A fluorescent sterol probe study of cholesterol/phospholipid membranes

The behavior of dehydroergosterol in -α-dimyristoylphosphatidylcholine (DMPC) unsonicated multilamellar liposomes was characterized by absorption spectroscopy and fluorescence measurements. Dehydroergosterol exhibited a lowered absorption coefficient in multilamellar liposomes whiel the steady-state fluorescence anisotropy of dehydroergosterol in these membranes decreased significantly with increasing dehydroergosterol concentration, suggesting membrane sterol-sterol interactions. The comparative steady-state anisotropy of 0.9 mole percent dehydroergosterol in multilamellar liposomes was lower than in small unilamellar vesicles suggesting different sterol environments for dehydroergosterol. Dehydroergosterol fluorescence lifetime was relatively independent of membrane sterol content and yielded similar values in sonicated and unsonicated model membranes. In multilamellar liposomes containing 5 mole percent cholesterol, the gel-to-liqui crystalline phase transition of DMPC detected by 0.9 mole percent dehydroergosterol was significantly broadened when compared to the phase transition detected by dehydroergosterol in the absence of membrane cholesterol (Smutzer, G. et al. (1986) Biochim. Biophys. Acta 862, 361–371). In multilamellar liposomes containing 10 mole percent cholesterol, the major fluorescence lifetime of dehydroergosterol did not detect the gel-to-liquid crystalline phase transition of DMPC. Time-correlated fluorescence anisotropy decays of dehydroergosterol in DMPC multilamellar liposomes in the absence and presence of 5 mole percent cholesterol exhibited a single rotational correlation time near one nanosecond that was relatively independent of temperature and low concentrations of membrane cholesterol. The limiting anisotropy of 0.9 mole percent dehydroergosterol decreased above the gel-to-liquid crystalline phase transition in membranes without cholesterol and was not significantly affected by the phase transition in membranes containing 5 mole percent cholesterol. These results suggested hindered rotational diffusion of dehydroergosterol in multilamellar liposomes. Lifetime and time-correlated fluorescence measurements of 0.9 mole percent dehydroergosterol in multilamellar liposomes further suggested this fluorophore was detecting physical properties of the bulk membrane phospholipids in membranes devoid of cholesterol and was detecting sterol-rich regions in membranes of low sterol concentration.     

Fluorescence properties of cholestatrienol in phosphatidylcholine bilayer vesicles

The fluorescent sterol delta 5,7,9,(11)-cholestatrien-3 beta-ol (cholestatrienol) was incoporated into 1-palmitoyl-2-oleoyl-phosphatidylcholine (POPC) small unilamellar vesicles (SUV) with and without cholesterol in order to monitor sterol-sterol interactions in model membranes. Previously another fluorescent sterol, dehydroergosterol (F. Schroeder, Y. Barenholz, E. Gratton and T.E. Thompson. Biochemistry 26 (1987) 2441), was used for this purpose. However, there is some concern that dehydroergosterol may not be the best analogue for cholesterol. Fluorescence properties of cholestatrienol in POPC SUV were highly sensitive to cholestatrienol purity. The fluorescence decay of cholestatrienol in the POPC SUV was analyzed by assuming either that the decay is comprised of a discrete sum of exponential components or that the decay is made up of one or more component's distribution of lifetimes. The decay for cholestatrienol in POPC SUV analyzed using distributions had a lower chi 2 value and was described by a two-component Lorentzian function with centers near 0.86 and 3.24 ns, and fractional intensities of 0.96 and 0.04, respectively. Both distributions were quite narrow, i.e., 0.05 ns full-width at half-maximum peak height. It is proposed that the two lifetime distributions are generated by separate continua of environments for the cholestatrienol molecule described by different dielectric constants. In the range 0-6 mol% cholestatrienol, the cholestatrienol underwent a concentration-dependent relaxation. This process was characterized by red-shifted absorption and maxima and altered ratios of absorption and fluorescence excitation maxima. Fluorescence quantum yield, lifetime, steady-state anisotropy, limiting anisotropy and rotational rate remained constant. In contrast, in POPC vesicles containing between 6 and 33 mol% cholestatrienol, the fluorescent cholestatrienol partially segregated, resulting in quenching. Thus, below 6 mol% cholestatrienol, the cholestatrienol appeared to behave in part as monomers exposed to some degree to the aqueous solvent in a sterol-poor domain within POPC bilayers. Since the lifetime did not decrease above 6 mol% cholestatrienol, the fluorescence at high mol% values of cholestatrienol was due to cholestatrienol in the sterol-poor domain. The fluorescence intensity, quantum yield, steady-state anisotropy, and limiting anisotropy of cholestatrienol in the sterol-poor domain decreased to limiting, nonzero values while the rotational rate increased to a limiting value. Thus, the sterol-poor domain became more disordered when it coexisted with the sterol-rich domain.(ABSTRACT TRUNCATED AT 400 WORDS)     

