Fluorescence studies of dehydroergosterol in phosphatidylethanolamine/phosphatidylcholine bilayers

Our previous fluorescence study has provided indirect evidence that lipid headgroup components tend to adopt regular, superlattice-like lateral distribution in fluid phosphatidylethanolamine/phosphatidylcholine (PE/PC) bilayers (, Biophys. J. 73:1967-1976). Here we have further studied this intriguing phenomenon by making use of the fluorescence properties of a sterol probe, dehydroergosterol (DHE). Fluorescence emission spectra, fluorescence anisotropy (r), and time-resolved fluorescence intensity decays of DHE in 1-palmitoyl-2-oleoyl-PC (POPC)/1-palmitoyl-2-oleoyl-PE (POPE) mixtures were measured as a function of POPE mole fraction (X(PE)) at 23 degrees C. Deviations, including dips or kinks, in the ratio of fluorescence peak intensity at 375 nm/fluorescence peak intensity at 390 nm (I(375)/I(390)), fluorescence decay lifetime (tau), or rotational correlation time (rho) of DHE versus PE composition plots were found at X(PE) approximately 0.10, 0.25, 0.33, 0.65, 0.75, and 0.88. The critical values at X(PE) approximately 0.33 and 0.65 were consistently observed for all measured parameters. In addition, the locations, but not the depth, of the dips for X(PE) < 0.50 did not vary significantly over 10 days of annealing at 23 degrees C. The observed critical values of X(PE) coincide (within +/-0.03) with some of the critical mole fractions predicted by a headgroup superlattice model proposing that the PE and PC headgroups tend to be regularly distributed in the plane of the bilayer. These results agree favorably with those obtained in our previous fluorescence study using dipyrenylPC and Laurdan probes and thus support the proposition that 1) regular arrangement within a domain exists in fluid PE/PC bilayers, and 2) superlattice formation may play a significant role in controlling the lipid composition of cellular membranes (, Proc. Natl. Acad. Sci. USA. 95:4964-4969). The present data provide new information on the physical properties of such superlattice domains, i.e., the dielectric environment and rotational motion of membrane sterols appear to change abruptly as the lipid headgroups exhibit regular superlattice-like distributions in fluid bilayers.     

Permeability of phosphatidylcholine and phosphatidylethanolamine bilayers

Sodium and glucose effluxes were measured in liposomes formed from a series of saturated phosphatidylcholines (PC) and phosphatidylethanolamines (PE). Vesicles composed of a saturated PC display a local permeability maximum in the region of the lipid transition temperature. The height of this maximum is predominantly a function of the thickness of the hydrocarbon chain region. Liposomes formed from a saturated PE do not display such a permeability maximum and in these vesicles the permeability process appears to be controlled by the head group region. It is postulated that the control exerted by the ethanolamine group is due to the reorganization of water structure it induces at the bilayer surface.     

The intrinsic pKa values for phosphatidylserine and phosphatidylethanolamine in phosphatidylcholine host bilayers.

Potentiometric titrations and surface potential measurements have been used to determine the intrinsic pKa values of both the carboxyl and amino groups of phosphatidylserine (PS) in mixed vesicles of PS and phosphatidylcholine (PC), and also of the amino group of phosphatidylethanolamine (PE) in mixed PE-PC vesicles. The pKa of the carboxyl group of PS in liposomes with different PS/PC lipid ratios measured by the two different methods is 3.6 +/- 0.1, and the pKa of its amino group is 9.8 +/- 0.1. The pKa of the amino group of PE in PE-PC vesicles, determined solely by surface potential measurements, is 9.6 +/- 0.1. These pKa values are independent of the aqueous phase ionic strength and of the effect of the liposome's surface potential due to the presence of these partially charged lipids.     

