Detection and characterization of laterally phase separated cholesterol domains in model lipid membranes

Abstract

We present evidence that laterally phase separated cholesterol domains constitute a new, equilibrium phase in biological membranes. The domains are characterized in multi-lamellar vesicles (MLV) made of cholesterol and dimyristoylphosphatidylcholine (DMPC) but are also shown to exist in biologically relevant, egg lecithin systems containing a mixture of phospholipids. This work utilizes the fluorescent membrane probes 1-acyl-2-[12-[(5-dimethylamino-1-naphthalenesufonyl)amino]dodecanoyl]-sn- glycero-3-phosphocholine (DANSYL), and ergosta-5,7,9(11),22-tetraen-3β-ol (ERGO), which have been shown to be minimally invasive mimics of native membrane lipids. The highlight of the work is a heating-induced alleviation of a DANSYL blue shift at relatively high (but undersaturated) cholesterol loadings, which is reversible through at least three heating and cooling cycles. Comparison of the DANSYL spectral shifts with published DMPC-cholesterol phase diagrams shows unequivocally that the spectral results cannot be explained in terms of previously understood phase behavior. Rather, a lateral phase separation occurs within the vesicle bilayer, giving rise to cholesterol micro-domains. The cholesterol domains appear to coexist with, and should not be confused with, the well-known liquid-order phase that arises because of the cholesterol condensation effect. Additional studies involving ERGO-DANSYL energy transfer show a sequestration of probes within the bilayer, confirming the DANSYL spectral data, and a model that includes domains provides the best description of measured energy transfer efficiencies. Best fits of the energy transfer data, using a mathematical model developed to account for the presence of domains, indicates the domain size to be in the range 10-20 nm. © 2003 Elsevier Science B.V. All rights reserved.