Sarah L. Keller

Separation of liquid phases in giant vesicles of ternary mixtures of phospholipids and cholesterol.

We use fluorescence microscopy to directly observe liquid phases in giant unilamellar vesicles. We find that a long list of ternary mixtures of high melting temperature (saturated) lipids, low melting temperature (usually unsaturated) lipids, and cholesterol produce liquid domains. For one model mixture in particular, DPPC/DOPC/Chol, we have mapped phase boundaries for the full ternary system. For this mixture we observe two coexisting liquid phases over a wide range of lipid composition and temperature, with one phase rich in the unsaturated lipid and the other rich in the saturated lipid and cholesterol. We find a simple relationship between chain melting temperature and miscibility transition temperature that holds for both phosphatidylcholine and sphingomyelin lipids. We experimentally cross miscibility boundaries both by changing temperature and by the depletion of cholesterol with beta-cyclodextrin. Liquid domains in vesicles exhibit interesting behavior: they collide and coalesce, can finger into stripes, and can bulge out of the vesicle. To date, we have not observed macroscopic separation of liquid phases in only binary lipid mixtures.

Liquid Domains in Vesicles Investigated by NMR and Fluorescence Microscopy

We use (2)H-NMR, (1)H-MAS NMR, and fluorescence microscopy to detect immiscibility in three particular phospholipid ratios mixed with 30% cholesterol: 2:1 DOPC/DPPC, 1:1 DOPC/DPPC, and 1:2 DOPC/DPPC. Large-scale (>>160 nm) phase separation into liquid-ordered (L(o)) and liquid-crystalline (L(alpha)) phases is observed by both NMR and fluorescence microscopy. By fitting superimposed (2)H-NMR spectra, we quantitatively determine that the L(o) phase is strongly enriched in DPPC and moderately enriched in cholesterol. Tie-lines estimated at different temperatures and membrane compositions are based on both (2)H-NMR observations and a previously published ternary phase diagram. (2)H- and (1)H-MAS NMR techniques probe significantly smaller length scales than microscopy experiments (submicron versus micron-scalp), and complex behavior is observed near the miscibility transition. Fluorescence microscopy of giant unilamellar vesicles shows micrometer-scale domains below the miscibility transition. In contrast, NMR of multilamellar vesicles gives evidence for smaller ( approximately 80 nm) domains just below the miscibility transition, whereas large-scale demixing occurs at a lower temperature, T(low). A transition at T(low) is also evident in fluorescence microscopy measurements of the surface area fraction of ordered phase in giant unilamellar vesicles. Our results reemphasize the complex phase behavior of cholesterol-containing membranes and provide a framework for interpreting (2)H-NMR experiments in similar membranes.

Critical fluctuations in domain-forming lipid mixtures

Critical fluctuations are investigated in lipid membranes near miscibility critical points in bilayers composed of dioleoylphosphatidylcholine, chain perdeuterated dipalmitoylphosphatidylcholine, and cholesterol. Phase boundaries are mapped over the temperature range from 10°C to 60°C by deuterium NMR. Tie-lines and three-phase triangles are evaluated across two-phase and three-phase regions, respectively. In addition, a line of miscibility critical points is identified. NMR resonances are broadened in the vicinity of critical points, and broadening is attributed to increased transverse relaxation rates arising from modulation of chain order with correlation times on a microsecond time scale. We conclude that spectral broadening arises from composition fluctuations in the membrane plane with dimensions of <50 nm and speculate that similar fluctuations are commonly found in cholesterol-containing membranes.

Line Tensions, Correlation Lengths, and Critical Exponents in Lipid Membranes Near Critical Points

Membranes containing a wide variety of ternary mixtures of high chain-melting temperature lipids, low chain-melting temperature lipids, and cholesterol undergo lateral phase separation into coexisting liquid phases at a miscibility transition. When membranes are prepared from a ternary lipid mixture at a critical composition, they pass through a miscibility critical point at the transition temperature. Since the critical temperature is typically on the order of room temperature, membranes provide an unusual opportunity in which to perform a quantitative study of biophysical systems that exhibit critical phenomena in the two-dimensional Ising universality class. As a critical point is approached from either high or low temperature, the scale of fluctuations in lipid composition, set by the correlation length, diverges. In addition, as a critical point is approached from low temperature, the line tension between coexisting phases decreases to zero. Here we quantitatively evaluate the temperature dependence of line tension between liquid domains and of fluctuation correlation lengths in lipid membranes to extract a critical exponent, nu. We obtain nu = 1.2 +/- 0.2, consistent with the Ising model prediction nu = 1. We also evaluate the probability distributions of pixel intensities in fluorescence images of membranes. From the temperature dependence of these distributions above the critical temperature, we extract an independent critical exponent of beta = 0.124 +/- 0.03, which is consistent with the Ising prediction of beta = 1/8.