Maximum solubility of cholesterol in phosphatidylcholine and phosphatidylethanolamine bilayers

In any lipid bilayer membrane, there is an upper limit on the cholesterol concentration that can be accommodated within the bilayer structure; excess cholesterol will precipitate as crystals of pure cholesterol monohydrate. This cholesterol solubility limit is a well-defined quantity. It is a first-order phase boundary in the phospholipid/cholesterol phase diagram. There are many different solubility limits in the literature, but no clear picture has emerged that can unify the disparate results. We have studied the effects that different sample preparation methods can have on the apparent experimental solubility limit. We find that artifactual demixing of cholesterol can occur during conventional sample preparation and that this demixed cholesterol may produce artifactual cholesterol crystals. Therefore, phospholipid/cholesterol suspensions which are prepared by conventional methods may manifest variable, falsely low cholesterol solubility limits. We have developed two novel preparative methods which are specifically designed to prevent demixing during sample preparation. For detection of the cholesterol crystals, X-ray diffraction has proven to be quantitative and highly sensitive. Experiments based on these methods yield reproducible and precise cholesterol solubility limits: 66 mol% for phosphatidylcholine (PC) bilayers and 51 mol% for phosphatidylethanolamine (PE) bilayers. We present evidence that these are true, equilibrium values. In contrast to the dramatic headgroup effect (PC vs. PE), acyl chain variations had no effect on the cholesterol solubility limit in four different PC/cholesterol mixtures.     

Differences in hydrocarbon chain tilt between hydrated phosphatidylethanolamine and phosphatidylcholine bilayers. A molecular packing model.

Both wide-angle and lamellar x-ray diffraction data are interpreted in terms of a difference in hydrocarbon chain tilt between fully hydrated dipalmitoyl phosphatidylcholine (DPPC) and dipalmitoyl phosphatidylethanolamine (DPPE). Although the hydrocarbon chains of multilayers of DPPC tilt ty approximately 30 degrees relative to the normal to the plane of the bilayer, as previously reported by others, the hydrocarbon chains of DPPE appear to be oriented approximately normal to the plane of the bilayer. It is found that the chain tilt in DPPC bilayers can be reduced by either: (a) adding an n-alkane to the bilayer interiors or (b) adding lanthanum ions to the fluid layers between bilayers. A molecular packing model is presented which accounts for these data. According to this model, DPPC chains tilt because of the size and conformation of the PC polar head group.     

Lateral interactions among phosphatidylcholine and phosphatidylethanolamine head groups in phospholipid monolayers and bilayers

We develop theory for the lateral interactions among the zwitterionic head groups of phospholipids in monolayers and bilayers, particularly phosphatidylcholine (PC) and phosphatidylethanolamine (PE). With the P- end of the head group anchored at the water/hydrocarbon interface, a balance of two effects dictates the angle that the P--N+ dipole makes with respect to the plane of the bilayer: N+ is driven toward water due to the (Born) electrostatic free energy, but the hydrophobic effect drives the methyl and methylene groups around the N+ charge toward the hydrocarbon. The only adjustable parameter of the model is the average fluctuation of the oil/water interface or, alternatively, the dielectric constant of the hydrocarbon phase. The model predicts that at 5 degrees C the head group dipole should lie largely in the bilayer plane, in accord with X-ray, neutron diffraction, and NMR studies. The theory makes the novel prediction that the N+ end of the dipole becomes increasingly submerged in hydrocarbon with increasing temperature, leading to strongly enhanced lateral repulsion between PC head groups. This prediction is in good agreement with second and third viral coefficients of monolayer lateral pressures, and with the temperature dependence of the former. The theoretical model is consistent with head group fluctuations measured by neutron diffraction of PC and PE bilayers. Because PE has a smaller hydrophobic cluster near N+, its lateral repulsion should be much smaller and less temperature dependent than for PC, also in agreement with equation-of-state measurements. This suggests why at high density PE monolayers have higher melting temperatures than PC monolayers and more propensity for reversed curvature.     

Direct measurements of forces between phosphatidylcholine and phosphatidylethanolamine bilayers in aqueous electrolyte solutions

