1-2-oleoylphosphatidylcholine and laurdan

Using LAURDAN fluorescence we observed that water dynamics measured at the interface of DOPC bilayers can be differentially regulated by the presence of crowded suspensions of different proteins (HSA, IgG, Gelatin) and PEG, under conditions where the polymers are not in direct molecular contact with the lipid interface. Specifically, we found that the decrease in water dipolar relaxation at the membrane interface correlates with an increased fraction of randomly oriented (or random coil) configurations in the polymers, as Gelatin > PEG > IgG > HSA. By using the same experimental strategy, we also demonstrated that structural transitions from globular to extended conformations in proteins can induce transitions between lamellar and non-lamellar phases in mixtures of DOPC and monoolein. Independent experiments using Raman spectroscopy showed that aqueous suspensions of polymers exhibiting high proportions of randomly oriented conformations display increased fractions of tetracoordinated water, a configuration that is dominant in ice. This indicates a greater capacity of this type of structure for polarizing water and consequently reducing its chemical activity. This effect is in line with one of the tenets of the Association Induction Hypothesis, which predicts a long-range dynamic structuring of water molecules via their interactions with proteins (or other polymers) showing extended conformations. Overall, our results suggest a crucial role of water in promoting couplings between structural changes in macromolecules and supramolecular arrangements of lipids. This mechanism may be of relevance to cell structure/function when the crowded nature of the intracellular milieu is considered.

Topics: 2-Naphthylamine; Gelatin; Glycerides; Immunoglobulin G; Laurates; Lipids; Molecular Conformation; Phosphatidylcholines; Polyethylene Glycols; Polymers; Serum Albumin, Human; Water

Fluidity of lipid membranes is known to play an important role in the functioning of living organisms. The fluorescent probe Laurdan embedded in a lipid membrane is typically used to assess the fluidity state of lipid bilayers by utilizing the sensitivity of Laurdan emission to the properties of its lipid environment. In particular, Laurdan fluorescence is sensitive to gel vs liquid⁻crystalline phases of lipids, which is demonstrated in different emission of the dye in these two phases. Still, the exact mechanism of the environment effects on Laurdan emission is not understood. Herein, we utilize dipalmitoylphosphatidylcholine (DPPC) and dioleoylphosphatidylcholine (DOPC) lipid bilayers, which at room temperature represent gel and liquid⁻crystalline phases, respectively. We simulate absorption and emission spectra of Laurdan in both DOPC and DPPC bilayers with quantum chemical and classical molecular dynamics methods. We demonstrate that Laurdan is incorporated in heterogeneous fashion in both DOPC and DPPC bilayers, and that its fluorescence depends on the details of this embedding.

Topics: 1,2-Dipalmitoylphosphatidylcholine; 2-Naphthylamine; Fluorescence; Laurates; Lipid Bilayers; Models, Chemical; Molecular Dynamics Simulation; Phosphatidylcholines; Quantum Theory

Imaging lipid organization in cell membranes requires advanced fluorescent probes. Here, we show that a recently synthesized push-pull pyrene (PA), similarly to popular probe Laurdan, changes the emission maximum as a function of lipid order, but outperforms it by spectroscopic properties. In addition to red-shifted absorption compatible with common 405 nm diode laser, PA shows higher brightness and much higher photostability than Laurdan in apolar membrane environments. Moreover, PA is compatible with two-photon excitation at wavelengths >800 nm, which was successfully used for ratiometric imaging of coexisting liquid ordered and disordered phases in giant unilamellar vesicles. Fluorescence confocal microscopy in Hela cells revealed that PA efficiently stains the plasma membrane and the intracellular membranes at >20-fold lower concentrations, as compared to Laurdan. Finally, ratiometric imaging using PA reveals variation of lipid order within different cellular compartments: plasma membranes are close to liquid ordered phase of model membranes composed of sphingomyelin and cholesterol, while intracellular membranes are much less ordered, matching well membranes composed of unsaturated phospholipids without cholesterol. These differences in the lipid order were confirmed by fluorescence lifetime imaging (FLIM) at the blue edge of PA emission band. PA probe constitutes thus a new powerful tool for biomembrane research.

