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Making Giant Unilamellar Vesicles via Hydration of a Lipid Film

Suliana Manley1,  Vernita D. Gordon2

1National Institutes of Health, Cell Biology and Metabolism Branch, Bethesda, Maryland
2University of Illinois Urbana‐Champaign, Department of Materials Science and Engineering, Urbana, Illinois


Publication Name: 
Current Protocols in Cell Biology
Unit Number: 
Unit 24.3
DOI: 
10.1002/0471143030.cb2403s40
Online Posting Date: 
September, 2008
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Abstract

This unit describes protocols for making giant unilamellar vesicles (GUVs) based on rehydration of dried lipid films. These model membranes are useful for determining the impact of membrane and membrane-binding components on lipid bilayer stiffness and phase behavior. Due to their large size, they are especially amenable to studies using fluorescence and light microscopy, and may also be manipulated for mechanical measurements with optical traps or micropipets. In addition to their use in encapsulation, GUVs have proven to be useful model systems for studying many cellular processes, including tubulation, budding, and fusion, as well as peptide insertion. The introduction of enzymes or proteins can result in reorganization, leading to such diverse behavior as vesicle aggregation, fusion, and fission. Curr. Protoc. Cell Biol. 40:24.3.1-24.3.13. © 2008 by John Wiley & Sons, Inc.

Keywords: liposomes; giant vesicles; electroformation; liposome swelling; model membranes

     
 
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Table of Contents

  • Introduction
  • Basic Protocol 1: Preparing GUVs by Electroformation on Indium Tin Oxide–Coated Plates
  • Support Protocol 1: Making Lipid Mixtures
  • Support Protocol 2: Design of a PTFE (Teflon) Holder for Electroformation on ITO-Coated Plates
  • Alternate Protocol 1: Preparing GUVs by Electroformation on Platinum Wires
  • Support Protocol 3: Design of a Chamber for Electroformation on Platinum Wires
  • Basic Protocol 2: Preparing GUVs by Swelling off of PTFE (Teflon)
  • Alternate Protocol 2: Preparing GUVs by Swelling Off a Uniform Film on Glass
  • Alternate Protocol 3: Preparing GUVs by Dehydration and Rehydration of SUVs
  • Commentary
  • Literature Cited
  • Figures
     
 
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Materials

Basic Protocol 1: Preparing GUVs by Electroformation on Indium Tin Oxide–Coated Plates

 Materials
  • 100 µl lipid in chloroform, at 5 to 10 mg/ml (see Support Protocol 1)
  • Up to 600 mM sucrose, or sterile deionized H2O
  • 25-cm2 ITO-coated plates (e.g., Delta Technologies, Ltd., part no. CG-511IN-50x50/1.1; http://www.delta-technologies.com/)
  • Sealed chamber (e.g., vacuum desiccator) connected to dry nitrogen or vacuum source
  • Vacuum grease
  • Teflon holder and spacers for ITO-coated plates (see Support Protocol 2)
  • Oven with hole to introduce electrodes or heating bath (for lipid systems in which at least one component has a chain-melting transition temperature, Tm, above room temperature)
  • Electrodes (“Mini-Plunger to BNC Male” or equivalent; e.g., Radio Shack)
  • Function generator with readout for voltage and frequency (Stanford Research Systems, Ltd., http://www.thinksrs.com/)

Support Protocol 1: Making Lipid Mixtures

 Materials
  • Lipid (Avanti Polar Lipids; when received, store at –20°C or lower)
  • Chloroform
  • Methanol (optional)
  • Argon or nitrogen gas
  • Glass vials with Teflon closures
  • Microdispensers with glass bores (Drummond), or Hamilton syringes
  • Teflon tape

Alternate Protocol 1: Preparing GUVs by Electroformation on Platinum Wires

 Additional Materials (also see Basic Protocol 1)
  • 5 to 10 µl of lipid solution in chloroform (Support Protocol 1) at concentration of 0.5 to 0.66 mM (~0.5 mg/ml)
  • Up to 600 mM sucrose, or sterile deionized H2O
  • Platinum wires, 0.5- to 2.0-mm diameter
  • Microdispensers with glass bores (Drummond), or Hamilton syringes
  • Sealed chamber (e.g., vacuum desiccator) connected to dry nitrogen or vacuum source
  • Electroformation chamber (see Support Protocol 3), assembled with platinum wires

