Tiziana Parasassi

Quantitation of lipid phases in phospholipid vesicles by the generalized polarization of Laurdan fluorescence.

The sensitivity of Laurdan (6-dodecanoyl-2-dimethylaminonaphthalene) excitation and emission spectra to the physical state of the membrane arises from dipolar relaxation processes in the membrane region surrounding the Laurdan molecule. Experiments performed using phospholipid vesicles composed of phospholipids with different polar head groups show that this part of the molecule is not responsible for the observed effects. Also, pH titration in the range from pH 4 to 10 shows that the spectral variations are independent of the charge of the polar head. A two-state model of dipolar relaxation is used to qualitatively explain the behavior of Laurdan. It is concluded that the presence of water molecules in the phospholipid matrix are responsible for the spectral properties of Laurdan in the gel phase. In the liquid crystalline phase there is a relaxation process that we attribute to water molecules that can reorientate during the few nanoseconds of the excited state lifetime. The quantitation of lipid phases is obtained using generalized polarization which, after proper choice of excitation and emission wavelengths, satisfies a simple addition rule.

Laurdan and Prodan as Polarity-Sensitive Fluorescent Membrane Probes

The steady-state and dynamic fluorescence spectral properties of 2-dimethylamino-6-lauroylnaphthalene (LAURDAN) and several other naphthalene derivatives are summarized to illustrate their sensitivity to the polarity of the environment. Results obtained both in solvents of different polarity and in phospholipid vesicles in two phase states are presented. The emission red shift observed in polar solvents and in the phospholipid liquid–crystalline phase is explained on the basis of dipolar relaxation of solvent molecules surrounding the fluorescent naphthalene moiety of these probes. In phospholipid environments, experimental evidence is shown that excludes the intramolecular relative reorientation of the dimethylamino and carbonyl groups in the naphthalene and the reorientation of the entire fluorescent moiety. The solvent dipolar relaxation observed for LAURDAN and PRODAN in phospholipid bilayers has been attributed to a small number of water molecules present at the membrane interface. A comparison between LAURDAN emission in phospholipid vesicles prepared in D2O and in H2O is also presented. The definition and the derivation of the generalized polarization function are also discussed.

Two-photon fluorescence microscopy of laurdan generalized polarization domains in model and natural membranes.

Two-photon excitation microscopy shows coexisting regions of different generalized polarization (GP) in phospholipid vesicles, in red blood cells, in a renal tubular cell line, and in purified renal brushborder and basolateral membranes labeled with the fluorescent probe laurdan. The GP function measures the relative water content of the membrane. In the present study we discuss images obtained with polarized laser excitation, which selects different molecular orientations of the lipid bilayer corresponding to different spatial regions. The GP distribution in the gel-phase vesicles is relatively narrow, whereas the GP distribution in the liquid-crystalline phase vesicles (DOPC and DLPC) is broad. Analysis of images obtained with polarized excitation of the liquid-crystalline phase vesicles leads to the conclusion that coexisting regions of different GP must have dimensions smaller than the microscope resolution (approximately 200 nm radially and 600 nm axially). Vesicles of an equimolar mixture of DOPC and DPPC show coexisting rigid and fluid domains (high GP and low GP), but the rigid domains, which are preferentially excited by polarized light, have GP values lower than the pure gel-phase domains. Cholesterol strongly modifies the domain morphology. In the presence of 30 mol% cholesterol, the broad GP distribution of the DOPC/DPPC equimolar sample becomes narrower. The sample is still very heterogeneous, as demonstrated by the separations of GP disjoined regions, which are the result of photoselection of regions of different lipid orientation. In intact red blood cells, microscopic regions of different GP can be resolved, whereas in the renal cells GP domains have dimensions smaller than the microscope resolution. Preparations of renal apical brush border membranes and basolateral membranes show well-resolved GP domains, which may result from a different local orientation, or the domains may reflect a real heterogeneity of these membranes.

