Asymmetric phase diagram of DPPC/DOPC to investigate inter-leaflet coupling.

Abstract

Cell membranes are asymmetric, meaning that the bilayer is composed of leaflets with different lipid composition. Simplified models for the exoplasmic leaflet typically form coexisting Ld (liquid disordered) + Lo (liquid ordered) phases in symmetric bilayers. In contrast, the cytoplasmic leaflet lacks high-melting lipids, and models for this leaflet always form a single fluid phase in symmetric bilayers. Thus, among the major open questions is how the phase behavior of these two leaflets is coupled in an asymmetric bilayer. Here, we report the first experimentally determined asymmetric phase diagram for a binary lipid mixture, DPPC/DOPC (1,2-dipalmitoyl-sn-glycero-3-phosphocholine/1,2-dioleoyl-sn-glycero-3-phosphocholine) at room temperature. 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 calcium-induced hemifusion 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 theoretical phase diagrams calculated from mean field theory, where phase boundaries and tieline orientations depend on the relative strength of in-plane and out-of-plane lipid interaction energies. The experimental DPPC/DOPC asymmetric phase diagram is consistent with an intermediate coupling strength whereby anti-registered domains coexist with registered domains at lower levels of asymmetry, but phase separation is completely suppressed at higher levels of asymmetry. Our results allow us to estimate a mismatch free energy of 0.2-0.3 kT per square nanometer for a gel domain directly opposite a fluid domain.