Organization and interaction of cholesterol and phosphatidylcholine in model bilayer membranes

The molecular organization of sterols in liposomes of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) at 37 degrees C is examined by utilizing the fluorescent analogue of cholesterol cholesta-5,7,9-trien-3 beta-ol (cholestatrienol). (1) Cholestatrienol is shown to be indistinguishable from native cholesterol in terms of its ability to condense POPC, as determined by (i) pressure/area studies of mixed-lipid monolayers and (ii) its ability to increase the order of POPC bilayers (determined by electron spin resonance studies) whether on its own or admixed with cholesterol at various ratios. (2) By analysis of the perturbation of the absorption spectra, cholestatrienol was found to be freely miscible in aggregates of cholesterol in buffer. In contrast, a lack of any detectable direct interaction of the sterol molecules in POPC bilayers was detected. (3) Fluorescence intensity and lifetime measurements of POPC/sterol (1:1 mol/mol) at various cholesterol/cholestratrienol molar ratios (0.5:1 up to 1:1 cholestatrienol/POPC) confirmed that sterol molecules in the membrane matrix were not associated to any great degree. (4) A quantitative estimate of how close sterol molecules approach each other in the membrane matrix was evaluated from the concentration dependence of the steady-state depolarization of fluorescence and was found to be 10.6 A. From geometrical considerations, the sterol/phospholipid phase at 1:1 mol/mol is depicted as each sterol having four POPC molecules as nearest neighbors. We term this arrangement of the lipid matrix an "ordered bimolecular mesomorphic lattice". (5) The concentration dependence of depolarization of fluorescence of cholestatrienol in POPC liposomes in the absence of cholesterol yielded results that were consistent with the cholestatrienol molecules being homogeneously dispersed throughout the phospholipid phase at sterol/POPC ratios of less than 1:1. (6) From qualitative calculations of the van der Walls' hydrophobic interactions of the lipid species, the phospholipid condensing effect of cholesterol is postulated to arise from increased interpenetration of the flexible methylene segments of the acyl chains, as a direct result of their greater mutual attraction compared to their attraction for neighboring sterol molecules. (7) The interdependence of the ordered bimolecular mesomorphic lattice and the acyl chain condensation is discussed in an effort to understand the ability of cholesterol to modulate the physical and mechanical properties of biological membranes.     

Influence of sterol structure on phospholipid phase behavior as detected by parinaric acid fluorescence spectroscopy

Phospholipid-sterol interactions were investigated using parinaric acid fluorescence spectroscopy. Cholesterol and cholesterol analogues which were modified in the sterol nucleus or side chain were added at 50 mol % to multilamellar vesicles of model phospholipids selected to be representative of major components in an LM cell plasma membrane. These included sphingomyelins and saturated and monounsaturated phosphatidylcholines and phosphatidylethanolamines. Based on the changes in cis-parinaric acid steady-state fluorescence polarization observed with addition of sterol, 50 mol % cholesterol abolished the phase transition of all the model phospholipids. Dihydrocholesterol and trans-22-dehydrocholesterol behaved like cholesterol in the two systems studied. 24-Methylcholesterols interacted well with all phospholipids except phosphatidylethanolamine which contained an unsaturated fatty acid. 24-Alkyl,trans-22-dehydrocholesterols abolished the phase transition in only two systems: sphingomyelins and phosphatidylcholines possessing relatively short saturated acyl chains. Since steady-state anisotropy is a function of fluorescence lifetime, rotational diffusion rates, and limiting anisotropy, we determined these parameters for two of the phospholipid systems. The results show that steady-state anisotropy values for phospholipid-sterol interactions correlate closely with limiting anisotropy and to a lesser extent with rotational relaxation time. The behavior of the sterols in the model phospholipids are used to interpret 1) fluorescence polarization measurements made with phospholipids extracted from LM cell plasma membranes, and 2) changes in membrane lipid composition which accompany growth of LM cells on various sterols.     