Support Protocol 3: Design of a Chamber for Electroformation on Platinum Wires

 Materials
  • Platinum wires, 0.5- to 2.0-mm diameter
  • Aluminum block (machined; typical dimensions, 10 × 4 ×1 cm)
  • Electrical block fittings or terminal block fittings (McMaster-Carr, http://www.mcmaster.com)
  • Optional items:
    • PTFE seals
    • Cover glass for microscopy
    • Norland optical adhesive
    • Resistive heating wire
    • Ceramic insulators and fittings for electrical attachment
    • Thermocouple

Basic Protocol 2: Preparing GUVs by Swelling off of PTFE (Teflon)

 Materials
  • 20 mg/ml lipid in chloroform (Support Protocol 1)
  • Chloroform
  • Nitrogen gas
  • Swelling solution: 100 mM sucrose or glucose prepared using sterile deionized H2O
  • Microdispensers with glass bores (Drummond), or Hamilton syringes
  • PTFE (Teflon) sheet, ~2 × 2–cm square, roughened with fine-grain sandpaper (McMaster-Carr, http://www.mcmaster.com)
  • Desiccator chamber, attached to vacuum pump
  • Glass beaker
  • Incubator or oven

Alternate Protocol 2: Preparing GUVs by Swelling Off a Uniform Film on Glass

 Materials
  • 80 µl lipids in chloroform (Support Protocol 1) at 10 mM, (~10 mg/ml)
  • Chloroform
  • Nitrogen gas
  • Swelling solution: 100 mM sucrose in water (or other aqueous swelling solution), prepared using sterile deionized H2O
  • Microdispensers with glass bores (Drummond), or Hamilton syringes
  • 50- to 100-ml glass flask with pointed bottom
  • Rotary evaporator
  • Vacuum oven
  • Oven or incubator

Alternate Protocol 3: Preparing GUVs by Dehydration and Rehydration of SUVs

 Materials
  • 100 to 300 µl of lipid solution in chloroform at 20 mg/ml (Support Protocol 1)
  • 100 mM NaCl (prepared using sterile deionized H2O) containing 1 vol% glycerol
  • 100 mM NaCl (prepared using sterile deionized H2O), without glycerol
  • Hamilton syringe
  • Small glass vial
  • Desiccator chamber, attached to vacuum pump
  • Bath sonicator (43 kHz; L&R Ultrasonics Model T21, http://www.lrultrasonics.com)
  • Glass coverslips
Looking for materials?  Suppliers Guide
     
 
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Figures

  • Figure 24.3.1
    Teflon holder for ITO-coated plates, made from two pieces (shown left and right), front view and side view (shown top and bottom).

    View Image
  • Figure 24.3.2
    Electroformation assembly for platinum wires, showing optional water reservoir and harvesting chamber. The central chamber is essential, and contains the electrodes where the electroformation takes place.

    View Image
  • Figure 24.3.3
    Giant vesicles, viewed with microscopy. (A) By incorporating 0.1 mol% of a fluorescently labeled lipid, vesicles are readily viewed with fluorescence microscopy. Multilamellar vesicles appear brighter, as demonstrated by the upper vesicle. (B) Vesicles may also be viewed using phase microscopy. Scale bars = 10 µm.

