Towards a better understanding of interleaflet coupling in asymmetric membranes: the optimization and utilization of asymmetric model systems

The plasma membrane (PM) is a crucial membrane in all cells that acts as a protective barrier and is involved in many cell processes. Composed of diverse lipids and proteins, the PM's chemical complexity leads to the formation of functional microdomains called lipid rafts. The two PM leaflets exhibit transbilayer lipid compositional asymmetry. Simplified mixtures mimicking the composition of either leaflet have been widely used as biophysical models for the PM. However, symmetric bilayers have until recently been difficult to make and characterize, and consequently much less is known about their properties. A gap in our understanding of the PM concerns how the properties of the two leaflets are coupled and how this coupling influences raft properties. Calcium-induced hemifusion, a method for producing asymmetric giant unilamellar vesicles (aGUVs), offers a promising technique for probing interleaflet coupling in asymmetric membranes. This method produces aGUVs with varying degrees of asymmetry, useful for examining asymmetric phase boundaries. This dissertation aims to elucidate interleaflet coupling in asymmetric membranes and the molecular determinants of coupling strength. We optimized the hemifusion technique to produce aGUVs for our studies. To understand how systematic changes in lipid composition influence interleaflet coupling strength, we studied ternary mixtures of cholesterol, DPPC, and an unsaturated lipid with varied chain length (14:1-PC, 16:1-PC, 18:1-PC, 20:1-PC, or 22:1-PC). Using several techniques sensitive to lipid heterogeneity, we first investigated the phase behavior of symmetric mixtures and found the three systems with shorter unsaturated chains were macroscopically phase separated, the 20:1-PC system showed nanoscopic heterogeneity, and the 22:1-PC system was uniform. We then prepared aGUVs from the 16:1-PC ternary mixture hemifused to a supported lipid bilayer composed of 16:1-PC and cholesterol to investigate how asymmetry affects phase behavior. Inducing asymmetry abolished liquid-liquid phase separation, suggesting strong interleaflet coupling effects. Lastly, we replaced the unsaturated lipid in the system with 14:1-PC and showed that shorter unsaturated lipid chains shift the phase boundary in a manner consistent with weaker coupling. These findings enhance our understanding of domain formation in symmetric membranes and interleaflet coupling strength in asymmetric membranes, highlighting the impact of lipid composition on membrane behavior.