Utilizing asymmetric giant unilamellar vesicles to determine how lowmelting phospholipids affect interleaflet coupling strength

Model membranes that mimic cell membrane asymmetry can be used to study coupling between lateral and transverse lipid organization. Theory predicts that, in a leaflet composed of lipids that would otherwise phase separate in a symmetric bilayer, domain formation can be suppressed by strong coupling to a uniformly mixed leaflet. Conversely, when coupled to a leaflet with a tendency to demix, an otherwise miscible leaflet can be induced to demix. Each of these outcomes has been observed in model asymmetric membranes of different composition, suggesting that cells may be able to control domain formation through changes in lipid content and/or asymmetric lipid distribution. We have explored the influence of the low-melting (low-TM) lipid in a ternary mixture on macroscopic domain formation in asymmetric giant unilamellar vesicles (aGUVs). Symmetric GUVs composed of DPPC/low-TM lipid/Chol (39/39/22 mol%) with varied low-TM chain length (14:1-PC, 16:1-PC, 18:1-PC, 20:1-PC, and 22:1-PC) were investigated using confocal fluorescence microscopy; the mixtures containing 14:1-PC, 16:1-PC, and 18:1-PC showed macroscopic phase separation at room temperature, while the mixtures containing 20:1-PC and 22:1-PC did not. Cryogenic electron microscopy, small-angle neutron scattering, and Förster resonance energy transfer confirmed the presence of domains in the first three systems and revealed nanoscopic heterogeneity in the mixture containing 20:1-PC, while the 22:1-PC mixture was uniform. aGUVs were then prepared by calcium-induced hemifusion of the GUVs to a supported lipid bilayer composed of the corresponding low-TM lipid and Chol (80/20 mol%). The aGUVs showed a range of outer leaflet exchange, which revealed a correlation between phase behavior and extent of asymmetry. Domains in the 16:1-PC and 18:1-PC systems were suppressed in aGUVs with more than 50 and 60% asymmetry, respectively, indicating a relatively large free energy cost for antiregistered domains.