Presence of Salt and Solution Asymmetry Across Charged Membranes Influences Their Phase State

Abstract

Liquid-liquid phase separation in giant unilamellar vesicles (GUVs) leads to the formation of intramembrane domains or lipid rafts. In order to mimic charged biological membranes, we studied phase separation and domain formation in GUVs of ternary lipid mixtures, composed of egg sphingomyelin (eSM), cholesterol (Chol) and the negatively charged lipid dioleoyl-phosphatidylglycerol (DOPG). The GUVs were exposed to solutions of sucrose and high-saline buffer. The phase diagram was mapped using epifluorescence microscopy for vesicle populations with symmetric and asymmetric solution compositions across the membrane. Interestingly, solution asymmetry was also found to affect the membrane phase state. Furthermore, compared to the case of symmetric sucrose conditions, the phase diagram in the presence of high-saline buffer (both symmetrically or asymmetrically distributed across the membrane) was found to exhibit a significantly extended coexistence region for liquid ordered (Lo) and liquid disordered (Ld) phases. These observations were confirmed on single GUVs using microfluidics and confocal laser scanning microscopy. Moreover, we showed that the miscibility temperatures markedly increased for vesicles in the presence of symmetric and asymmetric salt solutions. Our results indicate a substantial effect of salt on the phase behavior of charged membranes and demonstrate that one has to control the solution conditions in order to appropriately map the phase diagram of lipid mixtures.This work is part of the MaxSynBio consortium which is jointly funded by the Federal Ministry of Education and Research of Germany and the Max Planck Society.