Giant unilamellar vesicles electroformed from native membranes and organic lipid mixtures under physiological conditions

Abstract

In recent years, giant unilamellar vesicles (GUVs) have become objects of intense scrutiny by chemists, biologists, and physicists who are interested in the many aspects of biological membranes. In particular, this "cell size" model system allows direct visualization of particular membrane-related phenomena at the level of single vesicles using fluorescence microscopy-related techniques. However, this model system lacks two relevant features with respect to biological membranes: 1), the conventional preparation of GUVs currently requires very low salt concentration, thus precluding experimentation under physiological conditions, and 2), the model system lacks membrane compositional asymmetry. Here we show for first time that GUVs can be prepared using a new protocol based on the electroformation method either from native membranes or organic lipid mixtures at physiological ionic strength. Additionally, for the GUVs composed of native membranes, we show that membrane proteins and glycosphingolipids preserve their natural orientation after electroformation. We anticipate our result to be important to revisit a vast variety of findings performed with GUVs under low- or no-salt conditions. These studies, which include results on artificial cell assembly, membrane mechanical properties, lipid domain formation, partition of membrane proteins into lipid domains, DNA-lipid interactions, and activity of interfacial enzymes, are likely to be affected by the amount of salt present in the solution.

FIGURE 1

Fluorescent images (false color representation) of DiI_C18-labeled red blood cell (A), RSO ghost (B), and RSO G-ghosts (C and D). All the images were obtained at 20°C in 25 mM HEPES, 150 mM NaCl, pH 7.2. The white bars correspond to 5 μm.

FIGURE 2

(A) Multicolor fluorescence image of RSO G-ghosts in the presence of band III (red, A2) and glycophorin A (magenta, A3) specific immunofluorescence markers (false color representation); the G-ghosts are labeled with DiI_C18 lipophilic fluorescent probe (yellow, A1). (B) Multicolor fluorescence image of IO G-ghosts in the presence of band III (red, B2) and glycophorin A (magenta, B3) specific markers. As showed in A, the G-ghosts are labeled with DiI_C18 lipophilic fluorescent probe (yellow, B1). The white bars correspond to 30 μm.

FIGURE 3

Fluorescence intensity representative images of RSO (A) and IO (B) G-ghosts in the presence of blood group A specific marker (blue); the G-ghosts are labeled with DiI_C18 lipophilic fluorescent probe (green). (C) Normalized average fluorescence intensity observed in RSO and IO G-ghost preparations (obtained over 30 individual vesicles) on addition of the immunofluorescence marker against blood group A (right panel). All the images were obtained at 20°C in 25 mM HEPES, 150 mM NaCl, pH 7.2. The white bar corresponds to 20 μm.

FIGURE 4

Laurdan GP images and the corresponding GP histograms obtained for red blood cells (A) and RSO G-ghosts (B). The white bars correspond to 10 μm. All the images were obtained at 20°C in 25 mM HEPES, 150 mM NaCl, pH 7.2.

FIGURE 5

(A) Fluorescence intensity image (false color representation) of DiI_C18-labeled RSO G-ghosts (yellow) electroformed from erythrocyte ghosts loaded with Alexa 488 dextran (3000 mol wt, green). Notice that the fluorescent dextran remains entrapped in the G-ghosts after electroformation. (B and C) Sketches of the possible mechanisms proposed for G-ghost electroformation. Based on the results reported in Figs. 2, 3, and 5 A, the mechanism described in panel C is excluded (see text). The white bar corresponds to 20 μm.

FIGURE 6

Fluorescence images (false color representation) of DiI_C18-labeled GUVs composed of red blood cell membrane lipid extracts (A). Laurdan-labeled GUVs composed of (DOPC:DPPC)/cholesterol ((1:1)/20 mol %); red and green areas correspond to liquid-ordered and liquid-disordered phases, respectively (B). DiI_C18-labeled GUVs composed of POPC/DPPC 3:2 mol (C); the high-fluorescence-intensity areas correspond to DPPC-rich gel phase. All the images were obtained at 20°C in 25 mM HEPES, 150 mM NaCl, pH 7.2. The white bar corresponds to 10 μm.