Tuning lipid mixtures to induce or suppress domain formation across leaflets of unsupported asymmetric bilayers

Plasma membranes of cells are asymmetric in both lipid and protein composition. The mechanism by which proteins on both sides of the membrane colocalize during signaling events is unknown but may be due to the induction of inner leaflet domains by the outer leaflet. Here we show that liquid domains form in asymmetric Montal–Mueller planar bilayers in which one leaflets composition would phase-separate in a symmetric bilayer and the others would not. Equally important, by tuning the lipid composition of the second leaflet, we are able to suppress domains in the first leaflet. When domains are present in asymmetric membranes, each leaflet contains regions of three distinct lipid compositions, implying strong interleaflet interactions. Our results show that mechanisms of domain induction between the outer and inner leaflets of cell plasma membranes do not necessarily require the participation of membrane proteins. Based on these findings, we suggest mechanisms by which cells could actively regulate protein function by modulating local lipid composition or interleaflet interactions.

An introduction to critical points for biophysicists; observations of compositional heterogeneity in lipid membranes

Scaling laws associated with critical points have the power to greatly simplify our description of complex biophysical systems. We first review basic concepts and equations associated with critical phenomena for the general reader. We then apply these concepts to the specific biophysical system of lipid membranes. We recently reported that lipid membranes can contain composition fluctuations that behave in a manner consistent with the two-dimensional Ising universality class. Near the membranes critical point, these fluctuations are micron-sized, clearly observable by fluorescence microscopy. At higher temperatures, above the critical point, we expect to find submicron fluctuations. In separate work, we have reported that plasma membranes isolated directly from cells exhibit the same Ising behavior as model membranes do. We review other models describing submicron lateral inhomogeneity in membranes, including microemulsions, nanodomains, and mean field critical fluctuations, and we describe experimental tests that may distinguish these models.

Nonequilibrium Behavior in Supported Lipid Membranes Containing Cholesterol

We investigate lateral organization of lipid domains in vesicles versus supported membranes and monolayers. The lipid mixtures used are predominantly DOPC/DPPC/Chol and DOPC/BSM/Chol, which have been previously shown to produce coexisting liquid phases in vesicles and monolayers. In a monolayer at an air-water interface, these lipids have miscibility transition pressures of approximately 12-15 mN/m, which can rise to 32 mN/m if the monolayer is exposed to air. Lipid monolayers can be transferred by Langmuir-Schäfer deposition onto either silanized glass or existing Langmuir-Blodgett supported monolayers. Micron-scale domains are present in the transferred lipids only if they were present in the original monolayer before deposition. This result is valid for transfers at 32 mN/m and also at lower pressures. Domains transferred to glass supports differ from liquid domains in vesicles because they are static, do not align in registration across leaflets, and do not reappear after temperature is cycled. Similar static domains are found for vesicles ruptured onto glass surfaces. Although supported membranes on glass capture some aspects of vesicles in equilibrium (e.g., gel-liquid transition temperatures and diffusion rates of individual lipids), the collective behavior of lipids in large liquid domains is poorly reproduced.

