BPS2026 – Role of cation identity in hemifusion driven assembly of asymmetric giant unilamellar vesicles

The plasma membrane is an asymmetric bilayer with distinct lipid compositions in the inner and outer leaflets, essential for processes like cell signaling and apoptosis. Phosphatidylcholine (PC), a zwitterionic lipid, predominantly resides in the outer leaflet, forming a rigid barrier. Hemifusion, facilitated by divalent cations, is used to create asymmetric giant unilamellar vesicles (aGUVs) for studying lipid-mediated protein functions. Previous studies demonstrated the fusogenic efficiency of Ca 2+ and Mg 2+ in fabricating aGUVs with outer leaflets composed of anionic lipids, such as phosphatidylserine (PS) and phosphatidylinositol 4,5-bisphosphate (PI(4,5)P 2 ). This study evaluates the efficiency of additional divalent cations, Be 2+ , Ba 2+ , Sr 2+ , and Zn 2+ in exchanging PC from a supported lipid bilayer (SLB) with lipids from the outer leaflet of a symmetrical GUV (sGUV). These cations are of interest due to their differences in their kosmotropic or chaotropic nature, which causes differences in the dehydration of the thin water layer between the sGUV and SLB, necessary for initiating hemifusion. We find that the more chaotropic a fusogen is, the better is the hemifusion efficiency identified by higher quality aGUVs. Given PC’s zwitterionic nature, this lipid is more likely to electrostatically interact with chaotropic agents based on the law of matching water affinities leading to improved efficiency in dehydrating the thin water layer between sGUVs and the SLB. Further studies will identify if a similar trend between fusogen hemifusion efficiency can be identified when fabricating aGUVs with outer leaflets partially composed of anionic lipids, e.g., PS, providing clarity on the physiochemical characteristics of the hemifusion process.