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 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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Fig. 1: Illustration of different aLUV preparation protocols.
Fig. 2: Gas chromatography (GC) assay for quantifying vesicle composition.
Fig. 3: 1H NMR assay for quantifying lipid asymmetry.

References

  1. 1.

    Lorent, J. H. et al. Structural determinants and functional consequences of protein affinity for membrane rafts. Nat. Commun. 8, 1219 (2017).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  2. 2.

    Lingwood, D. & Simons, K. Lipid rafts as a membrane-organizing principle. Science 327, 46–50 (2010).

    Article  PubMed  CAS  PubMed Central  Google Scholar 

  3. 3.

    Doktorova, M. et al. Cholesterol promotes protein binding by affecting membrane electrostatics and solvation properties. Biophys. J. 113, 2004–2015 (2017).

    Article  PubMed  CAS  Google Scholar 

  4. 4.

    Op den Kamp, J. A. F. Lipid asymmetry in membranes. Annu. Rev. Biochem. 48, 47–41 (1979).

    Article  PubMed  CAS  Google Scholar 

  5. 5.

    Verkleij, A. J. 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 323, 178–193 (1973).

    Article  PubMed  CAS  Google Scholar 

  6. 6.

    Fadok, V. A. et al. Exposure of phosphatidylserine on the surface of apoptotic lymphocytes triggers specific recognition and removal by macrophages. J. Immunol. 148, 2207–2216 (1992).

    PubMed  CAS  Google Scholar 

  7. 7.

    Perillo, V. L., Penalva, D. A., Vitale, A. J., Barrantes, F. J. & Antollini, S. S. Transbilayer asymmetry and sphingomyelin composition modulate the preferential membrane partitioning of the nicotinic acetylcholine receptor in Lo domains. Arch. Biochem. Biophys. 591, 76–86 (2016).

    Article  PubMed  CAS  Google Scholar 

  8. 8.

    Vitrac, H., MacLean, D. M., Jayaraman, V., Bogdanov, M. & Dowhan, W. Dynamic membrane protein topological switching upon changes in phospholipid environment. Proc. Natl. Acad. Sci. USA 112, 13874–13879 (2015).

    Article  PubMed  CAS  Google Scholar 

  9. 9.

    Hussain, N. F., Siegel, A. P., Ge, Y., Jordan, R. & Naumann, C. A. Bilayer asymmetry influences integrin sequestering in raft-mimicking lipid mixtures. Biophys. J. 104, 2212–2221 (2013).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  10. 10.

    Lin, Q. & London, E. The influence of natural lipid asymmetry upon the conformation of a membrane-inserted protein (perfringolysin O). J. Biol. Chem. 289, 5467–5478 (2014).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  11. 11.

    St. Clair, J. R., Wang, Q., Li, G. & London, E. in The Biophysics of Cell Membranes Vol. 19 (eds. Epand, R. & Ruysschaert, J. M.) 1–27 (Springer, Singapore, 2017).

  12. 12.

    Pautot, S., Frisken, B. J. & Weitz, D. A. Engineering asymmetric vesicles. Proc. Natl. Acad. Sci. USA 100, 10718–10721 (2003).

    Article  PubMed  CAS  Google Scholar 

  13. 13.

    Redelmeier, T. E., Hope, M. J. & Cullis, P. R. On the mechanism of transbilayer transport of phosphatidylglycerol in response to transmembrane pH gradients. Biochemistry 29, 3046–3053 (1990).

    Article  PubMed  CAS  Google Scholar 

  14. 14.

    Hope, M. J., Redelmeier, T. E., Wong, K. F., Rodrigueza, W. & Cullis, P. R. Phospholipid asymmetry in large unilamellar vesicles induced by transmembrane pH gradients. Biochemistry 28, 4181–4187 (1989).

    Article  PubMed  CAS  Google Scholar 

  15. 15.

    Denkins, Y. M. & Schroit, A. J. Phosphatidylserine decarboxylase: generation of asymmetric vesicles and determination of the transbilayer distribution of fluorescent phosphatidylserine in model membrane systems. Biochim. Biophys. Acta 862, 343–351 (1986).

