Effect of Counter Electrode in Electroformation of Giant Vesicles
Electroformation of cell-sized lipid membrane vesicles (giant vesicles, GVs), from egg yolk phosphatidylcholine, was examined varying the shape of the counter electrode. Instead of a planar ITO (indium tin oxide) electrode commonly used, platinum wire mesh was employed as a counter electrode facing lipid deposit on a planar formation electrode. The modification did not significantly alter GV formation, and many GVs of 30–50 μm, some as large as 100 μm, formed as with the standard setup, indicating that a counter electrode does not have to be a complete plane. When the counter electrode was reduced to a set of two parallel platinum wires, GV formation deteriorated. Some GVs formed, but only in close proximity to the counter electrode. Lower electric voltage with this setup no longer yielded GVs. Instead, a large onion-like multilamellar structure was observed. The deteriorated GV formation and the formation of a multilamellar structure seemed to indicate the weakened effect of the electric field on lipid deposit due to insufficient coverage with a small counter electrode. Irregular membranous objects formed by spontaneous swelling of lipid without electric voltage gradually turned into multilamellar structure upon following application of voltage. No particular enhancement of GV formation was observed when lipid deposit on a wire formation electrode was used in combination with a large planar counter electrode.
Giant unilamellar vesicle electroformation from lipid mixtures to native membranes under physiological conditions.
Giant unilamellar vesicles (GUVs) are well-known model systems, especially because they are easily observable using optical microscopy. In this chapter, we revisit in detail the versatile GUV electroformation protocol. We demonstrate how GUV electroformation can be adapted to various membrane systems including synthetic lipid mixtures, natural lipid extracts, and bilayers containing membrane proteins. Further, we show how to adjust this protocol to a given aqueous environment and prove that GUVs can be obtained under physiologically relevant conditions, that is, in the presence of electrolytes. Finally, we provide firm evidence that electroformation is a method of choice to produce giant vesicles from native cell membranes. This is illustrated with the example of GUV electroformation from red blood cell ghosts in a physiologically pertinent buffer. GUVs obtained in this manner maintain the native membrane asymmetry, thereby validating the physiological relevance of GUV electroformation.
Formation of tubules and giant vesicles from large multilamellar vesicles.
This study reports an observation of submicrometer multilamellar vesicles (MLVs) prepared by simply freeze-thawing a phospholipid dispersion at full hydration that transformed into giant vesicles (GVs) and tubules (TUs) when confined between microscope glass slides. Cover slide cleaning and surface treatment did not hamper the formation of GVs or TUs. However, when small unilamellar vesicles (SUV) were prepared or when MLVs were not confined but rather freely moved between the glass slides or when the phospholipid was in its gel phase, neither GVs nor TUs were observed. Altogether, our results suggested that MLVs would play a role as a lipid reservoir and that the liquid flow between the glass slides induces the peeling of the external bilayers, yielding the formation of tubules and giant unilamellar vesicles.