Investigating the Transbilayer Distribution of Cholesterol in Asymmetric Unilamellar Vesicles using Small-Angle Scattering

Abstract

Cholesterol is the most abundant component of eukaryotic plasma membranes (PMs), accounting for 30-50% of the total lipid. In addition to modulating the structural and mechanical properties of lipid bilayers, cholesterol promotes the formation of ordered+disordered fluid phase separation in model membranes, and may similarly contribute to microdomain or “raft” formation in the PM. A major unresolved question is how PM properties and functions are influenced by the asymmetric transbilayer distribution of lipids, including cholesterol. While the distribution of most phospholipids and sphingolipids is known, determining the average transbilayer distribution of cholesterol has been challenging due to its rapid translocation between the PM leaflets. Previous attempts to probe the cholesterol distribution in cell membranes using fluorescence-based methods have yielded conflicting results. Moreover, studies using model vesicles have been limited due to the difficulty of preparing asymmetric liposomes. Making use of recent advancements enabling the production of asymmetric large unilamellar vesicles (aLUVs) via cyclodextrin-mediated lipid exchange, we prepared cholesterol-containing aLUVs in which one leaflet was enriched in egg sphingomyelin (eSM), and the other in phosphatidylethanolamine (POPE). We then used small-angle neutron and x-ray scattering combined with isotopic labeling strategies to probe the average cholesterol location within these bilayers. An analysis of scattering data, together with complementary Molecular Dynamics simulations, provides insight into the transbilayer distribution of cholesterol in asymmetric membranes.