    Article  PubMed  CAS  Google Scholar 

  16. 16.

    Takaoka, R., Kurosaki, H., Nakao, H., Ikeda, K. & Nakano, M. Formation of asymmetric vesicles via phospholipase D-mediated transphosphatidylation. Biochim. Biophys. Acta 1860, 245–249 (2017).

    Article  PubMed  CAS  Google Scholar 

  17. 17.

    Bloj, B. & Zilversmit, D. B. Asymmetry and transposition rates of phosphatidylcholine in rat erythrocyte ghosts. Biochemistry 15, 1277–1283 (1976).

    Article  PubMed  CAS  Google Scholar 

  18. 18.

    Herrmann, A., Zachowski, A. & Devaux, P. F. Protein-mediated phospholipid translocation in the endoplasmic reticulum with a low lipid specificity. Biochemistry 29, 2023–2027 (1990).

    Article  PubMed  CAS  Google Scholar 

  19. 19.

    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. 100, 2671–2678 (2011).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  20. 20.

    Szejtli, J. Introduction and general overview of cyclodextrin chemistry. Chem. Rev. 98, 1743–1754 (1998).

    Article  PubMed  CAS  Google Scholar 

  21. 21.

    Bozelli, J. C. Jr., Hou, Y. H. & Epand, R. M. Thermodynamics of methyl-beta-cyclodextrin-induced lipid vesicle solubilization: effect of lipid headgroup and backbone. Langmuir 33, 13882–13891 (2017).

    Article  PubMed  CAS  Google Scholar 

  22. 22.

    Heberle, F. A. et al. Subnanometer structure of an asymmetric model membrane: interleaflet coupling influences domain properties. Langmuir 32, 5195–5200 (2016).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  23. 23.

    Mui, B. L., Cullis, P. R., Evans, E. A. & Madden, T. D. Osmotic properties of large unilamellar vesicles prepared by extrusion. Biophys. J. 64, 443–453 (1993).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  24. 24.

    Evans, E., Heinrich, V., Ludwig, F. & Rawicz, W. Dynamic tension spectroscopy and strength of biomembranes. Biophys. J. 85, 2342–2350 (2003).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  25. 25.

    Homan, R. & Pownall, H. J. Transbilayer diffusion of phospholipids: dependence on headgroup structure and acyl chain length. Biochim. Biophys. Acta 938, 155–166 (1988).

    Article  PubMed  CAS  Google Scholar 

  26. 26.

    Marsh, D. Handbook of Lipid Bilayers 2nd edn (CRC Press, Boca Raton, FL, 2013).

  27. 27.

    Pomorski, T. G. & Menon, A. K. Lipid somersaults: uncovering the mechanisms of protein-mediated lipid flipping. Prog. Lipid Res. 64, 69–84 (2016).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  28. 28.

    Son, M. & London, E. The dependence of lipid asymmetry upon polar headgroup structure. J. Lipid Res. 54, 3385–3393 (2013).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  29. 29.

    Son, M. & London, E. The dependence of lipid asymmetry upon phosphatidylcholine acyl chain structure. J. Lipid Res. 54, 223–231 (2013).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  30. 30.

    Nakano, M. et al. Flip-flop of phospholipids in vesicles: kinetic analysis with time-resolved small-angle neutron scattering. J. Phys. Chem. B 113, 6745–6748 (2009).

    Article  PubMed  CAS  Google Scholar 

  31. 31.

    Eicher, B. et al. Joint small-angle X-ray and neutron scattering data analysis of asymmetric lipid vesicles. J. Appl. Crystallogr. 50, 419–429 (2017).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  32. 32.

    Markones, M. et al. Engineering asymmetric lipid vesicles: accurate and convenient control of the outer leaflet lipid composition. Langmuir 34, 1999–2005 (2018).

    Article  PubMed  CAS  Google Scholar 

  33. 33.

