4-4-difluoro-4-bora-3a-4a-diaza-s-indacene and 1-2-oleoylphosphatidylcholine

Fluorescence measurements of the sterol analog 23-(dipyrrometheneboron difluoride)-24-norcholesterol (BODIPY-cholesterol) are used to compare the effects of cholesterol (Chol) in monolayers of 1,2-dipalmitoyl-sn-glycero-3-phosphatidylcholine (DPPC)/1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC)/Chol and chicken egg sphingomyelin (SM)/DOPC/Chol. Monolayers are formed using the Langmuir-Blodgett technique and compared at surface pressures of 8 and 30 mN/m. In particular, these ternary lipid mixtures are compared using both ensemble and single-molecule fluorescence measurements of BODIPY-cholesterol. In mixed monolayers incorporating 0.10 mol % BODIPY-cholesterol, fluorescence microscopy measurements as a function of cholesterol added reveal similar trends in monolayer phase structure for both DPPC/DOPC/Chol and SM/DOPC/Chol films. With a probe concentration reduced to ∼10(-8) mol % BODIPY-cholesterol, single-molecule fluorescence measurements using defocused polarized total internal reflection microscopy are used to characterize the orientations of BODIPY-cholesterol in the monolayers. Population histograms of the BODIPY emission dipole tilt angle away from the membrane normal reveal distinct insertion geometries with a preferred angle observed near 78°. The measured angles and populations are relatively insensitive to added cholesterol and changes in surface pressure for monolayers of SM/DOPC/Chol. For monolayers of DPPC/DOPC/Chol, however, the single-molecule measurements reveal significant changes in the BODIPY-cholesterol insertion geometry when the surface pressure is increased to 30 mN/m. These changes are discussed in terms of a squeeze-out mechanism for BODIPY-cholesterol in these monolayers and provide insight into the partitioning and arrangement of BODIPY-cholesterol in ternary lipid mixtures.

Topics: 1,2-Dipalmitoylphosphatidylcholine; Animals; Boron Compounds; Chickens; Cholesterol; Fluorescent Dyes; Microscopy, Fluorescence; Microscopy, Polarization; Phosphatidylcholines; Phosphatidylethanolamines; Sphingomyelins; Xanthenes

In this paper we have investigated the behaviour of newly synthesised mono-palmitoyl- and dipalmitoyl-phosphatidylethanolamine probes (abbreviated as mPE and dPE, respectively) labelled in the polar headgroup region by either the FL-BODIPY or the 564/570-BODIPY fluorophore and solubilised in lipid systems that exhibit different curvatures. Because of the bulky BODIPY-groups, the monoacyl-form derivatives have a conic-like shape, whereas that for the diacyl derivatives is rather cylindrical. A careful analysis of time-resolved resonance energy transfer experiments by means of analytical models as well as Monte Carlo simulations shows that the mPE derivatives have a comparable affinity to highly curved bilayer regions (torroidal pores formed by magainin-2 in lipid bilayers, or the rims of discoid bicelles) and to planar bilayer regions (i.e. the flat region of lipid bilayers and bicelles). Furthermore, the monoacyl-probes are as compared to the diacyl-probes effectively closer to each other in a lipid bilayer, while none of these probes seems to be randomly distributed. Self-aggregation is most efficiently induced by the larger aromatic 564/570-BODIPY chromophore, but it is suppressed when using the diacyl instead of the monoacyl-form, and/or by attaching BODIPY-groups to the acyl-chain.

Topics: Animals; Boron Compounds; Energy Transfer; Lipid Bilayers; Magainins; Monte Carlo Method; Phosphatidylcholines; Phosphatidylethanolamines; Spectrometry, Fluorescence; Xenopus; Xenopus Proteins

Determining the local structure, dynamics, and conformational requirements for protein-protein and protein-lipid interactions in membranes is critical to understanding biological processes ranging from signaling to the translocating and membranolytic action of antimicrobial peptides. We report here the application of a combined polarized total internal reflection fluorescence microscopy-in situ atomic force microscopy platform. This platform's ability to image membrane orientational order was demonstrated on DOPC/DSPC/cholesterol model membranes containing the fluorescent membrane probe, DiI-C(20) or BODIPY-PC. Spatially resolved order parameters and fluorophore tilt angles extracted from the polarized total internal reflection fluorescence microscopy images were in good agreement with the topographical details resolved by in situ atomic force microscopy, portending use of this technique for high-resolution characterization of membrane domain structures and peptide-membrane interactions.