Domain formation and stability in complex lipid bilayers as reported by cholestatrienol

In this study, we used cholestatrienol (CTL) as a fluorescent reporter molecule to study sterol-rich L(o) domains in complex lipid bilayers. CTL is a fluorescent cholesterol analog that mimics the behavior of cholesterol well. The ability of 12SLPC to quench the fluorescence of cholestatrienol gives a measure of the amount of sterol included in L(o) domains in mixed lipid membranes. The stability of sterol-rich domains formed in complex lipid mixtures containing saturated sphingomyelins, phosphatidylcholines, or galactosylceramide as potential domain-forming lipids were studied. The amount of sterol associated with sterol-rich domains seemed to always increase with increasing temperature. The quenching efficiency was highly dependent on the domain-forming lipid present in complex lipid mixtures. Sphingomyelins formed stable sterol-enriched domains and were able to shield CTL from quenching better than the other lipids included in this study. The saturated phosphatidylcholines also formed sterol-rich domains, but the quenching efficiency in membranes with these was higher than with sphingomyelins and the domains melted at lower temperatures. PGalCer was not able to form sterol-enriched domains. However, we found that PGalCer stabilized sterol-rich domains formed in PSM-containing bilayers. Using a fluorescent ceramide analog, we also demonstrated that N-palmitoyl-ceramide displaced the sterol from sphingolipid-rich domains in mixed bilayer membranes.     

Reevaluation of the wobbling dynamics of diphenylhexatriene in phosphatidylcholine and cholesterol/phosphatidylcholine membranes

The dynamics of lipid hydrocarbon chains in phosphatidylcholine (dimyristoyl- or dipalmitoyl-) and cholesterol/dimyristoylphosphatidylcholine membranes were investigated by nanosecond time-resolved fluorescence depolarization measurements on a lipophilic fluorescent probe 1,6-diphenyl-1,3,5-hexatriene embedded in the membranes. In the pure lipid membranes, both the range (amplitude) and the rate of the wobbling motion of the probe increased sigmoidally with temperature reflecting the thermotropic phase transition of the lipid. The rise in the rate slightly preceded the increase in the range, suggesting that the fluctuation of lipid chains is activated to a high level before the ordered array of chains melt into the liquid-crystalline phase. Above the transition temperature, incorporation of cholesterol resulted in a dramatic decrease in the range of wobbling motion while the rate remained high. Below the transition, on the other hand, cholesterol had little effect on the range, whereas the rate was greatly increased. These effects of cholesterol are remarkably similar to the effects of cytochrome oxidase on lipid chain dynamics (Kinosita, K., Jr., Kawato, S., Ikegami, A., Yoshida, S. and Orii, Y. (1981) Biochim. Biophys. Acta 647, 7–17).     

Dynamics and heterogeneity of bovine hippocampal membranes: Role of cholesterol and proteins

The structural and dynamic consequence of alterations in membrane lipid composition (specifically cholesterol) in neuronal membranes is poorly understood. Previous work from our laboratory has established bovine hippocampal membranes as a convenient natural source for studying neuronal receptors. In this paper, we have explored the role of cholesterol and proteins in the dynamics and heterogeneity of bovine hippocampal membranes using fluorescence lifetime distribution analysis of the environment-sensitive fluorescent probe Nile Red incorporated into such membranes by the maximum entropy method (MEM), and time-resolved fluorescence anisotropy measurements. The peak position and the width of the lifetime distribution of Nile Red show a progressive reduction with increasing cholesterol depletion from native hippocampal membranes indicating that the extent of heterogeneity decreases with decrease in membrane cholesterol content. This is accompanied by a concomitant decrease of the fluorescence anisotropy and rotational correlation time. Our results point out that the microenvironment experienced by Nile Red is relatively insensitive to the presence of proteins in hippocampal membranes. Interestingly, Nile Red lifetime distribution in liposomes of lipid extracts is similar to that of native membranes indicating that proteins do not contribute significantly to the high level of heterogeneity observed in native membranes. These results could be relevant in understanding the neuronal diseases characterized by defective membrane lipid metabolism.     

