Asymmetric Supported Lipid Bilayer Formed Via Methyl-β-Cyclodextrin Mediated Lipid Exchange: a Membrane Model System to Study Phase Separation and Transbilayer Lipid Movement

Supported lipid bilayers (SLBs) are broadly used as a membrane model system and commonly produced by vesicle fusion (VF). Despite its advantages, VF does not allow the controlled formation of asymmetric bilayers that mimic the asymmetry in lipid composition normally found in biological systems. Here we present a simple, quick and versatile method to produce stable SLBs with a desired asymmetric lipid composition. We apply a methyl-β-cyclodextrin (MβCD) mediated lipid exchange method to SLBs formed by VF to enrich the distal leaflet of the bilayer with sphingomyelin (SM). Bilayer asymmetry is assessed by fluorescence correlation spectroscopy (FCS), measuring lipid mobility separately in each leaflet. To check the compatibility of the method with the most common protein reconstitution approaches, we also report the production of asymmetric SLBs (aSLBs) in presence of a membrane protein, reconstituted both via direct protein insertion (i.e. directional insertion) and proteoliposomes fusion. aSLBs are a suitable membrane model system to study phase separation and transbilayer lipid movement of raft-mimicking lipid mixtures. Although their phase behavior was extensively investigated, the effect of compositional asymmetry on phase separation remains unclear. To address this question, we compared SLBs and aSLBs with the same overall lipid composition. The observed differences in terms of phase separation provide further experimental evidence that the transversal lipid distribution affect the overall lipid miscibility and allow to temporally investigate leaflet mixing (i.e. lipid flip-flop). Additionally, cholesterol distribution between cytosolic and extracellular side of the plasma membrane is of great importance in determining protein-membrane interaction and the formation of raft-like domains in vivo. Therefore, we use a combined FCS and mass spectrometric approach to investigate cholesterol distribution between inner and outer leaflet of SM-containing aSLBs.