Topics: Boron Compounds; Cholesterol; Fluorescent Dyes; Lipid Bilayers; Microscopy, Atomic Force; Microscopy, Fluorescence; Phosphatidylcholines

Cholesterol-rich, liquid-ordered (L(o)) domains are believed to be biologically relevant, and yet detailed knowledge about them, especially in live cells under physiological conditions, is elusive. Although these domains have been observed in model membranes, understanding cholesterol-lipid interactions at the molecular level, under controlled lipid mixing, remains a challenge. Further, although there are a number of fluorescent lipid analogs that partition into liquid-disordered (L(d)) domains, the number of such analogs with a high affinity for biologically relevant L(o) domains is limited. Here, we use a new Bodipy-labeled cholesterol (Bdp-Chol) derivative to investigate membrane fluidity, lipid order, and partitioning in various lipid phases in giant unilamellar vesicles (GUVs) as a model system. GUVs were prepared from mixtures of various molar fractions of dioleoylphosphatidylcholine, cholesterol, and egg sphingomyelin. The L(d) phase domains were also labeled with 1,1'-didodecyl-3,3,3',3'-tetramethylindocarbocyanine (DiI-C(12)) for comparison. Two-photon fluorescence lifetime and anisotropy imaging of Bdp-Chol are sensitive to lipid phase domains in GUVs. The fluorescence lifetime of Bdp-Chol in liquid-disordered, single-phase GUVs is 5.50 +/- 0.08 ns, compared with 4.1 +/- 0.4 ns in the presence of DiI-C(12). The observed reduction of fluorescence lifetime is attributed to Förster resonance energy transfer between Bdp-Chol (a donor) and DiI-C(12) (an acceptor) with an estimated efficiency of 0.25 and donor-acceptor distance of 2.6 +/- 0.2 nm. These results also indicate preferential partitioning (K(p) = 1.88) of Bdp-Chol into the L(o) phase. One-photon, time-resolved fluorescence anisotropy of Bdp-Chol decays as a triexponential in the lipid bilayer with an average rotational diffusion coefficient, lipid order parameter, and membrane fluidity that are sensitive to phase domains. The translational diffusion coefficient of Bdp-Chol, as measured using fluorescence correlation spectroscopy, is (7.4 +/- 0.3) x 10(-8) cm(2)/s and (5.0 +/- 0.2) x 10(-8) cm(2)/s in the L(d) and L(o) phases, respectively. Experimental translational/rotational diffusion coefficient ratios are compared with theoretical predictions using the hydrodynamic model (Saffman-Delbrück). The results suggest that Bdp-Chol is likely to form a complex with other lipid molecules during its macroscopic diffusion in GUV lipid bilayers at room temperature. Our integrated, multiscale results

Topics: Boron Compounds; Cholesterol; Diffusion; Fluorescence; Fluorescence Polarization; Fluorescence Resonance Energy Transfer; Lipid Bilayers; Lipid Metabolism; Membrane Fluidity; Phosphatidylcholines; Sphingomyelins; Time Factors; Unilamellar Liposomes

