Asymmetric phosphoinositide lipid bilayers generated by spontaneous lipid insertion

Phosphatidylinositol phosphate (PIP) lipids, enriched on the cytoplasmic leaflet of the plasma membrane, are key regulators of diverse cellular processes, often through interactions with partner proteins that regulate actin assembly. Supported lipid bilayers (SLBs) provide a powerful model system to study the interactions of PIP lipids with their partner proteins. However, despite advances in SLB preparation methods, it remains a challenge to robustly obtain fluid SLBs in which PIP lipids are both mobile and asymmetrically distributed. In this study, we report a simple and robust method to generate asymmetric SLBs containing tunable amounts of PI(4,5)P₂. By dissolving PI(4,5)P₂ below its critical micelle concentration (CMC), we enable its spontaneous insertion into the upper leaflet of SLBs exposed to bulk solution. The mobility of PI(4,5)P₂ is confirmed using fluorescence recovery after photobleaching (FRAP). Furthermore, we demonstrate that PI(4,5)P₂ incorporated using this method retains its functionality, recruiting binding partners, actin-membrane linker ezrin, and myosin 1 motors capable of sliding actin filaments on the SLBs. Our method offers a straightforward strategy to generate asymmetric PI(4,5)P₂-containing SLBs and is applicable to other lipid species with high CMC values.