Laurdan Generalized Polarization Research Articles

Novel bioactive lipids enhanced HDL-mediated cholesterol efflux from macrophages through the ABCA1 receptor pathway

High-density lipoprotein (HDL) has traditionally been acknowledged as "good cholesterol" owing to its significant association with a decreased risk of atherosclerosis. This association is primarily attributed to HDL's direct involvement in cholesterol efflux capacity, which plays a pivotal role in reverse cholesterol transport. A novel active compound from Nannochloropsis microalgae termed lyso-DGTS, a lipid that contains EPA fatty acids, was previously isolated and found to increase paraoxonase 1 activity and enhance HDL-mediated cholesterol efflux and HDL-induced endothelial nitric oxide release. Here, the effect of different lyso-DGTS derivatives and analogs on HDL-mediated cholesterol efflux from macrophages was examined, and the mechanism was explored. Structure–activity relationships were established to characterize the essential lipid moieties responsible for HDL-mediated cholesterol efflux from macrophages. Lyso-DGTS, 1-carboxy-N-N-N-trimethyl-3-oleamidopropan-1-aminium, and lyso-platelet-activating factor increased HDL-mediated cholesterol efflux from macrophages dose-dependently, mainly via the ABCA1-mediated cholesterol efflux pathway. The effect of lyso-DGTS derivatives and analogs on the surface polarity of HDL was examined using the Laurdan generalized polarization (GP) assay. A reverse Pearson linear regression was obtained between Laurdan GP values and HDL-mediated cholesterol efflux. Because the incorporation of bioactive lipids into the surface phospholipid layer of HDL leads to a decrease in Laurdan GP, these bioactive lipids may induce lower phospholipid ordering and greater free space on the HDL particle surface, thereby enhancing apolipoprotein A1 binding to the ABCA1 receptor and improving ABCA1 cholesterol-mediated efflux. Our findings suggest a beneficial effect of lyso-DGTS and its bioactive lipid derivatives on increasing HDL-mediated cholesterol efflux activity from macrophages, which may impact atherosclerosis attenuation.

Laurdan in live cell imaging: Effect of acquisition settings, cell culture conditions and data analysis on generalized polarization measurements.

Cell function is highly dependent on membrane structure, organization, and fluidity. Therefore, methods to probe the biophysical properties of biological membranes are required. Determination of generalized polarization (GP) values using Laurdan in fluorescence microscopy studies is one of the most widely-used methods to investigate changes in membrane fluidity in vitro and in vivo. In the last couple of decades, there has been a major increase in the number of studies using Laurdan GP, where several different methodological approaches are used. Such differences interfere with data interpretation inasmuch as it is difficult to validate if Laurdan GP variations actually reflect changes in membrane organization or arise from biased experimental approaches. To address this, we evaluated the influence of different methodological details of experimental data acquisition and analysis on Laurdan GP. Our results showed that absolute GP values are highly dependent on several of the parameters analyzed, showing that incorrect data can result from technical and methodological inconsistencies. Considering these differences, we further analyzed the impact of cell variability on GP determination, focusing on basic cell culture conditions, such as cell confluency, number of passages and media composition. Our results show that GP values can report alterations in the biophysical properties of cell membranes caused by cellular adaptation to the culture conditions. In summary, this study provides thorough analysis of the factors that can lead to Laurdan GP variability and suggests approaches to improve data quality, which would generate more precise interpretation and comparison within individual studies and among the literature on Laurdan GP.

LAURDAN since Weber: The Quest for Visualizing Membrane Heterogeneity.

