Disruption of liquid/liquid phase separation in asymmetric GUVs prepared by hemifusion.

We used calcium-induced hemifusion to create an asymmetric lipid distribution in giant unilamellar vesicles that are models for the mammalian plasma membrane. Confocal fluorescence micrographs of asymmetric vesicles showed that coexisting liquid-ordered and liquid-disordered domains initially present in symmetric vesicles were disrupted after 75% of the saturated lipid in their outer leaflets was replaced with unsaturated lipid. We developed quantitative models for extracting valuable information from the data, including the location of the phase boundary and the compositional uncertainty of individual asymmetric vesicles. The methodology we describe can help reveal the molecular determinants of interleaflet coupling of phase behavior and thus contribute to a better understanding of lipid raft phenomena.