Use of laurdan fluorescence intensity and polarization to distinguish between changes in membrane fluidity and phospholipid order

Sialyltransferase activity has been determined in membrane preparations containing the Golgi apparatus that were isolated from atherosclerotic and normal human aortic intima as well as in plasma of patients with documented atherosclerosis and healthy donors by measuring the transfer of N-acetylneuraminic acid (NeuAc) from CMP-NeuAc to asialofetuin. The asialofetuin sialyltransferase activity was found to be 2 times higher in the atherosclerotic intima as compared to the normal intima and 2-fold higher in patients' plasma than in that from healthy donors. The mean values of the apparent Michaelis constant (K(m)) for the sialylating enzyme for both tissues did not differ and were close for the intima and plasma. In contrast, the maximal velocity (V(max)) was 2 times higher for the atherosclerotic intima than for the normal intima and 3 times higher for patients' plasma than for that of the donors. These results suggest that the activity of asialofetuin sialyltransferases of aortal intima is enhanced in atherosclerosis as is the secretion of their soluble forms into patients' plasma.     

Alterations in membrane fluidity during keratinocyte differentiation measured by fluorescence polarization

Summary The epidermis shows a distinctive pattern of differentiation wherein keratinocytes proliferate in the basal cell layer and mature into spinous and granular cells. Using a discontinuous density-gradient centrifugation method, guinea-pig keratinocytes were separated into high (HDF), intermediate (IDF), and low (LDF) density fractions. Morphological and flow cytometrical observations demonstrated that HDF, IDF, and LDF were basal, spinous, and granular cell-rich fractions, respectively. Membrane fluidity of the fractionated keratinocytes was measured by diphenylhexatriene fluorescence polarization. Polarization (p)-value of keratinocytes was negatively correlated with temperature. At each temperature, HDF cells showed a lower p-value than IDF or HDF cells except at 40° C. Since a low p-value indicates a high degree of Brownian motion, membrane fluidity is higher in basal cells and lower in spinous and granular cells. Our results indicate that membrane fluidity of guinea-pig keratinocytes decreases during their maturation.     

A fluorescence polarization study of calcium and phase behaviour in synaptosomal lipids

Steady-state fluorescence polarization of the fluorescent probe 1,6-diphenyl-1,3,5-hexatriene reported temperature-dependent lipid order in l-α-dimyristoylphosphatidylcholine, egg phosphatidylcholine and synaptosomal membranes. No change in lipid order was detected after depolarization of synaptosomes by veratridine (150 μM) even in the presence of 2 mM CaCl₂. However, Ca²⁺ reduced the mobility of a second probe, dansylated dipalmitoylphosphatidylethanolamine, in dispersions of synaptosomal lipids. This effect, which was seen at a Ca²⁺/total phospholipid ratio as low as 0.1, may represent an interaction between the cation and negatively-charged phospholipids. It is suggested that Ca²⁺ promotes a phase separation in synaptosomal lipids which may be relevant to the process of neurotransmitter release.     

Lateral organization of mixed, two-phosphatidylcholine liposomes as investigated by GPS, the slope of Laurdan generalized polarization spectra

The effect of the excitation or emission wavelengths on Laurdan generalized polarization (GP) can be evaluated by GPS, a quantitative, simplified determination of the GP spectrum slope, the thermotropic dependence of which allows the assessment of phospholipid lamellar membrane phase, as shown in a recent publication of our laboratory [J.B. Velázquez, M.S. Fernández, Arch. Biochem. Biophys. 455 (2006) 163-174]. In the present work, we applied Laurdan GPS to phase transition studies of mixed, two-phosphatidylcholine liposomes prepared from variable proportions of dimyristoyl- and dipalmitoylphosphatidylcholine (DMPC and DPPC, respectively). We have found that the GPS function reports a clear limit between the gel/liquid-crystalline phase coexistence region and the liquid-crystalline state, not only at a certain temperature T_c for liposomes of constant composition submitted to temperature scans, but also at a defined mole fraction X_c, for two-component liposomes of variable composition at constant temperature. The T_c or the X_c values obtained from GPS vs. temperature or GPS vs. composition plots, respectively, allow the construction of a partial phase diagram for the DMPC-DPPC mixtures, showing the boundary between the two-phase coexisting region and the liquid-crystalline state. Likewise, at the onset of the transition region, i.e., the two-phase coexisting region as detected by GPS, it is possible to determine, although with less precision, a temperature T_o or a mole fraction X_o defining a boundary located below but near the limit between the gel and ripple phase, reported in the literature. These GPS results are consistent with the proposal by several authors that a fraction of L_α phospholipids coexists with gel phospholipids in the rippled phase.     

