Conformational state diagram of DOPC/DPPCd62/cholesterol mixtures

Raman spectra of aqueous suspensions of vesicles composed of 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), deuterated 1,2-dipalmitoyl-d62-sn-glycero-3-phosphocholine (DPPC_d62), and cholesterol (Chol) were studied at room temperature to determine the conformational states of the phospholipid hydrocarbon chains. Deuteration of DPPC_d62 allowed us to characterize the conformational states of DOPC and DPPC_d62 independently. The parameters of Raman peaks, which are sensitive to the conformational order, were studied in a wide range of compositions. It was found that the DOPC molecules are conformationally disordered for all compositions. The conformational state of the DPPC_d62 molecules changes with composition. Their conformational state is influenced by cholesterol-induced partial disordering and DOPC solvation, transforming the DPPC molecules into the disordered state. The conformational state diagram from the Raman experiment was compared with outcomes from the differential scanning calorimetry (DSC) experiment. The Raman spectra also revealed that the DPPC molecules coexist in the disordered and all-trans ordered states for the DOPC/DPPC_d62/Chol mixtures except for the pure liquid-disordered phase.     

Ternary phase diagram of dipalmitoyl-PC/dilauroyl-PC/cholesterol: nanoscopic domain formation driven by cholesterol

A ternary phase diagram is proposed for the hydrated lamellar lipid mixture dipalmitoylphosphatidylcholine/dilauroylphosphatidylcholine/cholesterol (DPPC/DLPC/cholesterol) at room temperature. The entire composition space has been thoroughly mapped by complementary experimental techniques, revealing interesting phase behavior that has not been previously described. Confocal fluorescence microscopy shows a regime of coexisting DPPC-rich ordered and DLPC-rich fluid lamellar phases, having an upper boundary at apparently constant cholesterol mole fraction chi(chol) approximately 0.16. Fluorescence resonance energy transfer experiments confirm the identification and extent of this two-phase regime and, furthermore, reveal a 1-phase regime between chi(chol) approximately 0.16 and 0.25, consisting of ordered and fluid nanoscopic domains. Dipyrene-PC excimer/monomer measurements confirm the new regime between chi(chol) approximately 0.16 and 0.25 and also show that rigidly ordered phases seem to disappear around chi(chol) approximately 0.25. This study should be considered as a step toward a more complete understanding of lateral heterogeneity within biomembranes. Cholesterol may play a role in domain separation on the nanometer scale.     

Cholesterol-induced fluid membrane domains: A compendium of lipid-raft ternary phase diagrams

The biophysical underpinning of the lipid-raft concept in cellular membranes is the liquid-ordered phase that is induced by moderately high concentrations of cholesterol. Although the crucial feature is the coexistence of phase-separated fluid domains, direct evidence for this in mixtures of cholesterol with a single lipid is extremely sparse. More extensive evidence comes from ternary mixtures of a high chain-melting lipid and a low chain-melting lipid with cholesterol, including those containing sphingomyelin that are taken to be a raft paradigm. There is, however, not complete agreement between the various phase diagrams and their interpretation. In this review, the different ternary phase diagrams of cholesterol-containing systems are presented in a uniform way, using simple x,y-coordinates to increase accessibility for the non-specialist. It is then possible to appreciate the common features and examine critically the discrepancies and hence what direct biophysical evidence there is that supports the raft concept.     

Domain formation in sphingomyelin/cholesterol mixed membranes studied by spin-label electron spin resonance spectroscopy

Interactions of palmitoylsphingomyelin with cholesterol in multilamellar vesicles have been studied over a wide range of compositions and temperatures in excess water by using electron spin resonance (ESR) spectroscopy. Spin labels bearing the nitroxide free radical group on the 5 or 14 C-atom in either the sn-2 stearoyl chain of phosphatidylcholine (predominantly 1-palmitoyl) or the N-stearoyl chain of sphingomyelin were used to determine the mobility and ordering of the lipids in the different phases. Two-component ESR spectra of the 14-position spin labels demonstrate the coexistence first of gel (L(beta)) and liquid-ordered (L(o)) phases and then of liquid-ordered and liquid-disordered (L(alpha)) phases, with progressively increasing temperature. These phase coexistences are detected over a limited range of cholesterol contents. ESR spectra of the 5-position spin labels register an abrupt increase in ordering at the L(alpha)-L(o) transition and a biphasic response at the L(beta)-L(o) transition. Differences in outer splitting between the C14-labeled sphingomyelin and phosphatidylcholine probes are attributed to partial interdigitation of the sphingomyelin N-acyl chains across the bilayer plane in the L(o) state. In the region where the two fluid phases, L(alpha) and L(o), coexist, the rate at which lipids exchange between phases (<7 x 10(7) s(-)(1)) is much slower than translational rates in the L(alpha) phase, which facilitates resolution of two-component spectra.     