Any chemist studying the interaction of molecules with lipid assemblies will eventually be confronted by the topic of membrane bilayer heterogeneity and may ultimately encounter the heterogeneity of natural membranes. In artificial bilayers, heterogeneity is defined by phase segregation that can be in the nano- and micrometer range. In biological bilayers, heterogeneity is considered in the context of small (10-200 nm) sterol and sphingolipid-enriched heterogeneous and highly dynamic domains. Several techniques can be used to assess membrane heterogeneity in living systems. Our approach is to use a fluorescent reporter molecule immersed in the bilayer, which, by changes in its spectroscopic properties, senses physical-chemistry aspects of the membrane. This dye in combination with microscopy and fluctuation techniques can give information about membrane heterogeneity at different temporal and spatial levels: going from average fluidity to number and diffusion coefficient of nanodomains. LAURDAN (6-dodecanoyl-2-(dimethylamino) naphthalene), is a fluorescent probe designed and synthesized in 1979 by Gregorio Weber with the purpose to study the phenomenon of dipolar relaxation. The spectral displacement observed when LAURDAN is either in fluid or gel phase permitted the use of the technique in the field of membrane dynamics. The quantitation of the spectral displacement was first addressed by the generalized polarization (GP) function in the cuvette, a ratio of the difference in intensity at two wavelengths divided by their sum. In 1997, GP measurements were done for the first time in the microscope, adding to the technique the spatial resolution and allowing the visualization of lipid segregation both in liposomes and cells. A new prospective to the membrane heterogeneity was obtained when LAURDAN fluorescent lifetime measurements were done in the microscope. Two channel lifetime imaging provides information on membrane polarity and dipole relaxation (the two parameters responsible for the spectral shift of LAURDAN), and the application of phasor analysis allows pixel by pixel understanding of these two parameters in the membrane. To increase temporal resolution, LAURDAN GP was combined with fluctuation correlation spectroscopy (FCS) and the motility of nanometric highly packed structures in biological membranes was registered. Lately the application of phasor analysis to spectral images from membranes labeled with LAURDAN allows us to study the full spectra pixel by pixel in an image. All these methodologies, using LAURDAN, offer the possibility to address different properties of membranes depending on the question being asked. In this Account, we will focus on the principles, advantages, and limitations of different approaches to orient the reader to select the most appropriate technique for their research.

Correlated flickering of erythrocytes membrane observed with dual time resolved membrane fluctuation spectroscopy under different d-glucose concentrations

A correlated human red blood cell membrane fluctuation dependent on d-glucose concentration was found with dual time resolved membrane fluctuation spectroscopy (D-TRMFS). This new technique is a modified version of the dual optical tweezers method that has been adapted to measure the mechanical properties of red blood cells (RBCs) at distant membrane points simultaneously, enabling correlation analysis. Mechanical parameters under different d-glucose concentrations were obtained from direct membrane flickering measurements, complemented with membrane fluidity measurements using Laurdan Generalized Polarization (GP) Microscopy. Our results show an increase in the fluctuation amplitude of the lipid bilayer, and a decline in tension value, bending modulus and fluidity as d-glucose concentration increases. Metabolic mechanisms are proposed as explanations for the results.

Hybrid lipid/block copolymer vesicles display broad phase coexistence region

The fluidity and polar environment of ~100 nm hybrid vesicles combining dipalmitoylphosphatidylcholine (DPPC) and poly(1,2-butadiene)-block-polyethylene oxide (PBd-PEO, average molecular weight 950 g/mol) were studied upon vesicle heating using the fluorescence spectroscopy techniques of DPH anisotropy and laurdan generalized polarization (GP). These techniques indicated PBd-PEO membranes are less ordered than solid DPPC, but slightly more ordered than fluid DPPC or dioleoylphosphatidylcholine (DOPC) membranes. We find the DPH anisotropy values are less than expected from additivity of the components' anisotropies in the fluid phase mixture of DPPC and PBd-PEO, inferring that DPPC strongly fluidizes the PBd-PEO. We use transitions in DPH anisotropy and laurdan GP to create a temperature/composition phase diagram for DPPC/PBd-PEO which we find displays a significantly broader solid/fluid phase coexistence region than DPPC/DOPC, showing that DPPC partitions less readily into fluid PBd-PEO than into fluid DOPC. The existence of a broad solid/fluid phase coexistence region in DPPC/PBd-PEO vesicles is verified by Förster resonance energy transfer results and the visualization of phase separation in giant unilamellar vesicles containing up to 95% PBd-PEO and a single phase in 100% PBd-PEO vesicles at room temperature. These results add to the limited knowledge of phase behavior and phase diagrams of hybrid vesicles, and should be useful in understanding and tailoring membrane surface architecture toward biomedical applications such as drug delivery or membrane protein reconstitution.