Measuring molecular order for lipid membrane phase studies: Linear relationship between Laurdan generalized polarization and deuterium NMR order parameter

Two dimensional phase separation in lipid membranes and cell membranes is of interest to biology because of the idea of membrane rafts — compositionally heterogeneous liquid crystal domains with cellular functions. Few quantitative tools exist for characterizing and differentiating coexisting phases on a molecular scale. Lipid acyl chain order can be measured directly using deuterium nuclear magnetic resonance spectroscopy (²H NMR), or inferred using fluorescence microscopy along with the environment-sensitive probe Laurdan. We found a linear relationship between the ²H NMR order parameter and Laurdan generalized polarization. This observed correlation supports the idea that lipid chain order is tightly associated with the amount and dynamics of water molecules at the glycerol backbone level of the membrane.     

Phase separations induced by melittin in negatively-charged phospholipid bilayers as detected by fluorescence polarization and differential scanning calorimetry

Interactions between melittin and a variety of negatively-charged lipid bilayers have been investigated by intrinsic fluorescence, fluorescence polarization of 1,6-diphenylhexatriene and differential scanning calorimetry. (1) Intrinsic fluorescence of the single tryptophan residue of melittin shows that binding of this peptide to negatively-charged phospholipids is directly related to the surface charge density, but is unaffected by the physical state of lipids, fluid or gel, single-shell vesicles or unsonicated dispersions. (2) Changes in the thermotropic properties of negatively-charged lipids upon melittin binding allow to differentiate two groups of lipids: (i) A progressive disappearance of the transition, without any shift in temperature, is observed with monoacid C₁₄ lipids such as dimyristoylphosphatidylglycerol and -serine (group 1). (ii) With a second group of lipids (group 2), a transition occurs even at melittin saturation, and two transitions are detected at intermediate melittin content, one corresponding to remaining unperturbed lipids, the other shifted downward by 10–20°C. This second group of lipids is constituted by monoacid C₁₆ lipids, dipalmitoylphosphatidylglycerol and -serine. Phosphatidic acids also enter this classification, but it is the net charge of the phosphate group which allows to discriminate: singly charged phosphatidic acids belong to group 2, whereas totally ionized ones behave like group 1 lipids, whatever the chain length. (3) It is concluded that melittin induces phase separations between unperturbed lipid regions which give a transition at the same temperature as pure lipid, and peptide rich domains in which the stoichiometry is 1 toxin per 8 phospholipids. The properties of such domains depend on the bilayer stability: in the case of C₁₆ aliphatic chains and singly charged polar heads, the lipid-peptide domains have a transition at a lower temperature than the pure lipid. With shorter C₁₄ chains or with two net charges by polar group, the bilayer structure is probably totally disrupted, and the new resulting phase can no longer lead to a cooperative transition.     

Phase transitions and cholesterol effects in phospholipid liposomes A new method employing the enhancement of the O-O vibronic transition of pyrene

The enhancement of the weakly allowed O-O vibronic transition in the fluorescence spectrum of the probe pyrene, which we previously showed to result from ground-state complexation with polar groups, has been shown in the present study to offer a new method for determining phase transition temperatures of liposomes and for studying the effects of cholesterol on the structure of their semipolar glycerol backbone. For dipalmitoylphosphatidylcholine it is found that small cholesterol contents (～9 mol%) induce an increase in the polarity of the microenvironment of the probe, whereas contents ?13 mol% induce a decrease in the polarity. The results are discussed in terms of cholesterol effects on the frequency and extent of thermally-induced structural fluctuations which, in turn, affect the penetration of the probe into the bilayer.     