Phase diagram of stigmasterol-dipalmitoylphosphatidylcholine mixtures dispersed in excess water

As a simple model of rafts in plant cells, the effect of stigmasterol, one of the predominant sterols in plant plasma membranes, on the phase behavior of dipalmitoylphosphatidylcholine (DPPC) multilayers has been studied by X-ray diffraction (XRD), differential scanning calorimetry (DSC), and freeze-fracture electron microscopy (FFEM) techniques. A partial phase diagram of the binary system has been constructed. Particularly, the stigmasterol concentrations of the “left endpoint” and “right endpoint” of the three-phase line have been determined using the newly developed linear and nonlinear fitting method. They are 6.2 and 23.7 mol%, respectively. Furthermore, the resemblance and difference of phase diagrams of DPPC/stigmasterol, DPPC/cholesterol, and DPPC/ergosterol have been compared and the efficiency of these sterols in promoting the formation of the liquid-ordered domains (rafts) have also been discussed.     

Phase diagram of stigmasterol-dipalmitoylphosphatidylcholine mixtures dispersed in excess water

As a simple model of rafts in plant cells, the effect of stigmasterol, one of the predominant sterols in plant plasma membranes, on the phase behavior of dipalmitoylphosphatidylcholine (DPPC) multilayers has been studied by X-ray diffraction (XRD), differential scanning calorimetry (DSC), and freeze-fracture electron microscopy (FFEM) techniques. A partial phase diagram of the binary system has been constructed. Particularly, the stigmasterol concentrations of the "left endpoint" and "right endpoint" of the three-phase line have been determined using the newly developed linear and nonlinear fitting method. They are 6.2 and 23.7 mol%, respectively. Furthermore, the resemblance and difference of phase diagrams of DPPC/stigmasterol, DPPC/cholesterol, and DPPC/ergosterol have been compared and the efficiency of these sterols in promoting the formation of the liquid-ordered domains (rafts) have also been discussed.     

Erythrocyte spectrin maintains its segmental motions on oxidation: a spin-label EPR study.

The segmental motions of cross-linked erythrocyte skeletal protein (spectrin-actin-protein 4.1) samples, labeled with nitroxide spin labels, were monitored by conventional first-harmonic and saturation transfer second-harmonic electron paramagnetic resonance methods. Skeletal proteins were extracted from human red blood cells and treated with three oxidative reagents (diamide, hydrogen peroxide, and phenylhydrazine) to cross-link sulfhydryl groups and with one fixative reagent (glutaraldehyde) to cross-link lysine residues. The treatments provided extensive cross-linking between spectrin-actin-protein 4.1 molecules, as determined by gel electrophoresis, and surface charge modification, as determined by pl measurements. However, segmental motions of the cross-linked skeletal proteins remained generally similar to those in normal skeletal proteins. Both the weakly immobilized and the strongly immobilized motions were similar in cross-linked and control samples. Small differences in some motional components were detected. In some cases, faster mobilities were observed, with approximately 5% of the strongly immobilized motions converted to the weakly immobilized motions in the cross-linked samples. It is often believed that the consequence of membrane protein oxidation is restricted protein dynamics, giving membrane rigidity. However, our studies provide needed experimental evidence to indicate that segmental motions are maintained with very little modification even in the presence of extensive cross-linking. Thus cross-linking does not restrict the internal molecular flexibility that gives rise to segmental motions.     

