Abstract
Giant unilamellar vesicles (GUVs) are routinely used to study coexisting liquid phases in bilayer membranes. Liquid domains are observed in a wide variety of ternary GUV membranes containing phospholipids and cholesterol, and are thought to model raft domains in cell membranes. GUVs are attractive model systems because vesicles are easily prepared using standard and inexpensive laboratory equipment, and phase-separated vesicles can be visualized using optical microscopy. In this chapter, a detailed method is presented to form and view 10–100-µm diameter single-walled vesicles of charged or uncharged lipid mixtures. GUVs can be visualized by fluorescence microscopy and methods are presented to measure miscibility transition temperatures and to distinguish solid (gel) and liquid domains. Numerous experimental artifacts associated with GUV preparation and viewing are discussed, including the effects of nonideal growth conditions and perturbations of fluorescent probes and other impurities.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
References
Bagatolli, L. A. and Gratton, E. (2000) Two Photon Fluoreescence Microscopy of Coexisting lipid Domains in Giant Unilamellar Vesicles of Binary Phospholipid Mixtures. Biophys. J. 78(1), 290–305.
Dietrich, C., Bagatolli, L. A., Volovyk, Z. N., et al. (2001) Lipid Rafts Reconstituted in Model Membranes. Biophys. J. 80, 1417–1428.
Feigenson, G. W. and Buboltz, J. T. (2001) Ternary phase diagram of dipalmitoyl-PC/dilauroyl-PC/cholesterol: nanoscopic domain formation driven by cholesterol. Biophys. J. 80(6), 2775–2788.
Korlach, J., Schwille, P., Webb, W. W., and Feigenson, G. W. (1999) Characterization of lipid bilayer phases by confocal microscopy and fluorescence correlation spectroscopy. Proc. Natl. Acad. Sci. USA 96(15), 8461–8466.
Parasassi, T., Gratton, E., Yu, W. M., Wilson, P., and Levi, M. (1997) Two-photon fluorescence microscopy of laurdan generalized polarization domains in model and natural membranes. Biophys. J. 72(6), 2413–2429.
Veatch, S. L. and Keller, S. L. (2005) Seeing Spots: Complex phase behaviour in simple membranes. Biochem. Biophys. Acta 1746(3),172–185.
Baumgart, T., Hess, S. T., and Webb, W. W. (2003) Imaging coexisting fluid domains in biomembrane models coupling curvature and line tension. Nature 425(6960), 821–824.
Kaizuka, Y. and Groves, J. T. (2004) Structure and dynamics of supported inter-membrane junctions. Biophys. J. 86(2), 905–912.
Puff, N., Lamaziere, A., Seigneuret, M., Trugnan, G., and Angelova, M. I. (2005) HDLs induce raft domain vanishing in heterogeneous giant vesicles. Chem. Phys. Lipids 133(2), 195–202.
Veatch, S. L. and Keller, S. L. (2002) Lateral Organisation in Lipid Membranes Containing Cholesterol. Phys. Rev. Lett. 89(26), 268, 101.
Veatch, S. L. and Keller, S. L. (2003) Seperation of Liquid Phases in Giant Vesicles of Ternary Mixtures of Phospholipids and Cholesterol. Biophys. J. 85(5), 3074–3083.
Veatch, S. L., Polozov, I. V., Gawrisch, K., and Keller, S. L. (2004) Liquid domains in vesicles investigated by NMR and fluorescence Microscopy. Biophys. J. 86(5), 2910–2922.
Kahya, N., Scherfeld, D., Bacia, K., Poolman, B., and Schwille, P. (2003) Probing lipid mobility of raft-exhibiting model membranes by fluorescence correlation spectroscopy. J. Biol. Chem. 278(30), 28,109–28,115.
Baumgart, T., Das, S., Webb, W. W., and Jenkins, J. T. (2005) Membrane elasticity in giant vesicles with fluid phase coexistence. Biophys. J. 89(2), 1067–1080.
Kahya, N., Brown, D. A., and Schwille, P. (2005) Raft partitioning and dynamic behaviour of human placental alkaline phosphatase in giant unilamellar vesicles. Biochemistry 44(20), 7479–7489.
Bacia, K., Schuette, C. G., Kahya, N., Jahn, R., and Schwille, P. (2004) SNAREs prefer liquid-disodered over “raft” (liquid-ordered) domains when reconstituted into giant unilamellar vesicles. J. Biol. Chem. 279(36), 37,951–37,955.
