Experimentally determined leaflet–leaflet phase diagram of an asymmetric lipid bilayer

Significance

We report an experimentally determined phase diagram of an asymmetric lipid bilayer. We studied mixtures of a saturated and unsaturated lipid that separate into gel and fluid phases in symmetric bilayers. Consistent with theoretical predictions for strongly coupled bilayers, we found that increasing the concentration of unsaturated lipid in one of the leaflets abolished phase separation in the opposing leaflet. This scenario roughly mimics the phospholipid asymmetry of an animal cell plasma membrane, in which one leaflet contains a mixture of ordered and disordered lipids, while the other leaflet contains predominantly disordered lipids. Leaflet–leaflet phase diagrams reveal how cells can control membrane phase behavior by changing both the composition and asymmetric distribution of lipids.

Abstract

We have determined the partial leaflet–leaflet phase diagram of an asymmetric lipid bilayer at ambient temperature using asymmetric giant unilamellar vesicles (aGUVs). Symmetric GUVs with varying amounts of 1,2-dipalmitoyl-sn-glycero-3-phosphocholine and DOPC (1,2-dioleoyl-sn-glycero-3-phosphocholine) were hemifused to a supported lipid bilayer (SLB) composed of DOPC, resulting in lipid exchange between their outer leaflets. The GUVs and SLB contained a red and green lipid fluorophore, respectively, thus enabling the use of confocal fluorescence imaging to determine both the extent of lipid exchange (quantified for individual vesicles by the loss of red intensity and gain of green intensity) and the presence or absence of phase separation in aGUVs. Consistent with previous reports, we found that hemifusion results in large variation in outer leaflet exchange for individual GUVs, which allowed us to interrogate the phase behavior at multiple points within the asymmetric composition space of the binary mixture. When initially symmetric GUVs showed coexisting gel and fluid domains, aGUVs with less than ~50% outer leaflet exchange were also phase-separated. In contrast, aGUVs with greater than 50% outer leaflet exchange were uniform and fluid. In some cases, we also observed three coexisting bilayer-spanning phases: two registered phases and an anti-registered phase. These results suggest that a relatively large unfavorable midplane interaction between ordered and disordered phases in opposing leaflets (i.e., a midplane surface tension) can overwhelm the driving force for lateral phase separation within one of the leaflets, resulting in an asymmetric bilayer with two uniformly mixed leaflets that is poised to phase-separate upon leaflet scrambling.