Using spin-label electron paramagnetic resonance (EPR) to discriminate and characterize the cholesterol bilayer domain

Electron paramagnetic resonance (EPR) spin-labeling methods make it possible not only to discriminate the cholesterol bilayer domain (CBD) but also to obtain information about the organization and dynamics of cholesterol molecules in the CBD. The abilities of spin-label EPR were demonstrated for Chol/POPC (cholesterol/1-palmitoyl-2-oleoylphosphatidylcholine) membranes, with a Chol/POPC mixing ratio that changed from 0 to 3. Using the saturation-recovery (SR) EPR approach with cholesterol analogue spin labels, ASL and CSL, and oxygen or NiEDDA relaxation agents, it was confirmed that the CBD was present in all membrane suspensions when the mixing ratio exceeded the cholesterol solubility threshold (CST). Conventional EPR spectra of ASL and CSL in the CBD were similar to those in the surrounding POPC bilayer (which is saturated with cholesterol), indicating that in both domains, cholesterol exists in the lipid-bilayer-like structures. The behavior of ASL and CSL (and, thus, the behavior of cholesterol molecules) in the CBD was compared with that in the surrounding POPC-cholesterol domain (PCD). In the CBD, ASL and CSL molecules are better ordered than in the surrounding PCD. This difference is small and can be compared to that induced in the surrounding domain by an ∼10 °C decrease in temperature. Thus, cholesterol molecules are unexpectedly dynamic in the CBD, which should enhance their interaction with the surrounding domain. The polarity of the water/membrane interface of the CBD is significantly greater than that of the surrounding PCD, which significantly enhances penetration of the water-soluble relaxation agent, NiEDDA, into that region. Hydrophobicity measured in the centers of both domains is similar. The oxygen transport parameter (oxygen diffusion-concentration product) measured in the center of the CBD is about ten times smaller than that measured in the center of the surrounding domain. Thus, the CBD can form a significant barrier to oxygen transport. The results presented here point out similarities between the organization and dynamics of cholesterol molecules in the CBD and in the surrounding PCD, as well as significant differences between CBDs and cholesterol crystals.     

Temperature and composition dependence of the interaction of delta-lysin with ternary mixtures of sphingomyelin/cholesterol/POPC

The kinetics of carboxyfluorescein efflux induced by the amphipathic peptide delta-lysin from vesicles of porcine brain sphingomyelin (BSM), 1-palmitoyl-2-oleoyl-phosphatidylcholine (POPC), and cholesterol (Chol) were investigated as a function of temperature and composition. Sphingomyelin (SM)/Chol mixtures form a liquid-ordered (L(o)) phase whereas POPC exists in the liquid-disordered (L(d)) phase at ambient temperature. delta-Lysin binds strongly to L(d) and poorly to L(o) phase. In BSM/Chol/POPC vesicles the rate of carboxyfluorescein efflux induced by delta-lysin increases as the POPC content decreases. This is explained by the increase of delta-lysin concentration in L(d) domains, which enhances membrane perturbation by the peptide. Phase separations in the micrometer scale have been observed by fluorescence microscopy in SM/Chol/POPC mixtures for some SM, though not for BSM. Thus, delta-lysin must detect heterogeneities (domains) in BSM/Chol/POPC on a much smaller scale. Advantage was taken of the inverse variation of the efflux rate with the L(d) content of BSM/Chol/POPC vesicles to estimate the L(d) fraction in those mixtures. These results were combined with differential scanning calorimetry to obtain the BSM/Chol/POPC phase diagram as a function of temperature.     

Lipid dynamics and domain formation in model membranes composed of ternary mixtures of unsaturated and saturated phosphatidylcholines and cholesterol

In recent years, the implication of sphingomyelin in lipid raft formation has intensified the long sustained interest in this membrane lipid. Accumulating evidences show that cholesterol preferentially interacts with sphingomyelin, conferring specific physicochemical properties to the bilayer membrane. The molecular packing created by cholesterol and sphingomyelin, which presumably is one of the driving forces for lipid raft formation, is known in general to differ from that of cholesterol and phosphatidylcholine membranes. However, in many studies, saturated phosphatidylcholines are still considered as a model for sphingolipids. Here, we investigate the effect of cholesterol on mixtures of dioleoyl-phosphatidylcholine (DOPC) and dipalmitoyl-phosphatidylcholine (DPPC) or distearoyl-phosphatidylcholine (DSPC) and compare it to that on mixtures of DOPC and sphingomyelin analyzed in previous studies. Giant unilamellar vesicles prepared from ternary mixtures of various lipid compositions were imaged by confocal fluorescence microscopy and, within a certain range of sterol content, domain formation was observed. The assignment of distinct lipid phases and the molecular mobility in the membrane bilayer was investigated by fluorescence correlation spectroscopy. Cholesterol was shown to affect lipid dynamics in a similar way for DPPC and DSPC when the two phospholipids were combined with cholesterol in binary mixtures. However, the corresponding ternary mixtures exhibited different spatial lipid organization and dynamics. Finally, evidences of a weaker interaction of cholesterol with saturated phosphatidylcholines than with sphingomyelin (with matched chain length) are discussed.     

