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.
Highlights
Lipid bilayer sheets in aqueous environments can deform and close into vesicles to shield free edges from water
Giant vesicles made of DOPC/DOPG (8/2 molar ratio) and pure DOPC were prepared via the standard electroformation protocol in HEPES buffer, see Material and Methods
The results presented here indicate that the conditions of electroformation of Giant unilamellar vesicles (GUVs) with charged lipids should be carefully scrutinized, especially when bilayer surface charge is important or the effects of membrane asymmetry are investigated
Summary
Lipid bilayer sheets in aqueous environments can deform and close into vesicles to shield free edges from water. Considering the involvement of electrostatic interactions in the electroformation protocol, we questioned the effect it might have on the distribution of charged lipids across the membrane of electroformed GUVs. Asymmetric distribution of the lipid species in the two leaflets can have two major consequences. The composition and charge of the two leaflets will differ from the one for an (assumed) symmetric distribution. Apart from advancing the understanding of GUV electroformation, membrane asymmetry (even though mostly undesired or unanticipated in model membrane studies) can be turned into an advantage for guiding and controlling the supramolecular (self-) assembly and organization of charged lipids at the nanometer scale of lipid membrane leaflets. Our study demonstrates membrane reshaping by compositional bilayer asymmetry, a feature ubiquitous in biological membranes
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