Asymmetric phosphoinositide lipid bilayers generated by spontaneous lipid insertion

Phosphatidylinositol phosphate (PIP) lipids, located on the inner leaflet of the membrane facing the cytoplasm, are key regulators of a wide range of cellular events at the plasma membrane, in some cases by interacting with partner proteins that regulate actin assembly. Supported lipid bilayers (SLBs) have been recognized as a powerful model membrane to dissect the mechanisms underlying the clustering and interactions of PIP lipids with their partner proteins. However, despite advances in methods to generate SLBs, it remains a challenge to robustly obtain fluid SLBs in which PIP lipids are mobile and asymmetrically distributed. In this study, we report a simple and robust method to generate asymmetric-SLBs with controllable amounts of PI(4,5)P2 and PI by dissolving them at a concentration below their critical micellar concentration (CMC) to facilitate their insertion into the upper leaflet of the SLB exposed to the bulk solution. We validated the mobility of PI(4,5)P2 and PI lipids in the SLBs using fluorescence recovery after photobleaching (FRAP). We demonstrated the utility of PI(4,5)P2-asymmetric SLBs by showing that they can recruit actin-membrane linker protein ezrin and myosin 1 motors, which are capable of sliding actin filaments on the SLBs. We expect our new method for preparing asymmetric-SLB to be applicable to the incorporation of other high CMC lipid species.