ArticlebioRxiv : the preprint server for biology2026
Quantifying interleaflet coupling of phase behavior and observing anti-registered phases in asymmetric lipid bilayers.
Article in bioRxiv : the preprint server for biology, 2026. The graph could read no effect estimate from its abstract, so it casts no vote on the map. Not yet cited in PubMed.
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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,
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