Investigation of polyene macrolide antibiotic-induced permeability changes in vesicles by 31P nuclear magnetic resonance

The permeability of egg yolk lecithin (EYL) vesicles to Pr3+ has been measured by 31P nuclear magnetic resonance (nmr) spectroscopy. Measurable Pr3+ leakage into the internal aqueous compartment of EYL vesicles at ambient (21 degrees C) temperature required the presence of small (7--10 mol%) amounts of dicetyl phosphate (DCP). The permeability of DCP-containing vesicles is decreased by incorporation of sterol (cholesterol greater than ergosterol approximately 5.6-dihydroergosterol greater than zymosterol) into the lipid bilayer. Addition of the polyene macrolide antibiotic, nystatin, to DCP-containing EYL vesicles with and without sterol resulted in increased Pr3+ permeability at the three temperatures studied (21--37.5 degrees C). Permeability changes observed upon addition of nystatin to sterol-impregnated, DCP-containing vesicles varied with sterol structure: ergosterol approximately 5,6-dihydroergosterol greater than cholesterol approximately zymosterol. These results are compared with other polyene macrolide induced permeability changes on model and natural membrane systems. Permeability changes induced by nystatin in sterol-free EYL vesicles were generally greater than for comparable sterol-containing vesicles. This is attributed to a nonspecific interaction of the antibiotic with the latter vesicles.     

On the existence of an amphotericin B--sterol complex in lipid vesicles and in propanol-water systems

The amphotericin B (AmB) - ergosterol complex, formed by interaction of the antibiotic with ergosterol-containing phospholipid vesicles, is associated with the lipid bilayer. It has been shown by circular dichroism studies that the AmB-ergosterol complex formed in water-propanol binary mixtures has a similar structure to that observed in phospholipid vesicles. A positive cooperativity is found for the interaction of AmB with ergosterol. The similar AmB-cholesterol complex is much less stable and rearranges rapidly to a different conformation.     

One-sided action of amphotericin B on cholesterol-containing membranes is determined by its self-association in the medium

The inducement of K+ permeability through membranes by the polyene antibiotic amphotericin B (AmB) has been analyzed as a measure of the antibiotic activity. Dose-response curves have been obtained with cholesterol- and ergosterol-containing egg yolk phosphatidylcholine large unilamellar vesicles (LUVs), human erythrocytes, and Saccharomyces cerevisiae cells. Conductance changes induced by AmB in sterol-containing planar bilayer membranes have also been studied. AmB self-association in aqueous buffer was determined by circular dichroism (CD) as a function of the antibiotic concentration. Electronic absorption and CD spectra of AmB were recorded in the presence of LUVs. For given AmB concentrations, the extent of permeability inducement is dependent on the lipid concentration. On the other hand, for cholesterol-containing LUVs or erythrocytes, a critical AmB concentration had to be reached before any permeability is observed. Independent of lipid concentration, this concentration was directly related to antibiotic self-association in the aqueous buffer. The same observation was made for erythrocytes and nystatin. The AmB absorption and CD spectra were totally different for ergosterol- and cholesterol-containing LUVs. Formation of single channels by one-sided addition of AmB could be observed only in ergosterol-containing membranes. These data lead us to propose that the permeability pathways induced by amphotericin B or nystatin, in ergosterol- and in cholesterol-containing membranes, are of different natures. In the latter case the antibiotics are only active, by single-sided addition, in the self-associated form. These findings offer important clues for the design of less toxic derivatives of AmB: they should have a low degree of self-association in water.     

Interaction of the polyene antibiotic amphotericin B with model membranes: differences between small and large unilamellar vesicles

The interaction of the polyene antibiotic amphotericin B (AmB) (Fig. 1) with large unilamellar vesicles (LUV) was monitored by circular dichroism (CD) and carboxyfluorescein (CF) release. LUV afford a far better model for biological membranes than small unilamellar vesicles (SUV) which have been used until now. With dimyristoyl phosphatidyl choline (DMPC) LUV (i.e., containing saturated acyl chains), a strong and not saturable binding for AmB/lipid ratios up to 0.5 was observed both above and below the phase transition temperature. Incorporation of cholesterol into the vesicles did not significantly change the interaction. With egg PC (EPC) LUV (i.e., containing unsaturated acyl chains), quite a different picture emerged: the binding reached saturation for AmB/lipid ratios of about 5 x 10(-3), a result not observed with EPC SUV. When sterols were introduced into membranes, the CD spectral features obtained in the presence of ergosterol were different from those obtained in the presence of cholesterol. Such a different behavior was not observed with SUV. We suggest that species whose CD spectrum was observed after 15 min in the presence of ergosterol-containing EPC LUV is the particular one which forms wide channels and induces a Ca2+ release. (H. Ramos, A. Attias, B.E. Cohen and J. Bolard, submitted for publication). The CF release from EPC LUV induced by AmB was very low, even at very high concentrations of the antibiotic (3 x 10(-4)M). In contrast, an important release of the fluorescent dye was observed with DMPC LUV at concentrations of approximately 10(-5)M.(ABSTRACT TRUNCATED AT 250 WORDS)     

