Fluorescence lifetime distributions of diphenylhexatriene-labeled phosphatidylcholine as a tool for the study of phospholipid-cholesterol interactions.

Fluorescence lifetimes of 1-palmitoyl-2-diphenylhexatrienylpro-pionyl-phosphatidylc hol ine in vesicles of palmitoyloleoyl phosphatidylcholine (POPC) (1:300, mol/mol) in the liquid crystalline state were determined by multifrequency phase fluorometry. On the basis of statistic criteria (chi 2red) the measured phase angles and demodulation factors were equally well fitted to unimodal Lorentzian, Gaussian, or uniform lifetime distributions. No improvement in chi 2red could be observed if the experimental data were fitted to bimodal Lorentzian distributions or a double exponential decay. The unimodal Lorentzian lifetime distribution was characterized by a lifetime center of 6.87 ns and a full width at half maximum of 0.57 ns. Increasing amounts of cholesterol in the phospholipid vesicles (0-50 mol% relative to POPC) led to a slight increase of the lifetime center (7.58 ns at 50 mol% sterol) and reduced significantly the distributional width (0.14 ns at 50 mol% sterol). Lifetime distributions of POPC-cholesterol mixtures containing greater than 20 mol% sterol were within the resolution limit and could not be distinguished from monoexponential decays on the basis of chi 2red. Cholesterol stabilizes and rigidifies phospholipid bilayers in the fluid state. Considering its effect on lifetime distributions of fluorescent phospholipids it may also act as a membrane homogenizer.     

Effect of proteins on fluorophore lifetime heterogeneity in lipid bilayers.

The effect of three different membrane proteins on the fluorescence lifetime heterogeneity of 1,6-diphenyl-1,3,5-hexatriene (DPH) in phospholipid vesicle systems was investigated. For large unilamellar vesicles of dimyristoylphosphatidylcholine (DMPC) and 1-palmitoyl-2-oleoylphosphatidylcholine (POPC) at 37 degrees C, the fluorescence decay was essentially monoexponential (8.6 and 8.2 ns, respectively) except for a minor component typical of DPH. For gramicidin D reconstituted into DMPC vesicles at a protein/lipid molar ratio of 1/7, the most appropriate analysis of the data was found to be in the form of a bimodal Lorentzian distribution. Centers of the major lifetime components were almost identical with those recovered for vesicles without proteins, while broad distributional widths of some 4.0 ns were recovered. Variation of the protein/lipid molar ratio in sonicated POPC vesicles revealed an abrupt increase in distributional width at ratios approximating 1/15-1/20, which leveled off at about 2.5 ns. For bacteriorhodopsin in DMPC vesicles and cytochrome b5 in POPC, the most appropriate analysis of the data was again found to be in the form of a bimodal Lorentzian also with broad distributional widths in the major component. Lifetime centers were decreased for these proteins due to fluorescence energy transfer to the retinal of the bacteriorhodopsin and heme of the cytochrome b5. Fluorescence energy transfer is distance dependent, and since a range of donor-acceptor distances would be expected in a membrane, lifetime distributions should therefore be recovered independently of other effects for proteins possessing acceptor chromophores.(ABSTRACT TRUNCATED AT 250 WORDS)     

Time-resolved fluorescence investigation of membrane cholesterol heterogeneity and exchange

The fluorescent sterol delta 5,7,9(11),22-ergostatetraen-3 beta-ol (dehydroergosterol) was investigated as a cholesterol analogue to examine sterol domains in and spontaneous exchange of sterol between 1-palmitoyl-2-oleoylphosphatidylcholine (POPC) small unilamellar vesicles (SUV). Fluorescence lifetime, acrylamide quenching analyses, and intermembrane exchange kinetics were consistent with the presence of at least two sterol domains in POPC. Fluorescence lifetime was determined by phase and modulation fluorescence spectroscopy and analyzed by nonlinear least-squares as well as continuous distributional analyses. Both methods demonstrated that pure dehydroergosterol in POPC SUV had two lifetime components (C) and fractional intensities (F) near C1 = 0.851 ns (F1 0.96) and C2 = 2.668 ns (F2 0.004). In contrast to component C1, the center of lifetime distribution, fractional intensity, and peak width of dehydroergosterol lifetime component C2 was dependent on the polarity of the medium and vesicle curvature. The sterol domain corresponding to dehydroergosterol component C2 was preferentially quenched by acrylamide. Acrylamide quenching of dehydroergosterol fluorescence demonstrated that the two lifetime components of dehydroergosterol were not due to transbilayer sterol domains with different lifetimes. In a spontaneous exchange assay not requiring separation of donor and acceptor SUV, the lifetime component C2, but not C1, shifted to a shorter lifetime with altered distributional width. The kinetics of these lifetime and distributional width changes best fitted a two-exponential function, with a fast exchange rate constant K1 = 0.0325 min-1, t1/2 = 21.3 min, and a slow rate constant k2 = 0.00275 min-1, t1/2 = 261 min. The fast exchanging pool correlates with the longer lifetime component C2. These kinetics were confirmed both by dehydroergosterol exchange measured with fluorescence intensity and by [3H]cholesterol exchange. In summary, lifetime, distributional width, acrylamide quenching, and classical exchange assay data are consistent with the presence of at least two pools of sterol in POPC SUV.     