A closer look at the canonical 'raft mixture' in model membrane studies

Recently, Dietrich et al. (2001)xLipid rafts reconstituted in model membranes. Dietrich, C., Bagatolli, L.A., Volovyk, Z.N., Thompson, N.L., Levi, M., Jacobson, K., and Gratton, L.A. Biophys. J. 2001; 80: 1417–1428Abstract | Full Text | Full Text PDF | PubMedSee all ReferencesDietrich et al. (2001) reported lateral separation of liquid phases in model bilayer membranes of two lipid mixtures. One of their mixtures, 1:1:1 DOPC/brain sphingomyelin (BSM)/Cholesterol, has since become a canonical ‘raft mixture’ on which other groups have based their research. A recent example from this journal is a paper by Gandhavadi et al. (2002)xStructure, composition, and peptide binding properties of detergent soluble bilayers and detergent resistant rafts. Gandhavadi, M., Allende, D., Vidal, A., Simon, S.A., and McIntosh, T.J. Biophys. J. 2002; 82: 1469–1482Abstract | Full Text | Full Text PDF | PubMedSee all ReferencesGandhavadi et al. (2002). One result presented by the authors is that lateral separation into two liquid phases could not be observed in bilayers of this mixture using x-ray diffraction techniques. The goal of this letter is to provide a possible explanation for this discrepancy between the two papers.In recent work from our laboratory, we have shown that lateral separation into liquid phases in bilayer membranes is not limited to the small set of previously published model mixtures, but in fact can be attained over a large composition and temperature range (Veatch and Keller, 2002xSee all ReferencesVeatch and Keller, 2002). In experiments using ternary lipid mixtures of equimolar saturated and unsaturated phosphatidylcholines mixed with cholesterol, coexistence of two liquid phases is observed in giant unilamillar vesicles (GUVs) with cholesterol compositions between 10–50 mol%. Domains are seen by fluorescence microscopy using 0.8 mol% Texas Red di(16:0)PE as a dye which partitions into the cholesterol-poor phase (Veatch and Keller, 2002xSee all ReferencesVeatch and Keller, 2002). At high temperatures all lipids are in one uniform liquid phase so that no domains are observed. As temperature decreases through the miscibility transition, lipid domains form spontaneously.A typical phase diagram of the 1:1 DOPC/BSM + cholesterol mixture is shown in Fig. 1 aFig. 1 a. At low cholesterol concentrations (∼10–25 mol%), miscibility transition temperatures are highest and do not vary greatly with cholesterol composition. At higher cholesterol, (∼30–35 mol% cholesterol) the miscibility transition temperature decreases sharply until a composition is reached (>35–40 mol%) at which domains are no longer observed in vesicles over accessible temperatures. Phase diagrams for similar ternary lipid mixtures (including sphingomyelin) follow this general trend with minor differences (Veatch and Keller, 2002xSee all ReferencesVeatch and Keller, 2002).Figure 1(a) Simplified sketch of anticipated miscibility phase diagram for mixtures of 1:1 DOPC/BSM with varying cholesterol composition. The location of the popular mixture of 1:1:1 DOPC/BSM/cholesterol at room temperature (RT) is denoted by a gray circle. (b, c) GUVs of 1:1:1 DOPC/BSM/cholesterol at 25°C (b) and 20°C (c). The same field of view is shown in both frames. In both cases, laterally separated liquid domains are observed in some but not all vesicles. Vesicles with domains are shown with arrows. At 20°C, more vesicles exhibit coexisting domains than at 25°C. (d) All vesicles made with 1:1 DOPC/BSM and 20% cholesterol exhibit phase separation at 25°C. All experiments use the fluorescent dye Texas Red DPPE which preferentially partitions into the cholesterol-poor phase.View Large Image | View Hi-Res Image | Download PowerPoint SlideConsidering the general phase behavior of these types of mixtures, there are three major reasons why the canonical 1:1:1 DOPC/BSM/Cholesterol ‘raft mixture’ can be problematic. First, this mixture is poised close to the high cholesterol edge of the miscibility transition. Since this edge is steep, slight changes in lipid composition at a given laboratory temperature will change the phase behavior. The second problem arises because different experimental methods produce bilayers with slightly different distributions of compositions. Again, since the high cholesterol edge of the miscibility transition is steep, this slight variation in composition will result in a large variation in observed phase behavior between experiments. Third, since miscibility transition temperatures are generally lower at higher cholesterol composition, it is likely that the transition in the 1:1:1 mixture will occur near or below room temperature. Hence, coexisting liquid domains will not necessarily be observed under normal experimental conditions. (In Dietrich et al. (2001)xLipid rafts reconstituted in model membranes. Dietrich, C., Bagatolli, L.A., Volovyk, Z.N., Thompson, N.L., Levi, M., Jacobson, K., and Gratton, L.A. Biophys. J. 2001; 80: 1417–1428Abstract | Full Text | Full Text PDF | PubMedSee all ReferencesDietrich et al. (2001), 1% GM1 was included in their 1:1:1 vesicles.) Combining all of these trends, it is clear that small changes in experimental conditions lead to large changes in the observed phase behavior for this particular mixture.To illustrate this point, and to support the use of Fig. 1 aFig. 1 a, we provide fluorescence micrographs of 1:1:1 DOPC/BSM/cholesterol GUVs at 25°C in Fig. 1 bFig. 1 b and at 20°C in Fig. 1 cFig. 1 c. The same field of view is shown in both frames. In both cases, lateral separation of domains is observed in some but not all vesicles. Vesicles with domains are shown with arrows. Coexisting domains on the surface of a vesicle are clearly distinguished from small vesicles in the interior. First, when the focus is adjusted to be midway through a giant vesicle, surface domains are no longer seen although interior vesicles are. Second, domains on the surface disappear as temperature is raised and interior vesicles remain. In Fig. 1 bFig. 1 b at 25°C many vesicles are in one uniform phase (above the transition) whereas in Fig. 1 cFig. 1 c at 20°C the majority exhibit coexisting liquid phases on their surfaces (below the transition). The range in transition temperatures is due to the slight variation in composition between vesicles. For our method of making GUVs (Angelova et al., 1992xPreparation of giant vesicles by external AC electric fields. Kinetics and applications. Angelova, M.I., Soleau, S., Meleard, P., Faucon, J.F., and Bothorel, P. Progr. Colloid Polym. Sci. 1992; 89: 127–131CrossrefSee all ReferencesAngelova et al., 1992), we estimate a variation of ±2 mol% cholesterol between individual vesicles. For this reason, we always examine large populations of vesicles.The observations above confirm that, indeed, at room temperature this 1:1:1 DOPC/BSM/cholesterol mixture is close to the miscibility transition temperature and that small changes in vesicle composition have a large effect on the observed phase behavior. In contrast, all vesicles made from 1:1 DOPC/BSM with 20% cholesterol exhibit two liquid phases at 25°C (Fig. 1 dFig. 1 d) indicating that this mixture may be a better choice for most experiments. In addition, the cholesterol-poor phase makes up the majority of these membranes. This may more closely resemble biological membranes in which cholesterol rich ‘rafts’ are islands in a background of liquid disordered phase.These results indicate that it is important to consider the lipid composition and corresponding miscibility transition temperature of ‘raft forming mixtures’ used in experiments. This lesson is especially important at this time when many groups are working to characterize and better understand the mechanism and physical nature of these laterally phase separated systems.