The effects of amphotericin B on pure and ergosterol- or cholesterol-containing dipalmitoylphosphatidylcholine bilayers as viewed by 2H NMR

Amphotericin B (AmB) is a widely used polyene antibiotic to treat systemic fungal infections. This drug is known to be lethal to fungal cells but it has also side effect toxicity on mammalian cells. The mechanism of action of AmB is thought to be related to the difference of the main sterol present in the mammalian and the fungal cells, namely cholesterol and ergosterol, respectively. The effect of AmB has been investigated on pure dipalmitoylphosphatidylcholine (DPPC) and on cholesterol- and ergosterol-containing DPPC bilayers by 2H NMR spectroscopy. The 2H NMR results first confirm that AmB forms a complex with sterol-free DPPC bilayers, the interaction causing the structurization of the lipids and the increase of the gel-to-lamellar fluid DPPC phase transition temperature with increasing concentration of the antibiotic. The results also show that the effects of AmB on cholesterol- and ergosterol-containing DPPC bilayers are remarkably different. On one hand, the drug causes an increase of the orientational order of the lipid acyl chains in cholesterol-containing membranes, mostly in high cholesterol content membranes. On the other hand, the addition of AmB disorders the DPPC acyl chains when ergosterol is present. This is thought to be due to the direct complexation of the ergosterol by AmB, causing the sterol ordering effect to be weaker on the lipids.     

Dissociation kinetics and equilibrium binding properties of polyene antibiotic complexes with phosphatidylcholine/sterol vesicles

The interactions of sonicated vesicles with the polyene antibiotics amphotericin B, candicidin, mediocidin , and a water-soluble, guanidine derivative of amphotericin B were examined by UV-visible spectroscopy at concentrations below which the polyenes become self-associated. The association constants, Kapp, and the numbers of binding sites per sterol or phospholipid molecule (n) were determined at 30 degrees C and pH 7.4. A single class of binding sites was found, with no evidence of cooperativity. For the binding of mediocidin , amphotericin B, and the guanidine derivative with phosphatidylcholine (PC), PC/cholesterol, and PC/ergosterol vesicles, Kapp was in the range of (1.0-3.0) X 10(6) M-1; Kapp was higher for candicidin-vesicle interaction, reaching 9.0 X 10(6) M-1 with PC/ergosterol vesicles. Binding of the guanidine derivative of amphotericin B to PC vesicles lacking sterol was extensive (n = 0.46); since the other polyenes, which have low aqueous solubilities, had n less than 0.05, positive charges in the mycosamine moiety appear to enhance the extent of polyene antibiotic interaction with the glycerophospholipid head group. Higher values of n (and, therefore, of nKapp ) were found with sterol-containing than with sterol-free vesicles, suggestive of penetration of the polyenes toward the interior of the bilayer when sterol is present. For binding to PC/sterol vesicles, nKapp followed the order of candicidin greater than guanidine derivative of amphotericin B greater than amphotericin B much greater than mediocidin . The values of n and nKapp were appreciably higher for amphotericin B-ergosterol than for amphotericin B-cholesterol interaction in vesicles.(ABSTRACT TRUNCATED AT 250 WORDS)     

Interactions of the drug amphotericin B with phospholipid membranes containing or not ergosterol: new insight into the role of ergosterol.