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)     

Time-resolved fluorescence of apoferritin and its subunits

The decay of the intrinsic fluorescence of the apoferritin polymer and its subunits has been studied by pulse and phase shift techniques. Both techniques show that the fluorescence decay of all the samples tested cannot be described by a single exponential function. The fluorescence decay data of the apoferritin subunits obtained with either technique can be fitted satisfactorily with a function resulting from the sum of two exponential components. However, the polymer data obtained with the high resolution phase shift technique operated either by synchrotron radiation or by a mode-locked argon ion laser can be fitted better using a bimodal gaussian continuous distribution of lifetime components. The molecular basis for this distribution of lifetime values may lie in the heterogeneity of the tryptophan environment generated by the assembly of the subunits into the polymer. The binding of the first 100 irons to apoferritin quenches the intrinsic fluorescence without affecting the lifetimes in a proportional way. This finding may be taken as an indication that the quenching of the tryptophan fluorescence induced by the binding of iron has both static and dynamic components.     

Fluorescence lifetime distributions of 1,6-diphenyl-1,3,5-hexatriene in phospholipid vesicles

The fluorescence emission properties of 1,6-diphenyl-1,3,5-hexatriene (DPH) in 1,2-dipalmitoyl-3-sn-phosphatidylcholine and 1,2-dimyristoyl-3-sn-phosphatidylcholine multilamellar vesicles have been measured by using multifrequency phase fluorometry. The fluorescence decay of DPH in the phospholipid vesicles has been analyzed by assuming either that the decay is made up of a discrete sum of exponential components or that the decay is made up of one or more continuous distributions of lifetime components. The fit of the decay curve using exponentials required at least two terms, and the reduced X2 was relatively large. The fit using a continuous distribution of lifetime values used two continuous components. Several symmetric distribution functions were used: uniform, Gaussian, and Lorentzian. The distribution function that best described the decay was the Lorentzian. The full width at half-maximum of the Lorentzian distribution was about 0.6 ns at temperatures below the phase transition temperature. At the phospholipid phase transition and at higher temperatures, the distribution became quite narrow, with a width of about 0.1 ns. It is proposed that the lifetime distribution is generated by a continuum of different environments of the DPH molecule characterized by different dielectric constants. Below the transition temperature in the gel phase, the dielectric constant gradient along the membrane normal determines the distribution of decay rates. Above the transition, in the liquid-crystalline phase, the translational and rotational mobility of the DPH molecule increases, and the DPH experiences an average environment during the excited-state lifetime. Consequently, the distribution becomes narrower.(ABSTRACT TRUNCATED AT 250 WORDS)     

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.     

Interaction of sphingomyelins and phosphatidylcholines with fluorescent dehydroergosterol

The fluorescent sterol dehydroergosterol was used as a cholesterol analogue in conjunction with multifrequency phase and modulation (1-250 MHz) fluorometry to examine whether sterols (1) interact preferentially with fluid- or solid-phase phospholipids and (2) interact preferentially with sphingomyelin in phase-separated or phase-miscible cosonicated phospholipid membranes. Cosonicated small unilamellar vesicles (SUV) were produced by mixing lipids in organic solvents, drying the mixture, adding buffer, sonicating, and separating SUV. Phospholipids of synthetic as well as biological origin were utilized. In phase-separated, cosonicated SUV of dimyristoylphosphatidylcholine/distearoylphosphatidylcholine (DMPC/DSPC, 1:1 molar ratio), the fluorescent sterol (0.5 mol %) interacted preferentially with the fluid-phase lipid (partition coefficient, Kf/s = 2.6-3.4) according to four criteria. First, dehydroergosterol detected only the phase transition of DMPC, the phospholipid with the lower phase transition temperature. Second, the dehydroergosterol fluorescence polarization, limiting anisotropy, order parameter, and rotational relaxation time in the cosonicated vesicle were similar to those of dehydroergosterol in SUV composed only of DMPC. Third, the number of dehydroergosterol fluorescence lifetime components as well as the distribution in the cosonicated SUV was similar to that of dehydroergosterol in SUV composed of DMPC. Fourth, dehydroergosterol concentration-dependent self-quenching was detected in DSPC SUV at much lower dehydroergosterol concentration than in DMPC SUV. Preference of dehydroergosterol for fluid-phase lipids was also observed by monitoring dehydroergosterol exchange between individually sonicated DMPC SUV and DSPC SUV after the two types of vesicles were mixed in equal proportions. In these SUV mixtures, the dehydroergosterol also partitioned into the more fluid SUV, 99:1.(ABSTRACT TRUNCATED AT 250 WORDS)     