Topics: 2-Naphthylamine; Cell Membrane; Cholesterol; Fluorescent Dyes; HeLa Cells; Humans; Intracellular Membranes; Laurates; Microscopy, Confocal; Microscopy, Fluorescence; Phosphatidylcholines; Pyrenes; Sensitivity and Specificity; Sphingomyelins; Unilamellar Liposomes

Microphase separation behaviors of cationic liposomes have been investigated using a pH-sensitive fluorescent probe with 4-heptadecyl-7-hydroxycoumarin (HHC), 1,6-diphenyl-1,3,5-hexatriene, and 6-lauroyl-2-dimethylaminonaphthalene, and to estimate localized electrostatic potentials. Shifts of the apparent pKa values of HHC were observed in cationic liposomes in proportion to the amount of cationic lipids. Two pKa values were obtained with 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC)/3β-[N(N',N'-dimethylaminoethane)-carbamoyl] cholesterol hydrochloride (DC-Ch) liposomes, while only one pKa value was generated with either DOPC/1,2-dioleoyl-3-trimethylammonium-propane (DOTAP) or DOPC/dimethyldioctadecylammonium-bromide (DODAB) liposomes. The physicochemical membrane property analyses, focusing on membrane fluidity and membrane polarity, revealed heterogeneity among DOPC/DC-Ch liposomes. By analyzing the pH titration curves using sigmoidal fitting, the localized electrostatic potentials were estimated. For DOPC/DOTAP = (7/3), the membrane was in the liquid-disordered phase and the density of cationic molecules was 0.41 cation/nm(2). For DOPC/DC-Ch = (7/3), the membrane was heterogeneous and the densities of cationic molecules in liquid-disordered and liquid-ordered phases were 0.25 and 1.24 cation/nm(2), respectively. We thereby conclude that the DC-Ch molecules can form nanodomains when these molecules are concentrated to 59%.

Topics: 2-Naphthylamine; Cholesterol; Diphenylhexatriene; Fatty Acids, Monounsaturated; Fluorescent Dyes; Hydrogen-Ion Concentration; Laurates; Liposomes; Membrane Fluidity; Phosphatidylcholines; Quaternary Ammonium Compounds; Spectrometry, Fluorescence; Umbelliferones

The fluorescent marker Laurdan and its new derivative, C-Laurdan, have been investigated by means of theoretical calculations in a DOPC lipid bilayer membrane at room temperature, and a comparison is made with results from fluorescence experiments. Experimentally, the latter probe is known to have a higher sensitivity to the membrane polarity at the lipid headgroup region and has higher water solubility. Results from Molecular Dynamics (MD) simulations show that C-Laurdan is oriented with the carboxyl group toward the head of the membrane, with an angle of 50° between the molecular backbone and the normal to the bilayer, in contrast to the orientation of the Laurdan headgroup whose carbonyl group is oriented toward the polar regions of the membrane and which describes an angle of ca. 70-80° with the membrane normal. This contrast in orientation reflects the differences in transition dipole moment between the two probes and, in turn, the optical properties. QM/MM results of the probes show little differences for one- (OPA) and two-photon absorption (TPA) spectra, while the second harmonic generation (SHG) beta component is twice as large in Laurdan with respect to C-Laurdan probe. The fluorescence anisotropy decay analysis of the first excited state confirms that Laurdan has more rotational freedom in the DOPC membrane, while C-Laurdan experiences a higher hindrance, making it a better probe for lipid membrane phase recognition.

Topics: 2-Naphthylamine; Fluorescent Dyes; Gases; Laurates; Lipid Bilayers; Molecular Dynamics Simulation; Phosphatidylcholines; Quantum Theory; Water

Cholesteryl hemisuccinate (CHS) is one of the cholesterol-mimicking detergents not observed in nature. It is, however, widely used in protein crystallography, in biochemical studies of proteins, and in pharmacology. Here, we performed an extensive experimental and theoretical study on the behavior of CHS in lipid membranes rich in unsaturated phospholipids. We found that the deprotonated form of CHS (that is the predominant form under physiological conditions) does not mimic cholesterol very well. The protonated form of CHS does better in this regard, but also its ability to mimic the physical effects of cholesterol on lipid membranes is limited. Overall, although ordering and condensing effects characteristic to cholesterol are present in systems containing any form of CHS, their strength is appreciably weaker compared to cholesterol. Based on the considerable amount of experimental and atomistic simulation data, we conclude that these differences originate from the fact that the ester group of CHS does not anchor it in an optimal position at the water-membrane interface. The implications of these findings for considerations of protein-cholesterol interactions are briefly discussed.