Amphotericin B (AmB) is an amphipathic polyene antibiotic which permeabilizes ergosterol-containing membranes, supposedly by formation of pores. In water, AmB forms chiral aggregates, modelled as stacks of planar dimers in which the joined polyene chains in each dimer turn round, from one dimer to the following in these stacks, by forming a helical array. Studies of the binding of AmB with L-dipalmitoylphosphatidylcholine (L-DPPC) and L-dilauroylphosphatidylcholine (L-DLPC) bilayers disclose the main following results. (1) An inversion of the helicity of the L-DPPC-bound AmB aggregates, when the L-DPPC bilayers are in the gel phase, is inferred from the evolution of the circular dichroism spectra of AmB+L-DPPC mixtures. (2) An AmB-induced gel-to-subgel transformation of L-DPPC bilayers, in the previous mixtures, is revealed by a differential scanning calorimetry study. (3) The role played by ergosterol in the location of phospholipid-bound AmB aggregates with respect to a phospholipid bilayer is directly demonstrated from atomic force microscopy observations of mica-supported AmB+L-DLPC mixtures, in the presence or absence of ergosterol. While in the absence of ergosterol AmB aggregates remained at the surface of the bilayer, in the presence of ergosterol they appeared embedded within this bilayer and became hollow-centered. As such an embedding in the hydrophobic core of a bilayer requires a rearrangement of the aggregates with respect to their architecture in water, this rearrangement is held responsible for the hollowing of aggregates. The hollow-centered sublayer-embedded AmB aggregates are thought to be the precursors of the formation of AmB pores.     

Interactions of the drug amphotericin B with phospholipid membranes containing or not ergosterol: new insight into the role of ergosterol

Amphotericin B (AmB) is an amphipathic polyene antibiotic which permeabilizes ergosterol-containing membranes, supposedly by formation of pores. In water, AmB forms chiral aggregates, modelled as stacks of planar dimers in which the joined polyene chains in each dimer turn round, from one dimer to the following in these stacks, by forming a helical array. Studies of the binding of AmB with L-dipalmitoylphosphatidylcholine (L-DPPC) and L-dilauroylphosphatidylcholine (L-DLPC) bilayers disclose the main following results. (1) An inversion of the helicity of the L-DPPC-bound AmB aggregates, when the L-DPPC bilayers are in the gel phase, is inferred from the evolution of the circular dichroism spectra of AmB+L-DPPC mixtures. (2) An AmB-induced gel-to-subgel transformation of L-DPPC bilayers, in the previous mixtures, is revealed by a differential scanning calorimetry study. (3) The role played by ergosterol in the location of phospholipid-bound AmB aggregates with respect to a phospholipid bilayer is directly demonstrated from atomic force microscopy observations of mica-supported AmB+L-DLPC mixtures, in the presence or absence of ergosterol. While in the absence of ergosterol AmB aggregates remained at the surface of the bilayer, in the presence of ergosterol they appeared embedded within this bilayer and became hollow-centered. As such an embedding in the hydrophobic core of a bilayer requires a rearrangement of the aggregates with respect to their architecture in water, this rearrangement is held responsible for the hollowing of aggregates. The hollow-centered sublayer-embedded AmB aggregates are thought to be the precursors of the formation of AmB pores.     

Comparative molecular dynamics simulations of amphotericin B-cholesterol/ergosterol membrane channels

Amphotericin B (AmB) is a very effective anti-fungal polyene macrolide antibiotic whose usage is limited by its toxicity. Lack of a complete understanding of AmB's molecular mechanism has impeded attempts to design less toxic AmB derivatives. The antibiotic is known to interact with sterols present in the cell membrane to form ion channels that disrupt membrane function. The slightly higher affinity of AmB toward ergosterol (dominant sterol in fungal cells) than cholesterol (mammalian sterol) is regarded as the most essential factor on which antifungal chemotherapy is based. To study these differences at the molecular level, two realistic model membrane channels containing molecules of AmB, sterol (cholesterol or ergosterol), phospholipid, and water were studied by molecular dynamics (MD) simulations. Comparative analysis of the simulation data revealed that the sterol type has noticeable effect on the properties of AmB membrane channels. In addition to having a larger size, the AmB channel in the ergosterol-containing membrane has a more pronounced pattern of intermolecular hydrogen bonds. The interaction between the antibiotic and ergosterol is more specific than between the antibiotic and cholesterol. These observed differences suggest that the channel in the ergosterol-containing membrane is more stable and, due to its larger size, would have a higher ion conductance. These observations are in agreement with experiments.     

Natamycin blocks fungal growth by binding specifically to ergosterol without permeabilizing the membrane

Natamycin is a polyene antibiotic that is commonly used as an antifungal agent because of its broad spectrum of activity and the lack of development of resistance. Other polyene antibiotics, like nystatin and filipin are known to interact with sterols, with some specificity for ergosterol thereby causing leakage of essential components and cell death. The mode of action of natamycin is unknown and is investigated in this study using different in vitro and in vivo approaches. Isothermal titration calorimetry and direct binding studies revealed that natamycin binds specifically to ergosterol present in model membranes. Yeast sterol biosynthetic mutants revealed the importance of the double bonds in the B-ring of ergosterol for the natamycin-ergosterol interaction and the consecutive block of fungal growth. Surprisingly, in strong contrast to nystatin and filipin, natamycin did not change the permeability of the yeast plasma membrane under conditions that growth was blocked. Also, in ergosterol containing model membranes, natamycin did not cause a change in bilayer permeability. This demonstrates that natamycin acts via a novel mode of action and blocks fungal growth by binding specifically to ergosterol.     