    View Image

Literature Cited

Literature Cited
    Angelova, M.I., Soleau, S., Meleard, P., Faucon, J.F., and Bothorel, P. 1992. Preparation of giant vesicles by external AC electric fields: Kinetics and applications. Progr. Colloid & Polymer Sci. 89:127-131.
    Bacia, K., Schwille, P., and Kurzchalia, T.V. 2005. Sterol structure determines the separation of phases and the curvature of the liquid-ordered phase in model membranes. Proc. Natl. Acad. Sci. U.S.A. 102:3272-3277.
    Bagatolli, L.A. 2006. To see or not to see: Lateral organization of biological membranes and fluorescence microscopy. Biochim. Biophys. Acta 1758:1541-1556.
    Bagatolli, L.A. and Gratton, E. 2000. Two photon fluorescence microscopy of coexisting lipid domains in giant unilamellar vesicles of binary phospholipid mixtures. Biophys. J. 78:290-305.
    Baumgart, T., Hess, S.T., and Webb, W.W. 2003. Imaging coexisting fluid domains in biomembrane models coupling curvature and line tension. Nature 425:821-824.
    Beattie, M., Veatch, S.L., Stottrup, B., and Keller, S.L. 2005. Sterol structure determines miscibility vs. melting transitions in lipid vesicles. Biophys. J. 89:1760-1768.
    Bigay, J., Gounon, P., Robineau, S., and Antonny, B. 2003. Lipid packing sensed by ArfGAP1 couples COPI coat disassembly to membrane bilayer curvature. Nature 426:563-566.
    Dietrich, C., Bagatolli, L.A., Volovyk, Z.N., Thompson, N.L., Levi, M., Jacobson, K., and Gratton, E. 2001. Lipid rafts reconstituted in model membranes. Biophysical Journal 80:1417-1428.
    Estes, D.J. and Mayer, M. 2005. Electroformation of giant liposomes from spin-coated films of lipids. Colloids and Surfaces B: Biointerfaces 42:115-123.
    Gordon, V.D., Beales, P.A., Shearman, G.C., Zhao, Z., Seddon, J.M., Egelhaaf, S.U., and Poon, W.C.K. Solid-like domains in mixed lipid bilayers: Effects of lipid phase behavior, transition pathway, and membrane lamellarity. Manuscript in preparation.
    Holopainen, J.M., Angelova, M.I., and Kinnunen, P.K.J. 2000. Vectorial budding of vesicles by asymmetrical enzymatic formation of ceramide in giant liposomes. Biophys. J. 78:830-838.
    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. U.S.A. 96:8461-8466.
    Koster, G., VanDujin, M., Hofs, B., and Dogterom, M. 2003. Membrane tube formation from giant vesicles by dynamic association of motor proteins. Proc. Natl. Acad. Sci. U.S.A. 100:15583-15588.
    Moscho, A., Orwar, O., Chiu, D.T., Biren, M.P., and Zare, R.N. 1996. Rapid preparation of giant unilamellar vesicles. Proc. Natl. Acad. Sci. U.S.A. 93:11443-11447.
    Mueller, P., Chien, T.F., and Rudy, B. 1983. Formation and properties of cell-size lipid bilayer vesicles. Biophys. J. 44:375-381.
    Nurminen, T.A., Holopainen, J.M., Zhao, H., and Kinnunen, P.K.J. 2001. Observation of topical catalysis by sphingomyelinase coupled to microspheres. J. Amer. Chem. Soc. 124:12129-12134.
    Roux, A., Cappello, G., Cartaud, J., Prost, J., Goud, B., and Bassereau, P. 2002. A minimal system allowing tubulation with molecular motors pulling on giant liposomes. Proc. Natl. Acad. Sci. U.S.A. 99:5394-5399.
    Simons, K. and Ikonen, E. 1997. Functional rafts in cell membranes. Nature 387:569-572.
    Staneva, G., Angelova, M.I., and Koumanov, K. 2004. Phospholipase A2 promotes raft budding and fission from giant liposomes. Chem. Phys. Lipids 129:53-62.
    Thoren, P.E.G., Persson, D., Esbjorner, E.K., Goksor, M., Lincoln, P., and Norden, B. 2004. Membrane binding and translocation of cell-penetrating peptides. Biochemistry 43:3471-3489.
    Veatch, S.L. and Keller, S.L. 2002. Organization in lipid membranes containing cholesterol. Phys. Rev. Lett. 89:268101.
    Veatch, S.L. and Keller, S.L. 2003. Separation of liquid phases in giant vesicles of ternary mixtures of phospholipids and cholesterol. Biophys. J. 85:3074-3083.
     
 
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