Charged giant unilamellar vesicles prepared by electroformation exhibit nanotubes and transbilayer lipid asymmetry

Abstract

Giant unilamellar vesicles (GUVs) are increasingly used as a versatile research tool to investigate membrane structure, morphology and phase state. In these studies, GUV preparation is typically enhanced by an externally applied electric field, a process called electroformation. We find that upon osmotic deflation, GUVs electroformed from charged and neutral lipids exhibit inward pointing lipid nanotubes, suggesting negative spontaneous curvature of the membrane. By quenching a fluorescent analog of the charged lipid, zeta potential measurements and experiments with the lipid marker annexin A5, we show that electroformed GUVs exhibit an asymmetric lipid distribution across the bilayer leaflets. The asymmetry is lost either after storing electroformed GUVs at room temperature for one day or by applying higher voltages and temperatures during electroformation. GUVs having the same lipid composition but grown via gel-assisted swelling do not show asymmetric lipid distribution. We discuss possible mechanisms for the generation and relaxation of lipid asymmetry, as well as implications for studies using electroformed vesicles. The observed effects allow to control the molecular assembly of lipid bilayer leaflets. Vesicle tubulation as reported here is an example of protein-free reshaping of membranes and is caused by compositional lipid asymmetry between leaflets.

Highly Efficient Protein-free Membrane Fusion: A Giant Vesicle Study

“…Lira, R. Dimova, K.A. Riske, unpublished data; see also (44)). This effect is negligible for high POPG mol%, at which fusion is very efficient.…”

“…GUVs were formed by the electroformation method with few modifications (43); see Supporting Materials and Methods. The vesicles were used fresh (the same day), and thus for vesicles made of POPC and POPG mixtures, slightly higher POG concentration on the vesicle surface may be expected (44). Alternatively, the gel-assisted method (45) was used for GUVs with 100 mol% POPG.…”

Switchable Lipid Provides pH-Sensitive Properties to Lipid and Hybrid Polymer/Lipid Membranes

“…We incorporated CSL into giant unilamellar vesicles (GUV-CSL) or giant hybrid unilamellar vesicles (GHUV-CSL) prepared by electroformation, since their micrometric size is better adapted to confocal microscopy [20]. Their morphological alterations upon global decrease of pH was experimentally examined under confocal microscopy ( Figures S6-S9, Supplementary Materials) and posteriorly categorized as vesicles with inward or outward structures (tubular protrusions, membrane-attached aggregation), internalized vesicles, or membrane fluctuation (non-round shaped vesicles) [23] and assembled in a table with values presented as the relative percentage of total vesicles counted (Table 2). As previously, non-switchable giant unilamellar vesicles (GUV-POPC) or giant hybrid unilamellar vesicles (GHUV-POPC) were used as control.…”

“…GUV-CSL 20% and GUV-CSL 50% vesicles responded to acidification by projecting outward tubular protrusions, since 20% and 14% of the vesicles analyzed presented such structures, respectively (Table 2). Membrane-derived structures pointing outward are commonly referred as positive curvatures [23], whilst negative curvature denotates membrane-arisen structures pointing inward. In the images, we could observe positive membrane curvatures in GUV-CSL 20% and 50% as a result of treatment with HCl but not NaCl ( Table 2, Figures S6 and S7, Supplementary Materials).…”

Particle engulfment by strongly asymmetric membranes with area reservoirs

“…spontaneous curvature, may be generated in many ways, 20 such as by an asymmetric concentration of lipids in each of the monolayers, 21,22 or by asymmetric concentrations of particles in solution that create adsorption or depletion layers near the membrane. 21,23,24 Thus, spontaneous formation of nanotubes has been observed for membranes with lipid asymmetry, 25 in particular of lipids with bulky headgroups such as GM-1; 26,27 for asymmetric concentrations of ions or sugars in the inner and outer aqueous solutions; 28,29 for asymmetric concentrations of polymers such as PEG that adsorb to the membrane; 30,31 or for membranes in contact with specialized curvature-inducing proteins such as BAR domains. 32 Importantly, it has been shown 26 that these artificial membrane reservoirs confer cell-like mechanical responses to the vesicles, such as the liquid-droplet-like instability in micropipette aspiration undergone by white blood cells.…”