Three-phase coexistence in binary charged lipid membranes in hypotonic solution

TL;DR

This study reveals the coexistence of three distinct lipid phases in charged lipid membranes under osmotic stress, combining experimental microscopy and molecular dynamics simulations to understand the stabilization mechanisms.

Contribution

It demonstrates the first observation of three-phase coexistence in DOPS/DPPC membranes and elucidates the role of ionization and hydrogen bonding in stabilization.

Findings

Three-phase coexistence observed in charged lipid membranes.

Ionization state of DOPS influences phase separation.

Hydrogen bonding stabilizes the three-phase system.

Abstract

We investigated the phase separation of dioleoylphosphatidylserine (DOPS) and dipalmitoylphosphatidylcholine (DPPC) in giant unilamellar vesicles in 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 three-phase coexistence even in 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.