Effect of betulin and betulonic acid on isolated rat liver mitochondria and liposomes

The paper considers the effects of plant triterpenoid betulin and its derivative betulonic acid on rat liver mitochondria and liposomes. It was found that betulonic acid and, to a lesser extent, betulin, activate mitochondrial respiration in states 2 and 4 and inhibit ADP- and DNP-stimulated (uncoupled) respiration. The effect of betulonic acid resulted in a significant decrease of the respiratory control and ADP/O ratios and decrease in Δψ. The effects of both compounds were most pronounced in the case of succinate-fueled mitochondrial respiration. This may include both the possible protonophore effect of betulonic acid and the inhibition of respiratory chain complexes by both compounds. Both agents enhanced H2O2 production in succinate-fueled mitochondria, while betulonic acid exerted an antioxidant effect with NAD-dependent substrates. Betulin was found to induce mitochondrial aggregation, but had no effect on membrane permeability. A similar pattern was found on liposomes. As revealed by the laurdan generalized polarization (GP) technique, betulin increased laurdan GP in lecithin liposomes, indicating a decrease in membrane fluidity. Measurements of GP as a function of fluorescence excitation wavelength gave an ascending line for high concentrations of betulin, which can be interpreted as phase heterogeneity of the lipid/betulin system. High concentrations of betulin (> 60 mol%) was also demonstrated to cause permeabilization of lecithin liposomes. Betulonic acid was much less effective in inducing the aggregation of mitochondria and liposomes and had no effect on membrane permeability. The possible mechanisms of betulin and betulonic acid effect on rat liver mitochondria and liposomes are discussed.

Biophysical characterization of the insertion of two potent antimicrobial peptides-Pin2 and its variant Pin2[GVG] in biological model membranes

The aim of this study was to investigate the factors that govern the activity and selectivity of two potent antimicrobial peptides (AMPs) using lipid membrane models of bacterial, erythrocyte and fungal cells. These models were used in calcein liposome leakage experiments to explore peptide efficiency. The AMPs (Pin2 and its variant Pin2[GVG]) showed highest affinity towards the bacterial models in the nanomolar range, followed by the erythrocyte and fungal systems. The presence of sterols modulated the variant's selectivity, while the wild type was unaffected. Liposome leakage experiments with Fluorescein Isothiocyanate-dextran (FITC)-dextran conjugates indicated that pore size depended on peptide concentration. Dynamic Light Scattering revealed peptide aggregation in aqueous solution, and that aggregate size was related to activity. The interacting peptides did not alter liposome size, suggesting pore forming activity rather than detergent activity. Atomic Force Microscopy showed differential membrane absorption, being greater in the bacterial model compared to the mammalian model, and pore-like defects were observed. Electrophysiological assays with the Tip-Dip Patch Clamp method provided evidence of changes in the electrical resistance of the membrane. Membrane potential experiments showed that liposomes were also depolarized in the presence of the peptides. Both peptides increased the Laurdan Generalized Polarization of the bacterial model indicating increased viscosity, on the contrary, no effect was observed with the erythrocyte and the fungal models. Peptide membrane insertion and pore formation was corroborated with Langmuir Pressure-Area isotherms and Brewster Angle Microscopy. Finally, molecular dynamics simulations were used to get an insight into the molecular mechanism of action.

Intragenic Antimicrobial Peptide Hs02 Hampers the Proliferation of Single- and Dual-Species Biofilms of P. aeruginosa and S. aureus: A Promising Agent for Mitigation of Biofilm-Associated Infections.