We report direct measurements of the full interbilayer force laws (force vs. distance) between bilayers of various phosphatidylcholines and phosphatidylethanolamine in aqueous solutions. Bilayers were first deposited on molecularly smooth (mica) surfaces and the interbilayer forces then measured at a resolution of 1 A. Three types of forces were identified: attractive van der Waals forces, repulsive electrostatic (double-layer) forces, and (at short range) repulsive steric hydration forces. Double-layer forces, which arise from ion binding, were insignificant in monovalent salt solutions, e.g., NaCl up to 1 M, but were already present in solutions containing millimolar levels of CaCl2 and MgCl2, giving rise to forces in excellent agreement with theory. Ca2+ binds more strongly than Mg2+, and both bind less to lecithin bilayers in the fluid state (T greater than Tc). The plane of charge coincides with the location of the negative phosphate groups, while the effective plane of origin of the van der Waals force is 4-5 A farther out. In water, the adhesion energies are in the range 0.10-0.15 erg/cm2 for lecithins and approximately 0.8 erg/cm2 for phosphatidylethanolamine. The adhesion energies vary on addition of salt due to changes in the repulsive double-layer and hydration forces rather than to a change in the attractive van der Waals force. The short-range repulsive forces which balance the van der Waals force at separations of 10-30 A are due to a combination of hydration and steric repulsions, the latter arising from thermal motions of head groups and thickness fluctuations of fluid bilayers (above Tc). It is also concluded that bilayer fusion is not simply related to the interbilayer force law.     

Influence of cholesterol on the polar region of phosphatidylcholine and phosphatidylethanolamine bilayers

The structural changes in the polar head group region of unsonicated bilayer membranes of 1,2-dipalmitoyl-sn-glycero-3-phosphocholine and 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine produced by addition of cholesterol have been determined using deuterium and phosphorus-31 NMR. Incorportion of up to 50 mol percent cholesterol produces little change in the phosphorus-31 chemical shielding anisotropies, compared with the values in pure bilayers above the phase transition temperatures, while some of the deuterium quadrupole splittings are reduced by almost a factor of two. Adjustment of the head group torsion angles by only a few degrees accounts for the observed spectral changes. Addition of cholesterol therefore has opposite effects on the hydrocarbon and polar regions of membranes: although cholesterol makes the hydrocarbon region gel-like, with an increased probability of trans conformations, the conformation of the polar head groups is very similar to that found in the liquid crystalline phase of pure phospholipid bilayers.     

Free volume model for lipid lateral diffusion coefficients. Assessment of the temperature dependence in phosphatidylcholine and phosphatidylethanolamine bilayers

The lipid lateral diffusion coefficients, DT, in fluid-phase phosphatidylcholine and phosphatidylethanolamine bilayers have been analysed in terms of the free-volume diffusion model by fitting the expression: DT = AT exp[- B/(T - T0)] to the observed temperature dependence, where A, B and T0 are the parameters to be optimized. Application of an unconstrained optimization procedure to data obtained from excimer formation (Galla et al. (1979) J. Membrane Biol. 48, 215-236) and from fluorescence photobleaching (Vaz et al. (1985) Biochemistry 24, 781-786) provides statistical evidence for a free-volume model as opposed to a simple Stokes-Einstein model (T0 = 0), only in certain cases. In the instances for which the parameter T0 can be determined with a reasonable degree of accuracy, it is found that this characteristic temperature at which the free volume extrapolates to zero lies below the bilayer gel-to-fluid phase transition temperature and does not coincide with the pre-transition temperature for phosphatidylcholines.     

Effects of diacylglycerols on conformation of phosphatidylcholine headgroups in phosphatidylcholine/phosphatidylserine bilayers.

The effects of five diacylglycerols (DAGs), diolein, 1-stearoyl,2-arachidonoyl-sn-glycerol, dioctanoylglycerol, 1-oleoyl,2-sn-acetylglycerol, and dipalmitin (DP), on the structure of lipid bilayers composed of mixtures of phosphatidylcholine and phosphatidylserine (4:1 mol/mol) were examined by 2H nuclear magnetic resonance (NMR). Dipalmitoylphosphatidylcholine deuterated at the alpha- and beta-positions of the choline moiety was used to probe the surface region of the membranes. Addition of each DAG except DP caused a continuous decrease in the beta-deuteron quadrupole splittings and a concomitant increase in the alpha-deuteron splittings indicating that DAGs induce a conformational change in the phosphatidylcholine headgroup. Additional evidence of conformational change was found at high DAG concentrations (> or = 20 mol%) where the alpha-deuteron peaks became doublets indicating that the two alpha-deuterons were not equivalent. The changes induced by DP were consistent with the lateral phase separation of the bilayers into gel-like and fluid-like domains with the phosphatidylcholine headgroups in the latter phase being virtually unaffected by DP. The DAG-induced changes in alpha-deuteron splittings were found to correlate with DAG-enhanced protein kinase C (PK-C) activity, suggesting that the DAG-induced conformational changes of the phosphatidylcholine headgroups are either directly or indirectly related to a mechanism of PK-C activation. 2H NMR relaxation measurements showed significant increase of the spin-lattice relaxation times for the region of the phosphatidylcholine headgroups, induced by all DAGs except DP. However, this effect of DAGs did not correlate with the DAG-induced activation of PK-C.     