    Eicher, B. et al. Intrinsic curvature-mediated transbilayer coupling in asymmetric lipid vesicles. Biophys. J. 114, 146–157 (2018).

    Article  PubMed  CAS  Google Scholar 

  34. 34.

    Marquardt, D. et al. 1H NMR shows slow phospholipid flip-flop in gel and fluid bilayers. Langmuir 33, 3731–3741 (2017).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  35. 35.

    Cheng, H. T., Megha & London, E. Preparation and properties of asymmetric vesicles that mimic cell membranes: effect upon lipid raft formation and transmembrane helix orientation. J. Biol. Chem. 284, 6079–6092 (2009).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  36. 36.

    Lin, Q. & London, E. Preparation of artificial plasma membrane mimicking vesicles with lipid asymmetry. PLoS ONE 9, e87903 (2014).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  37. 37.

    Li, G. et al. Efficient replacement of plasma membrane outer leaflet phospholipids and sphingolipids in cells with exogenous lipids. Proc. Natl. Acad. Sci. USA 113, 14025–14030 (2016).

    Article  PubMed  CAS  Google Scholar 

  38. 38.

    Kishimoto, Y. & Radin, N. S. A reaction tube for methanolysis; instability of hydrogen chloride in methanol. J. Lipid Res. 6, 435–436 (1965).

    PubMed  CAS  Google Scholar 

  39. 39.

    Kucerka, N., Pencer, J., Sachs, J. N., Nagle, J. F. & Katsaras, J. Curvature effect on the structure of phospholipid bilayers. Langmuir 23, 1292–1299 (2007).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  40. 40.

    Huang, Z. & London, E. Effect of cyclodextrin and membrane lipid structure upon cyclodextrin-lipid interaction. Langmuir 29, 14631–14638 (2013).

    Article  PubMed  CAS  Google Scholar 

  41. 41.

    Chiantia, S., Schwille, P., Klymchenko, A. S. & London, E. Asymmetric GUVs prepared by MbetaCD-mediated lipid exchange: an FCS study. Biophys. J. 100, L1–L3 (2011).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  42. 42.

    Lin, Q. & London, E. Ordered raft domains induced by outer leaflet sphingomyelin in cholesterol-rich asymmetric vesicles. Biophys. J. 108, 2212–2222 (2015).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

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Acknowledgements

The authors acknowledge support from University of Windsor startup funds (to D.M.); Natural Sciences and Engineering Research Council of Canada (NSERC) funding ref. no. 2018-04841 (to D.M.); Austrian Science Fund (FWF) project P27083 (to G.P.); U.S. National Science Foundation (NSF) grant DMR 1709035 (to E.L.); NSF grant MCB-1817929 (to F.A.H.); and the Laboratory Directed Research and Development Program of Oak Ridge National Laboratory (to F.A.H., J.K., and R.F.S.), managed by UT-Battelle for the U.S. Department of Energy under contract no. DE-AC05 00OR22725.

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M.D., F.A.H., and D.M. wrote the manuscript; M.D., F.A.H., B.E., G.P., E.L., R.F.S., J.K., and D.M. provided input and edited the manuscript; M.D., F.A.H., B.E., and D.M. conducted the experiments.

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Correspondence to Drew Marquardt.

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Key references using this protocol

1. Eicher, B. et al. Biophys. J. 114, 146–157 (2018): https://doi.org/10.1016/j.bpj.2017.11.009

2. Eicher, B. et al. J. Appl. Crystallogr. 50, 419–429 (2017): https://doi.org/10.1107/S1600576717000656

3. Marquardt, D. et al. Langmuir 33, 3731–3741 (2017): https://doi.org/10.1021/acs.langmuir.6b04485

4. Heberle, F. A. et al. Langmuir 32, 5195–5200 (2016): https://doi.org/10.1021/acs.langmuir.5b04562

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Doktorova, M., Heberle, F.A., Eicher, B. et al. Preparation of asymmetric phospholipid vesicles for use as cell membrane models. Nat Protoc 13, 2086–2101 (2018). https://doi.org/10.1038/s41596-018-0033-6

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