Sterol structure determines the separation of phases and the curvature of the liquid-ordered phase in model membranes

Abstract

The existence of lipid rafts in biological membranes in vivo is still debated. In contrast, the formation of domains in model systems has been well documented. In giant unilamellar vesicles (GUVs) prepared from ternary mixtures of dioleoyl-phosphatidylcholine/sphingomyelin/cholesterol, a clear separation of liquid-disordered and sphingomyelin-enriched, liquid-ordered phases could be observed. This phase separation can lead to the fission of the liquid-ordered phase from the vesicle. Here we show that in cholesterol-containing GUVs, the phase separation can involve dynamic redistribution of lipids from one phase into another as a result of a cross-linking perturbation. We found that the molecular structure of a sterol used for the preparation of GUVs determines (i) its ability to induce phase separation and (ii) the curvature (positive or negative) of the formed liquid-ordered phase. As a consequence, the latter can pinch off to the outside or inside of the vesicle. Remarkably, some mixtures of sterols induce liquid-ordered domains exhibiting both positive and negative curvature, which can lead to a new type of budding behavior in GUVs. Our findings could have implications for the role of sterols in various cell-biological processes such as budding of secretory vesicles, endocytosis, or formation of multivesicular bodies.

Fig. 1.

Temporal redistribution of GM1 from the l_d phase to the l_o phase after the addition of cholera toxin. (A, C, and E) Time course of the labeling of GUVs with AlexaFluor 488 cholera toxin B. (B, D, and F) Merge of labeling with Alexa Fluor 488 cholera toxin B (green) and DiI-C₁₈ (red). Notice the increase of cholera toxin labeling with time. Arrowheads and arrows show cholera toxin in the l_d and the l_o phase, respectively. The large GUV in the middle of each panel is the same one at all three time points.

Fig. 2.

The molecular structure of a sterol determines separation of phases in GUVs and the curvature of the l_o phase. Confocal images of GUVs produced from DOPC:SM:sterol (1:1:1) in 12 mM sucrose. The sterols used were cholesterol, 3-ketocholesterol, lanosterol, lophenol, or cholesteryl sulfonate. GUVs in Left images are labeled with DiI-C₁₈ (red) only. Center images were taken ≈30 minutes after cholera toxin (green) has been added. Right images were taken directly after injection of 26 mM sucrose solution. Same as in the case of cholesterol, DiI-C₁₈ labeling marks the l_d phase and cholera toxin labeling marks the l_o phase for all sterols that induce phase separation (see Figs. 3 and 6, which is published as supporting information on the PNAS web site). (Scale bar: 10 μm.) The ratio of vesicles showing outward bulging (or budding) behavior to vesicles showing an inward preference was at least 10 for cholesterol and lophenol, and <1/10th for lanosterol and cholesteryl sulfonate, as judged from overview scans with many vesicles.

Fig. 4.

Different proportions of cholesterol and CS in GUVs modulate domain size, domain curvatures, budding, and the formation of tubular structures. GUVs consisted of 42% DOPC, 42% SM, and 16% of sterol mixture (cholesterol and CS). Proportions of cholesterol and CS are indicated. Labeling is the same as in Fig. 2. A transition from clear phase separation (A; 16% cholesterol) to no observable domains (C; 0% cholesterol) occurs. At 8% cholesterol and 8% CS, a majority of GUVs show small domains that do not fuse to yield larger ones (B). At more CS, GUVs look mostly uniform (C). Confocal sections in D–G represent curvatures of GUVs at the cholesterol and CS concentrations indicated. At 16% cholesterol, curvature is predominantly positive. With an increasing fraction of CS, bell-shaped l_o domains with a negative curvature close to the borders with the l_d domains are observed (E and F). As seen in the time series (F), at the edges, small l_o phase vesicles bud inward (arrow). At higher CS concentrations (G), no domains but many tubular and reticular structures, are observed. (Scale bar: 10 μm.)

Fig. 3.

Sterol structure determines the dynamic properties of the l_o phase. FCS measurements of BODIPY-Chol in the more strongly labeled phase (black curves) and in the phase of lower probe enrichment (gray curves) of GUVs composed of DOPC:SM:sterol (1:1:1:), where the sterol is cholesterol, cholesteryl sulfonate, lophenol, or lanosterol. Diffusion times τ_diff can be read from the intersections of the broken lines with the abscissae and demonstrate that the probe mobility depends both on the type of phase and on the particular sterol. Diffusion is in all cases faster in the more strongly labeled phase (l_d) and slower in the other phase (l_o). BODIPY-Chol and DiI-C₁₈ exhibit the same qualitative partitioning (see Fig. 6). Hence, the DiI-C₁₈-enriched domains correspond in all cases to the l_d phase. Despite the qualitative resemblance, BODIPY-Chol partitioning was less pronounced than DiI-C₁₈ partitioning. BODIPY-Chol was therefore preferred for FCS measurements because a dye with more extreme partitioning (e.g., DiI-C₁₈) is more likely to produce artifacts in the correlation curve, in the event of a tiny vesicle of the brighter phase diffusing through the focus.