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

Abstract

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 phospholipidmore » 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.« less

Authors:
ORCiD logo [1];  [2];  [3];  [4]; ORCiD logo [5]; ORCiD logo [6];  [3];  [7]
+ Show Author Affiliations
  1. Weill Cornell Medical College, New York, NY (United States)
  2. Oak Ridge National Lab. (ORNL), Oak Ridge, TN (United States); Univ. of Tennessee, Knoxville, TN (United States)
  3. Univ. of Graz (Austria)
  4. Oak Ridge National Lab. (ORNL), Oak Ridge, TN (United States); East Tennessee State Univ., Johnson City, TN (United States)
  5. Oak Ridge National Lab. (ORNL), Oak Ridge, TN (United States)
  6. Stony Brook Univ., NY (United States)
  7. Univ. of Windsor, ON (Canada)
Publication Date:
Research Org.:
Oak Ridge National Laboratory (ORNL), Oak Ridge, TN (United States)
Sponsoring Org.:
USDOE Office of Science (SC), Basic Energy Sciences (BES); National Science Foundation (NSF)
OSTI Identifier:
1561603
Grant/Contract Number:  
AC05-00OR22725
Resource Type:
Accepted Manuscript
Journal Name:
Nature Protocols
Additional Journal Information:
Journal Volume: 13; Journal Issue: 9; Journal ID: ISSN 1754-2189
Publisher:
Nature Publishing Group
Country of Publication:
United States
Language:
English
Subject:
59 BASIC BIOLOGICAL SCIENCES

Citation Formats

Doktorova, Milka, Heberle, Frederick A., Eicher, Barbara, Standaert, Robert F., Katsaras, John, London, Erwin, Pabst, Georg, and Marquardt, Drew. Preparation of asymmetric phospholipid vesicles for use as cell membrane models. United States: N. p., 2018. Web. doi:10.1038/s41596-018-0033-6.
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Doktorova, Milka, Heberle, Frederick A., Eicher, Barbara, Standaert, Robert F., Katsaras, John, London, Erwin, Pabst, Georg, & Marquardt, Drew. Preparation of asymmetric phospholipid vesicles for use as cell membrane models. United States. https://doi.org/10.1038/s41596-018-0033-6
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Doktorova, Milka, Heberle, Frederick A., Eicher, Barbara, Standaert, Robert F., Katsaras, John, London, Erwin, Pabst, Georg, and Marquardt, Drew. Thu . "Preparation of asymmetric phospholipid vesicles for use as cell membrane models". United States. https://doi.org/10.1038/s41596-018-0033-6. https://www.osti.gov/servlets/purl/1561603.
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@article{osti_1561603,
title = {Preparation of asymmetric phospholipid vesicles for use as cell membrane models},
author = {Doktorova, Milka and Heberle, Frederick A. and Eicher, Barbara and Standaert, Robert F. and Katsaras, John and London, Erwin and Pabst, Georg and Marquardt, Drew},
abstractNote = {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.},
doi = {10.1038/s41596-018-0033-6},
journal = {Nature Protocols},
number = 9,
volume = 13,
place = {United States},
year = {Thu Sep 06 00:00:00 EDT 2018},
month = {Thu Sep 06 00:00:00 EDT 2018}
}
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Journal Article:
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Publisher's Version of Record
https://doi.org/10.1038/s41596-018-0033-6
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Cited by: 87 works
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Figures / Tables:

Fig. 1 Fig. 1: Illustration of the different aLUV preparation protocols. a, Chemical structure of methyl-β-cyclodextrin (mβCD), and the size and shape of its hydrophobic pocket. b, Heavy-acceptor strategy: (1) mβCD is incubated with donor lipid MLVs suspended in buffer; (2) mβCD facilitates the exchange of the outer leaflet of acceptor LUVsmore » (entrapped with sucrose) with donor lipid; (3) desired aLUVs are recovered from the pellet after ultracentrifugation through a sucrose cushion. c, Heavy-donor strategy: (1) mβCD is incubated with donor lipid MLVs entrapped with sucrose. The donor lipid is composed of the desired outer leaflet lipid; (2) mβCD facilitates the exchange of the outer leaflet of the acceptor LUVs with donor lipid; (3) the remaining sucrose-loaded donor MLVs are removed by centrifugation; (4) the aLUV sample is further purified through the removal of mβCD and mβCD–lipid complexes with a centrifugal concentrator. The desired aLUVs are then recovered from the retentate Adapted from Heberle et al. (original material licensed under a Creative Commons Attribution License).« less
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