Exploring the raft-hypothesis by probing planar bilayer patches of free-standing giant vesicles at nanoscale resolution, with and without Na,K-ATPase.
The structure of functional lipid domains (rafts) in biological membranes has for long time been unresolved due to their small length scales and transient nature. These cooperative properties of the lipid bilayer matrix are modelled by free-standing giant unilammellar vesicles (GUVs) with well-characterized lipid composition. We review a series of recent advances in preparation and analysis of GUVs, which allows for characterization of small domains by high-resolution imaging techniques. These includes a new GUV preparation method with a desired overall lipid composition achieved by mixing small unilammellar vesicles (SUVs), test of the lipids compositional uniformity in GUVs and swift adsorption of GUVs to solid support by kinetically arresting the lateral structure of membrane prior to collapse for subsequent imaging. The techniques are applied to the analysis of membrane domains in GUVs formed from mixtures of DOPC/DPPC/cholesterol with and without Na,K-ATPase (NKA), a transmembrane protein known to be associated with rafts. Two mechanisms of domain formation are revealed: 1) close to lo/ldphase coexistence, domains in size up to 100nm appear as thermally induced droplet fluctuations, 2) NKA shows interfacial activity and cluster in lo/ldmicro-emulsion droplets. Some perspectives for the application of the techniques and the understanding of the nature of raft domains are outlined.
MEMPHYS, University of Southern Denmark, Odense 5230, Denmark. Electronic address: tripta.bhatia@mpikg.mpg.de.
T
T
Bhatia
Department of Biomedicine, Aarhus University, 8000, Denmark.
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F
Cornelius
MEMPHYS, University of Southern Denmark, Odense 5230, Denmark.
J H
JH
Ipsen
D015060:1,2-Dipalmitoylphosphatidylcholine / Q000737:chemistry; D002784:Cholesterol / Q000737:chemistry; D008051:Lipid Bilayers / Q000737:chemistry*; D021962:Membrane Microdomains / Q000737:chemistry*; D053758:Nanoparticles; D010713:Phosphatidylcholines / Q000737:chemistry; D000254:Sodium-Potassium-Exchanging ATPase / Q000737:chemistry*
D008051:Lipid Bilayers; D010713:Phosphatidylcholines; D015060:1,2-Dipalmitoylphosphatidylcholine; D002784:Cholesterol; D000254:Sodium-Potassium-Exchanging ATPase; C017251:1,2-oleoylphosphatidylcholine
Giant unilamellar vesicles; Membrane lateral structure; Membrane rafts; Na,K-ATPase; l(o)-l(d) domains