Phase-separation in natural membranes provides a critical means to regulate the spatial distribution of molecules on membrane surface and is hence, central to an array of membrane-based processes. Research on this phenomenon has previously revealed that ternary lipid mixtures (e.g., composed of one lipid with saturated acyl chains, one with unsaturated acyl chains, and cholesterol) can undergo phase separation under certain conditions. Most of these studies have focused on mixtures of cholesterol and neutral lipids such as phosphatidylcholine (PC). However, natural membranes contain both neutral and charged lipids with important functional roles. Here, we aim to investigate if and how the presence of charged lipids affects the membrane’s phase behavior in ternary lipid compositions using electroformed giant unilamellar vesicles (GUVs) and fluorescence microscopy. To this end, negatively or positively charged lipids of similar acyl chain chemistry (DOPA and DOTAP, respectively) were used to replace the unsaturated lipids (DOPC) in a well-studied ternary mixture (DOPC: DPPC: Chol). These studies showed that the population of GUVs that exhibited detectable phase separation reduced as the amount of the charged lipids increased beyond half of the initial DOPC. These results were similar for both negative and positive charges. We also noted a decrease in the area fraction of liquid disordered phase on the phase-separated liposomes, presumably due to the smaller head groups of the examined charged lipids. Interestingly, inclusion of oppositely charged lipids in liposome formulation or presence of divalent cations, which reduced the charge repulsion in the membrane, restrained the suppressing effect of the negative lipid DOPA on phase separation. These results demonstrate that the electrostatic repulsion between the like charges on the head group may be utilized to regulate membrane phase behavior. The findings of this study may offer insight into the mechanisms underlying phase separation in biological membranes.

Membrane phase behavior in the presence of charged lipids

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