Lipid structural order parameters (reciprocal of fluidity) in biomembranes derived from steady-state fluorescence polarization measurements

This paper presents an interpretation of fluorescence polarization measurements in lipid membranes which are labelled with the apolar probe 1,6-diphenyl-1,3,5-hexatriene. The steady-state fluorescence anisotropy, $\text{r}{}_{\mathrm{<mtext>S</mtext>}}$, is resolved into a fast decaying or kinetic component, $\text{r}{}_{\mathrm{<mtext>f</mtext>}}$, and an infinitely slow decaying or static component, $\text{r}{}_{\mathrm{\infty }}$. The latter contribution, which predominates in biological membranes, is exclusively determined by the degree of molecular packing (order) in the apolar regions of the membrane; $\text{r}{}_{\mathrm{\infty }}$ is proportional to the square of the lipid order parameter. An empirical relation between $\text{r}{}_{\mathrm{<mtext>S</mtext>}}$ and $\text{r}{}_{\mathrm{\infty }}$ is presented, which is in agreement with a prediction based on a theory of rotational dynamics in liquid crystals. This relation enabled us to estimate a lipid structural order parameter directly from simple steady-state fluorescence polarization measurements in a variety of isolated biological membranes. It is shown that major factors determining the order parameter in biomembranes are the temperature, the cholesterol and sphingomyelin content and (in a few systems) the membrane intrinsic proteins.     

Implications of sterol structure for membrane lipid composition, fluidity and phospholipid asymmetry in Saccharomyces cerevisiae

Sterols are essential components of the plasma membrane in eukaryotic cells. Nystatin-resistant erg mutants were used in the present study to investigate the in vitro effects of altered sterol structure on membrane lipid composition, fluidity, and asymmetry of phospholipids. Quantitative analyses of the wild type and mutants erg2, erg3 and erg6 revealed that mutants have lower sterol (free)-to-phospholipid molar ratios than the wild type. Phosphatidylcholine content was decreased in erg2 and erg3 mutants; however, it was increased in erg6 strains as compared to normals. Phosphatidylserine content was increased in the erg6 mutant only. Fluorescence anisotropy decreased with temperature in both probes, and was lower for mutants than for the wild type, suggesting an increased freedom in rotational movement due to decreased membrane order. Investigation of changes in the aminophospholipid transbilayer distribution using two chemical probes, trinitrobenzene sulfonic acid and fluorescamine, revealed that the amounts of phosphatidylethanolamine derivatized by these probes were quite similar in both the wild type and various erg strains. The present findings suggest that adaptive responses in yeast cells with altered sterol structure are possibly manifested through changes in membrane lipid composition and fluidity, and not through transbilayer rearrangement of aminophospholipids.     

EPR and FTIR studies reveal the importance of highly ordered sterol-enriched membrane domains for ostreolysin activity

Ostreolysin is a cytolytic protein from the edible oyster mushroom (Pleurotus ostreatus), which recognizes specifically and binds to raft-like sterol-enriched membrane domains that exist in the liquid-ordered phase. Its binding can be abolished by micromolar concentrations of lysophospholipids and fatty acids. The membrane activity of ostreolysin, however, does not completely correlate with the ability of a certain sterol to induce the formation of a liquid-ordered phase, suggesting that the protein requires an additional structural organization of the membrane to exert its activity. The aim of this study was to further characterize the lipid membranes that facilitate ostreolysin binding by analyzing their lipid phase domain structure. Fourier-transformed infrared spectroscopy (FTIR) and electron paramagnetic resonance (EPR) were used to analyze the ordering and dynamics of membrane lipids and the membrane domain structure of a series of unilamellar liposomes prepared by systematically changing the lipid components and their ratios. Our results corroborate the earlier conclusion that the average membrane fluidity of ostreolysin-susceptible liposomes alone cannot account for the membrane activity of the protein. Combined with previous data computer-aided interpretation of EPR spectra strongly suggests that chemical properties of membrane constituents, their specific distribution, and physical characteristics of membrane nanodomains, resulting from the presence of sterol and sphingomyelin (or a highly ordered phospholipid, dipalmitoylphosphatidylcholine), are essential prerequisites for ostreolysin membrane binding and pore-formation.     

Analysis of early apoptotic events in individual cells by fluorescence intensity and polarization measurements

Apoptosis is a dynamic process of variable duration. The ability to continuously detect the death process occurring in single or subgroups of cells is therefore very important in identifying apoptotic cells within a complex population. The Individual Cell Scanner (ICS), a multiparametric, multilaser-based scanning static cytometer, was used in the present report for the continuous monitoring of the apoptosis process. Fluorescence intensity (FI), polarization (FP), kinetic measurements, and cluster analysis of subpopulations were carried out utilizing various fluorescent probes. Hydrogen peroxide-induced apoptosis was monitored online in intact live lymphocytes by continuous sequential measurements of intracellular hyperpolarization. Plasma membrane asymmetry, mitochondrial membrane potential, and lysosomal rupture were monitored in individual cells. Cytoplasmic condensations, due to cell shrinkage and early lysosomal rupture, were found to be very early events of apoptosis. The new analytical capabilities suggested here may provide simple and convenient methods for detecting apoptosis from its earlier stages.     

