Inter-Leaflet Coupling and Domain Formation in Asymmetric Giant Unilamellar Vesicles

Abstract

Cell membranes exhibit an asymmetric distribution of lipids across the two leaflets of the bilayer. While the sphingolipid-rich outer (exoplasmic) leaflet has a lipid composition prone to lateral segregation and domain formation, the situation in the sphingolipid-poor inner (cytosolic) leaflet is unclear. The interaction between the two leaflets, although involved in several biological processes, is not well understood. Although both supported and free-standing asymmetric bilayers have been previously produced, such approaches were based on a leaflet-by-leaflet assembly of the bilayer and, therefore, possibly incompatible with the reconstitution of transmembrane proteins. A highly reproducible solvent-free method that is fast, has a high yield and can be used with a wide variety of lipids and membrane proteins, is still needed. Here, we introduce a simple lipid exchange method to obtain stable asymmetric giant unilamellar vesicles (GUV) that satisfies the above-mentioned requirements. The asymmetry of the GUVs is investigated using fluorescence correlation spectroscopy (FCS) in order to probe dynamics of lipids in the two leaflets. It is shown that introduction of up to 50% sphingomyelin into the outer leaflet of GUV composed of an unsaturated phosphatidylcholine (PC) in the inner leaflet results in a larger decrease in outer leaflet lateral diffusion than inner leaflet lateral diffusion. The degree to which the inner leaflet later diffusion decreases depends on the identity of the inner leaflet PC. We also show that a transmembrane protein can be incorporated into the asymmetric GUV and its diffusion probed by FCS. Thus, this approach can be used to investigate the molecular mechanisms behind inter-leaflet coupling and the effect of membrane asymmetry on transmembrane proteins.