Dynamics and heterogeneity of bovine hippocampal membranes: role of cholesterol and proteins

The structural and dynamic consequence of alterations in membrane lipid composition (specifically cholesterol) in neuronal membranes is poorly understood. Previous work from our laboratory has established bovine hippocampal membranes as a convenient natural source for studying neuronal receptors. In this paper, we have explored the role of cholesterol and proteins in the dynamics and heterogeneity of bovine hippocampal membranes using fluorescence lifetime distribution analysis of the environment-sensitive fluorescent probe Nile Red incorporated into such membranes by the maximum entropy method (MEM), and time-resolved fluorescence anisotropy measurements. The peak position and the width of the lifetime distribution of Nile Red show a progressive reduction with increasing cholesterol depletion from native hippocampal membranes indicating that the extent of heterogeneity decreases with decrease in membrane cholesterol content. This is accompanied by a concomitant decrease of the fluorescence anisotropy and rotational correlation time. Our results point out that the microenvironment experienced by Nile Red is relatively insensitive to the presence of proteins in hippocampal membranes. Interestingly, Nile Red lifetime distribution in liposomes of lipid extracts is similar to that of native membranes indicating that proteins do not contribute significantly to the high level of heterogeneity observed in native membranes. These results could be relevant in understanding the neuronal diseases characterized by defective membrane lipid metabolism.     

Effect of cholesterol on molecular order and dynamics in highly polyunsaturated phospholipid bilayers.

The effect of cholesterol on phospholipid acyl chain packing in bilayers consisting of highly unsaturated acyl chains in the liquid crystalline phase was examined for a series of symmetrically and asymmetrically substituted phosphatidylcholines (PCs). The time-resolved fluorescence emission and decay of fluorescence anisotropy of 1,6-diphenyl-1,3,5-hexatriene (DPH) was used to characterize equilibrium and dynamic structural properties of bilayers containing 30 mol % cholesterol. The bilayers were composed of symmetrically substituted PCs with acyl chains of 14:0, 18:1n9, 20:4n6, or 22:6n3, containing 0, 1, 4, or 6 double bonds, respectively, and mixed-chain PCs with a saturated 16:0 sn-1 chain and 1, 4, or 6 double bonds in the sn-2 chain. DPH excited-state lifetime was fit to a Lorentzian lifetime distribution, the center of which was increased 1-2 ns by 30 mol % cholesterol relative to the cholesterol-free bilayers. Lifetime distributions were dramatically narrowed by the addition of cholesterol in all bilayers except the two consisting of dipolyunsaturated PCs. DPH anisotropy decay was interpreted in terms of the Brownian rotational diffusion model. The effect of cholesterol on both the perpendicular diffusion coefficient D perpendicular and the orientational distribution function f(theta) varied with acyl chain unsaturation. In all bilayers, except the two dipolyunsaturated PCs, 30 mol % cholesterol dramatically slowed DPH rotational motion and restricted DPH orientational freedom. The effect of cholesterol was especially diminished in di-22:6n3 PC, suggesting that this phospholipid may be particularly effective at promoting lateral domains, which are cholesterol-rich and unsaturation-rich, respectively. The results are discussed in terms of a model for lipid packing in membranes containing cholesterol and PCs with highly unsaturated acyl chains.     

