; Doktorova, Milka Langmuir, Vol. 32, Issue 20 https://doi.org/10.1021/acs.langmuir.5b04562	journal	May 2016
Transbilayer asymmetry and sphingomyelin composition modulate the preferential membrane partitioning of the nicotinic acetylcholine receptor in Lo domains Perillo, Vanesa L.; Peñalva, Daniel A.; Vitale, Alejandro J. Archives of Biochemistry and Biophysics, Vol. 591 https://doi.org/10.1016/j.abb.2015.12.003	journal	February 2016
Efficient replacement of plasma membrane outer leaflet phospholipids and sphingolipids in cells with exogenous lipids Li, Guangtao; Kim, JiHyun; Huang, Zhen Proceedings of the National Academy of Sciences, Vol. 113, Issue 49 https://doi.org/10.1073/pnas.1610705113	journal	November 2016
Effect of Cyclodextrin and Membrane Lipid Structure upon Cyclodextrin–Lipid Interaction Huang, Zhen; London, Erwin Langmuir, Vol. 29, Issue 47 https://doi.org/10.1021/la4031427	journal	November 2013
A reaction tube for methanolysis; instability of hydrogen chloride in methanol Kishimoto, Yasuo; Radin, Norman S. Journal of Lipid Research, Vol. 6, Issue 3 https://doi.org/10.1016/S0022-2275(20)39317-2	journal	July 1965

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