The distribution of Bodipy GM1 in monolayers of binary and ternary lipid mixtures with coexisting fluid and ordered phases has been examined using a combination of atomic force microscopy and near-field scanning optical microscopy. Monolayers deposited at high (30 mN/m) and low (5 or 10 mN/m) surface pressures were examined and compared to those containing the same concentration of unlabeled ganglioside. Measurements of monomer and dimer Bodipy emission were used to distinguish aggregated from dilute ganglioside levels. For binary DPPC/DOPC monolayers, Bodipy GM1 is distributed throughout both the fluid and ordered phases at low surface pressures, and both labeled and unlabeled gangliosides result in a reduction in the size of ordered DPPC domains at 0.4% and the appearance of small aligned ganglioside-rich domains at 4%. In agreement with earlier studies, GM1 is heterogeneously distributed in small islands in the condensed DPPC domains at high surface pressure. By contrast, Bodipy GM1 causes the disappearance of large DPPC domains at 0.4% and the formation of a new GM1-rich phase at 4%. The addition of both gangliosides leads to a comparable loss of large ordered domains at low surface pressure and the appearance of a new GM1-rich phase at 30 mN/m for ternary lipid mixtures containing cholesterol. The results demonstrate the complexity of GM1 partitioning and illustrate the utility of complementary AFM and high spatial resolution two-color fluorescence experiments for understanding Bodipy GM1 aggregation and distribution.

Topics: Boron Compounds; Cholesterol; Dimerization; Fluorescence; G(M1) Ganglioside; Membranes, Artificial; Phosphatidylcholines

Unsaturated trans fatty acids have been linked to a higher incidence of coronary artery disease, but not enough is known about the effect of trans lipids on membrane properties. Liquid-ordered (l(o)) and liquid-disordered (l(d)) membrane domains are implicated in various biological processes, such as endocytosis, adhesion, signaling, protein transport, apoptosis, and disease pathogenesis. The physical forces that induce domain formation and thus orchestrate cell function need to be further addressed and quantified. Here, we test the effect of trans DOPC (dielaidoyl phosphatidylcholine or DEPC) on the morphology of giant unilamellar vesicles (GUVs, used as a biomembrane model) made by electroformation with varying compositions of egg sphingomyelin, trans DOPC, cis DOPC, and cholesterol. GUVs were imaged by confocal fluorescence microscopy and then analyzed for changes in membrane morphology and properties such as l(o)/l(d) phase coexistence and area fractions, distribution of meridional curvature, and fluorescent-probe intensity distribution. BODIPY-FL-C(12)-sphingomyelin, Lissamine rhodamine B dioleoylphosphatidylethanolamine and BODIPY-TR-C(12)-sphingomyelin were used as fluorescent probes to differentially label the l(o) and l(d) phases. Trans DOPC induces some vesicles to form multidomain, invaginated morphologies that differ from the typical two-domain circular and truncated spherical shapes observed in its absence. Trans DOPC also alters the membrane curvature distribution; this is more pronounced in the l(o) phase near the phase boundary, where significantly negative curvatures (<-0.5 microm(-1)) are observed. A narrower distribution of meridional curvatures in GUVs with trans DOPC is suggestive of higher membrane bending rigidity. The ratio of average fluorescent intensities in the l(d)/l(o) phases indicates a greater concentration or brightness of the probes BODIPY-FL-C(12)-sphingomyelin and BODIPY-TR-C(12)-sphingomyelin in the l(o) phase in the presence of trans DOPC. Addition of trans DOPC does not alter the l(o)/l(d) area fractions, indicating that it does not act like egg sphingomyelin, a saturated lipid. These changes in membrane properties seen in the presence of trans lipids could significantly impact cell function.

Topics: Biophysical Phenomena; Biophysics; Boron Compounds; Cell Membrane; Fluorescent Dyes; Membrane Lipids; Models, Biological; Molecular Structure; Phosphatidylcholines; Phosphatidylethanolamines; Rhodamines; Stereoisomerism

