Abstract
Giant unilamellar vesicles (GUVs) constitute a cell-sized model membrane system that allows direct visualization of particular membrane-related phenomena, such as domain formation, at the level of single vesicles using fluorescence microscopy-related techniques. Currently available protocols for the preparation of GUVs work only at very low salt concentrations, thus precluding experimentation under physiological conditions. In addition, the GUVs thus obtained lack membrane compositional asymmetry. Here we show how to prepare GUVs using a new protocol based on the electroformation method either from native membranes or organic lipid mixtures at physiological ionic strength. Additionally, we describe methods to test whether membrane proteins and glycosphingolipids preserve their natural orientation after electroformation of GUVs composed of native membranes.
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References
Bagatolli LA (2006) To see or not to see: Lateral organization of biological membranes and fluorescence microscopy. Biochim Biophys Acta 1758:1541-1556
Menger FM, Keiper JS (1998) Chemistry and physics of giant vesicles as biomembrane models. Curr Opin Chem Biol 2:726-732
Veatch SL, Keller SL (2005) Seeing spots: Complex phase behavior in simple membranes. Biochim Biophys Acta 1746:172-185
Ambroggio EE, Separovic F, Bowie JH, Fidelio GD, Bagatolli LA (2005) Direct visualization of membrane leakage induced by the antibiotic peptides: Maculatin, citropin, and aurein. Biophys J 89:1874-1881
Bernardino de la Serna J, Perez-Gil J, Simonsen AC, Bagatolli LA (2004) Cholesterol rules: Direct observation of the coexistence of two fluid phases in native pulmonary surfactant membranes at physiological temperatures. J Biol Chem 279:40715-40722
Kahya N, Schwille P (2006) Fluorescence correlation studies of lipid domains in model membranes. Mol Membr Biol 23:29-39
Bagatolli LA, Parasassi T, Gratton E (2000) Giant phospholipid vesicles: Comparison among the whole lipid sample characteristics using different preparation methods: A two photon fluorescence microscopy study. Chem Phys Lipids 105:135-147
Akashi K, Miyata H, Itoh H, Kinosita K Jr (1996) Preparation of giant liposomes in physiological conditions and their characterization under an optical microscope. Biophys J 71:3242-3250
Baumgart T, Hammond AT, Sengupta PS, Hess T, Holowka DA, Baird BA, Webb WW (2007) Large scale fluid/fluid phase separation of proteins and lipids in giant plasma membrane vesicles. Proc Natl Acad Sci USA 104:3165-3170
Pautot S, Frisken BJ, Weitz DA (2003) Engineering asymmetric vesicles. Proc Natl Acad Sci USA 100:10718-10721
Pott T, Bouvrais H, Méléard P (2008) Giant unilamellar vesicle formation under physiologically relevant conditions. Chem Phys Lip 154:115-119
Montes LR, Alonso A, Goñi FM, Bagatolli LA (2007) Giant unilamellar vesicles electroformed from native membranes and organic lipid mixtures under physiological conditions. Biophys J 10:3548-3554
Steck TL, Kant JA (1974) Preparation of impermeable ghosts and insideout vesicles from human erythrocyte membranes. Meth Enzymol 31:172-180
Schow G, Passow H (1973) Preparation and properties of human erythrocyte ghosts. Mol Cell Biochem 2:197-217
Fiske HC, Subbarow Y (1925) The colorimetric determination of phosphorus. J Biol Chem 66:375-400
Böttcher C, van Gent C, Fries C (1961) A rapid and sensitive sub-micro phosphorous determination. Anal Chim Acta 1061:297-303
Bligh EG, Dyer WJ (1959) A rapid method of total lipid extraction and purification. Can J Biochem Physiol 37:911-917
Fidorra M, Duelund L, Leidy C, Simonsen AC, Bagatolli LA (2006) Absence of fluid-ordered/fluid-disordered phase coexistence in ceramide/POPC mixtures containing cholesterol. Biophys J 90:4437-4451
Kaufmann S, Tanaka M (2003) Cell adhesion onto highly curved surfaces: One-step immobilization of human erythrocyte membranes on silica beads. Chemphyschem 4:699-704
Sot J, Ibarguren M, Busto JV, Montes LR, Goñi FM, Alonso A (2008) Cholesterol displacement by ceramide in sphingomyelin-containing liquid-ordered domains, and generation of gel regions in giant lipidic vesicles. FEBS Lett 582:3230-3236
Montes LR, López DJ, Sot J, Bagatolli LA, Stonehouse MJ, Vasil ML, Wu BX, Hannun YA, Goñi FM, Alonso A (2008) Ceramide-enriched membrane domains in red blood cells and the mechanism of sphingomyelinase-induced hot-cold hemolysis. Biochemistry 47:11222-11230.
Acknowledgements
This work was supported by FNU, Denmark (272-06-0511), the Danish National Research Foundation, the Spanish Ministerio de Educación y Ciencia (BFU 2005-0695 and BFU 2004-02955), and the University of the Basque Country (GIU06/42).
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Montes, LR. et al. (2010). Electroformation of Giant Unilamellar Vesicles from Native Membranes and Organic Lipid Mixtures for the Study of Lipid Domains under Physiological Ionic-Strength Conditions. In: Weissig, V. (eds) Liposomes. Methods in Molecular Biology™, vol 606. Humana Press. https://doi.org/10.1007/978-1-60761-447-0_9
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DOI: https://doi.org/10.1007/978-1-60761-447-0_9
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