Muscovite (mica) allows the characterisation of supported bilayers by ellipsometry and confocal fluorescence correlation spectroscopy

We demonstrate for the first time that ellipsometry and confocal fluorescence correlation spectroscopy (FCS) are complementary methods for the characterisation of supported planar phospholipid bilayers (SPBs) formed on mica, a mineral used in atomic force microscopy investigations of SPBs. Addition of small unilamellar vesicles containing 20% dioleoyl-phosphatidylserine (DOPS) and 80% dioleoyl-phosphatidylcholine (DOPC) to an oxidised borosilicate surface, on the other hand, results in a planar lipid system characterised by lateral diffusion coefficients which are three time smaller than those obtained for SPBs. Moreover, seven labelled phospholipids were tested for their suitability in the FCS characterisation of vesicles as well as of SPBs.     

Local mobility in lipid domains of supported bilayers characterized by atomic force microscopy and fluorescence correlation spectroscopy

Fluorescence correlation spectroscopy (FCS) is used to examine mobility of labeled probes at specific sites in supported bilayers consisting of 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) lipid domains in 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC). Those sites are mapped beforehand with simultaneous atomic force microscopy and submicron confocal fluorescence imaging, allowing characterization of probe partitioning between gel DPPC and disordered liquid DOPC domains with corresponding topography of domain structure. We thus examine the relative partitioning and mobility in gel and disordered liquid phases for headgroup- and tailgroup-labeled GM1 ganglioside probes and for headgroup- and tailgroup-labeled phospholipid probes. For the GM1 probes, large differences in mobility between fluid and gel domains are observed; whereas unexpected mobility is observed in submicron gel domains for the phospholipid probes. We attribute the latter to domain heterogeneities that could be induced by the probe. Furthermore, fits to the FCS data for the phospholipid probes in the DOPC fluid phase require two components (fast and slow). Although proximity to the glass substrate may be a factor, local distortion of the probe by the fluorophore could also be important. Overall, we observe nonideal aspects of phospholipid probe mobility and partitioning that may not be restricted to supported bilayers.     

Continuous fluorescence microphotolysis and correlation spectroscopy using 4Pi microscopy

Continuous fluorescence microphotolysis (CFM) and fluorescence correlation spectroscopy (FCS) permit measurement of molecular mobility and association reactions in single living cells. CFM and FCS complement each other ideally and can be realized using identical equipment. So far, the spatial resolution of CFM and FCS was restricted by the resolution of the light microscope to the micrometer scale. However, cellular functions generally occur on the nanometer scale. Here, we develop the theoretical and computational framework for CFM and FCS experiments using 4Pi microscopy, which features an axial resolution of ∼100 nm. The framework, taking the actual 4Pi point spread function of the instrument into account, was validated by measurements on model systems, employing 4Pi conditions or normal confocal conditions together with either single- or two-photon excitation. In all cases experimental data could be well fitted by computed curves for expected diffusion coefficients, even when the signal/noise ratio was small due to the small number of fluorophores involved.     

Phospholipase A2 hydrolysis of supported phospholipid bilayers: a neutron reflectivity and ellipsometry study

We have investigated the phospholipase A(2) catalyzed hydrolysis of supported phospholipid bilayers using neutron reflection and ellipsometry. At the hydrophilic silica-water interface, hydrolysis of phosphatidylcholine bilayers by phospholipase A(2) from Naja mossambica mossambica venom is accompanied by destruction of the bilayer at an initial rate, which is comparable for DOPC and DPPC but is doubled for POPC. The extent of bilayer destruction at 25 degrees C decreases from DOPC to POPC and is dramatically reduced for DPPC. Neutron reflectivity measurements indicate that the enzyme penetrates into the bilayers in increasing order for DOPC, POPC, and DPPC, while the amount of enzyme adsorbed at the interface is smallest for DPPC and exhibits a maximum for POPC. Penetration into the hydrophobic chain region in the bilayer is further supported by the fact that the enzyme adsorbs strongly and irreversibly to a hydrophobic monolayer of octadecyltrichlorosilane. These results are rationalized in terms of the properties of the reaction products and the effect of their accumulation in the membrane on the kinetics of enzyme catalysis.     