Pseudomonas aeruginosa and Staphylococcus aureus are two major pathogens involved in a large variety of infections. Their co-occurrence in the same site of infection has been frequently reported and is linked to enhanced virulence and difficulty of treatment. Herein, the antimicrobial and antibiofilm activities of an intragenic antimicrobial peptide (IAP), named Hs02, which was uncovered from the human unconventional myosin 1H protein, were investigated against several P. aeruginosa and S. aureus strains, including multidrug-resistant (MDR) isolates. The antibiofilm activity was evaluated on single- and dual-species biofilms of P. aeruginosa and S. aureus. Moreover, the effect of peptide Hs02 on the membrane fluidity of the strains was assessed through Laurdan generalized polarization (GP). Minimum inhibitory concentration (MIC) values of peptide Hs02 ranged from 2 to 16 μg/mL against all strains and MDR isolates. Though Hs02 was not able to hamper biofilm formation by some strains at sub-MIC values, it clearly affected 24 h preformed biofilms, especially by reducing the viability of the bacterial cells within the single- and dual-species biofilms, as shown by confocal laser scanning microscopy (CLSM) and atomic force microscopy (AFM) images. Laurdan GP values showed that Hs02 induces membrane rigidification in both P. aeruginosa and S. aureus. Peptide Hs02 can potentially be a lead for further improvement as an antibiofilm agent.

Inorganic mercury and cadmium induce rigidity in eukaryotic lipid extracts while mercury also ruptures red blood cells

Hg and Cd are non-essential toxic heavy metals that bioaccumulate in the tissues of living systems but less is known about their interactions with Eukaryotic lipid bilayers. Microscopy experiments showed that Hg and Cd changed the cell morphology of rabbit erythrocytes while Hg also induced cell rupture. As membranes are one of the first available targets, our study aimed to better understand metal-lipid interactions that could lead to toxic effects. Fluorescence spectroscopy (Laurdan Generalized Polarization) and dynamic light scattering were used to analyze metal-induced changes in membrane fluidity and the size of liposomes composed of Brain (Porcine), Liver (Bovine), Heart (Bovine) and Yeast (S. cerevisiae) lipid extracts. Under physiological chloride and pH levels, Hg irreversibly cleaves plasmalogens resulting in an increase in membrane rigidity. These lipids are enriched in Brain, Heart and Erythrocyte membranes and are important in signalling and the protection against oxidative stress. Interestingly, Hg had a heavily reduced effect on the plasmalogen-free Yeast extract membrane. In contrast, Cd induced rigidity by targeting negatively charged phosphatidic acid, phosphatidylserine, phosphatidylinositol, phosphatidylglycerol and cardiolipin in these extracts. Metal-induced liposome aggregation depended on the proportion of negatively charged lipids/plasmalogen and even the order of metal addition. Our results show that data from model systems correlate with trends observed in complex biological extracts and red blood cells and serve as a predictive tool for analyzing metal-lipid interactions. The determination of the specific lipid targets for Hg and Cd provides new insights how these metals exert toxic effects on cell membranes.

Study of the mechanism of permeabilization of lecithin liposomes and rat liver mitochondria by the antimicrobial drug triclosan

