Bilayer charge asymmetry and oil residues destabilize membranes upon poration

Abstract

Membrane asymmetry is ubiquitous in cell membranes particularly with respect to lipids, whereby charged lipids are mainly restricted to the inner monolayer. We investigate the influence of anionic lipid asymmetry on the stability of giant unilamellar vesicles (GUVs), minimal plasma membrane models. To quantify asymmetry, we apply a fluorescence quenching assay, which is often difficult to reproduce and caution in handling the quencher is generally underestimated. Thus, we first optimize this assay and then apply it to GUVs prepared with the inverted emulsion transfer protocol using increasing fractions of anionic lipids restricted to one leaflet. This protocol is found to produce highly asymmetric bilayers, but with ~20% interleaflet mixing. To probe the stability of asymmetric vs symmetric membranes, we expose the GUVs to porating DC pulses and monitor the fraction of destabilized vesicles. The pulses open macropores, and the GUVs either completely recover their integrity or become destabilized exhibiting leakage or bursting/collapse. Destabilization is much more pronounced in asymmetrically charged membranes, which is corroborated by pore edge tension data showing considerable decrease with asymmetry. Rendering GUV membrane asymmetric from exposure to different transmembrane pH, we confirm that poration-triggered destabilization does not depend on the approach used to generate membrane asymmetry.

Effect of leaflet asymmetry on the stretching elasticity of lipid bilayers with phosphatidic acid

“…Of course, the degree of this outcome could depend on the type of lipids and oils used. The reduced leaflet coupling in the presence of oil (Figure 5C) might be related to observations showing that residual oil in GUVs prepared with the inverted emulsion method destabilize porated membranes [38].…”

“…Additionally, DMPC:DOPC (dimyristoyl phosphatidylcholine:dioleoyl phosphatidylcholine) vesicles obtained using custom-made microfluidic devices [37], were shown to exhibit increased bending rigidity and area compressibility of the investigated asymmetric system compared to the symmetric ones. Furthermore, membranes with higher charge asymmetry were shown to be more prone to destabilization when exposed to pore-inducing electric pulses, suggesting that asymmetrically charged membranes are less stable [38].…”

Disruption of liquid/liquid phase separation in asymmetric GUVs prepared by hemifusion

“…The hemifusion procedure offers some advantages compared to other aGUV preparation methods. Most importantly, it avoids the use of cyclodextrins, organic solvents, or oil that can potentially interact with the bilayer and alter its properties 25 .…”