Membrane rafts and lipid domains

It has been long realized that model systems of membranes prepared with phospholipid mixtures with different main transition temperatures (T_m) can exhibit lateral phase coexistence of gel/fluid phases. Depending on the ideality of the mixture, gel-fluid and even gel-gel and fluid-fluid phase separation can occur. Another group of lipid mixtures that have been intensely used are phospholipid (mainly PC)/cholesterol (chol) binary systems, for which liquid ordered (ld)/liquid disordered (lo) phase separation may occur. These mixtures' properties and interaction with peptides and proteins is of importance due to the high abundance of chol in mammalian plasma membranes.

The fact that phase separation occurs for mixtures of lipids coexisting in cell membranes under conditions close to physiological made the detection and characterization of this kind of lateral heterogeneity of considerable interest in the biophysical and biochemical communities. The composition-temperature phase diagrams, at constant pressure (and ionic strength, etc.), are a convenient way to represent this type of behavior for each pair of phospholipids. However, they do not present a complete description of the phase separation phenomenon, because, at variance with common 3D phase separation in binary mixtures, this process may not go to completion, but lead to the formation of smaller portions (domains) of the least abundant phase, dispersed in the other phase.

Lipid rafts are a special type of membrane domains. They were postulated in 1997, and are thought to consist of sphingolipid/chol- enriched and (unsaturated) phosphatidylcholine (PC)-depleted domains. They are involved in important cellular processes such as sorting and trafficking of lipids and proteins, and signal transduction. The simplest model for rafts is a mixture of a high T_m lipid with a low T_m lipid and chol. The rafts would correspond to the lo domains enriched in high T_m lipid and chol, dispersed in a ld matrix enriched in low T_m lipid. Apparently, rafts in a rest state in cell membranes are in general <100 nm.

In our group, the properties of several binary and ternary lipid mixtures (vesicles) have been studied by fluorescence spectroscopy. Concerning the binary mixtures (for which the phase diagram was previously known) the size of domains was estimated for mixtures of two phospholipids with gel/fluid phase separation (stemming from a large difference of acyl chain length in the two constituents). The slow kinetics of phase separation after sudden cooling from a temperature above the T_m of both lipids was also studied (Figures 1 and 2).

Figure 1 - DLPC/DSPC phase diagram describing the thermal quench experiments.

Figure 2 - Decay curves of steady-state fluorescence intensity (left) and anisotropy (right) of NBD-DLPE following the thermal quench, showing the slow phase separation kinetics.

PC/chol binary mixtures with ld/lo phase separation are usually more difficult to study, because the two coexisting phases are more similar than for gel/fluid separation. However, the use of fluorescence techniques (namely FRET) made it possible to study the domains for different experimental conditions (composition, temperature).

Figure 3 - PSM/POPC/chol phase diagram at 23°C (A) and at 37°C (B). The circles are experimental points. The red (quasi) tie-line on the tie-triangle describes the lo/ld composition at the right of which there is also so phase. The blue tie-lines are the interval for the possible tie-lines that contain the 1:1:1 composition. The purple point marks the 1:1:1 composition and the green point marks the 2:1:1 composition.

Recently, we published the phase diagram for the raft model ternary system sphingomyelin (SM)/PC/chol, obtained by fluorescence methods (Figure 3). The diagram gives the composition and boundaries of the rafts, explains apparently conflicting literature results, and, because the direction of some tie-lines was also determined, allows carrying out for the first time thermodynamically consistent studies on that ternary system, namely the interaction of other molecules with raft model membranes. The knowledge of the tie-line also allowed carrying out a FRET study similar to those carried out in the binary systems (Figure 4). Other ternary systems of considerable biological relevance are currently under study.

Figure 4 - PSM/POPC/chol phase diagram at 23ºC, showing also the boundaries and schematic illustrations of the size of lipid rafts. Rafts are present in the blue-shaded area (ld/lo coexistence). In the darker area, lo predominates over ld, and the reverse occurs for the light-shaded area. Rafts can also exist in the green-shaded area, where there is coexistence of three-phases, but the so phase is present only in very low amounts. Insets: A) region of large rafts; detected by microscopy and FRET (> 75-100 nm); B) region of intermediate size rafts: detected by FRET but not by microscopy (between ~20 nm and ~75-100 nm); C) region of small rafts: not detected by FRET nor microscopy (< 20 nm).

Ceramide (Cer), a biosynthetic precursor of sphingomyelin that can be also generated by the action of sphingomyelinase in cell membranes, has recently emerged as a key molecule in the modulation of several cellular processes such as apoptosis and stress-signalling cascades, probably by inducing the formation of large raft platforms. Cer is considered to act as a second messenger in various signalling pathways and its action is commonly ascribed to changes in the membrane physical properties after Cer generation. Increase in membrane order, microdomain formation (gel-fluid phase separation) and formation of non-lamellar lipid phases are frequently reported for this lipid. Both binary (Cer/PC) and ternary (Cer/PC/chol) mixtures are currently being studied.

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