Sarah L. Veatch

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.

Probing protein heterogeneity in the plasma membrane using PALM and pair correlation analysis

Photoactivated localization microscopy (PALM) is a powerful approach for investigating protein organization, yet tools for quantitative, spatial analysis of PALM datasets are largely missing. Combining pair-correlation analysis with PALM (PC-PALM), we provide a method to analyze complex patterns of protein organization across the plasma membrane without determination of absolute protein numbers. The approach uses an algorithm to distinguish a single protein with multiple appearances from clusters of proteins. This enables quantification of different parameters of spatial organization, including the presence of protein clusters, their size, density and abundance in the plasma membrane. Using this method, we demonstrate distinct nanoscale organization of plasma-membrane proteins with different membrane anchoring and lipid partitioning characteristics in COS-7 cells, and show dramatic changes in glycosylphosphatidylinositol (GPI)-anchored protein arrangement under varying perturbations. PC-PALM is thus an effective tool with broad applicability for analysis of protein heterogeneity and function, adaptable to other single-molecule strategies.

Critical Fluctuations in Plasma Membrane Vesicles

We demonstrate critical behavior in giant plasma membrane vesicles (GPMVs) that are isolated directly from living cells. GPMVs contain two liquid phases at low temperatures and one liquid phase at high temperatures and exhibit transition temperatures in the range of 15 to 25 degrees C. In the two-phase region, line tensions linearly approach zero as temperature is increased to the transition. In the one-phase region, micrometer-scale composition fluctuations occur and become increasingly large and long-lived as temperature is decreased to the transition. These results indicate proximity to a critical point and are quantitatively consistent with established theory. Our observations of robust critical fluctuations suggest that the compositions of mammalian plasma membranes are tuned to reside near a miscibility critical point and that heterogeneity corresponding to < 50 nm-sized compositional fluctuations are present in GPMV membranes at physiological temperatures. Our results provide new insights for plasma membrane heterogeneity that may be related to functional lipid raft domains in live cells.

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.

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.

Correlation functions quantify super-resolution images and estimate apparent clustering due to over-counting.

We present an analytical method using correlation functions to quantify clustering in super-resolution fluorescence localization images and electron microscopy images of static surfaces in two dimensions. We use this method to quantify how over-counting of labeled molecules contributes to apparent self-clustering and to calculate the effective lateral resolution of an image. This treatment applies to distributions of proteins and lipids in cell membranes, where there is significant interest in using electron microscopy and super-resolution fluorescence localization techniques to probe membrane heterogeneity. When images are quantified using pair auto-correlation functions, the magnitude of apparent clustering arising from over-counting varies inversely with the surface density of labeled molecules and does not depend on the number of times an average molecule is counted. In contrast, we demonstrate that over-counting does not give rise to apparent co-clustering in double label experiments when pair cross-correlation functions are measured. We apply our analytical method to quantify the distribution of the IgE receptor (FcεRI) on the plasma membranes of chemically fixed RBL-2H3 mast cells from images acquired using stochastic optical reconstruction microscopy (STORM/dSTORM) and scanning electron microscopy (SEM). We find that apparent clustering of FcεRI-bound IgE is dominated by over-counting labels on individual complexes when IgE is directly conjugated to organic fluorophores. We verify this observation by measuring pair cross-correlation functions between two distinguishably labeled pools of IgE-FcεRI on the cell surface using both imaging methods. After correcting for over-counting, we observe weak but significant self-clustering of IgE-FcεRI in fluorescence localization measurements, and no residual self-clustering as detected with SEM. We also apply this method to quantify IgE-FcεRI redistribution after deliberate clustering by crosslinking with two distinct trivalent ligands of defined architectures, and we evaluate contributions from both over-counting of labels and redistribution of proteins.

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.