A differential scanning calorimetry study of the interaction of lasalocid antibiotic with phospholipid bilayers

Interaction of lasalocid sodium salt (Las-Na) with dipalmitoylphosphatidylcholine (DPPC) as a membrane model was investigated by highly-sensitive differential scanning calorimetry (DSC). The insertion properties of the antibiotic were studied both in multilamellar suspensions and unilamellar vesicles, for Las-Na/DPPC molar ratios (r) ranging from 0.005 to 0.1. The effect of the antibiotic on the lipid thermotropic behavior is concentration dependent and drastically changes at a critical r of 0.04 in both model membranes. Below this ratio, Las-Na molecules interact with DPPC bilayers without disrupting the global organization of the membrane. In the multilamellar systems only the transition cooperativity is affected whereas for the mixed vesicles, a decrease in the enthalpy change suggests a different mode of insertion. Above this ratio, implantation of the antibiotic give rise to lateral phase separation in multilamellar systems. These structural modifications have repercussions on the formation of mixed LAS-Na/DPPC vesicles which seems limited to an r value of 0.04.     

Investigation of the interaction of myelin basic protein with phospholipid bilayers using solid-state NMR spectroscopy

Interaction of bovine myelin basic protein and its constituent charge isomers (C1-C3) with phospholipid bilayers was studied using solid-state NMR experiments on model membranes. 31P NMR experiments on multilamellar vesicles and mechanically aligned bilayers were used to measure the degree of protein-induced disorder in the lipid headgroup region while 2H NMR data provided the disorder caused by the protein in the hydrophobic core of the bilayers. Our results suggest that MBP and its charge isomers neither fragment nor significantly disrupt DMPC, POPC, POPC:POPG, and POPE bilayers. These results demonstrate that the MBP-induced fragmentation of POPC bilayers is due to the freeze-thaw cycles used in the preparation of multilamellar vesicles and not due to intrinsic protein-lipid interactions.     

Site-specific interaction of thrombin and inhibitors observed by fluorescence correlation spectroscopy.

We report on the application of fluorescence correlation spectroscopy (FCS) to observe the interaction between thrombin and thrombin inhibitors. Two site-specific fluorescent labels were used to distinguish between inhibitors directed to the active site, the exosite, or both binding sites of thrombin. For several well-known inhibitors of thrombin, the binding sites observed by FCS correspond to previous studies. The interaction of the recently discovered thrombin inhibitor ornithodorin from the tick Ornithodorus moubata with thrombin was investigated. It was found that this inhibitor, like hirudin and rhodniin, binds to both the active site and exosite of thrombin simultaneously. This study shows the feasibility of FCS as a sensitive and selective method for observing protein-ligand interactions. As an additional technique, simultaneous labeling with both fluorescent labels was successfully demonstrated.     

A correlation between lipid domain shape and binary phospholipid mixture composition in free standing bilayers: A two-photon fluorescence microscopy study

Giant unilamellar vesicles (GUVs) composed of different phospholipid binary mixtures were studied at different temperatures, by a method combining the sectioning capability of the two-photon excitation fluorescence microscope and the partition and spectral properties of 6-dodecanoyl-2-dimethylamino-naphthalene (Laurdan) and Lissamine rhodamine B 1,2-dihexadecanoyl-sn-glycero-3-phosphoethanolamine (N-Rh-DPPE). We analyzed and compared fluorescence images of GUVs composed of 1,2-dilauroyl-sn-glycero-3-phosphocholine/1, 2-dipalmitoyl-sn-glycero-3-phosphocholine (DLPC/DPPC), 1, 2-dilauroyl-sn-glycero-3-phosphocholine/1, 2-distearoyl-sn-glycero-3-phosphocholine (DLPC/DSPC), 1, 2-dilauroyl-sn-glycero-3-phosphocholine/1, 2-diarachidoyl-sn-glycero-3-phosphocholine (DLPC/DAPC), 1, 2-dimyristoyl-sn-glycero-3-phosphocholine/1, 2-distearoyl-sn-glycero-3-phosphocholine (DMPC/DSPC) (1:1 mol/mol in all cases), and 1,2-dimyristoyl-sn-glycero-3-phosphoethanolamine/1, 2-dimyristoyl-sn-glycero-3-phosphocholine (DMPE/DMPC) (7:3 mol/mol) at temperatures corresponding to the fluid phase and the fluid-solid phase coexistence. In addition, we studied the solid-solid temperature regime for the DMPC/DSPC and DMPE/DMPC mixtures. From the Laurdan intensity images the generalized polarization function (GP) was calculated at different temperatures to characterize the phase state of the lipid domains. We found a homogeneous fluorescence distribution in the GUV images at temperatures corresponding to the fluid region for all of the lipid mixtures. At temperatures corresponding to phase coexistence we observed concurrent fluid and solid domains in the GUVs independent of the lipid mixture. In all cases the lipid solid domains expanded and migrated around the vesicle surface as we decreased the temperature. The migration of the solid domains decreased dramatically at temperatures close to the solid-fluid-->solid phase transition. For the DLPC-containing mixtures, the solid domains showed line, quasicircular, and dendritic shapes as the difference in the hydrophobic chain length between the components of the binary mixture increases. In addition, for the saturated PC-containing mixtures, we found a linear relationship between the GP values for the fluid and solid domains and the difference between the hydrophobic chain length of the binary mixture components. Specifically, at the phase coexistence temperature region the difference in the GP values, associated with the fluid and solid domains, increases as the difference in the chain length of the binary mixture component increases. This last finding suggests that in the solid-phase domains, the local concentration of the low melting temperature phospholipid component increases as the hydrophobic mismatch decreases. At the phase coexistence temperature regime and based on the Laurdan GP data, we observe that when the hydrophobic mismatch is 8 (DLPC/DAPC), the concentration of the low melting temperature phospholipid component in the solid domains is negligible. This last observation extends to the saturated PE/PC mixtures at the phase coexistence temperature range. For the DMPC/DSPC we found that the nonfluorescent solid regions gradually disappear in the solid temperature regime of the phase diagram, suggesting lipid miscibility. This last result is in contrast with that found for DMPE/DMPC mixtures, where the solid domains remain on the GUV surface at temperatures corresponding to that of the solid region. In all cases the solid domains span the inner and outer leaflets of the membrane, suggesting a strong coupling between the inner and outer monolayers of the lipid membrane. This last finding extends previous observations of GUVs composed of DPPE/DPPC and DLPC/DPPC mixtures (, Biophys. J. 78:290-305).     