Phase equilibria in binary mixtures of phosphatidylcholine and cholesterol

The paramagnetic resonance spectra of two spin-labels, 2,2,6,6-tetramethylpiperadinyl-1-oxy and a head-group spin-labeled phosphatidylethanolamine (L-alpha-dipalmitoylphosphatidyl-N-ethanolamine), have been used to study solid-liquid and liquid-liquid phase separations in binary mixtures of dimyristoylphosphatidylcholine and cholesterol. A quantitative analysis of these resonance spectra supports the view that at temperatures below theta m, the chain-melting temperature of the phospholipid, and at cholesterol mole fractions Xc less than 0.2, these mixtures consist of two phases, a solid phase of essentially pure dimyristoylphosphatidylcholine and a fluid phase having a mole fraction of cholesterol equal to 0.2. The spin-label data also provide evidence for fluid-fluid immiscibility in the bilayer membrane at temperatures above the chain melting transition temperature of dimyristoylphosphatidylcholine.     

Phase separation is induced by phenothiazine derivatives in phospholipid/sphingomyelin/cholesterol mixtures containing low levels of cholesterol and sphingomyelin

Lipid rafts are membrane structures enriched in cholesterol, sphingomyelin and glycolipids. In majority raft-mimicking model systems high contents of cholesterol and sphingomyelin (approximately 30 mol%) are used. Existence of raft-like structures was, however, reported also in model and natural membranes containing low levels of cholesterol and sphingomyelin. In the present work differential scanning calorimetry and fluorescence spectroscopy with the use of Laurdan probe was employed to demonstrate the existence of phase separation in model systems containing DPPC with addition of 5 mol% or 10 mol% of both cholesterol and sphingomyelin. Additionally, the influence of three phenothiazine derivatives on phase separation in mixed DPPC/cholesterol/sphingomyelin bilayers was investigated. Chlorpromazine, thioridazine and trifluoperazine were able to induce phase separation in DPPC and DPPC/cholesterol/sphingomyelin bilayers in temperatures below lipid main phase transition. However, only trifluoperazine induced phase separation in temperatures close to or above main phase transition. Trifluoperazine also induced phase separation in bilayers composed of egg yolk PC or DOPC mixed with cholesterol and sphingomyelin. We concluded that presence of lipid domains can be observed in model membranes containing low levels of cholesterol and sphingomyelin. Among three phenothiazine derivatives studied, only trifluoperazine was able to induce a permanent phase separation in phosphatidylcholine/cholesterol/sphingomyelin systems.     

Phase equilibria of cholesterol/dipalmitoylphosphatidylcholine mixtures: 2H nuclear magnetic resonance and differential scanning calorimetry

