BPS2026 – Investigating the impact of mixed-chain saturated phospholipids on interleaflet coupling using asymmetric GUVs

Lipids with mixed hydrocarbon chains are abundant in biological membranes and can influence lateral organization, yet the biophysical effects of fully saturated mixed-chain lipids remain poorly understood. Previously, we studied two such lipids—MSPC (14:0–18:0 PC) and SMPC (18:0–14:0 PC)—in ternary mixtures with DOPC and cholesterol (chol). We found that MSPC mixtures exhibited coexistence of liquid-ordered (Lo) and liquid-disordered (Ld) phases with macroscopic domains at 22 °C, whereas SMPC mixtures were characterized by nanoscopic phase separation. Here, we extend this study to asymmetric membranes. We first prepared symmetric giant unilamellar vesicles (GUVs) from ternary mixtures of DOPC and Chol plus a high-melting lipid (DPPC, MSPC, PSM, or SSM), using compositions that produced macroscopic Ld+Lo phase separation. We then generated asymmetry via calcium-induced hemifusion with a supported lipid bilayer (SLB) composed of DOPC and Chol, effectively replacing the high-melting lipid in the GUV outer leaflet with DOPC. The GUVs and SLB were differentially labeled with red and green fluorophores, respectively, both to quantify the extent of outer-leaflet lipid exchange and interrogate the macroscopic phase behavior. We find that the phase behavior of the asymmetric GUVs (aGUVs) is influenced by the extent of outer leaflet exchange—aGUVs with lower levels of exchange retained macroscopic phase separation, whereas those with greater exchange were visibly uniform, consistent with inhibition of large-scale domain formation due to interleaflet coupling. We observe differences in the location of the asymmetric phase boundary depending on the high-melting lipid that are consistent with a major role for line tension in the coupling of leaflet phase behavior.