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Elucidating membrane structure and protein behavior using giant plasma membrane vesicles

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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Figure 1: Overview of the protocol.
Figure 2: GPMV visualization.
Figure 3: C-laurdan characterization of GPMVs.
Figure 4: Temperature-controlled imaging.
Figure 5: Quantification of component partitioning.

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References

  1. Simons, K. & Ikonen, E. Functional rafts in cell membranes. Nature 387, 569–572 (1997).

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

  5. Gaus, K., Chklovskaia, E., Fazekas de St Groth, B., Jessup, W. & Harder, T. Condensation of the plasma membrane at the site of T lymphocyte activation. J. Cell. Biol. 171, 121–131 (2005).

    Article  CAS  Google Scholar 

  6. Baumgart, T. 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).

    Article  CAS  Google Scholar 

  7. Lingwood, D., Ries, J., Schwille, P. & Simons, K. Plasma membranes are poised for activation of raft phase coalescence at physiological temperature. Proc. Natl. Acad. Sci. USA 105, 10005–10010 (2008).

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

  9. Scott, R.E., Perkins, R.G., Zschunke, M.A., Hoerl, B.J. & Maercklein, P.B. Plasma membrane vesiculation in 3T3 and SV3T3 cells. I. Morphological and biochemical characterization. J. Cell Sci. 35, 229–243 (1979).

    CAS  Google Scholar 

  10. Fridriksson, E.K. 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).

    Article  CAS  Google Scholar 

  11. Tank, D.W., Wu, E.S. & Webb, W.W. Enhanced molecular diffusibility in muscle membrane blebs: release of lateral constraints. J. Cell Biol. 92, 207–212 (1982).

    Article  CAS  Google Scholar 

  12. Ge, M. 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).

    Article  CAS  Google Scholar 

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

    CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

  15. Saalik, P. 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).

    Article  Google Scholar 

  16. Sengupta, P., Hammond, A., Holowka, D. & Baird, B. Structural determinants for partitioning of lipids and proteins between coexisting fluid phases in giant plasma membrane vesicles. Biochim. Biophys. Acta 1778, 20–32 (2008).

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

  18. Sezgin, E. 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. Levental, I., Lingwood, D., Grzybek, M., Coskun, U. & Simons, K. Palmitoylation regulates raft affinity for the majority of integral raft proteins. Proc. Natl. Acad. Sci. USA 107, 22050–22054 (2010).

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

  23. Roper, K., Corbeil, D. & Huttner, W.B. Retention of prominin in microvilli reveals distinct cholesterol-based lipid micro-domains in the apical plasma membrane. Nat. Cell Biol. 2, 582–592 (2000).

    Article  CAS  Google Scholar 

  24. Schuck, S., Honsho, M., Ekroos, K., Shevchenko, A. & Simons, K. Resistance of cell membranes to different detergents. Proc. Natl. Acad. Sci. USA 100, 5795–5800 (2003).

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

  26. Veatch, S.L. & Keller, S.L. Organization in lipid membranes containing cholesterol. Phys. Rev. Lett. 89, 268101 (2002).

    Article  Google Scholar 

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

    Article  CAS  Google Scholar 

  28. Levental, I., Grzybek, M. & Simons, K. Raft domains of variable properties and compositions in plasma membrane vesicles. Proc. Natl. Acad. Sci. USA 108, 11411–11416 (2011).

    Article  CAS  Google Scholar 

  29. Keller, H., Lorizate, M. & Schwille, P. PI(4,5)P2 degradation promotes the formation of cytoskeleton-free model membrane systems. Chemphyschem 10, 2805–2812 (2009).

    Article  CAS  Google Scholar 

  30. Holowka, D. & Baird, B. 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).

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

  32. Ayuyan, A.G. & Cohen, F.S. 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).

    Article  CAS  Google Scholar 

  33. Owen, D.M., Rentero, C., Magenau, A., Abu-Siniyeh, A. & Gaus, K. Quantitative imaging of membrane lipid order in cells and organisms. Nat. Protoc. 7, 24–35 (2011).

    Article  Google Scholar 

  34. 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, 093704 (2010).

    Article  Google Scholar 

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

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

  38. Shogomori, H. 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).

    Article  CAS  Google Scholar 

  39. Kahya, N., Brown, D.A. & Schwille, P. Raft partitioning and dynamic behavior of human placental alkaline phosphatase in giant unilamellar vesicles. Biochemistry 44, 7479–7489 (2005).

    Article  CAS  Google Scholar 

  40. Bacia, K., Schuette, C.G., Kahya, N., Jahn, R. & Schwille, P. SNAREs prefer liquid-disordered over 'raft' (liquid-ordered) domains when reconstituted into giant unilamellar vesicles. J. Biol. Chem. 279, 37951–37955 (2004).

    Article  CAS  Google Scholar 

  41. Baumgart, T., Hunt, G., Farkas, E.R., Webb, W.W. & Feigenson, G.W. Fluorescence probe partitioning between Lo/Ld phases in lipid membranes. BBA—Biomembranes 1768, 2182 (2007).

    Article  CAS  Google Scholar 

  42. Bacia, K., Scherfeld, D., Kahya, N. & Schwille, P. Fluorescence correlation spectroscopy relates rafts in model and native membranes. Biophys. J. 87, 1034–1043 (2004).

    Article  CAS  Google Scholar 

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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.

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Authors and Affiliations

Authors

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.

Corresponding author

Correspondence to Ilya Levental.

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Competing interests

The authors declare no competing financial interests.

Supplementary information

Supplementary Video 1

GPMV formation. Shown is the initial 1 h of GPMV formation as in Step 10. (MP4 979 kb)

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Sezgin, E., Kaiser, HJ., Baumgart, T. et al. Elucidating membrane structure and protein behavior using giant plasma membrane vesicles. Nat Protoc 7, 1042–1051 (2012). https://doi.org/10.1038/nprot.2012.059

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