Supported lipid bilayer array to study clathrin mediated endocytosis in vitro

We describe our effort to realize an assay system to study clathrin-mediated endocytosis (CME) in vitro. Since CME related proteins are known to bind phosphatidylinositol-4,5-bisphosphate (PI(4,5)P₂), we propose an accessible method to create an array of supported lipid bilayers (SLBs) with different lipid compositions, using a PDMS stamp and a microchannel. Formation of a uniform SLB of a complex lipid mixture containing PI(4,5)P₂ was confirmed by fluorescent imaging and fluorescence recovery after photobleaching (FRAP). Our technique should allow us to vary the concentration of PI(4,5)P₂ or other membrane constituents to study their requirements. In preliminary experiments we showed that dynamin binds to the planar bilayer containing PI(4,5)P₂, but without causing membrane tubulation as it does when dynamin binds liposomes.