The effect of the antimicrobial compound triclosan (5-chloro-2′-(2,4-dichlorophenoxy)phenol) on the permeability of lecithin liposomes and rat liver mitochondria was studied. It was found that triclosan was able to increase nonspecific permeability of liposomes in a dose-dependent manner, which was detected by the release of the fluorescent probe sulforhodamine B (SRB) from vesicles. A partial release of SRB occurs instantly at the moment of triclosan addition, which is followed by a slow leakage of the dye. The triclosan-induced release of SRB from liposomes grew as pH of the medium was decreased from 9.5 to 7.5. As revealed by the laurdan generalized polarization (GP) technique, triclosan increased laurdan GP in lecithin liposomes, indicating a decrease in membrane fluidity. Measurements of GP as a function of fluorescence excitation wavelength gave an ascending line for triclosan-containing liposomes, which can be interpreted as phase heterogeneity of the lipid/triclosan system. Dynamic light scattering experiments also showed that at a high triclosan-to-lipid molar ratio (~0.5), a population of smaller light-scattering particles (~0.4 of the size of liposomes) appear in the system. Experiments with rat liver mitochondria demonstrated that triclosan (10–70μM) induced a high-amplitude cyclosporin А-insensitive swelling of the organelles accompanied the release of cytochrome c. On the basis of the results obtained, possible mechanisms of the toxic effect of triclosan in eukaryotic cells are discussed.

Membrane properties of giant polymer and lipid vesicles obtained by electroformation and pva gel-assisted hydration methods

Giant Unilamellar Vesicles (GUV) are the most commonly used biomimetic membrane model. Their production in physiological conditions through the well-known electroformation process is complex and other gel-assisted hydration procedures have recently emerged to circumvent some limitations of the process. In this work, the membrane physical properties of Giant Polymer Unilamellar Vesicles (Polymersomes) and lipid vesicles (Liposomes) obtained from the gel-assisted hydration procedure and electroformation, have been evaluated thoroughly by different techniques: micropipettes aspiration (MPA), Fluorescence Recovery After Photo Bleaching (FRAP) and Laurdan Generalized Polarization (GP). The study was performed on GUVs made of either phospholipids (POPC and DPPC) or amphiphilic copolymers based on Poly(dimethylsiloxane) and poly(ethyleneoxide). A significant deviation of stretching modulus, lateral diffusion coefficient and membrane packing was observed for GUVs formed by the PVA gel-assisted hydration method compared to the classical electroformation process. We were able to show that this deviation was due to the presence of PVA chains in the membrane and in the suspending medium. Globally our findings reveal that particular attention has to be paid on the gel assisted hydration process as the vesicles obtained present altered mechanical behaviour.

Enzymatic studies on planar supported membranes using a widefield fluorescence LAURDAN Generalized Polarization imaging approach

We introduce a custom-built instrument designed to perform fast LAURDAN Generalized Polarization (GP) imaging on planar supported membranes. It is mounted on a widefield fluorescence microscope and allows kinetic analysis of the GP function in the millisecond time scale, largely improving the temporal resolution previously achieved using laser scanning based microscopes. A dedicated protocol to calibrate LAURDAN GP data obtained with charge-coupled device (CCD) cameras as detectors is also presented, enabling reliable assignment of GP values in the field of view. Using this methodology we studied structural and dynamical transformations induced by Sphingomyelinase D (SM-D) on planar supported membranes composed of N-lauroyl sphingomyelin (C12SM). GP data show the evolution of an initially compositionally homogeneous symmetric bilayer existing in a single liquid disordered phase, to an intermediate configuration showing coexistence of liquid disordered and solid ordered domains, which are not always in-register across the axial plane of the bilayer. This intermediate state, caused by the transformation of C12SM to C12-ceramide-1-phosphate in the distal leaflet of the bilayer, evolved to a single solid ordered phase at longer time scales. Additionally, we comparatively studied this system using the membrane fluorophore DiIC18. The advantages and limitations of both fluorescent dyes are discussed, emphasizing the adequacy of LAURDAN GP imaging to explore this type of membrane phenomena.