Detection of lipid domains in model and cell membranes by fluorescence lifetime imaging microscopy of fluorescent lipid analogues

The presence of lipid domains in cellular membranes and their characteristic features are still an issue of dividing discussion. Several recent studies implicate lipid domains in plasma membranes of mammalian cells as short lived and in the submicron range. Measuring the fluorescence lifetime of appropriate lipid analogues is a proper approach to detect domains with such properties. Here, the sensitivity of the fluorescence lifetime of1-palmitoyl-2-[6-[(7-nitro-2-1,3-benzoxadiazol-4-yl)amino]-hexanoyl]-sn-glycero-3-phospholipid (C6-NBD-phospholipid) analogues has been employed to characterize lipid domains in giant unilamellar vesicles (GUVs) and the plasma membrane of mammalian cells by fluorescence lifetime imaging (FLIM). Fluorescence decay of C6-NBD-phosphatidylcholine is characterized by a short and long lifetime. For GUVs forming microscopically visible lipid domains the longer lifetime in the liquid disordered (ld) and the liquid ordered (lo) phase was clearly distinct, being approximately 7 ns and 11 ns, respectively. Lifetimes were not sensitive to variation of cholesterol concentration of domain-forming GUVs indicating that the lipid composition and physical properties of those lipid domains are well defined entities. Even the existence of submicroscopic domains can be detected by FLIM as demonstrated for GUVs of palmitoyloleoyl phosphatidylcholine/N-palmitoyl-d-sphingomyelin/cholesterol mixtures. A broad distribution of the long lifetime was found for C6-NBD-phosphatidylcholine inserted in the plasma membrane of HepG2 and HeLa cells centered around 11 ns. FLIM studies on lipid domains forming giant vesicles derived from the plasma membrane of HeLa cells may suggest that a variety of submicroscopic lipid domains exists in the plasma membrane of intact cells.     

A fluorescence study of dehydroergosterol in phosphatidylcholine bilayer vesicles

The fluorescent sterol delta 5,7,9(11),22-ergostatetraen-3 beta-ol (dehydroergosterol) was incorporated into 1-palmitoyl-2-oleoylphosphatidylcholine (POPC) small unilamellar vesicles (SUV) with and without cholesterol in order to monitor sterol-sterol interactions in model membranes. In the range 0-5 mol % fluorescent sterol, dehydroergosterol underwent a concentration-dependent relaxation characterized by red-shifted wavelengths of maximum absorption as well as altered ratios of absorbance maxima and fluorescence excitation maxima at 338 nm/324 nm. Fluorescence intensity per mole of dehydroergosterol increased up to 5 mol % in POPC vesicles. In contrast, quantum yield, steady-state anisotropy, limiting anisotropy, lifetime, and rotational rate remained relatively constant in this concentration range. Similarly, addition of increasing cholesterol in the range 0-5 mol % in the presence of 3 mol % dehydroergosterol also increased the fluorescence intensity per mole of dehydroergosterol, red-shifted wavelengths of maximum absorption, and altered ratios of absorbance maxima. In POPC vesicles containing between 5 and 33 mol % dehydroergosterol, the fluorescent dehydroergosterol interacted to self-quench, thereby decreasing the fluorescence intensity, quantum yield, steady-state anisotropy, and limiting anisotropy and increasing the rotational rate (decreased rotational relaxation time) of the fluorescent sterol. The fluorescence lifetime of dehydroergosterol remained unchanged. The results were in accord with the interpretation that below 5 mol% sterol, the sterols behaved as monomers exposed to some degree to the aqueous solvent in POPC bilayers.(ABSTRACT TRUNCATED AT 250 WORDS)     

