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

Abstract Model asymmetric lipid bilayers provide a powerful platform for probing how lateral phase behavior in one leaflet is coupled to that of the opposing leaflet. 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 composed of the low-melting lipid and cholesterol selectively altered the outer leaflet composition, producing aGUVs with controlled but variable asymmetry. Quantification of outer leaflet exchange using both probe-exit and probe-entry fluorescence measurements revealed substantial vesicle-to-vesicle variability within a given preparation, resulting in overlapping populations of phase-separated and uniformly mixed aGUVs. To account for this variability, we developed a population-based, coupled-distributions framework that enables robust determination of the asymmetric miscibility boundary, defined as the outer leaflet composition at which macroscopic phase separation is suppressed. Independent analyses of probe-exit and probe-entry data yielded consistent boundary locations. Comparing the two lipid systems, 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 both symmetric and asymmetric miscibility boundaries in a common fractional-coordinate framework, we introduce a phenomenological parameter, Δ∗, that quantifies the direction and strength of interleaflet coupling of phase behavior. Together, these results demonstrate that modest changes in lipid chain length can markedly alter asymmetric miscibility boundaries and provide a quantitative link between experimental observations, leaflet dominance concepts, and coupled-leaflet theories of membrane organization. Statement of Significance Membrane asymmetry is a defining feature of eukaryotic cells whose influence on lateral membrane organization remains unclear. Using asymmetric giant vesicles, we find that coexisting liquid-ordered and liquid-disordered domains transition to a uniform appearance as saturated lipid in the outer leaflet is replaced with unsaturated lipid. The extent of exchange required to disrupt phase separation increased with acyl-chain length mismatch, revealing a compositional dependence of interleaflet coupling. In mixtures with greater hydrophobic mismatch, we also observe coexisting anti-registered phases predicted by theory but rarely observed experimentally, providing new constraints for models of coupled-leaflet behavior. By accounting for vesicle-to-vesicle compositional variability, these results provide a framework for measuring asymmetric miscibility boundaries and for connecting asymmetric membrane organization to lipid raft phenomena. Competing Interest Statement The authors have declared no competing interest.