Structural differences among phosphatidylcholine, phosphatidylethanolamine, and mixed phosphatidylcholine/phosphatidylethanolamine multilayers: an infrared absorption study

We have examined the infrared absorption spectra from 4000 to 250 cm^?1 of multilayers of phosphatidylcholine, phosphatidylethanolamine, and phosphatidylcholine/phosphatidylethanolamine (1:1 m/m) as a function of hydration, pH, and fatty acid composition. Characteristic splittings of the CH₂ bending and rocking modes and the position of the phosphoryl absorption at ca. 1240 cm^?1 reveal differences in acyl chain packing and head group conformation in the various films. Spectra demonstrate the importance of NH → O hydrogen bonding of the ethanolamine head group and the prerequisite head group conformation (tangent to the multilayer plane) in establishing these structural differences. The general appearance of the P-O-C stretching region (~1050 cm^?1) in the pure and mixed films further supports these conclusions and shows that the spectra clearly distinguish among the different head group orientations. Self-association of phosphatidylethanolamine is sometimes sufficient to prevent formation of mixed phases with phosphatidylcholine at neutral pH. The amount of fine structure, particularly in the low-frequency (800?200 cm^?1) region, in spectra of films of anhydrous, saturated-chain phospholipids decreases considerably when the films are monohydrated, when mixed phases exist, or when there are unsaturations in the acyl chains. These changes likely result from decreased crystal field effects in the spectra as the phosphatide packing density is decreased by any of the above procedures. Furthermore, the absence of other changes upon complete hydration of phosphatidylcholine films suggests that only the initial water is tightly bound to the lipid.     

Growth rate of myelin figures for phosphatidylcholine and phosphatidylethanolamine.

The growth length of myelin figures (or myelin tubes) was measured for several kinds of phospholipids using optical microscopy. The measurements were done for myelin figures with various thickness of tube wall. In spite of remarkable differences in morphology between the myelin figures of phosphatidylcholine and those of phosphatidylethanolamine, the growth rates for both were adapted to the expression proposed previously. The initial rate and the damping factor of the growth were inversely proportional to the wall thickness of myelin tubes.     

Solid- and liquid-phase equilibria in phosphatidylcholine/phosphatidylethanolamine mixtures. A calorimetric study

Phase diagrams have been determined for mixing of binary mixtures of phosphatidylethanolamines (PE) with phosphatidylcholines (PC), using high-sensitivity differential scanning calorimetry and allowing extensive incubation times to equilibrate samples in the solid phase. All of the PE-PC systems examined, which contained saturated or trans-unsaturated PC components, showed limited solid-phase miscibility, chiefly because the PC component can adopt more solid phases than the PE component. For the dielaidoyl PE-PC system, the lamellar-to-hexagonal II transition endotherm seen at 63.5 degrees C for the pure PE is shifted to considerably higher temperatures upon incorporation of even low mole fractions of PC. All of the PE-PC systems examined here reveal a complete miscibility in the liquid phase, including the dipalmitoyl PE-dielaidoyl PC system for which limited liquid-phase miscibility had previously been suggested (Wu, S-H. and McConnell, H.M. (1975) Biochemistry 14, 847-854). However, PE-PC mixing appears to be less nearly ideal than the mixing of either PE or PC with anionic phospholipids. Our results demonstrate that calorimetry can be useful in determining accurate phase diagrams for lipid mixtures of this type, but only if proper attention is given to the existence and the proper equilibration of multiple solid phases in these systems.     