Topics: 2-Naphthylamine; Cholesterol; Cholesterol Esters; Dihydropyridines; Laurates; Lipid Bilayers; Liposomes; Molecular Dynamics Simulation; Phosphatidylcholines; Protons; Water

Membrane nanodomains are dynamic liquid entities surrounded by another type of dynamic liquid. Diffusion can take place inside, around and in and out of the domains, and membrane components therefore continuously shift between domains and their surroundings. In the plasma membrane, there is the further complexity of links between membrane lipids and proteins both to the extracellular matrix and to intracellular proteins such as actin filaments. In addition, new membrane components are continuously delivered and old ones removed. On top of this, cells move. Taking all of this into account imposes great methodological challenges, and in the present chapter we discuss some methods that are currently used for membrane nanodomain studies, what information they can provide and their weaknesses.

Topics: 2-Naphthylamine; Actin Cytoskeleton; beta-Cyclodextrins; Cell Membrane; Cholesterol; Cytoskeleton; Fluorescent Dyes; Humans; Jurkat Cells; Laurates; Membrane Proteins; Octoxynol; Phosphatidylcholines; Sphingomyelins; Unilamellar Liposomes

This study was conducted to explore how the nature of the acyl chains of sphingomyelin (SM) influence its lateral distribution in the ternary lipid mixture SM/cholesterol/1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC), focusing on the importance of the hydrophobic part of the SM molecule for domain formation. Atomic force microscopy (AFM) measurements showed that the presence of a double bond in the 24:1 SM molecule in mixtures with cholesterol (CHO) or in pure bilayers led to a decrease in the molecular packing. Confocal microscopy and AFM showed, at the meso- and nanoscales respectively, that unlike 16:0 and 24:0 SM, 24:1 SM does not induce phase segregation in ternary lipid mixtures with DOPC and CHO. This ternary lipid mixture had a nanomechanical stability intermediate between those displayed by liquid-ordered (Lo) and liquid-disordered (Ld) phases, as reported by AFM force spectroscopy measurements, demonstrating that 24:1 SM is able to accommodate both DOPC and CHO, forming a single phase. Confocal experiments on giant unilamellar vesicles made of human, sheep, and rabbit erythrocyte ghosts rich in 24:1 SM and CHO, showed no lateral domain segregation. This study provides insights into how the specific molecular structure of SM affects the lateral behavior and the physical properties of both model and natural membranes. Specifically, the data suggest that unsaturated SM may help to keep membrane lipids in a homogeneous mixture rather than in separate domains.

Topics: 2-Naphthylamine; Animals; Cholesterol; Erythrocyte Membrane; Humans; Laurates; Lipid Bilayers; Mechanical Phenomena; Microscopy, Atomic Force; Nanoparticles; Phosphatidylcholines; Rabbits; Sheep; Sphingomyelins

Time-dependent fluorescence shift (TDFS) of Laurdan embedded in phospholipid bilayers reports on hydration and mobility of the phospholipid acylgroups. Exchange of H2O with D2O prolongs the lifetime of lipid-water and lipid-water-lipid interactions, which is reflected in a significantly slower TDFS kinetics. Combining TDFS measurements in H2O and D2O hydrated bilayers with atomistic molecular dynamics (MD) simulations provides a unique tool for characterization of the hydrogen bonding at the acylgroup level of lipid bilayers. In this work, we use this approach to study the influence of fluoride anions on the properties of cationic bilayers composed of trimethylammonium-propane (DOTAP). The results obtained for DOTAP are confronted with those for neutral phosphatidylcholine (DOPC) bilayers. Both in DOTAP and DOPC H2O/D2O exchange prolongs hydrogen-bonding lifetime and does not disturb bilayer structure. These results are confirmed by MD simulations. TDFS experiments show, however, that for DOTAP this effect is cancelled in the presence of fluoride ions. We interpret these results as evidence that strongly hydrated fluoride is able to steal water molecules that bridge lipid carbonyls. Consequently, when attracted to DOTAP bilayer, fluoride disrupts the local hydrogen-bonding network, and the differences in TDFS kinetics between H2O and D2O hydrated bilayers are no longer observed. A distinct behavior of fluoride is also evidenced by MD simulations, which show different lipid-ion binding for Cl(-) and F(-).