Membrane Lipid Domains and Dynamics as Detected by Laurdan Fluorescence

Abstract2-Dimethylamino-6-lauroylnaphthalene (Laurdan) is a membrane probe of recent characterization, which shows high sensitivity to the polarity of its environment. Steady-state Laurdan excitation and emission spectra have different maxima and shape in the two phospholipid phases, due to differences in the polarity and in the amount of dipolar relaxation. In bilayers composed of a mixture of gel and liquid-crystalline phases, the properties of Laurdan excitation and emission spectra are intermediate between those obtained in the pure phases. These spectral properties are analyzed using the generalized polarization (GP). TheGP value can be used for the quantitation of each phase. The wavelength dependence of theGP value is used to ascertain the coexistence of different phase domains in the bilayer. Moreover, by following the evolution of Laurdan emission vs. time after excitation, the kinetics of phase fluctuation in phospholipid vesicles composed of coexisting gel and liquid-crystalline phases was determined.GP measurements performed in several cell lines did not give indications of coexistence of phase domains in their membranes. In natural membranes, Laurdan parameters indicate a homogeneously fluid environment, with restricted molecular motion in comparison with the phospholipid liquid-crystalline phase. The influence of cholesterol on the phase properties of the two phospholipid phases is proposed to be the cause of the phase behavior observed in natural membranes. In bilayers composed of different phospholipids and various cholesterol concentrations, Laurdan response is very similar to that arising from cell membranes. In the absence of cholesterol, from the steady-state and time-resolved measurements of Laurdan in phospholipid vesicles, the condition for the occurrence of separate coexisting domains in the bilayer has been determined: the molecular ratio between the two phases must be in the range between 30% and 70%. Below and above this range, a single homogeneous phase is observed, with the properties of the more concentrated phase, slightly modified by the presence of the other. Moreover, in this concentration range, the calculated dimension of the domains is very small, between 20 and 50 Å.

INFLUENCE OF CHOLESTEROL ON PHOSPHOLIPID-BILAYERS PHASE DOMAINS AS DETECTED BY LAURDAN FLUORESCENCE

Coexisting gel and liquid-crystalline phospholipid phase domains can be observed in synthetic phospholipid vesicles during the transition from one phase to the other and, in vesicles of mixed phospholipids, at intermediate temperatures between the transitions of the different phospholipids. The presence of cholesterol perturbs the dynamic properties of both phases to such an extent as to prevent the detection of coexisting phases. 6-Lauroyl-2-dimethylaminopahthalene (Laurdan) fluorescence offers the unique advantage of well resolvable spectral parameters in the two phospholipid phases that can be used for the detection and quantitation of coexisting gel and liquid-crystalline domains. From Laurdan fluorescence excitation and emission spectra, the generalized polarization spectra and values were calculated. By the generalized polarization phospholipid phase domain coexistence can be detected, and each phase can be quantitated. In the same phospholipid vesicles where without cholesterol domain coexistence can be detected, above 15 mol% and, remarkably, at physiological cholesterol concentrations, > or = 30 mol%, no separate Laurdan fluorescence signals characteristic of distinct domains can be observed. Consequences of our results on the possible size and dynamics of phospholipid phase domains and their biological relevance are discussed.

Cholesterol modifies water concentration and dynamics in phospholipid bilayers: a fluorescence study using Laurdan probe

The effect of cholesterol on the gel, the liquid-crystalline, and mixed phospholipid phases has been studied using the fluorescence properties of 2-dimethylamino-6-lauroylnaphthalene (Laurdan). Laurdan sensitivity to the polarity and to the dynamics of its environment reveals that cholesterol affects phospholipid bilayers in the gel phase by expelling water and by increasing the amount of dipolar relaxation. In the liquid-crystalline phase, the effect of cholesterol is a reduction of both water concentration and amount of dipolar relaxation. Detailed studies of Laurdan excitation and emission spectral contours as a function of cholesterol concentration show that there are some cholesterol concentrations at which Laurdan spectral properties changes discontinuously. These peculiar cholesterol concentrations are in agreement with recent observations of other workers showing the formation of local order in the liquid-crystalline phase of phospholipids upon addition of phospholipid derivatives of pyrene. A local organization of phospholipids around cholesterol molecule seems to be produced by the presence of peculiar concentrations of cholesterol itself. This local organization is stable enough to be observed during the excited state lifetime of Laurdan of approximately 5-6 ns.

Giant phospholipid vesicles: Comparison among the whole lipid sample characteristics using different preparation methods - A two photon fluorescence microscopy study

Several methods for the preparation of giant unilamellar vesicles (GUVs) using synthetic phosphatidylcholine phospholipids were evaluated. We compared the physical characteristics--in terms of lamellarity and morphology--of the whole lipid sample for each different lipid preparation using the sectioning capability of the two-photon excitation fluorescence microscope. From the evaluation of the entire lipid sample we determined that vesicle size, internal shape and shell thickness distributions depend on the vesicles preparation method. Our results show that the preparation of giant unilamellar vesicles by the application of external electric fields offers several advantages among the other methods tested here. Using this method a high yield (approximately 95%) of giant unilamellar vesicles with a narrow size distribution was obtained. Independently of the preparation method, some lipid structures, which are held together by lipid tethers, were identified and resolved. These particular lipid structures show shell thickness and size heterogeneity. Labeling the lipid samples with 6-lauroyl-2-(N,N-dimethylamino)naphtalene (LAURDAN) and using the LAURDAN generalized polarization function we show that the lipid packing in these tethers or tubes is similar to those found in the phospholipid vesicles. The fact that both vesicles and tethers are found in the lipid preparations indicates similar stability between these structures.