Solid- and liquid-phase equilibria in phosphatidylcholine / phosphatidylethanolamine mixtures. A calorimetric study

Phase diagrams have been determined for mixing of binary mixtures of phosphatidylethanolamines (PE) with phosphatidylcholines (PC), using high-sensitivity differential scanning calorimetry and allowing extensive incubation times to equilibrate samples in the solid phase. All of the PE-PC systems examined, which contained saturated or trans-unsaturated PC components, showed limited solid-phase miscibility, chiefly because the PC component can adopt more solid phases than the PE component. For the dielaidoyl PE-PC system, the lamellar-to-hexagonal II transition endotherm seen at 63.5°C for the pure PE is shifted to considerably higher temperatures upon incorporation of even low mode fractions of PC. All of the PE-PC systems examined here reveal a complete miscibility in the liquid phase, including the dipalmitoyl PE-dielaidoyl PC system for which limited liquid-phase miscibility had previously been suggested (Wu, S-H. and McConnell, H.M. (1975) Biochemistry 14, 847–854). However, PE-PC mixing appears to be less nearly ideal than the mixing of either PE or PC with anionic phospholipids. Our results demonstrate that calorimetry can be useful in determining accurate phase diagrams for lipid mixtures of this type, but only if proper attention is given to the existence and the proper equilibration of multiple solid phases in these systems.     

Landau theory of two-component phospholipid bilayers. I. Phosphatidylcholine/phosphatidylethanolamine mixtures

Priest's phenomenological model (Mol. Cryst. Liq. Cryst. 60 (1980) 167.) on one- and two-component PC bilayers is extended here. We constructed a new excess free energy term in the state function to describe the thermodynamic properties of the two-component phospholipid bilayers where the chain lengths and the polar heads of the components can be different simultaneously. By means of this generalized state function, we can calculate the phase diagrams of DPPC/DPPE, DMPC/DMPE, DMPC/DPPE, DPPC/DMPE and DSPC/DMPE mixtures. We obtained complete miscibility both in the liquid crystalline and in the gel phase if the chain lengths of the components were the same. If the chain length of the PE component was longer than that of the PC component, we obtained a peritectic system. A eutectic system was obtained in the reverse case. The results of the model were compared with the experimental data available. Applying the quasichemical approximation, we determined the molecular meaning of the phenomenological model parameters. Namely, sigma and gamma are proportional to the sublimation heat of the CH2 group in the long-chain alkanes and to the hydrogen-bonding energy between the polar heads of the ethanolamines; otherwise the model resulted in--1.94 kcal/mol per CH2 for the sublimation heat and --1.4 kcal/mol for the hydrogen-bond energy.     

31P and 2H NMR studies of structure and motion in bilayers of phosphatidylcholine and phosphatidylethanolamine

The structural and motional properties of mixed bilayers of phosphatidylcholine (PC) and phosphatidylethanolamine (PE) have been examined by using wide-line 31P, 14N, and 2H NMR. 2H and 14N NMR data showed that in mixed bilayers containing both PC and PE the conformations of the head-group moieties are essentially identical with those observed for bilayers containing a single phospholipid species. Equimolar amounts of cholesterol induce also only a small change in head-group conformation. 31P T1 relaxation measurements (at 300 MHz) at various temperatures of bilayers containing phospholipids with a mixture of phosphocholine and phosphoethanolamine head-groups and unsaturated fatty acid residues revealed in all cases a clearly defined minimum corresponding to the condition omega O tau C-1 approximately 1. For all phospholipid mixtures studied, the 31P T1 relaxation was homogeneous over the whole powder spectrum and could be fitted to a single-exponential decay. The 31P vs temperature profiles were analyzed by a simple correlation model following the analysis of Seelig et al. (1981) [Seelig, J., Tamm, L., Hymel, L., & Fleischer, S. (1981) Biochemistry 20, 3922-3932]. Rotational diffusion of the phosphate moiety in bilayers of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) was slower than that of 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), and the activation energy was increased by a factor of 1.7 to 31.4 kJ mol-1.(ABSTRACT TRUNCATED AT 250 WORDS)     

Atomic force microscopy of hydrated phosphatidylethanolamine bilayers.

We present images of the polar or headgroup regions of bilayers of dimyristoyl-phosphatidylethanolamine (DMPE), deposited by Langmuir-Blodgett deposition onto mica substrates at high surface pressures and imaged under water at room temperature with the optical lever atomic force microscope. The lattice structure of DMPE is visualized with sufficient resolution that the location of individual headgroups can be determined. The forces are sufficiently small that the same area can be repeatedly imaged with a minimum of damage. The DMPE molecules in the bilayer appear to have relatively good long-range orientational order, but rather short-range and poor positional order. These results are in good agreement with x-ray measurements of unsupported lipid monolayers on the water surface, and with electron diffraction of adsorbed monolayers.     