Topics: 2-Naphthylamine; Fatty Acids, Monounsaturated; Fluorescent Dyes; Fluorides; Hydrogen Bonding; Laurates; Lipid Bilayers; Molecular Dynamics Simulation; Phosphatidylcholines; Quaternary Ammonium Compounds; Water

Surfactin, a bacterial amphiphilic lipopeptide is attracting more and more attention in view of its bioactive properties which are in relation with its ability to interact with lipids of biological membranes. In this work, we investigated the effect of surfactin on membrane structure using model of membranes, vesicles as well as supported bilayers, presenting coexistence of fluid-disordered (DOPC) and gel (DPPC) phases. A range of complementary methods was used including AFM, ellipsometry, dynamic light scattering, fluorescence measurements of Laurdan, DPH, calcein release, and octadecylrhodamine B dequenching. Our findings demonstrated that surfactin concentration is critical for its effect on the membrane. The results suggest that the presence of rigid domains can play an essential role in the first step of surfactin insertion and that surfactin interacts both with the membrane polar heads and the acyl chain region. A mechanism for the surfactin lipid membrane interaction, consisting of three sequential structural and morphological changes, is proposed. At concentrations below the CMC, surfactin inserted at the boundary between gel and fluid lipid domains, inhibited phase separation and stiffened the bilayer without global morphological change of liposomes. At concentrations close to CMC, surfactin solubilized the fluid phospholipid phase and increased order in the remainder of the lipid bilayer. At higher surfactin concentrations, both the fluid and the rigid bilayer structures were dissolved into mixed micelles and other structures presenting a wide size distribution.

Topics: 1,2-Dipalmitoylphosphatidylcholine; 2-Naphthylamine; Bacterial Physiological Phenomena; Calorimetry; Cell Membrane; Diphenylhexatriene; Fluoresceins; Laurates; Light; Lipid Bilayers; Lipids; Lipopeptides; Liposomes; Micelles; Microscopy, Atomic Force; Microscopy, Fluorescence; Peptides, Cyclic; Phosphatidylcholines; Phospholipids; Rhodamines; Scattering, Radiation; Spectrometry, Fluorescence

The toxicity of Loxosceles spider venom has been attributed to a rare enzyme, sphingomyelinase D, which transforms sphingomyelin to ceramide-1-phosphate. The bases of its inflammatory and dermonecrotic activity, however, remain unclear. In this work the effects of ceramide-1-phosphate on model membranes were studied both by in situ generation of this lipid using a recombinant sphingomyelinase D from the spider Loxosceles laeta and by pre-mixing it with sphingomyelin and cholesterol. The systems of choice were large unilamellar vesicles for bulk studies (enzyme kinetics, fluorescence spectroscopy and dynamic light scattering) and giant unilamellar vesicles for fluorescence microscopy examination using a variety of fluorescent probes. The influence of membrane lateral structure on the kinetics of enzyme activity and the consequences of enzyme activity on the structure of target membranes containing sphingomyelin were examined. The findings indicate that: 1) ceramide-1-phosphate (particularly lauroyl ceramide-1-phosphate) can be incorporated into sphingomyelin bilayers in a concentration-dependent manner and generates coexistence of liquid disordered/solid ordered domains, 2) the activity of sphingomyelinase D is clearly influenced by the supramolecular organization of its substrate in membranes and, 3) in situ ceramide-1-phosphate generation by enzymatic activity profoundly alters the lateral structure and morphology of the target membranes.

