bioRxiv2022
The asymmetry of membranes has a significant impact on their biophysical characteristics and behavior. This study investigates the composition and mechanical properties of symmetric and asymmetric membranes in giant unilamellar vesicles (GUVs) made of phosphatidylcholine (POPC) and phosphatidic acid (POPA). A combination of fluorescence quantification, zeta potential measurements, micropipette aspiration and bilayer molecular dynamics simulations are used to characterize these membranes. The outer leaflet composition in vesicles is found consistent across the two preparation methods we employed, namely electroformation and inverted emulsion transfer. However, characterizing the inner leaflet poses challenges. Micropipette aspiration of GUVs show that oil residues do not substantially alter membrane elasticity, but simulations reveal increased membrane thickness and decreased interleaflet coupling in the presence of oil. Asymmetric membranes with a POPC:POPA mixture in the outer leaflet and POPC in the inner leaflet display similar stretching elasticity values to symmetric POPC:POPA membranes, suggesting potential POPA insertion into the inner leaflet during vesicle formation and suppressed asymmetry. The inverse compositional asymmetry, with POPC in the outer leaflet and POPC:POPA in the inner yield less stretchable membranes with higher compressibility modulus compared to their symmetric counterparts. Challenges in achieving and predicting compositional correspondence highlight the limitations of phase-transfer-based methods. Additionally, caution is advised when using fluorescently labeled lipids (even at low fractions of 0.5 mol%), as unexpected gel-like domains in symmetric POPC:POPA membranes were observed only with a specific type of labeled DOPE (dioleoylphosphatidylethanolamine) and the same fraction of unlabeled DOPE. The latter suggest that such phase separation may result from interactions between lipids and membrane fluorescent probes. Overall, this study underscores the complexity of factors influencing GUV membrane asymmetry, emphasizing the need for further research and improvement of characterization techniques. SIGNIFICANCEAsymmetrically charged lipid bilayer models are superior to commonly used symmetrical ones, exhibiting naturally present asymmetry, thereby exhibiting a more adequate range of biophysical membrane characteristics better reflecting biological membranes. This study focuses on the mechanical properties of phosphatidic acid (PA)-enriched membranes, a crucial lipid for cellular lipid metabolism, e.g. glycerophospholipid synthesis, and for signal transduction. Micropipette aspiration, fluorescent PA-sensor, and zeta potential studies demonstrate that asymmetric membranes are less stretchable than symmetric ones. Accompanying in silico studies on the symmetric membranes confirm that oil impurities do not influence the membrane stretching elasticity but increase its thickness and decrease the coupling of the two leaflets, which sheds light on the elastic behavior of experimental models of asymmetric lipid bilayers.