Susceptibilities of phospholipid vesicles containing different sterols to amphotericin B-loaded lysophosphatidylcholine micelles

To investigate the susceptibilities of fungal and mammalian cells to amphotericin B (AmB), AmB-loaded lysophosphatidylcholine (LPC)micelles as drug delivery vehicles were incubated at 37 degrees C with phosphatidylcholine vesicles containing different sterols as model systems for fungal and mammalian cells. The binding and kinetics of AmB to sterols in the membranes were judged by UV-visible spectroscopy. In the 91% monomeric form, AmB interacted rapidly with ergosterol and slowly with 7-dehydrocholesterol (7-DHC), while it did not interact with cholesterol. In the 50% monomeric form, AmB formed complexes more rapidly with ergosterol or 7-DHC than in the monomeric form, whereas it did not still interact with cholesterol. The interaction was also characterized by resonance energy transfer between the fluorescent probe trimethylammonium diphenylhexatriene (TMA-DPH) and AmB. In the 91% monomeric form, AmB caused initial fluorescence quenching in bilayer membranes containing any sterol as well as sterol-free bilayer membranes due to the release of AmB and its incorporation within the membranes. However, a second phase of increasing fluorescence was found in the case of ergosterol alone. On the other hand, in the 47% monomeric form, AmB gave a biphasic intensity profile in membranes containing any sterol as well as sterol-free membranes. However, the extent of the second phase of increasing fluorescence intensity was markedly dependent upon sterol composition. Studies using sterol-containing vesicles provide important insights into the role of the aggregation state of AmB in its effects on cells.     

Pressure effects on the lateral distribution of cholesterol in lipid bilayers: a time-resolved spectroscopy study.

The effects of hydrostatic pressure and temperature on the phase behavior and physical properties of the binary mixture palmitoyloleoylphosphatidylcholine/cholesterol, over the 0-40 molar % range of cholesterol compositions, were determined from the changes in the fluorescence lifetime distribution and anisotropy decay parameters of the natural lipid trans-parinaric acid (t-PnA). Pressurized samples were excited with a Ti-sapphire subpicosecond laser, and fluorescence decays were analyzed by the quantified maximum entropy method. Above the transition temperature (T(T) = -5 degrees C), at atmospheric pressure, two liquid-crystalline phases, alpha and beta, are formed in this system. At each temperature and cholesterol concentration below the transition pressure, the fluorescence lifetime distribution pattern of t-PnA was clearly modulated by the pressure changes. Pressure increased the fraction of the liquid-ordered beta-phase and its order parameter, but it decreased the amount of cholesterol in this phase. Palmitoyloleoylphosphatidylcholine/cholesterol phase diagrams were also determined as a function of temperature and hydrostatic pressure.     

Effect of cholesterol on interaction of dibucaine with phospholipid vesicles: a fluorescence study

Interaction of the local anesthetic dibucaine with small unilamellar vesicles of dimyristoylphosphatidylcholine (DMPC) and dioleoyl phosphatidylcholine (DOPC) containing different mol percents of cholesterol has been studied by fluorescence spectroscopy. Fluorescence measurements on dibucaine in presence of phospholipid vesicles containing various amounts of cholesterol yielded a pattern of variation of wavelength at emission maximum and steady-state anisotropy which indicated that the microenvironment of dibucaine is more polar and flexible in membranes that contain cholesterol than in membranes without cholesterol. Experiments on quenching of fluorescence from membrane-associated dibucaine by potassium iodide showed a marked increase in quenching efficiency as the cholesterol content of the vesicles was increased, demonstrating increased accessibility of the iodide quenchers to dibucaine in the presence of cholesterol, when compared to that in its absence. Total emission intensity decay profiles of dibucaine yielded two lifetime components of approximately 1 ns and approximately 2.8--3.1 ns with mean relative contributions of approximately 25 and approximately 75%, respectively. The mean lifetime in vesicles was 20--30% smaller than in the aqueous medium and showed a moderate variation with cholesterol content. Fluorescence measurements at two different temperatures in DMPC SUVs, one at 33 degrees C, above the phase transition temperature and another at 25 degrees C, around the main phase transition, indicated two different mode of dibucaine localization. At 25 degrees C dibucaine partitioned differentially in presence and absence of cholesterol. However, at 33 degrees C the apparent partition coefficients remained unaltered indicating differences in the microenvironment of dibucaine in presence and absence of cholesterol in the phospholipid 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.     

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).     

