The Many Faces of Lipid Rafts

Recent experimental observations in raft-forming lipid bilayers demonstrated a phase behavior unsuspected just a few years ago (1,2). Mixtures of Chol (cholesterol) with disordered (unsaturated) and ordered (saturated) phospholipids change their phase behavior from apparently uniform phases to modulated phases to complete phase separation by small variations in bilayer composition. In this issue of Biophysical Journal, Shlomovitz et al. (3) study a model of a lipid bilayer that displays this rich behavior. The beauty of the model resides in emphasizing only those aspects that are essential to explain the experimental observations. But why is this important? The organization of biological membranes depends on small differential lipid-lipid interactions, defined by ωAB = eAB –(eAA + eBB)/2, where the e are interaction free energies between unlike and like lipids (4). Among the various ideas proposed over the years, the raft hypothesis has dominated the field, and elicited the most controversy. As a membrane-organizing principle (5), the raft hypothesis brings together a plethora of observations in cell membranes on the common basis of a simple physical idea: that Chol interacts favorably (ωAB 0) with unsaturated lipids in a liquid-disordered (Ld) state. Membrane lipids thus separate into rafts and disordered regions. Membrane proteins are sensitive to different environments, and preferentially partition into either rafts or disordered regions. In giant unilamellar vesicles (GUVs), phospholipid-Chol interactions result in macroscopic separation into two liquid phases, one rich in lipids in the Lo state, the other, in lipids in the Ld state. Such phase separation has been observed by fluorescence microscopy for over a decade. But rafts in cell membranes appear to be small, indeed invisible by light microscopy. Why? One explanation is that some membrane components behave as two-dimensional surfactants, or linactants. Hybrid lipids, such as POPC (1-palmitoyl-2-oleoylphosphatidylcholine), could play this role (6). The palmitic chain would favor ordered lipids and the oleic chain, disordered; thus, hybrid lipids would orient themselves around a lipid domain just like a surfactant orients its hydrophilic head to water and its hydrophobic tail to oil in stabilizing an emulsion. Recently, however, it was shown that the effects of POPC and the short but saturated DLPC (dilauroylphosphatidylcholine) on the phase behavior of mixtures of DSPC (distearoylphosphatidylcholine), DOPC (dioleoylphosphatidylcholine), and Chol are indistinguishable (7). POPC and DLPC reduce the amount of unfavorable interactions (ωAB > 0) between DSPC and DOPC, thus favoring smaller domains by a mechanism independent of the hybrid nature of the lipid. An exciting development occurred when modulated phases were observed in GUVs (1,2). Modulated (Mod) phases consist of regular periodic arrangements of two kinds of domains, which in this case are rich either in disordered lipids or in Chol and ordered phospholipids. Most often, these arrangements are either alternating stripes (Fig. 1 C) or a hexagonal array of small domains of one kind in a background of the other (Fig. 1 D). The observation of Mod phases provides a connection between complete phase separation and apparently uniform phases. Figure 1 Qualitative phase diagram of DSPC/(DOPC+POPC)/Chol at room temperature (drawn by the author). Regions containing gel phases, below the Ld + Lo coexistence region, are not represented for clarity. (Solid triangle above Ld + Lo region) Coexistence of L ... This is where the model by Shlomovitz et al. (3) comes in. The authors use a Ginzburg-Landau expansion of the free energy as function of an order parameter. This parameter does not refer to lipid chain order, but is the difference in local composition ϕ(r) in a region centered on position r in the plane of the bilayer. The various phase behaviors observed appear naturally in this mean-field model: regular uniform phases, microemulsions, modulated phases, and macroscopic phase separation. Incorporation of fluctuations breaks the long-range order of the Mod stripe phase, and changes slightly the topology of the phase diagram, allowing for direct transitions from the two-phase region to the microemulsion. The model is consistent with the experimental observations in quaternary mixtures of DSPC/(DOPC+POPC)/Chol (2). Fig. 1 shows a qualitative phase diagram of this system at room temperature, drawn by the author based on Goh et al. (2) but according to the model of Shlomovitz et al. (3), with the simplification that no distinction is made