Cyclodextrin-catalyzed extraction of fluorescent sterols from monolayer membranes and small unilamellar vesicles

This study examined the kinetics of sterol desorption from monolayer and small unilamellar vesicle membranes to 2-hydroxypropyl-beta-cyclodextrin. The sterols used include cholesterol, dehydroergosterol (ergosta-5,7,9,(11),22-tetraen-3beta-ol) and cholestatrienol (cholesta-5,7,9,(11)-trien-3beta-ol). Desorption rates of dehydroergosterol and cholestatrienol from pure sterol monolayers were faster (3.3-4.6-fold) than the rate measured for cholesterol. In mixed monolayers (sterol: 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine 30:70 mol%), both dehydroergosterol and cholestatrienol desorbed faster than cholesterol. clearly indicating a difference in interfacial behavior of these sterols. In vesicle membranes desorption of dehydroergosterol was slower than desorption of cholestatrienol, and both rates were markedly affected by the phospholipid composition. Desorption of sterols was slower from sphingomyelin as compared to phosphatidylcholine vesicles. Desorption of fluorescent sterols was also faster from vesicles prepared by ethanol-injection as compared to extruded vesicles. The results of this study suggest that dehydroergosterol and cholestatrienol differ from cholesterol in their membrane behavior, therefore care should be exercised when experimental data derived with these probes are interpreted.     

Sterol and squalene carrier protein interactions with fluorescent delta 5,7,9(11)-cholestatrien-3 beta-ol

The fluorescent sterol delta 5,7,9(11)-cholestatrien-3 beta-ol (cholestatrienol) was used as an analogue of cholesterol to determine the properties of the sterol in aqueous buffer and the interaction of cholesterol with sterol and squalene carrier protein (SCP). Cholestatrienol was synthesized and purified to a stable product by reverse phase high performance liquid chromatography. The critical micelle concentration of cholestatrienol in aqueous buffer was 1 nM while its maximum solubility was 1.15 microM as ascertained from fluorescence polarization and light scattering properties, respectively. Several lines of evidence indicated a close molecular interaction of cholestatrienol with purified rat liver SCP. The fluorescence emission spectrum of monomeric cholestatrienol in aqueous buffer was blue shifted upon addition of SCP. The fluorescence lifetime of monomeric cholestatrienol in aqueous buffer was increased by SCP from 5 to 12 ns. The SCP increased the fluorescence polarization of monomeric cholestatrienol from 0.002 to 0.38 in aqueous buffer. The close molecular interaction of cholestatrienol with SCP was also demonstrated by energy transfer experiments. Fluorescence energy transfer from tyrosine residues of SCP to the conjugated triene fluorophore in cholestatrienol had a transfer efficiency of 59%. R, the apparent distance between the tyrosine energy donor and the cholestatrienol energy acceptor, was 16.3 A. Binding analysis indicated that cholestatrienol interacted with SCP with an apparent KD = 0.5 microM and a Bmax = 3.54 microM. One mol of cholestatrienol was bound per mol of SCP. These results demonstrate the utility of cholestatrienol not only as a membrane sterol probe molecule but also as a probe for sterol-protein interactions.     

Fluidity and lipid composition of oat and rye shoot plasma membrane: effect of sterol perturbation by xenobiotics

Oat and rye plants were treated with either tetcyclacis (an experimental plant growth regulator), nuarimol (a fungicide) or gamma-ketotriazole (an experimental herbicide). These treatments reduced shoot growth and changed the lipid composition of the shoot plasma membranes. In oat, both tetcyclacis and nuarimol treatments increased plasma membrane cholesterol and increased the phosphatidylethanolamine/phosphatidylcholine (PE/PC) ratio, whereas gamma-ketotriazole treatment reduced cholesterol and the PE/PC ratio. In rye, all treatments reduced the PE/PC ratio. Generally, the sterol/phospholipid ratio was less in oat than in rye but the cholesterol/phospholipid ratio was greater. With all treatments in oat and rye, increases were observed in unsaturation of the phospholipid acyl chains. The fluidity of membranes was measured by steady-state fluorescence polarisation of the probe diphenylhexatriene; oat membranes were more fluid than rye. Membrane fluidity was greater in plasma membranes from plants treated with the xenobiotics than the controls. The results are discussed in the context of the effect of plasma membrane lipid composition on membrane fluidity, and it is concluded that there appears to be no overall simple relationship between membrane lipid composition and fluidity that holds for all treatments in both species.     