On the Binding Preference of Human Groups IIA and X Phospholipases A2 for Membranes with Anionic Phospholipids

Mammals contain 9–10 secreted phospholipases A2 (sPLA2s) that display widely different affinities for membranes, depending on the phospholipid composition. The much higher enzymatic activity of human group X sPLA2(hGX) compared with human group IIA sPLA2 (hGIIA) on phosphatidylcholine (PC)-rich vesicles is due in large part to the higher affinity of the former enzyme for such vesicles; this result also holds when vesicles contain cholesterol and sphingomyelin. The inclusion of anionic phosphatidylserine in PC vesicles dramatically enhances interfacial binding and catalysis of hGIIA but not of hGX. This is the result of the large number of lysine and arginine residues scattered over the entire surface of hGIIA, which cause the enzyme to form a supramolecular aggregate with multiple vesicles. Thus, high affinity binding of hGIIA to anionic vesicles is a complex process and cannot be attributed to a few basic residues on its interfacial binding surface, as is also evident from mutagenesis studies. The main reason hGIIA binds poorly to PC-rich vesicles is that it lacks a tryptophan residue on its interfacial binding surface, a residue that contributes to the high affinity binding of hGX to PC-rich vesicles. Results show that the lag in the onset of hydrolysis of PC vesicles by hGIIA is due in part to the poor affinity of this enzyme for these vesicles. Binding affinity of hGIIA, hGX, and their mutants to PC-rich vesicles is well correlated to the ability of these enzymes to act on the PC-rich outer plasma membrane of mammalian cells.

Small concentrations of alamethicin induce a cubic phase in bulk phosphatidylethanolamine mixtures.

Under normal conditions, excess water dispersions of liquid crystalline 1,2-dielaidoyl-sn-glycero-3- phosphoethanolamine (DEPE) are known to convert from a liquid crystalline lamellar (L alpha) to inverse hexagonal (HII) phase at about 60 degrees Celsius. The nonlamellar phase behavior of lipid systems is also known to depend on the monolayer spontaneous curvature. The single-channel activity of alamethicin in black lipid bilayer membranes has been shown to be dependent upon the lipid composition of the membrane. Since the monolayer spontaneous curvature properties (e.g., the monolayer spontaneous curvature, curvature coefficients and bilayer thickness) vary with lipid composition, the single-channel activity of alamethicin presumably also correlates with the monolayer spontaneous curvature properties. Accordingly, we reasoned that if alamethicin couples to the curvature properties of a lipid film, then the curvature properties must, in turn, be perturbed by the presence of alamethicin and that this perturbation should be observable in the lipid phase behavior. Here X-ray diffraction and NMR are used to show that the presence of as little as 1% alamethicin introduces a large region of cubic phase into the thermal phase diagram. This suggests that perturbation of the nonlamellar phase behavior of a lipid system may be a method to survey different channel-forming molecules for possible behavior that indicates that the ion channel is sensitive to the monolayer spontaneous curvature properties.