Unilamellar-Vesicle Systems as Model for Studying Membrane Permeabilization by Polyene Antibiotics

Abstract

Permeabilization of phospholipid/sterol unilamellar vesicles by polyene antibiotics (amphotericin B and lucensomycin) was studied by measuring proton leakage with a pH-stat method. The percentage of proton release was directly related to the antibiotic concentration. Using ergosterol-containing vesicles, a relevant proton efflux was induced by micromolar concentrations of amphotericin B, whereas lucensomycin caused membrane permeabilization at higher concentrations (0.1 mM). Cholesterol-containing vesicles were less sensible to the lytic action of polyenes. When amphotericin B was carried in cholesterol-containing liposomes, the selectivity towards ergosterol-containing vesicles was enhanced. An increase in drug selectivity was also observed by dissolving amphotericin B in fresh human plasma. At concentrations one order of magnitude lower than those necessary to induce a detectable proton efflux, lucensomycin seemed to protect the vesicles from the subsequent permeabilizing action of amphotericin B.     

Elasticity and phase behavior of DPPC membrane modulated by cholesterol, ergosterol, and ethanol

Giant vesicles formed of 1,2-dipalmitoylphosphatidylcholine (DPPC) and sterols (cholesterol or ergosterol) in water and water/ethanol solutions have been used to examine the effect of sterol composition and ethanol concentration on the area compressibility modulus (K(a)), overall mechanical behavior, vesicle morphology, and induction of lipid alkyl chain interdigitation. Our results from micropipette aspiration suggest that cholesterol and ergosterol impact the order and microstructure of the gel (L(beta)') phase DPPC membrane. At low concentration (10-15 mol%) these sterols disrupt the long-range lateral order and fluidize the membrane (K(a) approximately 300 mN/m). Then at 18 mol%, these sterols participate in the formation of a continuous cohesive liquid-ordered (L(o)) phase with a sterol-dependent membrane density (K(a) approximately 750 for DPPC/ergosterol and K(a) approximately 1100 mN/m for DPPC/cholesterol). Finally at approximately 40 mol% both cholesterol and ergosterol impart similar condensation to the membrane (K(a) approximately 1200 mN/m). Introduction of ethanol (5-25 vol%) results in drops in the magnitude of K(a), which can be substantial, and sometimes individual vesicles with lowered K(a) reveal two slopes of tension versus apparent area strain. We postulate that this behavior represents disruption of lipid-sterol intermolecular interactions and therefore the membrane becomes interdigitation prone. We find that for DPPC vesicles with sterol concentrations of 20-25 mol%, significantly more ethanol is required to induce interdigitation compared to pure DPPC vesicles; approximately 7 vol% more for ergosterol and approximately 10 vol% more for cholesterol. For lower sterol concentrations (10-15 mol%), interdigitation is offset, but by <5 vol%. These data support the idea that ergosterol and cholesterol do enhance survivability for cells exposed to high concentrations of ethanol and provide evidence that the appearance of the interdigitated (L(beta)I) phase bilayer is a major factor in the disruption of cellular activity, which typically occurs between approximately 12 and approximately 16 vol% ethanol in yeast fermentations. We summarize our findings by producing, for the first time, "elasticity/phase diagrams" over a wide range of sterol (cholesterol and ergosterol) and ethanol concentrations.     