Prodan as a Membrane Surface Fluorescence Probe: Partitioning between Water and Phospholipid Phases

Fluorescence spectral features of 6-propionyl-2-dimethylaminonaphthalene (Prodan) in phospholipid vesicles of different phase states are investigated. Like the spectra of 6-lauroyl-2-dimethylaminonaphthalene (Laurdan), the steady-state excitation and emission spectra of Prodan are sensitive to the polarity of the environment, showing a relevant shift due to the dipolar relaxation phenomenon. Because of the different lengths of their acyl residues, the partitioning of the two probes between water and the membrane bilayer differs profoundly. To account for the contribution of Prodan fluorescence arising from water, we introduce a three-wavelength generalized polarization method that makes it possible to separate the spectral properties of Prodan in the lipid phase and in water, and to determine the probe partitioning between phospholipid and water and between the gel and the liquid-crystalline phases of phospholipids. In contrast to Laurdan, Prodan preferentially partitions in the liquid-crystalline phase with respect to the gel and is sensitive to the polar head pretransition, and its partition coefficient between the membrane and water depends on the phase state, i.e., on the packing of the bilayer. Prodan is sensitive to polarity variations occurring closer to the bilayer surface than those detected by Laurdan.

ABRUPT MODIFICATIONS OF PHOSPHOLIPID-BILAYER PROPERTIES AT CRITICAL CHOLESTEROL CONCENTRATIONS

The fluorescence generalized polarization (GP) of 2-dimethylamino-6-lauroylnaphthalene (Laurdan) reveals different effects of cholesterol on the phase behavior of phospholipid bilayers. Phospholipid vesicles composed of gel, liquid-crystalline, and coexisting domains of the two phases have been studied at temperatures from 1 to 65 degrees C, without cholesterol and with cholesterol concentrations of 3-50 mol %. Laurdan GP measurements show the general effect of cholesterol of increasing the molecular dynamics of the gel and of decreasing the molecular dynamics of the liquid-crystalline phase. In the liquid-crystalline phase, the increased order yields Laurdan GP values close to those obtained in the gel phase. At cholesterol concentrations > 15 mol % a phase transition cannot be detected. Using the wavelength dependence of the excitation and emission GP spectra we determine that differences between the two phospholipid phases cannot be detected. In particular, in vesicles composed of coexisting gel and liquid-crystalline phases the GP wavelength dependence characteristic of coexisting domains cannot be observed at cholesterol concentrations > or = 15 mol %. Cholesterol causes the decrease in both the polarity and the dipolar relaxation effects on the neighborhood of the fluorescent naphthalene moiety of Laurdan. Probably because of a cholesterol-induced increase in the bilayer packing, these effects do not occur continuously with the increase of cholesterol concentration in the bilayer. Cholesterol concentrations inducing higher Laurdan GP values have been determined at about 5, 10, 15, 30, and 45 mol % with respect to phospholipids. We propose that the formation of ordered molecular microdomains at critical cholesterol concentrations can explain the occurrence of the observed discontinuities.

MODULATION AND DYNAMICS OF PHASE PROPERTIES IN PHOSPHOLIPID MIXTURES DETECTED BY LAURDAN FLUORESCENCE

Steady‐state and dynamic fluorescence properties of 6‐lauroyl‐2‐dimethylaminonaphthalene (Laurdan) have been used to ascertain the coexistence of separate phase domains and their dynamic properties in phospholipid vesicles composed of different mole ratios of dilauroyl‐ and dipalmitoyl‐phosphatidylcholine (DLPC and DPPC, respectively). The recently introduced generalized polarization together with time‐resolved emission spectra have been utilized for detecting changes. The results indicate the coexistence of phospholipid phase domains in vesicle compositions in the range between 30 mol% and 70 mol% DPPC in DLPC. Below and above these concentrations a homogeneous phase is observed, with averaged properties. In the case of coexisting phase domains, the properties of each individual phase are largely influenced by the presence of the other phase. Implications on fluctuations between the coexisting phases and on the size and shape of domains are discussed.