The use of hemifusion to create asymmetric giant unilamellar vesicles: Insights on induced order domains.

The natural asymmetry of the lipid bilayer in biological membranes is, in part, a testament to the complexity of the structure and function of this barrier limiting and protecting cells (or organelles). These lipid bilayers consist of two lipid leaflets with different lipid compositions, resulting in unique interactions within each leaflet. These interactions, combined with interactions between the two leaflets, determine the overall behavior of the membrane. Model membranes provide the most suitable option for investigating the fundamental interactions of lipids. This report describes a comprehensive method to make asymmetric giant unilamellar vesicles (aGUVs) using the technique of hemifusion. In this method, calcium ions induce the hemifusion of giant unilamellar vesicles (GUVs) with a supported lipid bilayer (SLB), both having different lipid compositions. During hemifusion, a stalk, or a more commonly seen hemifusion diaphragm, connects the outer leaflets of GUVs and the SLB. The lateral diffusion of lipids naturally promotes the lipid exchange between the connected outer leaflets. After calcium chelation to prevent further fusion, a mechanical shear detaches aGUVs from the SLB. A fluorescence quench assay is employed to test the extent of bilayer asymmetry. A fluorescence quenching assay tests bilayer asymmetry and verifies dye and lipid migration to a GUV's outer leaflet.