Investigation of temperature-induced phase transitions in DOPC and DPPC phospholipid bilayers using temperature-controlled scanning force microscopy

Under physiological conditions, multicomponent biological membranes undergo structural changes which help define how the membrane functions. An understanding of biomembrane structure-function relations can be based on knowledge of the physical and chemical properties of pure phospholipid bilayers. Here, we have investigated phase transitions in dipalmitoylphosphatidylcholine (DPPC) and dioleoylphosphatidylcholine (DOPC) bilayers. We demonstrated the existence of several phase transitions in DPPC and DOPC mica-supported bilayers by both atomic force microscopy imaging and force measurements. Supported DPPC bilayers show a broad L(beta)-L(alpha) transition. In addition to the main transition we observed structural changes both above and below main transition temperature, which include increase in bilayer coverage and changes in bilayer height. Force measurements provide valuable information on bilayer thickness and phase transitions and are in good agreement with atomic force microscopy imaging data. A De Gennes model was used to characterize the repulsive steric forces as the origin of supported bilayer elastic properties. Both electrostatic and steric forces contribute to the repulsive part of the force plot.     

The interaction of bacterial lipopolysaccharide with phospholipid bilayers and monolayers

The association of bacterial lipopolysaccharide with artificial membranes was studied in an attempt to understand the mechanism of binding of lipopolysaccharide to cell surfaces and to look for an effect on membrane stability. The membrane models used were phospholipid bilayers and monolayers. As measured by survival time, lipopolysaccharide was found to decrease the stability of bilayers at a concentration of 300 μg/ml. When assayed by dielectric breakdown, an effect of lipopolysaccharide was noticeable at concentrations of 50 μg/ml. In studies involving the penetration of monomolecular films of various phospholipids, native and alkali-treated lipopolysaccharide both caused increases in surface pressure, and therefore penetrated the films. However, alkali-treated lipopolysaccharide was at least ten times more efficient than the native product in penetration. Alkali-treated lipopolysaccharide had a greater degree of surface activity than native lipopolysaccharide, since alkali-treated lipopolysaccharide formed monomolecular films by itself, whereas native lipopolysaccharide did not. The changes in the surface pressure and surface potential of phospholipid films produced by lipopolysaccharide in the subsolution suggested that the interaction of lipopolysaccharide with phospholipid monolayers was by a combination of penetration and adsorption to the undersurface.     