Membrane Heterogeneity in Erythrocytes Studied by Laurdan GP and Flim-Phasor

Detergents can selectively solubilize membrane constituents by incorporating them into small micelles, whereas other components may remain in the so-called detergent-resistant membrane fragments. This differential interaction provides a unique tool to selectively isolate proteins and study their localization into specific membrane environments or domains. Laurdan Generalized Polarization (Laurdan GP), defined as [(I440-I490)/(I440+I490)] (1), measures the red spectral shift of the molecule due to polarity and dipole relaxation processes. This parameter gives valuable information about membrane heterogeneity; however there is more information veiled behind the Laurdan spectral shift that can be revealed by lifetime measurements at the two GP wavelengths (2). Lifetime measurements can isolate the two effects measured by Laurdan GP: the polarity of the environment (reported mainly by the intensity at 440 nm) and the rate of dipolar relaxation of water molecules that can reorient around laurdan's dipole during its lifetime (monitored at 490 nm)We used sucrose monoester of myristic acid, b-D-Fructofuranosyl-6-O-myristyl-a-D-Glucopyranoside (MMS) and Laurdan (6-lauroyl,1-2-dimethylamino naphthalene) to study the changes promoted in membrane heterogeneity in erythrocytes with different cholesterol content. We followed the hemolysis, the changes in size and the changes in fluidity on the membrane of erythrocytes treated with increasing amount of MMS. Our results indicate that the insertion of MMS molecules in the membrane produces a decrease in polarity of the bilayer (reported mainly by the intensity at 440 nm) together with an increase on dipolar relaxation of water molecules around Laurdan (reported at 490 nm).Financial support: Fondecyt #1140454 (S.S.), Fondecyt# 1120196 (G.G.) and Beca de Doctorado Conicyt 21120554 (C.S.). Technical Support: CMABioBio-UdeC.[1] Parasassi T, et al. Biophys J. 57; 1179, 1990.[2] Golfetto O, et al. Biophys J. 104, 1238-1247, 2013

Surface Properties and Membrane Packing in Hybrid Liposomes Composed of Tetraether and Diester Lipids

Archaeal bipolar tetraether lipids are extraordinarily stable against biochemical and physical stressors and non-toxic to animals; thus, they are appealing biomaterials that hold great promise for technological applications. Here, we used electrophoretic mobility measurements and TNS fluorescence to determine zeta potential and surface potential of liposomes (∼150 nm in diameter) composed of archaeal tetraether lipids and conventional diester lipids. We also used Laurdan fluorescence to explore membrane packing in the same membrane system. The polar lipid fraction E (PLFE) from the archaeon S. acidocaldarius was used as the tetraether component. PLFE lipids are asymmetric macrocyclic molecules carrying a negative charge on the phosphate moiety at one of the two polar ends at neutral pH. We found that zeta potential varies with PLFE content in a biphasic manner, being most negative when the PLFE content is ∼60 mol%. Surface potential is also PLFE content dependent, showing a minimum at ∼50 mol% PLFE. On the other hand, Laurdan GP (generalized polarization) data, inferentially membrane packing, displays a peculiar behavior at 20-40 mol% PLFE. In a separate study, we used the AAPH-induced Laurdan fluorescence intensity change to deduce information about membrane packing. AAPH (2,2'-azobis (2-amidinopropane) dihydrochloride) is a water-soluble, free-radical generator. Upon addition of AAPH, there was a lag time (t), followed by a steady decrease in Laurdan fluorescence intensity due to probe oxidation. The t values as well as the initial rate (R) of AAPH-induced Laurdan oxidation were found to change with PLFE mole fraction in a non-monotonic manner, showing an anomaly between 20-50 mol % PLFE. These data together suggest that, in the range 20-50 mol %, these hybrid liposomes undergo major structural changes engendered by PLFE content.

Unique thermal behavior of sphingomyelin species with nonhydroxy and 2-hydroxy very-long-chain (C28-C32) PUFAs

