Quantifying interleaflet coupling of phase behavior and observing anti-registered phases in asymmetric lipid bilayers

Biological membranes are compositionally asymmetric, with distinct lipid mixtures in each leaflet, yet how this asymmetry influences lateral membrane organization remains poorly understood. Here, we use calcium-induced hemifusion to generate asymmetric giant unilamellar vesicles (aGUVs) and investigate how lipid composition modulates interleaflet coupling of liquid-liquid phase separation. Symmetric GUVs composed of cholesterol, the high-melting lipid DPPC, and a low-melting phosphatidylcholine (either 14:1-PC or 16:1-PC) were prepared at compositions exhibiting coexisting liquid-ordered (Lo) and liquid-disordered (Ld) phases. Hemifusion with a uniformly mixed supported lipid bilayer selectively altered the outer leaflet composition, producing aGUVs with controlled but variable asymmetry. Fluorescence measurements of outer leaflet exchange revealed substantial vesicle-to-vesicle variability, resulting in overlapping populations of phase-separated and uniformly mixed aGUVs. To account for this variability, we developed a statistical framework that jointly models the distribution of exchange fractions and the location of a phase boundary in asymmetric composition space, allowing all observed vesicles to contribute to the analysis. We find that aGUVs containing 14:1-PC require significantly greater outer leaflet exchange to abolish phase separation than those containing 16:1-PC. Only in the 14:1-PC system do we observe vesicles exhibiting coexistence of distinct anti-registered phases, a theoretically predicted but rarely observed regime consistent with large hydrophobic mismatch. By expressing symmetric and asymmetric miscibility boundaries in a common compositional framework, we introduce a phenomenological parameter, Δ^∗, that quantifies the direction and strength of interleaflet coupling. These results demonstrate that modest changes in lipid chain length can markedly alter asymmetric phase boundaries and provide a quantitative link between experimental observations, leaflet dominance concepts, and coupled-leaflet theories of membrane organization.