The effect of osmotic stress on the biophysical behavior of the Bacillus subtilis membrane studied by dynamic and steady-state fluorescence anisotropy

The thermotropic behavior of intact bacterial membranes and vesicles prepared from total and polar lipids isolated from Bacillus subtilis cultures grown at 37 degrees C in normal (LB) and hyperosmotic (LBN) conditions was studied using 1,6-diphenyl-1,3,5-hexatriene (DPH), 1-(4-trimethylammoniumphenyl)-6-phenyl-1,3,5-hexatriene p-toluenesulfonate (TMA-DPH), and 2-diethylamino-6-lauroyl-naphthalene (Laurdan) as fluorescent probes. No phase transition of bulk lipids was observed in these preparations at the range of temperature studied. The anisotropy values (r(s)) for DPH and TMA-DPH in purified membranes showed significant differences between the LB and LBN conditions, suggesting that there was an increase in membrane packing during the adaptation to osmotic stress. Furthermore, generalized polarization (GP) parameters for Laurdan indicated small but significant changes in water relaxation at the membrane hydrophobic/hydrophilic interface. Membrane preparations showed r(s) higher values than those of lipid vesicles and a higher temperature dependence of the Laurdan GP parameter. This fact indicates that membrane proteins increase the lipid packing and keep the membrane more sensitive to temperature changes.     

Temperature-dependent changes in phospholipid and fatty acid composition and membrane lipid fluidity of Yersinia enterocolitica

Temperature-dependent compositional changes of phospholipids and their fatty acids were analysed in Yersinia enterocolitica grown at 5°, 25° and 37°C. The relative amounts of the four phospholipids, phosphatidylethanolamine (75–78%), phosphatidylglycerol (10–11%), cardiolipin (<7%) and lysophosphatidylethanolamine (<5%), were essentially the same at all growth temperatures. The degree of fatty acid unsaturation of the four phospholipids increased with decrease in growth temperature, mainly due to an increase of C_16:1 and C_18:1 and a corresponding decrease of C_16;0, C_18:0 and cyclo C_17:0. An electron spin resonance spectroscopic study of the membrane lipids showed that membrane lipid fluidity was enhanced by decreasing the growth temperatures. The changes in fatty acid composition of phospholipids in response to varied temperatures were consistent with the temperature-dependent changes in the membrane lipid fluidity of Y. enterocolitica , and were similar to those reported for other bacteria.     

Dynamic fluorescence polarization studies on lipid mobilities in phospholipid vesicles in the presence of calcium mediators

The influence of Ca²⁺ mediators (nifedipine, verapamil and prostaglandin F_2α) on fluorescence polarization of l-anilino-8-napthalene-sulphonate in dipalmitoyl phosphatidylcholine and dimyristoyl phosphatidylcholine liposomes was studied at various temperatures to understand the dynamic behaviour of membrane lipids. We also studied the effect of change in calcium concentration on the fluorescence polarization of the dye in the liposomes. Our results show increase in polarization (indicative of stiffening of the membrane) in the presence of Ca²⁺ ions. In the case of dimyristoyl phosphatidylcholine liposomes, all 3 drugs caused decrease in fluorescence polarization (increase in fluidity of the membrane) with or without Ca²⁺ ions in the medium. Contrary to this, in the case of dipalmitoyl phosphatidylcholine liposomes, the fluidization effect is observed for all the 3 drugs in the absence of Ca²⁺ ions; in the presence of Ca²⁺ ions stiffening is observed upon addition of nifedipine and verapamil which are antagonists, and fluidization is observed upon addition of prostaglandin F_2α. The role of drug-induced fluidity changes in membranes in therapy planning is discussed in the paper.     

Use of fluorescence polarization to monitor intracellular membrane changes during temperature acclimation. Correlation with lipid compositional and ultrastructural changes.