Tamoxifen perturbs lipid bilayer order and permeability: comparison of DSC, fluorescence anisotropy, laurdan generalized polarization and carboxyfluorescein leakage studies

The perturbation of the lipid bilayer structure by tamoxifen may contribute to its multiple mechanisms of anticancer action not related to estrogen receptors. This study evaluates the effect of tamoxifen on structural characteristics of model membranes using differential scanning calorimetry (DSC), fluorescence anisotropy of 1,6-diphenyl-1,3,5-hexatriene (DPH) and 1-[4-[trimethylammonium)phenyl]-6-phenylhexa-1,3,5-triene (TMA-DPH), as well as 6-dodecanoyl-2-dimethylaminonaphthalene (Laurdan) generalized polarization. The comparative measurements in multilammelar vesicles (MLV) prepared from dipalmitoylphosphatidylcholine (DPPC) revealed that tamoxifen decreases the phase transition temperature (Tm) paralleled by a broadening of the phase transition profile. In large unilamellar vesicles (LUV) prepared from egg yolk phosphatidylcholine (EPC), tamoxifen increased the lipid bilayer order predominantly in the outer bilayer region. From membrane permeability measurements, we conclude that the tamoxifen-induced release of entrapped carboxyfluorescein (CF) results from a permanent bilayer disruption and the formation of transient holes in the lipid bilayer.     

Influence of cholesterol and ergosterol on membrane dynamics: a fluorescence approach

Sterols are essential membrane components of eukaryotic cells and are important for membrane organization and function. Cholesterol is the most representative sterol present in higher eukaryotes. It is often found distributed non-randomly in domains or pools in biological and model membranes. Cholesterol-rich functional microdomains (lipid rafts) are often implicated in cell signaling and membrane traffic. Interestingly, lipid rafts have also recently been isolated from organisms such as yeast and Drosophila, which have ergosterol as their major sterol component. Although detailed biophysical characterization of the effect of cholesterol on membranes is well documented, the effect of ergosterol on the organization and dynamics of membranes is not very clear. We have monitored the effect of cholesterol and ergosterol on the dynamic properties of both fluid (POPC) and gel (DPPC) phase membranes utilizing the environment-sensitive fluorescent membrane probe DPH. Our results from steady state and time-resolved fluorescence measurements show, for the first time, differential effects of ergosterol and cholesterol toward membrane organization. These novel results are relevant in the context of lipid rafts in ergosterol-containing organisms such as Drosophila which maintain a low level of sterol compared to higher eukaryotes.     

Amphotericin B channel conductance inactivation

Effects induced in bilayer lipid membranes by amphotericin B and its alkyl derivatives was analysed. Inactivation of the antibiotic-dependent multichannel membrane conductance was discovered. Kinetics of membrane conductivity was shown to depend on the antibiotic concentration in the membrane. At concentrations between 10(-8) and 10(-7) M, the resulting conductance appeared to the transient. We suggest that the phenomenon of biphasic kinetics of membrane conductance is the result of a consecutive transformation of polyene channels in the membrane: half-pores are assembled on either side of membrane-nonconducting 1; two half-pores combine to build up a conducting channels-conducting 2, and the conducting channels are disassemled to monomers and nonconducting self-associated forms inside the membrane-disassembled state (nonconducting 3). To explain the transient characteristics of the induced conductance, it is proposed that the antibiotic, present in the solution under self-associated form, binds the membrane and forms pores, then dissociates in the bilayer in a non-active monomeric form. The existence of definite monomers and nonconducting self-associated forms of amphotericin B molecules inside the membrane was estimated from the dependence of kinetic conductance of lipid membranes of amphotericin B and its alkyl derivatives, when the antibiotics are washed out from aqueous medium. Equilibrium between different antibiotic assemblies inside the membrane was demonstrated by the kinetics of conductance decrease following washing the antibiotic. Using circular dichroism measurements, we observed that amphotericin B alkyl derivatives were in self-associated form being susceptible to form pores across cholesterol-containing membranes. The phenomenon of biophasic kinetics was observed only in the cholesterol-containing membrane. The substitution of membrane cholesterol for ergosterol provides monotonic kinetics of membrane conductance at any antibiotic concentration.     

Conformation and self-assembly of a nystatin nitrobenzoxadiazole derivative in lipid membranes

Nystatin is a polyene (tetraene) macrolide antibiotic presenting antifungal activity that acts at the cellular membrane level. In the present study, we report the interaction of this antibiotic labelled at its amine group with 7-nitrobenz-2-oxa-1,3-diazole (NBD-Nys) with sterol-free and ergosterol- and cholesterol-containing 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) large unilamellar vesicles (LUV). The mean tetraene to NBD separating distance determined from fluorescence energy transfer measurements increased from 18 to 25.6 A upon antibiotic binding to the lipid vesicles, indicating that the monomeric labelled antibiotic adopts a more extended conformation in its lipid-bound state than in aqueous solution. The oligomeric state of membrane-bound NBD-Nys was also studied by resonance energy homotransfer between the NBD fluorophores. The decrease measured in its steady state fluorescence anisotropy upon increasing the surface concentration of the NBD-Nys is shown to be consistent with a random distribution of molecules on the surface of the liposomes. This data contradicts the sharp increase measured for nystatin mean fluorescence lifetime in the presence of 10 mol% ergosterol-containing POPC LUV within the same antibiotic concentration range and which is known to report nystatin oligomerization in the lipid vesicles. Therefore, we conclude that the amine group of nystatin is an essential requisite for the supramolecular organization/pore formation of this antibiotic.     