Fluorescence studies of dehydroergosterol in phosphatidylethanolamine/phosphatidylcholine bilayers

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

Variations in dietary triacylglycerol saturation alter the lipid composition and fluidity of rat intestinal plasma membranes

Rats were maintained on nutritionally complete diets enriched in unsaturated (corn oil) or saturated (butter fat) triacylglycerols. After 6 weeks, significant differences in the lipid composition and fluidity of a number of intestinal membranes were observed. The corn oil diet (enriched mainly in linoleic acid) increased the overall unsaturation of the acyl chains and enhanced the lipid fluidity, as assessed by the fluorescence anisotropy of 1,6-diphenyl-1,3,5-hexatriene, of enterocyte microvillus and basolateral membranes and of colonocyte basolateral membranes. Concomitantly, the cholesterol content and the cholesterol/phospholipid molar ratio were increased in the microvillus but not in the basolateral membranes. The increased cholesterol in ileal microvillus membranes can result from enhanced cellular biosynthesis, since ileal slices from rats fed the unsaturated diet incorporated [14C]octanoate more rapidly into digitonin-precipitable sterol. Increased fluidity of the enterocyte microvillus and basolateral membranes, respectively, enhanced the enzyme specific activities of p-nitrophenylphosphatase and (Na+ + K+)-dependent adenosine triphosphatase. The results indicate that the lipid composition, fluidity and enzyme activities of intestinal plasma membranes can be altered by dietary means. Moreover, rat enterocytes possess regulatory mechanisms which modulate the cholesterol content of the microvillus membranes so as to mitigate changes in lipid fluidity.     

Physical properties of the fluorescent sterol probe dehydroergosterol

Spectroscopic studies were performed on the fluorescent sterol probes ergosta-5,7,9(11),22-tetraen-3 beta-ol (dehydroergosterol) and cholesta-5,7,9(11)-trien-3 beta-ol (cholestatrienol). In most isotropic solvents, these molecules exhibited a single lifetime near 300 ps. Fluorescence lifetimes in 2-propanol were independent of emission wavelength and independent of excitation wavelength. Excited state behavior of these probes appears relatively simple. In isotropic solvents, dehydroergosterol fluorescence emission underwent at most a small Stokes shift as solvent polarity was modified. Time-resolved anisotropy decays indicated that dehydroergosterol decay was monoexponential, with rotational correlation times dependent on solvent viscosity. When incorporated into L-alpha-dimyristoylphosphatidylcholine liposomes at a concentration of 0.9 mol%, dehydroergosterol fluorescence lifetime decreased at the phase transition of this phospholipid indicating that the sterol probe was detecting physical changes of the bulk phospholipids. Furthermore, total fluorescence decays and anisotropy decays were sensitive to the environment of the sterol. Dehydroergosterol and cholestatrienol are thus useful probes for monitoring sterol behavior in biological systems.     

Membrane effects of lysozyme amyloid fibrils

The influence of mature lysozyme fibrils on the structural and physical properties of model membranes composed of phosphatidylcholine (PC) and its mixtures with cardiolipin (CL) (10 mol%) and cholesterol (Chol) (30 mol%) was studied using fluorescent probes DPH, pyrene, Laurdan and MBA. Analysis of pyrene fluorescence spectra along with the measurements of DPH fluorescence anisotropy revealed that the structure of hydrocarbon chains region of lipid bilayer is not affected by the fibrillar aggregates of lysozyme. In contrast, probing the membrane effects by Laurdan and MBA showed the rise of both the generalized polarization of Laurdan and the MBA fluorescence anisotropy, suggesting that amyloid protein induces reduction of bilayer hydration and increase of lipid packing in the interfacial region of model membranes.     

Sterol affinity for bilayer membranes is affected by their ceramide content and the ceramide chain length

