Protocol | Published:

Elucidating membrane structure and protein behavior using giant plasma membrane vesicles

Nature Protocols volume 7, pages 10421051 (2012) | Download Citation

Abstract

The observation of phase separation in intact plasma membranes isolated from live cells is a breakthrough for research into eukaryotic membrane lateral heterogeneity, specifically in the context of membrane rafts. These observations are made in giant plasma membrane vesicles (GPMVs), which can be isolated by chemical vesiculants from a variety of cell types and microscopically observed using basic reagents and equipment available in any cell biology laboratory. Microscopic phase separation is detectable by fluorescent labeling, followed by cooling of the membranes below their miscibility phase transition temperature. This protocol describes the methods to prepare and isolate the vesicles, equipment to observe them under temperature-controlled conditions and three examples of fluorescence analysis: (i) fluorescence spectroscopy with an environment-sensitive dye (laurdan); (ii) two-photon microscopy of the same dye; and (iii) quantitative confocal microscopy to determine component partitioning between raft and nonraft phases. GPMV preparation and isolation, including fluorescent labeling and observation, can be accomplished within 4 h.

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References

  1. 1.

    & Functional rafts in cell membranes. Nature 387, 569–572 (1997).

  2. 2.

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

  3. 3.

    et al. Imaging of mobile long-lived nanoplatforms in the live cell plasma membrane. J. Biol. Chem. 285, 41765–41771 (2010).

  4. 4.

    et al. Direct observation of the nanoscale dynamics of membrane lipids in a living cell. Nature 457, 1159–1162 (2009).

  5. 5.

    , , , & Condensation of the plasma membrane at the site of T lymphocyte activation. J. Cell. Biol. 171, 121–131 (2005).

  6. 6.

    et al. Large-scale fluid/fluid phase separation of proteins and lipids in giant plasma membrane vesicles. Proc. Natl. Acad. Sci. USA 104, 3165–3170 (2007).

  7. 7.

    , , & Plasma membranes are poised for activation of raft phase coalescence at physiological temperature. Proc. Natl. Acad. Sci. USA 105, 10005–10010 (2008).

  8. 8.

    Plasma membrane vesiculation: a new technique for isolation of plasma membranes. Science 194, 743–745 (1976).

  9. 9.

    , , , & Plasma membrane vesiculation in 3T3 and SV3T3 cells. I. Morphological and biochemical characterization. J. Cell Sci. 35, 229–243 (1979).

  10. 10.

    et al. Quantitative analysis of phospholipids in functionally important membrane domains from RBL-2H3 mast cells using tandem high-resolution mass spectrometry. Biochemistry 38, 8056–8063 (1999).

  11. 11.

    , & Enhanced molecular diffusibility in muscle membrane blebs: release of lateral constraints. J. Cell Biol. 92, 207–212 (1982).

  12. 12.

    et al. Ordered and disordered phases coexist in plasma membrane vesicles of RBL-2H3 mast cells. An ESR study. Biophys. J. 85, 1278–1288 (2003).

  13. 13.

    & Lactoperoxidase-catalyzed iodination of the receptor for immunoglobulin E at the cytoplasmic side of the plasma membrane. J. Biol. Chem. 259, 3720–3728 (1984).

  14. 14.

    et al. Penetration of amphiphilic quantum dots through model and cellular plasma membranes. ACS Nano 6, 2150–2156 (2012).

  15. 15.

    et al. Penetration without cells: membrane translocation of cell-penetrating peptides in the model giant plasma membrane vesicles. J. Control Release 153, 117–125 (2011).

  16. 16.

    , , & Structural determinants for partitioning of lipids and proteins between coexisting fluid phases in giant plasma membrane vesicles. Biochim. Biophys. Acta 1778, 20–32 (2008).

  17. 17.

    et al. Cholesterol-dependent phase separation in cell-derived giant plasma-membrane vesicles. Biochem. J. 424, 163–167 (2009).

  18. 18.

    et al. Partitioning, diffusion, and ligand binding of raft lipid analogs in model and cellular plasma membranes. BBA—Biomembranes published online, doi:10.1016/j.bbamem.2012.03.007 (17 March 2012).

  19. 19.

    , , , & Palmitoylation regulates raft affinity for the majority of integral raft proteins. Proc. Natl. Acad. Sci. USA 107, 22050–22054 (2010).

  20. 20.

    et al. Temperature-dependent phase behavior and protein partitioning in giant plasma membrane vesicles. Biochim. Biophys. Acta 1798, 1427–1435 (2010).

  21. 21.

    et al. Critical fluctuations in plasma membrane vesicles. ACS Chem. Biol. 3, 287–293 (2008).

  22. 22.

    & Detergent resistance as a tool in membrane research. Nat. Protoc. 2, 2159–2165 (2007).

  23. 23.

    , & Retention of prominin in microvilli reveals distinct cholesterol-based lipid micro-domains in the apical plasma membrane. Nat. Cell Biol. 2, 582–592 (2000).

  24. 24.