An in vitro antioxidant assay has been developed to better reflect the in vivo conditions of antioxidants interacting with membrane and lipid surfaces. The lipid peroxidation inhibition capacity (LPIC) method measures the ability of both lipophilic and hydrophilic antioxidants to protect a lipophilic fluorescent probe 4, 4-difluoro-5-(4-phenyl-1,3-butadienyl)-4-bora-3a,4a-diaza-s-indacene-3-undecanoic acid, incorporated in the membrane, from 2,2'-azobis(2-amidinopropane)hydrochloride generated radicals in the surrounding aqueous solution. Antioxidant activities of test compounds were measured either after they were mixed with preformed liposomes (LPIC(Mixed)) or after they were incorporated into liposomes (LPIC(Inco)) as they were made. The results were analysed to determine how the method of mixing and the structures of the antioxidants influenced their protection of the membrane from free radical attack. The LPIC(Mixed) values were larger than the LPIC(Inco) values for a range of 12 structurally diverse antioxidant compounds. However, there was no linear correlation between the lipophilicities, as measured by their partition coefficient, log P and either LPIC(Inco) or LPIC(Mixed) values. A strong correlation was found between LPIC(Inco) and LPIC(Mixed) values.

Topics: alpha-Tocopherol; Antioxidants; beta Carotene; Boron Compounds; Chromans; Coumaric Acids; Flavonoids; Fluorescent Dyes; Free Radicals; Gallic Acid; Lipid Peroxidation; Liposomes; Oxidation-Reduction; Phase Transition; Phosphatidylcholines

Elucidating the role that charged membrane proteins play in determining cell membrane structure and dynamics is an area of active study. We have applied in situ correlated atomic force and confocal microscopies to characterize the interaction of the NAP-22 peptide with model membranes prepared as supported planar bilayers containing both liquid-ordered and liquid-disordered domains. Our results demonstrated that the NAP-22 peptide interacts with membranes in a concentration-dependent manner, preferentially inserting into DOPC (ld) domains. While at low peptide concentrations, the NAP-22 peptide formed aggregate-like structures within the ld domains, at high peptide concentrations, it appeared to sequester cholesterol into the ld domains and recruited phosphatidyl-myo-inositol 4,5-bisphosphate by inducing a blending effect that homogenizes the phase-segregated domains into one liquid-ordered domain. This study describes a possible mechanism by which the NAP-22 peptide can affect neuronal morphology.

Topics: Animals; Boron Compounds; Brain Chemistry; Calmodulin-Binding Proteins; Cytoskeletal Proteins; Diagnostic Imaging; Lipid Bilayers; Membranes; Microscopy, Atomic Force; Models, Biological; Nerve Tissue Proteins; Peptide Fragments; Phosphatidylcholines; Phosphotransferases (Alcohol Group Acceptor); Protein Binding; Swine

We report the synthesis and characterization of a novel thiourea derivative of sphingomyelin (AD2765). In vitro assays using pure enzyme and/or cell extracts revealed that this compound inhibited the hydrolysis of BODIPY-conjugated or 14C-labeled sphingomyelin by acid sphingomyelinase and Mg2+-dependent neutral sphingomyelinase. Studies in normal human skin fibroblasts further revealed that AD2765 was taken up by cells and inhibited the hydrolysis of BODIPY-conjugated sphingomyelin in situ. In situ and in vitro studies also showed that this compound inhibited the synthesis of sphingomyelin from BODIPY-conjugated ceramide. The specificity of AD2765 for enzymes involved in sphingomyelin metabolism was demonstrated by the fact that it had no effect on the hydrolysis of BODIPY-conjugated ceramide by acid ceramidase or on the synthesis of BODIPY-conjugated glucosylceramide from BODIPY-conjugated ceramide. The overall effect of AD2765 on sphingomyelin metabolism was concentration-dependent, and treatment of normal human skin fibroblasts or cancer cells with this compound at concentrations > 10 microM led to an increase in cellular ceramide and cell death. Thus, AD2765 might be used to manipulate sphingomyelin metabolism in various ways, potentially to reduce substrate accumulation in cells from types A and B Niemann-Pick disease patients, and/or to affect the growth of human cancer cells.