Fluorescence polarization of 1,6-diphenylhexatriene (DPH) was used to study the effects of temperature acclimation on Tetrahymena membranes. The physical properties of membrane lipids were found to be highly dependent on cellular growth temperature. DPH polarization in lipids from three different membrane fractions correlated well with earlier freeze-fracture and electron spin resonance observations showing that membrane fluidity progressively decreases in the order microsomes greater than pellicles greater than cilia throughout a wide range of growth temperatures. Changes in membrane lipid fluidity following a shift from high to low growth temperatures proceed rapidly in the microsomes, whereas there is a pronounced lag in the changes of peripheral cell membrane lipids. These data support previous observations that adaptive changes in membrane fluidity proceed via lipid modifications in the endoplasmic reticulum, followed by dissemination of lipid components to other cell membranes. The rapid changes in polarization observed in the microsomal lipids following a temperature shift correspond closely with the time-dependent alterations in both lipid fatty acid composition and freeze-fracture patterns of membrane particle distribution, suggesting that, in the endoplasmic reticulum, lipid phase separation is the primary cause of membrane particle rearrangements.     

The lack of relationship between fluorescence polarization and lateral diffusion in biological membranes

An investigation has been carried out of the relationship between changes in the fluorescence polarization of 1,6-diphenyl-1,3,5-hexatriene (DPH) and concomittant changes in the lateral diffusion of proteins and lipid probes in membranes. Plasma membranes from lymphocytes and a CH1 mouse lymphoma line were treated with up to 70 mol% (relative to the total membrane phospholipid) of oleic or linoleic fatty acids. Under these conditions the fluorescence polarization of DPH decreased by between 8 and 15% which, in the framework of the microviscosity approach, suggests a membrane fluidity change of between 20 and 50%. The lateral diffusion coefficients of surface immunoglobin and the lipid probes 3,3′-dioctadecylindocarbocyanine and pyrene were also measured in these membranes using the fluorescence photobleaching recovery technique and the rate of pyrene excimer formation. The diffusion rates were found to be unaffected by the presence of free fatty acids. Hence despite large ‘microviscosity’ changes as reported by depolarization of DPH fluorescence, lateral diffusion coefficients are essentially unchanged. This finding is consistent with the idea that perturbing agents such as free fatty acids do not cause a general fluidization of the membrane but act locally to alter, for example, protein function. It is also consistent with the suggestion that lateral mobility of membrane proteins is not modulated by the lipid viscosity.     

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.     

Liquid crystal-based detection of thrombin coupled to interactions between a polyelectrolyte and a phospholipid monolayer

Herein, we describe a real-time, label-free biosensing strategy for thrombin detection that uses the orientational properties of nematic liquid crystals (LCs) and the interactions between a polyelectrolyte and a phospholipid monolayer. The imaging principle is based on the disruption of the orientation of 4-cyano-4′-pentylbiphenyl by reorganized 1,2-dioleoyl-sn-glycero-3-phospho-rac-(1-glycerol) sodium salt (DOPG) at the aqueous/LC interface. Positively charged, multiple arginine peptides (poly-l-arginine hydrochloride) interacted with negatively charged DOPG at the aqueous/LC interface, which caused reorganization of the phospholipid layer and induced an orientational transition of LCs from a homeotropic to a planar state. As a result, a dark to bright shift in the optical response was observed. Thrombin cleaves poly-l-arginine hydrochloride into peptides. Thus, when thrombin was added, the optical signals generated by the LCs reverted from bright to dark because of the weakened ability of the fragments to induce electrostatic interactions. The limit of detection of the LC-based sensor was 0.25 ng/mL (6.7 pM) thrombin, and the sensor was fully reusable. The detection limit of our LC-based interface sensor is 600 times lower than that of a previously reported enzyme-linked aptamer assay for the detection of thrombin. Thus, we have established a new, simple thrombin biosensor with high sensitivity and low interference.     

Effects of pressure on the phase transition of bilayers in liposomes influence of cholesterol and α-tocopherol

The effects of presure on the gel-to-liquid crystalline phase transition temperature of dimyristoylphosphatidylcholine (DMPC) bilayers containing cholesterol, α-tocopherol and α-tocopheryl acetate were studied by fluorescence depolarizalion. The transition temperature of cholesterol mixtures ( > 7.5 mol%) was lower than that of 100% DMPC at atmospheric pressure, but it became higher than the latter on increase in pressure. The thermodynamic parameters of the transition (ΔV,ΔS,ΔH) were estimated and the functions of cholesterol and α-tocopherols in the bilayers are discussed.