    , , , & Resistance of cell membranes to different detergents. Proc. Natl. Acad. Sci. USA 100, 5795–5800 (2003).

  25. 25.

    & Separation of liquid phases in giant vesicles of ternary mixtures of phospholipids and cholesterol. Biophys. J. 85, 3074–3083 (2003).

  26. 26.

    & Organization in lipid membranes containing cholesterol. Phys. Rev. Lett. 89, 268101 (2002).

  27. 27.

    et al. Lipid rafts reconstituted in model membranes. Biophys. J. 80, 1417–1428 (2001).

  28. 28.

    , & Raft domains of variable properties and compositions in plasma membrane vesicles. Proc. Natl. Acad. Sci. USA 108, 11411–11416 (2011).

  29. 29.

    , & PI(4,5)P2 degradation promotes the formation of cytoskeleton-free model membrane systems. Chemphyschem 10, 2805–2812 (2009).

  30. 30.

    & Structural studies on the membrane-bound immunoglobulin E-receptor complex. 1. Characterization of large plasma membrane vesicles from rat basophilic leukemia cells and insertion of amphipathic fluorescent probes. Biochemistry 22, 3466–3474 (1983).

  31. 31.

    et al. Molecular convergence of bacterial and eukaryotic surface order. J. Biol. Chem. 286, 40631–40637 (2011).

  32. 32.

    & Lipid peroxides promote large rafts: effects of excitation of probes in fluorescence microscopy and electrochemical reactions during vesicle formation. Biophys. J. 91, 2172–2183 (2006).

  33. 33.

    , , , & Quantitative imaging of membrane lipid order in cells and organisms. Nat. Protoc. 7, 24–35 (2011).

  34. 34.

    & Precise and millidegree stable temperature control for fluorescence imaging: application to phase transitions in lipid membranes. Rev. Sci. Instrum. 81, 093704 (2010).

  35. 35.

    et al. Hemagglutinin of influenza virus partitions into the nonraft domain of model membranes. Biophys. J. 99, 489–498 (2010).

  36. 36.

    et al. A two-photon fluorescent probe for lipid raft imaging: C-laurdan. Chembiochem. 8, 553–559 (2007).

  37. 37.

    et al. Order of lipid phases in model and plasma membranes. Proc. Natl. Acad. Sci. USA 106, 16645–16650 (2009).

  38. 38.

    et al. Palmitoylation and intracellular domain interactions both contribute to raft targeting of linker for activation of T cells. J. Biol. Chem. 280, 18931–18942 (2005).

  39. 39.

    , & Raft partitioning and dynamic behavior of human placental alkaline phosphatase in giant unilamellar vesicles. Biochemistry 44, 7479–7489 (2005).

  40. 40.

    , , , & SNAREs prefer liquid-disordered over 'raft' (liquid-ordered) domains when reconstituted into giant unilamellar vesicles. J. Biol. Chem. 279, 37951–37955 (2004).

  41. 41.

    , , , & Fluorescence probe partitioning between Lo/Ld phases in lipid membranes. BBA—Biomembranes 1768, 2182 (2007).

  42. 42.

    , , & Fluorescence correlation spectroscopy relates rafts in model and native membranes. Biophys. J. 87, 1034–1043 (2004).

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Acknowledgements

The work was supported by the Max Planck Society; Humboldt Foundation Postdoctoral Fellowship, Technical University of Dresden; Deutsche Forschung Gemeinschaft (DFG) 'Schwerpunktprogramm1175' grant SI459/2-1, 'Transregio 83' grant TRR83 TP02; European Science Foundation 'LIPIDPROD' grant SI459/3-1; Bundesministerium für Bildung und Forschung 'ForMaT' grant 03FO1212; US National Institutes of Health grant R21AI073409; and the Klaus Tschira Foundation.

Author information

Affiliations

  1. Biophysics/BIOTEC, Technische Universität Dresden, Dresden, Germany.

    • Erdinc Sezgin
    •  & Petra Schwille
  2. Max Planck Institute for Molecular Cell Biology and Genetics, Dresden, Germany.

    • Erdinc Sezgin
    • , Kai Simons
    •  & Ilya Levental
  3. Max Delbrück Center for Molecular Medicine, Berlin, Germany.

    • Hermann-Josef Kaiser
  4. Department of Chemistry, University of Pennsylvania, Philadelphia, Pennsylvania, USA.

    • Tobias Baumgart

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Contributions

E.S. and I.L. gathered the data; E.S. and H.J.K. designed the laurdan experiments; I.L. and T.B. designed GPMV isolation, microscopy and partitioning protocols; and E.S., P.S., K.S. and I.L. wrote the manuscript.

Competing interests

The authors declare no competing financial interests.

Corresponding author

Correspondence to Ilya Levental.

Supplementary information

Videos

  1. 1.

    Supplementary Video 1

    GPMV formation. Shown is the initial 1 h of GPMV formation as in Step 10.

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DOI

https://doi.org/10.1038/nprot.2012.059

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