Topics: 2-Naphthylamine; Animals; Calorimetry, Differential Scanning; Ceramides; Cholesterol; Fluorescence Resonance Energy Transfer; Kinetics; Laurates; Light; Membranes, Artificial; Microscopy, Fluorescence; Phosphatidylcholines; Phosphoric Diester Hydrolases; Scattering, Radiation; Sphingomyelins; Spiders; Temperature; Unilamellar Liposomes

The feasibility of applying multiphoton excitation fluorescence microscopy-related techniques in planar membrane systems, such as lipid monolayers at the air-water interface (named Langmuir films), is presented and discussed in this paper. The non-linear fluorescence microscopy approach, allows obtaining spatially and temporally resolved information by exploiting the fluorescent properties of particular fluorescence probes. For instance, the use of environmental sensitive probes, such as LAURDAN, allows performing measurements using the LAURDAN generalized polarization function that in turn is sensitive to the local lipid packing in the membrane. The fact that LAURDAN exhibit homogeneous distribution in monolayers, particularly in systems displaying domain coexistence, overcomes a general problem observed when "classical" fluorescence probes are used to label Langmuir films, i.e. the inability to obtain simultaneous information from the two coexisting membrane regions. Also, the well described photoselection effect caused by excitation light on LAURDAN allows: (i) to qualitative infer tilting information of the monolayer when liquid condensed phases are present and (ii) to provide high contrast to visualize 3D membranous structures at the film's collapse pressure. In the last case, computation of the LAURDAN GP function provides information about lipid packing in these 3D structures. Additionally, LAURDAN GP values upon compression in monolayers were compared with those obtained in compositionally similar planar bilayer systems. At similar GP values we found, for both DOPC and DPPC, a correspondence between the molecular areas reported in monolayers and bilayers. This correspondence occurs when the lateral pressure of the monolayer is 26+/-2 mN/m and 28+/-3 mN/m for DOPC and DPPC, respectively.

Topics: 1,2-Dipalmitoylphosphatidylcholine; 2-Naphthylamine; Laurates; Membranes, Artificial; Microscopy, Fluorescence, Multiphoton; Phase Transition; Phosphatidylcholines

The nonsteroidal anti-inflammatory drug indomethacin exhibits diverse biological effects, many of which have no clear molecular mechanism. Membrane-bound receptors and enzymes are sensitive to their phospholipid microenvironment. Amphipathic indomethacin could therefore potentially modulate cell signaling by changing membrane properties. Here we examined the effect of indomethacin on membrane lateral heterogeneity. Fluorescence lifetime imaging of cells expressing lipid-anchored probes revealed that treatment of BHK cells with therapeutic levels of indomethacin enhances cholesterol-dependent nanoclustering, but not cholesterol-independent nanoclustering. Immuno-electron microscopy and quantitative spatial mapping of intact plasma membrane sheets similarly showed a selective effect of indomethacin on promoting cholesterol-dependent, but not cholesterol-independent, nanoclustering. To further evaluate the biophysical effects of indomethacin, we measured fluorescence polarization of the phase-sensitive probe Laurdan and FRET between phase-partitioning probes in model bilayers. Therapeutic levels of indomethacin enhanced phase separation in DPPC/DOPC/Chol (1:1:1) and DPPC/Chol membranes in a temperature-dependent manner, but had minimal effect on the phase behavior of pure DOPC at any temperature. Taken together, the imaging results on intact epithelial cells and the biophysical assays of model membranes suggest that indomethacin can enhance phase separation and stabilize cholesterol-dependent nanoclusters in biological membranes. These effects on membrane lateral heterogeneity may have significant consequences for cell signaling cascades that are assembled on the plasma membrane.

Topics: 2-Naphthylamine; Animals; Anti-Inflammatory Agents, Non-Steroidal; Cell Line; Cell Membrane; Cholesterol; Cricetinae; Fluorescence Polarization; Fluorescent Dyes; Humans; Indomethacin; Laurates; Membranes, Artificial; Phosphatidylcholines; Signal Transduction