It is known that ceramides can influence the lateral organization in biological membranes. In particular ceramides have been shown to alter the composition of cholesterol and sphingolipid enriched nanoscopic domains, by displacing cholesterol, and forming gel phase domains with sphingomyelin. Here we have investigated how the bilayer content of ceramides and their chain length influence sterol partitioning into the membranes. The effect of ceramides with saturated chains ranging from 4 to 24 carbons in length was investigated. In addition, unsaturated 18:1- and 24:1-ceramides were also examined. The sterol partitioning into bilayer membranes was studied by measuring the distribution of cholestatrienol, a fluorescent cholesterol analogue, between methyl-β-cyclodextrin and large unilamellar vesicle with defined lipid composition. Up to 15 mol% ceramide was added to bilayers composed of DOPC:PSM:cholesterol (3:1:1), and the effect on sterol partitioning was measured. Both at 23 and 37 °C addition of ceramide affected the sterol partitioning in a chain length dependent manner, so that the ceramides with intermediate chain lengths were the most effective in reducing sterol partitioning into the membranes. At 23 °C the 18:1-ceramide was not as effective at inhibiting sterol partitioning into the vesicles as its saturated equivalent, but at 37 °C the additional double bond had no effect. The longer 24:1-ceramide behaved as 24:0-ceramide at both temperatures. In conclusion, this work shows how the distribution of sterols within sphingomyelin-containing membranes is affected by the acyl chain composition in ceramides. The overall membrane partitioning measured in this study reflects the differential partitioning of sterol into ordered domains where ceramides compete with the sterol for association with sphingomyelin.     

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.     

Manipulation of cholesterol levels in rod disk membranes by methyl-beta-cyclodextrin: effects on receptor activation

The effect of cholesterol on rod outer segment disk membrane structure and rhodopsin activation was investigated. Disk membranes with varying cholesterol concentrations were prepared using methyl-beta-cyclodextrin as a cholesterol donor or acceptor. Cholesterol exchange followed a simple equilibrium partitioning model with a partition coefficient of 5.2 +/- 0.8 in favor of the disk membrane. Reduced cholesterol in disk membranes resulted in a higher proportion of photolyzed rhodopsin being converted to the G protein-activating metarhodopsin II (MII) conformation, whereas enrichment of cholesterol reduced the extent of MII formation. Time-resolved fluorescence anisotropy measurements using 1,6-diphenyl-1,3,5-hexatriene showed that increasing cholesterol reduced membrane acyl chain packing free volume as characterized by the parameter f(v). The level of MII formed showed a positive linear correlation with f(v) over the range of 4 to 38 mol % cholesterol. In addition, the thermal stability of rhodopsin increased with mol % of cholesterol in disk membranes. No evidence was observed for the direct interaction of cholesterol with rhodopsin in either its agonist- or antagonist-bound form. These results indicate that cholesterol mediates the function of the G protein-coupled receptor, rhodopsin, by influencing membrane lipid properties, i.e. reducing acyl chain packing free volume, rather than interacting specifically with rhodopsin.     

A fluorescence and radiolabel study of sterol exchange between membranes

The fluorescent sterols delta 5,7,9(11),22-ergostatetraen-3 beta-ol (dehydroergosterol) and delta 5,7,9,(11)-cholestatrien-3 beta-ol (cholestatrienol) as well as [1,2-3H]cholesterol were utilized as cholesterol analogues to examine spontaneous exchange of sterol between 1-palmitoyl-2-oleoylphosphatidylcholine (POPC) small unilamellar vesicles (SUV). Exchange of fluorescent sterols was monitored at 24 degrees C by release from self-quenching of polarization from the time of mixing without separation of donor and acceptor vesicles. The polarization curve for 35 mol% sterol in POPC best fitted a two-exponential function, with a fast-exchange rate constant k1 = 0.0217 min-1, 1t1/2 = 32 min, size pool 1 = 12%, and a slow rate constant k2 = 2.91.10(-3) min-1, 2t1/2 = 238 min, size pool 2 = 88%. In addition to the above two exchangeable pools of sterol, the data were consistent with the presence of a slowly or nonexchangeable pool, 42% of total sterol, that was highly dependent on sterol content. These results were confirmed by simultaneous monitoring of [1,2-3H]cholesterol radioactivity and dehydroergosterol fluorescence intensity after separation of donor and acceptor vesicles by ion-exchange column chromatography. Thus, dehydroergosterol or cholestatrienol exchange as measured by fluorescence parameters (polarization and/or intensity) provides two new methods to follow cholesterol spontaneous exchange. These methods allow resolution and quantitation of a shorter exchange t1/2 near 30 min previously not reported. Thus, the cholesterol desorption rate from membranes may be faster than previously believed. In addition, the presence of a slowly non-exchangeable pool was confirmed.