In rat germ cells and spermatozoa, sphingomyelin (SM) contains molecular species with nonhydroxy (n) and 2-hydroxy (h) very-long-chain polyunsaturated fatty acids (V), the most abundant being SMs with (n- and h-) 28:4n-6, 30:5n-6, and 32:5n-6 as acyl chains. The aim of this study was to gain information about their thermotropic behavior and interactions with other lipids. After isolation from rat testis, multilamellar and giant unilamellar vesicles from these SMs were examined using fluorescent probes. Only n-32:5 SM and h-32:5 SM displayed a gel-liquid transition temperature (Tt ∼ 21-22°C), the rest remaining in the liquid state in the 5°C-45°C range. The degree of order was larger in bilayers of any of the h-V SMs than in those of their chain-matched n-V SMs. Both, but n-V SM relatively more than h-V SM, decreased the Tt of dimyristoylphosphatidylcholine as their proportion increased in binary phosphatidylcholine:SM liposomes. In contrast to the established ability of 16:0 SM to form lateral cholesterol/SM-rich ordered domains in ternary dioleoylphosphatidylcholine:cholesterol:SM bilayers, neither n-V SM nor h-V SM showed a tendency to do so. Thus, these SMs are in the fluid state and are not involved in this type of domains in spermatozoa at physiological temperatures. However, this state could be altered at the very low temperatures at which these gametes are usually preserved.

Understanding Plasma Memebrane Organization and Cellular Homeostasis Relationship

Plasma membrane (PM) is a dynamic barrier which translates the outside signals to the cell. We are interested in how changes in the PM (composition, organization etc) influence cell homeostasis and vice versa. For this purpose we constructed two permanent Chinese Hamster Ovary (CHO KI) cell lines with permanent modifications in the PM lipid composition. The first line (SCD), over expressing Stearoyl CoA desaturase resulted in an increase of monounsaturated fatty acids at the PM phospholipids. The second cell line over expressing both Δ5 and Δ 6 desaturases (Δ 5/ Δ 6-cells), turned into a rise of poly unsaturated fatty acids and lower ratio cholesterol/phospholipids.

Effects of Alpha Hemolysin from E.coli on Erythrocytes from Different Species

Alpha-hemolysin (HlyA)is one of the key virulence factors released by E. coli strains. This toxin causes lysis of various mammalian cells, including erythrocytes of different animal species.The hemolytic activity of the toxin on rabbit and sheep erythrocytes was determined, showing that rabbit is the most susceptible specie. Calcium concentration inside the erythrocytes while incubated with sublytic concentrations of HlyA was monitored using two-photon fluorescence Lifetime Imaging (FLIM), the calcium indicator Calcium Green 1 and the phasor analysis method. HlyA induces an increase in calcium concentration in both erythrocytes, but the increment in rabbit is 4 times higher and faster than in sheep. Two-photon Laurdan Generalized Polarization (GP) was used to determine the fluidity of the membrane (measured as the membrane water content) in the presence and absence of HlyA in live erythrocytes. The GP value for the sheep erythrocytes membranes was higher that the ones from rabbit and after incubation of the erythrocytes with sublytic concentration of Hlyn, an increase on the GP value was observed only on the rabbit membranes. Membrane lipid composition showed similar content of Cholesterol and PE in the two cell type, however the content of Phosphatidylcholine (PC) and sphingomyelin (SM) showed differences: sheep erythrocytes contained 28% of SM and not PC and Rabbit erythrocyte membranes present 10% SM and 18% of PC.We concluded that at sublytical concentration, the initial interaction of HlyA with the erythrocyte and the mechanisms of calcium influx strongly depend on the membrane composition and fluidity of the target cell.Financial support NIH RR03155 for S.S. and E.G. and CONICET (International Collaboration) for V.H.

Lipid packing determines protein-membrane interactions: challenges for apolipoprotein A-I and high density lipoproteins.

