Liquid–liquid immiscibility under non-equilibrium conditions in a model membrane: An X-ray synchrotron study

Several non-random lipid mixtures have been proposed as models of lipid plasma membrane, as they mimic the ability of biomembranes to form lateral domains. Biological membranes are characterised by a succession of localised transient steady-state lipid organisations rather than stable equilibria. This suggests that several quasi at-equilibrium lipid organisations may exist at different times in the same local patch of membranes. Identification of the conditions which can mimic heterogeneous dynamic membrane states in a lipid membrane model is a challenge. This is of particular importance as the lateral organisation of lipids mixtures in fully equilibrated samples may differ from the arrangement found in quasi at-equilibrium conditions. To address this issue, we have performed a real-time synchrotron X-ray diffraction study in ternary mixtures of egg-phosphatidylcholine/egg-sphingomyelin and cholesterol using a 0.5 °C/15 s step within a 20–50–20 °C thermal cycle. In the present study, all ternary mixtures displayed lamellar phase separation. A d-spacing value was observed reversibly during the heating and cooling scan for each of the two coexisting phases. In mixtures with a cholesterol concentration from 20 to 50 mol%, a liquid-ordered (Lo) and liquid-disordered (Ld) phase separation was observed in the 20–50 °C thermal range. These results are discussed in terms of a specific interaction between lipid molecular aggregates.