BPS2026 – Investigating phosphatidylserine-lipid raft domain registration in asymmetric giant unilamellar vesicles

Biological membranes display both vertical asymmetry and lateral heterogeneity, with different lipid compositions distributed between and across leaflets. Depending on lipid type and acyl-chain composition, lipid bilayers can segregate into coexisting ordered and liquid-disordered phases, which facilitates the recruitment of proteins to specific membrane sites for diverse physiological functions. Phosphatidylserine (PS), for instance, is primarily localized in the inner leaflet of the plasma membrane and plays a critical role in signaling pathways such as apoptosis, whereas high-melting point lipids in the outer leaflet form raft domains that serve as platforms for protein trafficking and signaling. To investigate membrane asymmetry and lipid interactions, we developed an adapted hemifusion method to fabricate asymmetric giant unilamellar vesicles (aGUVs) containing lipid rafts in the inner bilayer leaflet and PS in the outer leaflet, thereby generating inverted cell membrane mimics. Fluorescence microscopy analysis of these aGUVs, compared with symmetric control GUVs, revealed key interactions between PS, cholesterol, Ca 2+ , and Mg 2+ during hemifusion. Notably, both PS registration and anti-registration were observed regardless of the cation used, and the yield of phase-separated aGUVs was significantly lower than that of non-phase-separated aGUVs.