Nystatin-induced lipid vesicles permeabilization is strongly dependent on sterol structure

The selectivity of the antibiotic nystatin towards ergosterol compared to cholesterol is believed to be a crucial factor in its specificity for fungi. In order to define the structural features of sterols that control this effect, nystatin interaction with ergosterol-, cholesterol-, brassicasterol- and 7-dehydrocholesterol-containing palmitoyloleoylphosphocholine vesicles was studied by fluorescence spectroscopy. Variations in sterol structure were correlated with their effect on nystatin photophysical and activity properties. Substitution of cholesterol by either 7-dehydrocholesterol or brassicasterol enhance nystatin ability to dissipate a transmembrane K⁺ gradient, showing that the presence of additional double bonds in these sterols-carbon C7 and C22, plus an additional methyl group on C-24, respectively-as compared to cholesterol, is fundamental for nystatin-sterol interaction. However, both modifications of the cholesterol molecule, like in the fungal sterol ergosterol, are critical for the formation of very compact nystatin oligomers in the lipid bilayer that present a long mean fluorescence lifetime and induce a very fast transmembrane dissipation. These observations are relevant to the molecular mechanism underlying the high selectivity presented by nystatin towards fungal cells (with ergosterol) as compared to mammalian cells (with cholesterol).     

Nystatin-induced lipid vesicles permeabilization is strongly dependent on sterol structure

The selectivity of the antibiotic nystatin towards ergosterol compared to cholesterol is believed to be a crucial factor in its specificity for fungi. In order to define the structural features of sterols that control this effect, nystatin interaction with ergosterol-, cholesterol-, brassicasterol- and 7-dehydrocholesterol-containing palmitoyloleoylphosphocholine vesicles was studied by fluorescence spectroscopy. Variations in sterol structure were correlated with their effect on nystatin photophysical and activity properties. Substitution of cholesterol by either 7-dehydrocholesterol or brassicasterol enhance nystatin ability to dissipate a transmembrane K+ gradient, showing that the presence of additional double bonds in these sterols-carbon C7 and C22, plus an additional methyl group on C-24, respectively-as compared to cholesterol, is fundamental for nystatin-sterol interaction. However, both modifications of the cholesterol molecule, like in the fungal sterol ergosterol, are critical for the formation of very compact nystatin oligomers in the lipid bilayer that present a long mean fluorescence lifetime and induce a very fast transmembrane dissipation. These observations are relevant to the molecular mechanism underlying the high selectivity presented by nystatin towards fungal cells (with ergosterol) as compared to mammalian cells (with cholesterol).     

Sterol structure determines miscibility versus melting transitions in lipid vesicles

Lipid bilayer membranes composed of DOPC, DPPC, and a series of sterols demix into coexisting liquid phases below a miscibility transition temperature. We use fluorescence microscopy to directly observe phase transitions in vesicles of 1:1:1 DOPC/DPPC/sterol within giant unilamellar vesicles. We show that vesicles containing the "promoter" sterols cholesterol, ergosterol, 25-hydroxycholesterol, epicholesterol, or dihydrocholesterol demix into coexisting liquid phases as temperature is lowered through the miscibility transition. In contrast, vesicles containing the "inhibitor" sterols androstenolone, coprostanol, cholestenone, or cholestane form coexisting gel (solid) and liquid phases. Vesicles containing lanosterol, a sterol found in the cholesterol and ergosterol synthesis pathways, do not exhibit coexisting phases over a wide range of temperatures and compositions. Although more detailed phase diagrams and precise distinctions between gel and liquid phases are required to fully define the phase behavior of these sterols in vesicles, we find that our classifications of promoter and inhibitor sterols are consistent with previous designations based on fluorescence quenching and detergent resistance. We find no trend in the liquid-liquid or gel-liquid transition temperatures of membranes with promoter or inhibitor sterols and measure the surface fraction of coexisting phases. We find that the vesicle phase behavior is related to the structure of the sterols. Promoter sterols have flat, fused rings, a hydroxyl headgroup, an alkyl tail, and a small molecular area, which are all attributes of "membrane active" sterols.