Probing the interaction of Arg9Cys mutated phospholamban with phospholipid bilayers by solid-state NMR spectroscopy

Phospholamban (PLB) is a 52 amino acid integral membrane protein that interacts with the sarcoplasmic reticulum Ca^2 + ATPase (SERCA) and helps to regulate Ca^2 + flow. PLB inhibits SERCA impairing Ca^2 + translocation. The inhibition can be relieved upon phosphorylation of PLB. The Arg9 to Cys (R9C) mutation is a loss of function mutation with reduced inhibitory potency. The effect R9C PLB has on the membrane surface and the hydrophobic region dynamics was investigated by ³¹P and ²H solid-state NMR spectroscopy in multilamellar vesicles (MLVs). The ³¹P NMR spectra indicate that, like the phosphorylated PLB (P-PLB), the mutated R9C-PLB protein has significantly less interaction with the lipid bilayer headgroup when compared to wild-type PLB (WT-PLB). Similar to P-PLB, R9C-PLB slightly decreases ³¹P T₁ values in the lipid headgroup region. ²H S_CD order parameters of ²H nuclei along the lipid acyl chain decrease less dramatically for R9C-PLB and P-PLB when compared to WT-PLB. The results suggest that R9C-PLB interacts less with the membrane surface and hydrophobic region than WT-PLB. Detachment of the cytoplasmic domain of R9C-PLB from the membrane surface could be related to its loss of function.     

Molecular interactions of peptides with phospholipid vesicle membranes as studied by fluorescence correlation spectroscopy

Interactions of the peptides melittin and magainin with phospholipid vesicle membranes have been studied using fluorescence correlation spectroscopy. Molecular interactions of melittin and magainin with phospholipid membranes are performed in rhodamine-entrapped vesicles (REV) and in rhodamine-labelled phospholipid vesicles (RLV), which did not entrap free rhodamine inside. The results demonstrate that melittin makes channels into vesicle membranes since exposure of melittin to vesicles causes rhodamine release only from REV but not from RLV. It is obvious that rhodamine can not be released from RLV because the inside of RLV is free of dye molecules. In contrast, magainin breaks vesicles since addition of magainin to vesicles results in rhodamine release from both REV and RLV. As the inside of RLV is free of rhodamine, the appearance of rhodamine in solution confirms that these vesicles are broken into rhodamine-labelled phospholipid fragments after addition of magainin. This study is of pharmaceutical significance since it will provide insights that fluorescence correlation spectroscopy can be used as a rapid protocol to test incorporation and release of drugs by vesicles.     

The structure and stability of phospholipid bilayers by atomic force microscopy.

Atomic force microscopy (AFM) was used to investigate the structure, stability, and defects of the hydrophilic surfaces of Langmuir-Blodgett bilayer films of distearoylphosphatidylcholine (DSPC) and dipalmitoylphosphatidylethanolamine (DPPE) in the solid phase, and dilinoleoylphosphatidylethanolamine (DLPE) in the fluid phase. Their relative resilience to external mechanical stress by the scanning tip and by fluid exchange were also investigated. DPPE monolayers showed parallel ridges at the surface with a period of 0.49 nm, corresponding to the rows of aligned headgroups consistent with the known crystallographic structure. DSPC and DLPE monolayers did not show any periodic order. The solid DSPC and DPPE monolayers were stable to continued rastering by the AFM tip; however, the stability of DLPE monolayers depended on the pH of the aqueous environment. Structural defects in the form of monolayer gaps and holes were observed after fluid exchange, but the defects in DLPE monolayer at pH 11 were stable during consecutive scanning. At pH 9 and below, the defects induced by fluid exchange over DLPE monolayers were more extensive and were deformed easily by consecutive scanning of the AFM tip at a force of 10 nN. The pH dependence of resilience was explained by the increasing bending energy or frustration due to the high spontaneous curvature of DLPE monolayers at low pH. The tangential stress exerted by the AFM tip on the deformable monolayers eventually produced a ripple pattern, which could be described as a periodic buckling known as Shallamach waves.     

Confocal laser scanning microscopy of whole mouse ovaries: excellent morphology, apoptosis detection, and spectroscopy.

BACKGROUND: Ovaries consist of numerous follicles, oocytes, and granulosa cells in different stages of development. Many of these follicles will undergo an apoptotic process during the lifetime of the animal. By using proper tissue preparation methods, the events within the whole ovary can be observed by using 3D confocal microscopy. METHODS: Whole ovaries were stained with LysoTracker Red (LT), fixed with 4% paraformaldehyde (PF) and 1% glutaraldehyde (Glut), dehydrated with methanol (MEOH), and cleared with benzyl alcohol and benzyl benzoate (BABB). Using this tissue preparation technique, the ovary becomes relatively transparent, allowing its morphology to be observed with confocal microscopes. A spectral imaging system (PARISS) located on a conventional microscope was used to interpret the LT dye spectra and fixation products in the tissues with different excitation wavelengths. RESULTS: Apoptosis in the follicle was detected as clusters of intensely stained granulosa cells located in close proximity to the oocytes. The fixation with Glut and PF preserved morphological details, increased tissue fluorescence, thus increased the signal to noise of the background image. CONCLUSIONS: Thick tissues can be imaged after they are properly stained, aldehyde fixed, and BABB cleared. LT intensely stained single cells or clusters of apoptotic cells in the follicles and the nucleolus. Spectral differences between LT as an indicator of apoptosis and Glut-PF fixation was used to visualize ovarian morphology and apoptosis. The PARISS spectrophotometer revealed spectral peaks for LT at 609.6 nm and for Glut-PF at 471.3 nm. The proper use of the spectra from these fluorescence molecules is the foundation for high quality morphological images of apoptosis. By sequentially imaging the two probes with a 488 nm laser and a 543/568 nm laser, there was a reduction in fluorescent cross talk and an increase in image quality.     