Deuterium nuclear magnetic resonance spectroscopy and differential scanning calorimetry are used to map the phase boundaries of mixtures of cholesterol and chain-perdeuteriated 1,2-dipalmitoyl-sn-glycero-3-phosphocholine at concentrations from 0 to 25 mol % cholesterol. Three distinct phases can be identified: the L alpha or liquid-crystalline phase, the gel phase, and a high cholesterol concentration phase, which we call the beta phase. The liquid-crystalline phase is characterized by highly flexible phospholipid chains with rapid axially symmetric reorientation; the gel phase has much more rigid lipid chains, and the motions are no longer axially symmetric on the 2H NMR time scale; the beta phase is characterized by highly ordered (rigid) chains and rapid axially symmetric reorientation. In addition, we identify three regions of two-phase coexistence. The first of these is a narrow L alpha/gel-phase coexistence region lying between 0 and about 6 mol % cholesterol at temperatures just below the chain-melting transition of the pure phospholipid/water dispersions, at 37.75 degrees C. The dramatic changes in the 2H NMR line shape which occur on passing through the phase transition are used to map out the boundaries of this narrow two-phase region. The boundaries of the second two-phase region are determined by 2H NMR difference spectroscopy, one boundary lying near 7.5 mol % cholesterol and running from 37 down to at least 30 degrees C; the other boundary lies near 22 mol % cholesterol and covers the same temperature range. Within this region, the gel and beta phases coexist. As the temperature is lowered below about 30 degrees C, the phospholipid motions reach the intermediate time scale regime of 2H NMR so that spectral subtractions become difficult and unreliable. The third two-phase region lies above 37 degrees C, beginning at a eutectic point somewhere between 7.5 and 10 mol % cholesterol and ending at about 20 mol %. In this region, the L alpha and beta phases are in equilibrium. The boundaries for this region are inferred from differential scanning calorimetry traces, for the boundary between the L alpha- and the two-phase region, and from a dramatic sharpening of the NMR peaks on crossing the boundary between the two-phase region and the beta-phase region. In this region, the technique of difference spectroscopy fails, presumably because the diffusion rate in both the L alpha- and beta-phase domains is so rapid that phospholipid molecules exchange rapidly between domains on the experimental time scale.     

Phospholipid/cholesterol model membranes: formation of cholesterol crystallites

Experimental data that define conditions under which cholesterol crystallites form in cholesterol/phospholipid model membranes are reviewed. Structural features of the phospholipids that determine cholesterol crystallization include the length and degree of unsaturation of the acyl chains, the presence of charge on the headgroups and interheadgroup hydrogen bonds.     

Phospholipid/cholesterol model membranes: formation of cholesterol crystallites

Experimental data that define conditions under which cholesterol crystallites form in cholesterol/phospholipid model membranes are reviewed. Structural features of the phospholipids that determine cholesterol crystallization include the length and degree of unsaturation of the acyl chains, the presence of charge on the headgroups and interheadgroup hydrogen bonds.     

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.     

Sphingomyelin/phosphatidylcholine/cholesterol phase diagram: boundaries and composition of lipid rafts

The ternary system palmitoylsphingomyelin (PSM)/palmitoyloleoylphosphatidylcholine (POPC)/cholesterol is used to model lipid rafts. The phase behavior of the three binary systems PSM/POPC, PSM/cholesterol, and POPC/cholesterol is first experimentally determined. Phase coexistence boundaries are then determined for ternary mixtures at room temperature (23 degrees C) and the ternary phase diagram at that temperature is obtained. From the diagram at 23 degrees C and the binary phase diagrams, a reasonable expectation is drawn for the ternary phase diagram at 37 degrees C. Several photophysical methodologies are employed that do not involve detergent extraction, in addition to literature data (e.g., differential scanning calorimetry) and thermodynamic rules. For the ternary phase diagrams, some tie-lines are calculated, including the one that contains the PSM/POPC/ cholesterol 1:1:1 mixture, which is often used in model raft studies. The diagrams here described are used to rationalize literature results, some of them apparently discrepant, and to discuss lipid rafts within the framework of liquid-ordered/liquid-disordered phase coexistence.     

Ceramide drives cholesterol out of the ordered lipid bilayer phase into the crystal phase in 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine/cholesterol/ceramide ternary mixtures

