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

Abstract

Model asymmetric bilayers are useful for studying the coupling between lateral and transverse lipid organization. Here, we used calcium-induced hemifusion to create asymmetric giant unilamellar vesicles (aGUVs) for exploring the phase behavior of 16:0-PC/16:1-PC/Cholesterol, a simplified model for the mammalian plasma membrane. Symmetric GUVs (sGUVs) were first prepared using a composition that produced coexisting liquid-disordered and liquid-ordered phases visible by confocal fluorescence microscopy. The sGUVs were then hemifused to a supported lipid bilayer (SLB) composed of uniformly mixed 16:1-PC/Cholesterol. The extent of outer leaflet exchange was quantified in aGUVs in two ways: (1) from the reduction in fluorescence intensity of a lipid probe initially in the sGUV ("probe exit"); or (2) from the gain in intensity of a probe initially in the SLB ("probe entry"). These measurements revealed a large variability in the extent of outer leaflet exchange in aGUVs within a given preparation, and two populations with respect to their phase behavior: a subset of vesicles that remained phase separated, and a second subset that appeared uniformly mixed. Moreover, a correlation between phase behavior and extent of asymmetry was observed, with more strongly asymmetric vesicles having a greater probability of being uniformly mixed. We also observed substantial overlap between these populations, an indication that the uncertainty in measured exchange fraction is high. We developed models to determine the position of the phase boundary (i.e., the fraction of outer leaflet exchange above which domain formation is suppressed) and found that the phase boundaries determined separately from probe-entry and probe-exit data are in good agreement. Our models also provide improved estimates of the compositional uncertainty of individual aGUVs. We discuss several potential sources of uncertainty in the determination of lipid exchange from fluorescence measurements. Statement of SignificanceWe 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.