Hammond, A. T., Heberle, F. A., Baumgart, T., Holowka, D., Baird, B., and Feigenson, G. W. (2005) Crosslinking a lipid raft component triggers liquid ordered-liquid disordered phase separation in model plasma membranes. Proc. Natl. Acad. Sci. USA 102(18), 6320–6325.
Liu, A. P. C. Personal communication.
Bernardino, S. J., Perez-Gil, J., Simonsen, A. C., and Bagatalli, L. A. (2004) Cholesterol rules: direct observation of the coexistence of two fluid phases in native pulmonary surfactant membranes at physiological temperatures. J. Biol. Chem. 279(39), 40,715–40,722.
Edidin, M. (2003) The State of Lipid Rafts: From Model Membranes to Cells. Annu. Rev. Biophys. Biomol. Struct. 32, 257–283.
Munro, S. (2003) Lipid rafts: elusive or illusive? Cell 115(4), 377–388.
Veatch, S. L., Gawrisch, K., and Keller, S. L. (2006) Closed-loop miscibility gap and quantitative tie-lines in ternary membranes containing diphytanoyl PC. Biophys. J. 90(12), 4428–4436.
Angelova, M. I., Soleau, S., Mekeard, P., Faucon, J. F., and Bothorel, P. (1992) Preparation of giant vesicles by external AC electric fields. Kinetics and application. Progr. Colloid Polym. Sci. 89, 127–131.
Mathivet, L., Cribier, S., and Devaux, F. (1996) Shape change and physical properties of giant phospholipid vesicles prepared in the presence of an AC electric field. Biophys. J. 70, 1112–1121.
Veatch, S. L. (2004) Liquid Immiscibility in Model Bilayer Lipid Membranes, University of Washington, Seattle, WA, 172 p.
Veatch. S. L. and Keller, S. L. (2005) Miscibility phase diagrams of giant vesicles containing sphingomyelin. Phys. Rev. Lett. 94(14), 148,101.
Eckfeldt, E. L. and Lucasse, W. W. (1943) The Liquid-Liquid Phase Equilibria of the System Cyclohexane-ethyl Alcohol in the Presence of Various Salts as Third Components. Phys. Chem. 47(2), 164–183.
Ambroggio, E. E., Kim, D. H., Separovic, F., et al. (2005) Surface behavior and lipid interaction of Alzheimer beta-amyloid peptide 1-42: a membrane-distupting peptide. Biophys. J. 88(4), 2706–2713.
Evans, E., Hwinrich, V., Ludwig, F., and Rawicz,W. (2003) Dynamic tension spectroscopy and strength of biomembranes. Biophys. J. 85(4), 2342–2350.
Bacia, K., Schwille, P., and Kurzchalia, T. (2005) Sterol structure determines the separation of phases and the curvature of the liquid-ordered phase in model membranes. Proc. Natl. Acad. Sci. USA 102(9), 3272–3277.
Bagatolli, L. A. (2003) Direct observation of lipid domains in free standing bilayers: from simple t complex lipid mixtures. Chem. Phys. Lipids 122(1–2), 137–145.
Jin, L., Millard, A. C., Wuskell, J. P., et al. (2006) Characterzation and application of a new optical probe for membrane lipid domains. Biophys. J. 90(7), 2563–2575.
Huang, J., Bubolts, J. T., and Feigenson, G. W. (1999) Maximum solubility of cholesterol in phosphatidylcholine and phosphatidylethanolamine bilayers. Biochim. Biophys. Acta 1417(1), 89–100.
Author information
Authors and Affiliations
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2007 Humana Press Inc., Totowa, NJ
About this protocol
Cite this protocol
Veatch, S.L. (2007). Electro-Formation and Fluorescence Microscopy of Giant Vesicles With Coexisting Liquid Phases. In: McIntosh, T.J. (eds) Lipid Rafts. Methods in Molecular Biology, vol 398. Humana Press. https://doi.org/10.1007/978-1-59745-513-8_6
Download citation
DOI: https://doi.org/10.1007/978-1-59745-513-8_6
Publisher Name: Humana Press
Print ISBN: 978-1-58829-729-7
Online ISBN: 978-1-59745-513-8
eBook Packages: Springer Protocols