Formation of supported phospholipid bilayers on molecular surfaces: role of surface charge density and electrostatic interaction

Electrostatic interaction is known to play important roles in the adsorption of charged lipids on oppositely charged surfaces. Here we show that, even for charge neutral (zwitterionic) lipids, electrostatic interaction is critical in controlling the adsorption and fusion of lipid vesicles to form supported phospholipid bilayers (SPBs) on surfaces. We use terminally functionalized alkanethiol self-assembled monolayers (SAMs) to systematically control the surface charge density. Charge neutral egg phophatidylcholine (eggPC) vesicles readily fuse into SPBs on either a positively charged 11-aminino-1-undecanethiol SAM or a negatively charged 10-carboxy-1-decanethiol SAM when the density of surface charge groups is > or = 80%. These processes depend critically on the buffer environment: fusion of adsorbed vesicles to form SPBs on each charged molecular surface does not occur when the molecular ion of the buffer used is of the opposite charge type. We attribute this to the high entropic repulsion (electric double layer repulsion) due to the large size of molecular counterions. On the other hand, such a critical dependence on buffer type is not observed when charged lipids are used. This study suggests the general importance of controlling electrostatic interaction in the formation of stable SPBs.     

Visualization and analysis of apolipoprotein A-I interaction with binary phospholipid bilayers

Apolipoprotein A-I (apoA-I) interaction with specific cell lipid domains was suggested to trigger cholesterol and phospholipid efflux. We analyzed here apoA-I interaction with dimyristoylphosphatidylcholine/distearoylphosphatidylcholine (DMPC/DSPC) bilayers at a temperature showing phase coexistence. Solid and liquid-crystalline domains were visualized by two-photon fluorescence microscopy on giant unilamellar vesicles (GUVs) labeled with 6-dodecanoyl-2-dimethyl-amino-naphthalene (Laurdan). A decrease of vesicle size was detected as long as they were incubated with lipid-free apoA-I, together with a shape deformation and a relative enrichment in DSPC. Selective lipid removal mediated by apoA-I from different domains was followed in real time by changes in the Laurdan generalized polarization. The data show a selective interaction of apoA-I with liquid-crystalline domains, from which it removes lipids, at a molar ratio similar to the domain compositions. Next, apoA-I was incubated with DMPC/DSPC small unilamellar vesicles, and products were isolated and quantified. Protein solubilized both lipids but formed complexes relatively enriched in the liquid component. We also show changes in the GUV morphology when cooling down. Our results suggest that the most efficient reaction between apoA-I and DMPC/DSPC occurs in particular bilayer conditions, probably when small fluid domains are nucleated within a continuous gel phase and interfacial packing defects are maximal.     

Analysis of interaction between chaperonin GroEL and its substrate using fluorescence correlation spectroscopy.

Fluorescence correlation spectroscopy (FCS) provides information about translational diffusion properties of fluorescent molecules in tiny detection volume and allows the analysis of binding processes of biomolecules in homogeneous solution. In this study, FCS was used to measure equilibrium binding constants of disulfide-reduced apo-alpha-lactalbumin (rLA), denatured pepsin, and apo-cytochrome c (apo-cyt c) bound by chaperonin GroEL at different salt concentrations. The results indicate that apo-cyt-c has a much stronger affinity to GroEL than denatured pepsin and rLA have. Titration experiments of GroEL to each substrate with various concentrations of four kinds of salts (K+, Na+, Ca2+, and Mg2+) show that the binding constant of denatured pepsin and rLA to GroEL depends on the salt concentration. The dependence of denatured pepsin binding to GroEL on salt concentration is much stronger than that of rLA. However, the interaction of positively charged apo-cyt c with GroEL is not affected by the salt concentration. Furthermore, the divalent cation promotes the binding of GroEL to denatured pepsin and rLA more strongly than does the monovalent cation.