The effect of brain ceramide on the maximum solubility of cholesterol in ternary mixtures of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC), cholesterol, and ceramide was investigated at 37 degrees C by a cholesterol oxidase (COD) reaction rate assay and by optical microscopy. The COD reaction rate assay showed a sharp increase in cholesterol chemical potential as the cholesterol mole fraction approaches the solubility limit. A decline in the COD reaction rate was found after the formation of cholesterol crystals. The maximum solubility of brain ceramide in POPC bilayers was determined to be 68 +/- 2 mol % by microscopy. We found that ceramide has a much higher affinity for the ordered bilayers than cholesterol, and the maximum solubility of cholesterol decreases with the increase in ceramide content. More significantly, the displacement of cholesterol by ceramide follows a 1:1 relation. At the cholesterol solubility limit, adding one more ceramide molecule to the lipid bilayer drives one cholesterol out of the bilayer into the cholesterol crystal phase, and cholesterol is incapable of displacing ceramide from the bilayer phase. On the basis of these findings, a ternary phase diagram of the POPC/cholesterol/ceramide mixture was constructed. The behaviors of ceramide and cholesterol can be explained by the umbrella model. Both ceramide and cholesterol have small polar headgroups and relatively large nonpolar bodies. In a PC bilayer, ceramide and cholesterol compete for the coverage of the headgroups of neighboring PC to prevent the exposure of their nonpolar bodies to water. This competition results in the 1:1 displacement as well as the displacement of cholesterol by ceramide from lipid raft domains.     

The thermotropism and prototropism of ternary mixtures of ceramide C16, cholesterol and palmitic acid. An exploratory study

Mixtures of ceramides with other lipids in the presence of water are key components of the structure of the lipid matrix of the stratum corneum and are involved in lateral phase separation processes occurring in lipid membranes. Besides their structural role, ceramides are functional for cell signaling and trafficking. We elected, as our object of study, a mixture of N-hexadecanoylceroyl-d-erythro-sphyngosine, C16-Cer, with cholesterol, Ch, in a molar proportion 54:46 in excess water to which palmitic acid, PA, is added in varying amounts. The chosen C16-Cer:Ch proportion replicates the relative abundance of ceramides and cholesterol found in the stratum corneum lipid matrix. For each lipidic composition, we identify the phases in equilibrium and study the thermotropism of the system, using differential scanning calorimetry and temperature-dependent small and wide-angle X-ray powder diffraction. Since the molecular aggregation of the system and its mesoscopic properties are affected by the degree of protonation of the PA, we explore mixtures with several PA contents at two extreme pH values, 9.0 and 4.0. A specific C16-Cer:Ch:PA composition forms at pH 9.0 a lamellar crystalline aggregate, to which we attribute the stoichiometry C16-Cer₅Ch₄PA₂, that melts at 88–90 °C to give a H_II phase. For pH values at which there is partial or total protonation of PA another L_C C16-Cer:Ch (2:3) stoichiometric aggregate is observed, identical to that previously reported for C16-Cer:Ch mixtures (Souza et al., 2009, J. Phys. Chem. B, 113, 1367–1375), coexisting with a lamellar fluid phase. For pH 4.0 and 7.0, the existing lamellar liquid crystalline converts into a isotropic fluid phase at high temperatures. It is also found that the miscibility of PA in the C16-Cer:Ch mixture at pH 4.0 does not exceed ca. 18 mol%, but for pH 9.0 no free PA is detected at least until 60 mol%.     

A fluorescence anisotropy study on the phase behavior of dimyristoylphosphatidylcholine/cholesterol mixtures

The phase behavior of L-alpha-dimyristoylphosphatidylcholine/cholesterol mixtures was studied in multilamellar vesicles by fluorescence polarization of the sterol molecule dehydroergosterol and of the polyene molecule alpha-parinaric acid. In the absence of cholesterol, dehydroergosterol exhibited an increase in polarization as DMPC vesicles were heated through the phase transition. This rise in polarization anisotropy was observed over a 0.6-1.0 degrees C increase in temperature with the midpoint of the phase transition occurring at 23.6 degrees C. Addition of 5 mol% cholesterol completely obliterated this change in polarization anisotropy through the phase transition of DMPC. alpha-Parinaric acid underwent a characteristic decrease in polarization anisotropy through the phase transition of DMPC. The change in anisotropy through the phase transition was over 4-fold greater than the values observed with dehydroergosterol. Vesicles containing 5 mol% cholesterol in the presence of alpha-parinaric acid underwent a decrease in polarization anisotropy that was over 75% of the original decrease in amplitude observed in the absence of any membrane cholesterol. The difference in sensitivity of the two fluorescent probes to the phase transition of DMPC as a function of membrane cholesterol content may be explained by a preferential partitioning of dehydroergosterol (and cholesterol) into a sterol-rich phase at low sterol concentrations. This partitioning allows dehydroergosterol to detect sterol-rich regions in the membrane bilayer.