Nonelectrolyte substitution for water in phosphatidylcholine bilayers

Glycerol substitutes for water in multilamellar phosphatidylcholine liposomes in that the fluid spaces between bilayers, as well as their main transition temperatures, heat capacities, and ethalpies are very similar in water and in pure glycerol. One major difference is that the gel state phase of dipalmitoylphosphatidylcholine (DPPC) in glycerol consists of bilayers with fully interdigitated hydrocarbon chains. Interdigitated DPPC phases are also formed in ethylene glycol or in methanol (at low methanol content). In solutions of glycerol and water, the fluid spacing between bilayers is a function of mole fraction of glycerol X_g, reaching maximum values at $\text{X}{}_{\mathrm{<mtext>g</mtext>}}\text{≌ 0.1}$ for lipid in the liquid crystalline phase and at $\text{X}{}_{\mathrm{<mtext>g</mtext>}}\text{≌ 0.3}$ for the gel phase. These changes are explained in terms of a modification of the long-range Van der Waals attractive forces by glycerol.     

Polylysine-induced 2H NMR-observable domains in phosphatidylserine/phosphatidylcholine lipid bilayers.

The interaction of three polylysines, Lys(5) (N = 5), Lys(30) (N = 30), and Lys(100) (N = 100), where N is the number of lysine residues per chain, with phosphatidylserine-containing lipid bilayer membranes was investigated using 2H NMR spectroscopy. Lys(30) and Lys(100) added to multilamellar vesicles composed of (70:30) (mol:mol) mixtures of choline-deuterated 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) + 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphoserine (POPS) produced two resolvable 2H NMR spectral components under conditions of low ionic strength and for cases where the global anionic lipid charge was in excess over the global cationic polypeptide charge. The intensities and quadrupolar splittings of the two spectral components were consistent with the existence of polylysine-bound domains enriched in POPS, in coexistence with polylysine-free domains depleted in POPS. Lys(5), however, yielded no 2H NMR resolvable domains. Increasing ionic strength caused domains to become diffuse and eventually dissipate entirely. At physiological salt concentrations, only Lys(100) yielded 2H NMR-resolvable domains. Therefore, under physiological conditions of ionic strength, pH, and anionic lipid bilayer content, and in the absence of other, e.g., hydrophobic, contributions to the binding free energy, the minimum number of lysine residues sufficient to produce spectroscopically resolvable POPS-enriched domains on the 2H NMR millisecond timescale may be fewer than 100, but is certainly greater than 30.     

Atomic force microscopy studies of ganglioside GM1 domains in phosphatidylcholine and phosphatidylcholine/cholesterol bilayers.

The distribution of ganglioside in supported lipid bilayers has been studied by atomic force microscopy. Hybrid dipalmitoylphosphatidylcholine (DPPC)/dipalmitoylphosphatidylethanolamine (DPPE) and (2:1 DPPC/cholesterol)/DPPE bilayers were prepared using the Langmuir Blodgett technique. Egg PC and DPPC bilayers were prepared by vesicle fusion. Addition of ganglioside GM1 to each of the lipid bilayers resulted in the formation of heterogeneous surfaces that had numerous small raised domains (30--200 nm in diameter). Incubation of these bilayers with cholera toxin B subunit resulted in the detection of small protein aggregates, indicating specific binding of the protein to the GM1-rich microdomains. Similar results were obtained for DPPC, DPPC/cholesterol, and egg PC, demonstrating that the overall bilayer morphology was not dependent on the method of bilayer preparation or the fluidity of the lipid mixture. However, bilayers produced by vesicle fusion provided evidence for asymmetrically distributed GM1 domains that probably reflect the presence of ganglioside in both inner and outer monolayers of the initial vesicle. The results are discussed in relation to recent inconsistencies in the estimation of sizes of lipid rafts in model and natural membranes. It is hypothesized that small ganglioside-rich microdomains may exist within larger ordered domains in both natural and model membranes.