PHASE DIAGRAM OF ASYMMETRIC LIPID BILAYER TO INVESTIGATE INTER-LEAFLET COUPLING

Cell membranes are asymmetric, meaning that the lipid bilayer is composed of leaflets with different lipid composition. The exoplasmic leaflet has a lipid composition that can form coexistence of Ld (liquid disordered) + Lo (liquid ordered) phases. In contrast, the cytoplasmic leaflet lacks high melting lipids, a kind of lipid that is enriched in the Lo phase along with cholesterol, and thereby this cytoplasmic leaflet would form a single fluid phase by itself. Among the major open questions is how these two leaflets are coupled. Theoretical studies have calculated phase diagrams of asymmetric bilayers, where phase boundaries and tieline orientations depend on the relative strength of intra- and inter-leaflet interaction energies. Here, we report the first experimentally determined phase diagram of asymmetric bilayers composed of a binary lipid mixture, DPPC/DOPC (1,2-dipalmitoyl-sn-glycero-3-phosphocholine / 1,2-dioleoyl-sn-glycero-3-phosphocholine). Different from cholesterol-containing model membranes that separate into coexisting liquid phases, this simpler mixture exhibits gel + fluid coexistence over a range of composition and temperature. We used the hemifusion method to engineer asymmetric GUVs (aGUVs), and we measured the extent of outer leaflet exchange through quantitative analysis of fluorescent probe intensities. Our experiments can be directly compared to the theoretical phase diagrams calculated from mean field theory. We suggest the DPPC/DOPC phase diagram for asymmetric bilayers reflects an intermediate coupling strength with features of a low coupling strength yielding partial anti-registration of domains, and features of a relatively high coupling, in which the phase separation is abolished beyond a threshold level of asymmetry. Our results also allow us to estimate the mismatch free energy of an anti-registered phase. We found a range of 0.2-0.3 kBT/ nm2, a value that is similar to theoretical calculations and molecular dynamic simulations.

This work was supported by National Science Foundation - NSF Grant MCB-1817929 (to F.A.H.) T.A.E acknowledges financial support from São Paulo Research Foundation - FAPESP (2022/04046-4 and 2023/05540-5).