Quantitative assessment of sterol traffic in living cells by dual labeling with dehydroergosterol and BODIPY-cholesterol

Cholesterol with BODIPY at carbon-24 of the side chain (BCh2) has recently been introduced as new cholesterol probe with superior fluorescence properties. We compare BCh2 with the intrinsically fluorescent dehydroergosterol (DHE), a well-established marker for cholesterol, by introducing simultaneous imaging of both sterols in model membranes and living cells. BCh2 had a lower affinity than DHE for the biologically relevant liquid-ordered phase in model membranes. Still, DHE and BCh2 trafficked from the plasma membrane to the endocytic recycling compartment (ERC) of BHK cells with identical kinetics. This transport pathway was strongly reduced after energy depletion of cells or expression of the dominant-negative clathrin heavy chain. The partitioning into lipid droplets of BHK and HeLa cells was higher for BCh2 than for DHE. Within droplets, the photodegradation of BCh2 was enhanced and followed a stretched exponential decay, while the fluorescence lifetime of BCh2 was comparable in various cellular regions. Our results indicate that BCh2 is suitable for analyzing sterol uptake pathways and inter-organelle sterol flux in living cells. The BODIPY-moiety affects lipid phase preference of the sterol probe and causes some differential targeting of BCh2 and DHE in cells with high fat content.     

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).     

Complexity of lipid domains and rafts in giant unilamellar vesicles revealed by combining imaging and microscopic and macroscopic time-resolved fluorescence

The application of fluorescence lifetime imaging microscopy to study gel/fluid and raftlike lipid domains in giant unilamellar vesicles (GUVs) is demonstrated here. Different regions of the ternary dipalmitoylphosphatidylcholine/dioleoylphosphatidylcholine/cholesterol phase diagram were studied. The head-labeled phospholipid Rhodamine-dioleoylphosphatidylethanolamine (Rhod-DOPE) was used as a fluorescent probe. Gel/fluid and liquid-ordered (l(o))/liquid-disordered (l(d)) phase separation were clearly visualized upon two-photon excitation. Fluorescence intensity decays in different regions of a GUV were also obtained with the microscope in fixed laser-beam configuration. The ensemble behavior of the system was studied by obtaining fluorescence intensity decays of Rhod-DOPE in nongiant vesicle suspensions. The fingerprints for gel/fluid coexistence and for the presence of l(o) raftlike phase, based on fluorescence lifetime imaging microscopy histograms and images, and on the fluorescence intensity decay parameters of Rhod-DOPE, are presented. The presence of three lipid phases in one single GUV is detected unequivocally. From the comparison of lifetime parameters, it can be concluded that the l(o) phase is formed in the binary dipalmitoylphosphatidylcholine/cholesterol but not in the dioleoylphosphatidylcholine/cholesterol mixture. The domains apparent in fluorescence intensity images have a more complex substructure revealed by analysis of the lifetime data. The potential applications of this combined imaging/microscopic/macroscopic methodology are discussed.     

Correlation of enzyme activities with fluorescence anisotropy of dansyl-labeled cytochrome b5/NADH-cytochrome-b5 reductase systems in phosphatidylcholine vesicles

The changes in steady-state fluorescence lifetimes and anisotropy decay parameters, as well as enzyme activities, of dansyl-labeled cytochrome b5 (DNS-cytochrome b5), on interaction with NADH-cytochrome-b5 reductase in DMPC vesicles, have been measured as a function of temperature. Steady-state fluorescence of DNS-cytochrome b5 in DMPC vesicles with and without cholesterol was increased on interaction with reductase at temperatures both above and below the DMPC phase transition. In all systems three fluorescence decay components of the dansyl label in DNS-cytochrome b5 were observed. In the reductase-containing system, the long (major) decay time component of DNS-cytochrome b5 and the fraction of the total fluorescence associated with this component increased over the temperature range 15-30 degrees C. In time-resolved anisotropy measurements, the order parameters of DNS-cytochrome b5 in DMPC vesicles increased on interaction with reductase at temperatures above the DMPC phase transition, and this increase was even more pronounced in cholesterol-containing vesicles, at temperatures from 15-30 degrees C. The enzyme activity of the DNS-cytochrome-b5 reductase system in DMPC vesicles was also greatly increased in the presence of cholesterol. These results show that interaction of vesicle-bound DNS-cytochrome b5 and NADH-cytochrome-b5 reductase leads to an increased degree of order of the dansyl-labeled cytochrome with little change in its rotational flexibility, and suggests that the increased order can be correlated with increased enzyme activity.