Morphology of Egg Phosphatidylcholine-cholesterole Single-bilayer Vesicles, Studied by Freeze-etch Electron Microscopy

Homogeneous, small, single-bilayer vesicles were prepared from egg phosphatidylcholine with various concentrations of cholesterol by ultrasonic dispersion in 0.1 M KCl, 0.01 M Tris, pH 8.0, buffer, followed by gel chromatography. The shape and size distributions of the fractionated vesicles were investigated for preparations with cholesterol compositions from 0 to 50 moles/100 moles, using freeze-etch electron microscopy. The size distribution was estimated from the shadow width of vesicles which were exposed by etching and the vesicle shape was checked by comparing the images obtained by tilting the replicas. The widths of the vesicle diameter distributions were relatively broad, corresponding to standard deviations in the range 60--90 A, but showing no systematic variation with cholesterol composition. In all cases it was found that 70% of the vesicle diameters lay within 150 A of the modal value. The apparent vesicle diameters remained constant for cholesterol compositions up to 20 moles/100 moles (modal diameter = 330 +/- 20 A, mean diameter = 350 +/- 3 A), but there was a sharp net increase in diameter at 30 moles cholesterol/100 moles reaching a model diameter of 430 +/- 20 A (mean diameter = 430 +/- 3 A) at 50 moles cholesterol/100 moles. Using the tilted microscope stage it was found that all vesicles were spherical at all cholesterol compositions studied, including those above 30 moles cholesterol/100 moles. The molecular mechanism by which cholesterol controls the vesicle size is discussed in terms of the asymmetric distribution of cholesterol across the vesicle bilayer.