between stripe and hexagonal Mod phases. The ordered lipid is DSPC, but the disordered is a mixture of POPC and DOPC, whose combined amount is given by the phase diagram. The central region is where inhomogeneities are observed. Goh et al. (2) consider this entire region, including the Mod phase area, as of coexistence of Ld and Lo phases. Fig. 1 A shows a GUV with coexistence between Mod and Ld phases; Fig. 1, B–D, shows various Mod phases. As the ratio of lipid-lipid interactions to temperature (T) decreases, the Mod phase melts to a uniform phase. But the interactions that gave rise to the Mod phase and its periodic structure persist, albeit weakened, and the fluid is a microemulsion (3). In a regular fluid, the compositional fluctuations decay exponentially with a certain correlation length. What characterizes a microemulsion is not that each small domain of one phase is coated by a linactant, but that the short-range order now decays as a damped oscillation of compositional fluctuations, determined by a second correlation length that measures the space between neighboring domains (8). The power of the model stems from its simplicity: 1. It applies to a symmetric bilayer; 2. There are only two lipid components; and 3. Only two parameters control the phase behavior: a and τ. The parameter a represents the strength of lipid-lipid interactions relative to kT, where k is the Boltzmann constant. Negative a corresponds to ωAB > ∼ kT; the result is phase separation or Mod phases. Positive a corresponds to lack of preferential interactions between lipids (ωAB << kT); the result is a homogeneous phase. The parameter τ determines the phase structure. If the system is macroscopically homogeneous, positive τ favors microscopically uniform mixtures, whereas negative τ favors microemulsions. If the system is inhomogeneous, positive τ favors complete phase separation, whereas negative τ favors Mod phases. What is τ physically? It may represent the coupling of variations in membrane composition to thickness or the natural curvature of phospholipid molecules (9); the effect of Chol distribution on local curvature (10), which is a consequence of its location in Ld and Lo domains (11); the effect of phospholipid dipoles (12,13); or the overall membrane curvature (14), which depends on vesicle size. Recent simulations make a compelling case for the role of curvature (12): unfavorable lipid interactions (ωAB > 0) between domains (line tension) favor complete phase separation. But the curvature energy required to bend a large Lo domain into a spherical vesicle favors small domains: the energy per unit area decreases if the large domain is broken into small flat patches, generating a Mod hexagonal phase, just as a soccer ball is produced from almost flat pentagons and hexagons. Negative τ corresponds to large overall curvature. At constant temperature (Fig. 1), transitions from a uniform phase (microemulsion) to a modulated phase to complete phase separation can occur by changing the composition. For example, increasing Chol concentration weakens the mean unfavorable interactions (ωAB) because Chol favors ordered phospholipid conformations that are intermediate between disordered and all-trans. Such transitions are also induced by changing the proportions of POPC and DOPC within the unsaturated phospholipid (2). Replacing DOPC by POPC reduces the mean ωAB, because DSPC interacts more favorably with POPC than with DOPC. Indeed, it is only within a limited range of ρ(%) = DOPC/(POPC+DOPC) that Mod phases are observed. But why? There is nothing in the model of Shlomovitz et al. (3) that requires a four-component, not even a three-component system, to observe this rich phase behavior: only two components are needed. Yet (to the best of my knowledge) Mod phases have only been seen directly in four-component GUVs. That the minimal requirement is a binary system is evident from the observation of Mod phases in DPPC/Chol monolayers (13). In principle, Mod phases could be observed in ternary mixtures (DSPC/DOPC/Chol). It appears that they have not been seen because the interactions have not been tuned in a sufficiently fine manner to balance the effect of other variables such as curvature (contained in τ). Differences in lipid interactions are small, ωAB ∼ 100 cal/mol at room temperature (4), but changes in ωAB by a few tens of calories bring about large changes in lipid domain size distributions (4). The controlled replacement of some DOPC by POPC in the four-component system (2) appears to provide the fine-tuning necessary to move smoothly between the various phases available to the system.