One key tenet of the raft hypothesis is that the formation of glycosphingolipid- and cholesterol-rich lipid domains can be driven solely by characteristic lipid-lipid interactions, suggesting that rafts ought to form in model membranes composed of appropriate lipids. In fact, domains with raft-like properties were found to coexist with fluid lipid regions in both planar supported lipid layers and in giant unilamellar vesicles (GUVs) formed from 1) equimolar mixtures of phospholipid-cholesterol-sphingomyelin or 2) natural lipids extracted from brush border membranes that are rich in sphingomyelin and cholesterol. Employing headgroup-labeled fluorescent phospholipid analogs in planar supported lipid layers, domains typically several microns in diameter were observed by fluorescence microscopy at room temperature (24 degrees C) whereas non-raft mixtures (PC-cholesterol) appeared homogeneous. Both raft and non-raft domains were fluid-like, although diffusion was slower in raft domains, and the probe could exchange between the two phases. Consistent with the raft hypothesis, GM1, a glycosphingolipid (GSL), was highly enriched in the more ordered domains and resistant to detergent extraction, which disrupted the GSL-depleted phase. To exclude the possibility that the domain structure was an artifact caused by the lipid layer support, GUVs were formed from the synthetic and natural lipid mixtures, in which the probe, LAURDAN, was incorporated. The emission spectrum of LAURDAN was examined by two-photon fluorescence microscopy, which allowed identification of regions with high or low order of lipid acyl chain alignment. In GUVs formed from the raft lipid mixture or from brush border membrane lipids an array of more ordered and less ordered domains that were in register in both monolayers could reversibly be formed and disrupted upon cooling and heating. Overall, the notion that in biomembranes selected lipids could laterally aggregate to form more ordered, detergent-resistant lipid rafts into which glycosphingolipids partition is strongly supported by this study.

Topics: 1,2-Dipalmitoylphosphatidylcholine; 2-Naphthylamine; Animals; Cholesterol; Fluorescent Dyes; G(M1) Ganglioside; Kidney Cortex; Laurates; Lipid Bilayers; Membrane Lipids; Microscopy, Fluorescence; Microvilli; Models, Biological; Models, Molecular; Molecular Conformation; Phosphatidylcholines; Phosphatidylethanolamines; Rats; Rats, Sprague-Dawley; Sphingomyelins

There is an increasing body of evidence to support the notion that the function of the nicotinic acetylcholine receptor (AChR) is influenced by its lipid microenvironment [see Barrantes, F. J. (1993) FASEB J. 7, 1460-1467]. We have recently made use of the so-called generalized polarization (GP) of the fluorescent probe Laurdan (6-dodecanoyl-2-(dimethylamino)naphthalene) to learn about the physical state of the lipids in Torpedo marmorata AChR native membrane [Antollini, S. S., Soto, M. A., Bonini de Romanelli, I., Gutiérrez Merino, C., Sotomayor, P., and Barrantes, F. J. (1996) Biophys. J. 70, 1275-1284] and cells expressing endogenous or heterologous AChR [Zanello, L. P., Aztiria, E., Antollini, S., and Barrantes, F. J. (1996) Biophys. J. 70, 2155-2164]. In the present work, Laurdan GP was measured in T. marmorata native AChR membrane by direct excitation or under energy transfer conditions in the presence of exogenous lipids. GP was found to diminish in these two regions upon addition of oleic acid and dioleoylphosphatidylcholine and not to vary significantly upon addition of cholesterol hemisuccinate, indicating an increase in the polarity of the single, ordered-liquid lipid phase in the two former cases. Complementary information about the bulk lipid order was obtained from measurements of fluorescence anisotropy of DPH and two of its derivatives. The membrane order diminished in the presence of oleic acid and dioleoylphosphatidylcholine. The location of Laurdan was determined using the parallax method. Laurdan lies at approximately 10 A from the center of the bilayer, i.e., at depth of approximately 5 A from the lipid-water interface. Exogenous lipids modified the energy transfer efficiency from the intrinsic fluorescence to Laurdan. This strategy is introduced as a new analytic tool that discloses for the first time the occurrence of discrete and independent sites for phospholipids and sterols, respectively, both accessible to fatty acids, and presumably located at a shallow depth close to the phospholipid polar head region in the native AChR membrane.

Topics: 2-Naphthylamine; Animals; Binding Sites; Cholesterol; Cholesterol Esters; Fatty Acids; Fluorescence Polarization; Fluorescent Dyes; Intracellular Membranes; Laurates; Lipid Metabolism; Lipids; Phosphatidylcholines; Phospholipids; Receptors, Nicotinic; Solvents; Spectrometry, Fluorescence; Sterols; Torpedo