Protein and protein-lipid interactions, with and within specific areas in the cell membrane, are critical in order to modulate the cell signaling events required to maintain cell functions and viability. Biological bilayers are complex, dynamic platforms, and thus in vivo observations usually need to be preceded by studies on model systems that simplify and discriminate the different factors involved in lipid-protein interactions. Fluorescence microscopy studies using giant unilamellar vesicles (GUVs) as membrane model systems provide a unique methodology to quantify protein binding, interaction, and lipid solubilization in artificial bilayers. The large size of lipid domains obtainable on GUVs, together with fluorescence microscopy techniques, provides the possibility to localize and quantify molecular interactions. Fluorescence Correlation Spectroscopy (FCS) can be performed using the GUV model to extract information on mobility and concentration. Two-photon Laurdan Generalized Polarization (GP) reports on local changes in membrane water content (related to membrane fluidity) due to protein binding or lipid removal from a given lipid domain. In this review, we summarize the experimental microscopy methods used to study the interaction of human apolipoprotein A-I (apoA-I) in lipid-free and lipid-bound conformations with bilayers and natural membranes. Results described here help us to understand cholesterol homeostasis and offer a methodological design suited to different biological systems.

Combining LAURDAN Generalized Polarization, Fluorescence Correlation Spectroscopy and Fluorescence Lifetime Imaging as a Tool in Skin Diagnostics

The outermost layer of the skin tissue, i.e. the stratum corneum (SC), plays a key role in the barrier function of the skin. SC (10 to 20 micrometer thick) is comprised of dead, keratin filled cells residing in a matrix of complex extracellular lipids. Physical related features of skin SC such as the lipid lateral packing in the extracellular matrix, local proton (water) activity, or local diffusion of particular substances are crucial in determining the permeability of skin towards exogenously added compounds (e.g. drugs and cosmetics). In addition, these parameters may be altered in abnormal skin and further on used as biomarkers for detecting skin anomalies. In this work we decided to map and compare proton activity, polarity (that in turn is related to lipid packing of skin SC membranes) and local diffusion of particular substances in excised skin samples, obtained from wild type mice and abnormal mice, showing increased transepidermal water loss. The experimental techniques utilized are based in multiphoton excitation microscopy techniques. LAURDAN Generalized Polarization (GP) measurements are used to evaluate the overall polarity of potential transepidermal pathways (1). Our results show markedly higher GP values in skin lipid membranes from mice with increased transepidermal water loss compared to controls. To further assess the properties of the skin lipid membranes at the two different physiological conditions, local diffusion through the barrier is evaluated by fluorescence correlation spectroscopy. The data obtained are related to the proton gradient across the SC, as observed by fluorescent lifetime imaging (2). This combined strategy is being tested for potential implementations in skin diagnostics.1) D.C. Carrer, et al, Journal of Controlled Release (2008), doi:10.1016/j.jconrel.2008.08.006.2) Hanson et al. 2002, Biophys. J. 83, pp. 1682-1690.

Laurdan GP Fluctuations In Membranes Of Intact Erythrocytes

Lipids in the cell membranes are believed to be organized in micro-domains, known as rafts. If they in fact exist, rafts may provide important boundaries for the organization and sequestering of membrane proteins. The prevailing theory holds that membrane rafts are as small structures (10-200 nm), heterogeneous in size, highly dynamic and sterol- and sphingolipid-enriched domains that compartmentalize cellular processes.The existence of these small domains is still under debate despite a great deal of work in the area. The most commonly used method to detect rafts is their resistance to solubilization by the nonionic detergent Triton X-100 and sensitivity to cholesterol depletion. These measurements are indirect and potentially open to alternative interpretations. Directly visualizing rafts in living cells has been a difficult task because they are extremely small. Their existence still remains controversial.Here we report the use of a new methodology were two powerful microscopic techniques are applied simultaneously. The first technique, Laurdan Generalized polarization (GP), can differentiate liquid ordered phases from liquid disordered phases based on the water content rather than on the partition properties of the probe. The second technique, Scanning Fluorescence Correlation Spectroscopy quantifies the GP heterogeneities in the membranes. Using these techniques we observed GP fluctuations in the plasma membrane of rabbit red blood cells that can be explained by a model that includes the existence of lipid micro-domains, which are heterogeneous in size and mobility. The properties of these GP microdomains are similar to the proposed properties of rafts. Financial support is provided by NIH RR03155.