Various model membrane systems have been used by physicists and chemists to study phase separation in lipid mixtures. They are either monolayers or bilayers. Monolayers are either assembled at an air-water interface with the packing density of the lipids being adjusted by applying lateral pressure, or on a supporting lipid monolayer that is fixed to a solid support. Bilayers are used in the supported version as described above, or in the form of vesicles. The most commonly used vesicles are large or giant unilamellar vesicles (LUV or GUV, respectively) composed of only a single bilayer, but also multilamellar vesicles (MLV) are used. The basic principles were first established in simple binary lipid mixtures, but recently ternary mixtures which more closely mimic the composition of the cell plasma membrane have been used. The mixtures usually contain one lipid with a high melting temperature (Tm), one with a low Tm, and cholesterol. GUVs are probably the system closest to a cell membrane, because artifacts from a support are excluded. Still, cell membranes are asymmetric with different lipid compositions of the outer versus the inner leaflet, while the GUVs used so far were all symmetric. Since maintaining an asymmetric lipid distribution is energy-consuming, perhaps by reconstituting lipid translocators into liposomes this drawback can be overcome in the future. Although model membrane systems produce very simplified pictures of cell membranes, there are many examples of a close correlation with experimental data obtained in living cells .
Ipsen et al. were the first to describe the formation of a liquid-ordered phase by cholesterol and saturated phospholipids [15,16]. This phase can coexist with other lipid phases, and its characteristics are described as follows: the translational order of lipid molecules within the liquid-ordered phase is similar to that in a fluid bilayer state, whereas the configurational order of the hydrocarbon chains compares more to that in a gel state. The formation of the liquid-ordered phase was attributed to the unique chemical nature of cholesterol (for a review, see ), but later it was shown that all natural sterols promote domain formation and that also small amounts of ceramide (3%) can stabilize domains formed in vesicles . Leventis and Silvius showed that the interaction of cholesterol with different lipid species is dependent on the nature of their hydrocarbon chains and, to a lesser extent, also on their headgroup. The interaction preference decreases with SM > PS > PC > PE and with increasing unsaturation of the acyl chains . Whereas the kink in unsaturated hydrocarbon chains is likely to hinder tight packing with the flat sterol ring of cholesterol, the reason for the preferential interaction of cholesterol with SM is still a debated issue.
The first visualization of "raft-like domains" in model membranes was achieved by Dietrich et al. . They visualized liquid ordered domains in supported bi-layers and GUVs composed not only of synthetic lipid mixtures but also of lipid extracts from brush border membrane, the apical membrane of intestinal cells. Domain formation was cholesterol-dependent, since domains disappeared after treatment with the cholesterol-extracting drug methyl-b-cyclodextrin. Another big step forward was the establishment of a ternary phase diagram of SM/PC/choles-terol at the physiological temperature of 37 °C . This predicts the coexistence of liquid-ordered and liquid-disordered phases for a wide range of compositions mimicking those occurring in the plasma membrane of cells. Most domains observed in model membranes are rather large (i.e., several micrometer in diameter) or they start small when they are being formed and then grow continuously by collision and fusion as the system reaches equilibrium . Contrary to this, raft domains in cells are believed to be small, most likely because the cell membrane is not at equilibrium (see below). Interestingly, fluorescence resonance energy transfer (FRET) measurements on vesicles composed of a ternary lipid mixture mimicking the outer leaflet of the plasma membrane revealed heterogeneities (i. e., domains) of sizes in the tens of nanometer range at 37 °C . Large domains were observed with the same lipid mixture only below 20 °C.
A slightly different interpretation of liquid-liquid immiscibility observed in model membranes was proposed by McConnell and colleagues. These authors argue for the formation of "condensed complexes" between cholesterol and SM rather than a liquid-ordered phase or domain. The name originates from the observation that cholesterol and SM occupy less surface area when mixed together compared to the sum of the areas occupied by each component alone before mixing. Such a complex is supposed to contain 15-30 molecules with a fixed stoichio-metry of 2:1 (SM :cholesterol). These complexes could exist in quite high concentration without necessarily leading to a phase separation (for a review, see ). However, the condensed complex theory was developed on monolayer membranes and has not yet been validated for bilayers.
Taken together, there is clear evidence for lipid-driven domain formation in model membrane systems mimicking the outer leaflet of the plasma membrane. On the contrary, domain formation could not be observed in lipid mixtures mimicking the inner leaflet of the plasma membrane . The intermolecular forces leading to phase separation are van der Waals interactions between saturated acyl chains and cholesterol, as well as forces such as hydrophobic shielding or the "umbrella effect", described for cholesterol filling the holes left between the acyl chains of glycosphingolipids with large headgroups . However, none of the systems described so far has included proteins in their analysis, and the question remains whether proteins choose the domain they partition into, or whether they organize a domain around them.
Partitioning experiments have been performed, in which proteins were reconstituted into model membranes, and their phase distribution was analyzed. In this way, glycosyl-phosphatidyl-inositol (GPI)-anchored placental alkaline phosphatase (PLAP; [27,28] and Thy-1  were shown to partition into the liquid-ordered phase, and the chain length of the GPI-anchor was shown to be important for partitioning of the protein . Similarly, peptides modified with prenyl groups were excluded from liquid-ordered domains, while peptides modified with cholesterol or palmityl chains partitioned significantly into the ordered phase . Partitioning studies with synthetic transmembrane peptides revealed that longer transmembrane domains are incorporated better into liquid-ordered domains than shorter versions . Another important determinant for the partitioning of a molecule is the size and orientation of its dipole moment . The membrane dipole moment is stronger in ordered phases where the dipoles are better aligned. Only molecules displaying a dipole moment with the same orientation as the dipolar potential of the membrane, are predicted to be able to enter the ordered phase. Nevertheless, our knowledge about lipid-transmembrane protein interactions is still scarce and this area of research is a major challenge.
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