Three-Phase Coexistence in Binary Charged Lipid Membranes in a Hypotonic Solution

We investigated the phase separation of dioleoylphosphatidylserine (DOPS) and dipalmitoylphosphatidylcholine (DPPC) in giant unilamellar vesicles in a hypotonic solution using fluorescence and confocal laser scanning microscopy. Although phase separation in charged lipid membranes is generally suppressed by the electrostatic repulsion between the charged headgroups, osmotic stress can promote the formation of charged lipid domains. Interestingly, we observed a three-phase coexistence even in the DOPS/DPPC binary lipid mixtures. The three phases were DPPC-rich, dissociated DOPS-rich, and nondissociated DOPS-rich phases. The two forms of DOPS were found to coexist owing to the ionization of the DOPS headgroup, such that the system could be regarded as quasi-ternary. The three formed phases with differently ionized DOPS domains were successfully identified experimentally by monitoring the adsorption of positively charged particles. In addition, coarse-grained molecular dynamics simulations confirmed the stability of the three-phase coexistence. Attraction mediated by hydrogen bonding between protonated DOPS molecules and reduction of the electrostatic interactions at the domain boundaries stabilized the three-phase coexistence.

Automated summary

The study found that even in binary lipid mixtures, DOPS and DPPC can form a three-phase coexistence due to the ionization of DOPS headgroups, making the system quasi-ternary. Through experiments and coarse-grained molecular dynamics simulations, it was determined that the stability of the three-phase coexistence is due to attraction mediated by hydrogen bonding between protonated DOPS molecules and reduction of electrostatic interactions at the domain boundaries.