Topics: Boron Compounds; Cell Death; Cell Line; Cell Line, Tumor; Ceramides; Fibroblasts; HL-60 Cells; Humans; Hydrolysis; Jurkat Cells; Lipids; Lysosomal-Associated Membrane Protein 2; Microscopy, Fluorescence; Models, Chemical; Niemann-Pick Diseases; Phosphatidylcholines; Skin; Sphingomyelin Phosphodiesterase; Sphingomyelins; Trypan Blue

The birefringence and linear dichroism of anisotropic thin films such as proteolipid membranes are related to molecular properties such as polarizability, shape, and orientation. Coupled plasmon-waveguide resonance (CPWR) spectroscopy is shown in the present work to provide a convenient means of evaluating these parameters in a single lipid bilayer. This is illustrated by using 1-10 mol % of an acyl chain chromophore-labeled phosphatidylcholine (PC) incorporated into a solid-supported PC bilayer deposited onto a hydrated silica surface. CPWR measurements were made of refractive index and extinction coefficient anisotropies with two exciting light wavelengths, one of which is absorbed by the chromophore and one of which is not. These results were used to calculate longitudinal and transverse molecular polarizabilities, the orientational order parameter and average angle between the longitudinal axis of the lipid molecule and the membrane normal, and the molecular shape factors of the lipid molecules. The values thereby obtained are in excellent agreement with parameters determined by other techniques, and provide a powerful tool for analyzing lipid-protein, protein-protein, and protein-ligand interactions in proteolipid films.

Topics: Anisotropy; Birefringence; Boron Compounds; Equipment Design; Fluorescent Dyes; Lipid Bilayers; Orientation; Phosphatidylcholines; Proteolipids; Surface Plasmon Resonance

A BODIPY-labelled sulfatide (N-(BODIPY-FL-pentanoyl)-galactosylcerebroside-sulfate, hereafter abbreviated as BD-Sulfatide) was solubilised at different concentrations in lipid vesicles of 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC). Time-correlated single photon counting experiments show that the fluorescence relaxation is mono-exponential (with a lifetime of 6.5 ns) at molar ratios of BD-Sulfatide: DOPC that are less than 1:100. The fluorescence steady-state anisotropy decreases monotonously at molar ratios smaller than 1:1000, which is compatible with donor-donor energy migration (DDEM) among the BODIPY groups. A model that assumes DDEM across the lipid bilayers, as well as in their planes, was used to analyse the time-resolved fluorescence anisotropy. Only two parameters appear in the model namely: the bilayer thickness (d) and the average number density (C2) distribution of BD-Sulfatide in the lipid bilayers. The extracted d-values vary between 35 and 40 A, which is about the reported thickness of a bilayer of DOPC (38 A). Hence, the BODIPY groups are preferentially located in the water-lipid interface. At low concentration the experimental C2-values and those independently calculated are in good agreement, while the experimental values gradually become lower with increasing BD-Sulfatide concentration. These results are compatible with an aggregation of the sulfatides and self-quenching of BODIPY, which is clearly established at higher concentrations of the BD-Sulfatide.

Topics: Boron Compounds; Energy Transfer; Fluorescence Polarization; Fluorescent Dyes; Lipid Bilayers; Mathematics; Molecular Structure; Phosphatidylcholines; Spectrometry, Fluorescence

The membrane topography of proteins that convert between soluble and membrane-inserted states has proven a challenging problem. In particular, it has been difficult to define both whether a transmembrane orientation is achieved and what are the boundaries of membrane-inserted segments. In this report the fluorescence of bimane-labeled Cys residues and the binding of anti-BODIPY antibodies to BODIPY-labeled Cys residues are combined to define these features for helices TH8 and TH9 of the T domain of diphtheria toxin. Using a series of labeled residues the topography of these helices was examined in both conformations of membrane-inserted T domain identified previously (Wang, Y., Malenbaum, S. E., Kachel, K., Zhan, H., Collier, R. J., and London, E. (1997) J. Biol. Chem. 272, 25091-25098). In the shallowly inserted conformation these helices are found to be aligned close to the cis surface of the bilayer all along their sequences. In contrast, in the more deeply inserted conformation most TH8 and TH9 residues examined located in a non-polar environment, with the boundaries of the membrane-inserted sequences close to residues 324 and 372-374 on the cis (insertion) side of the bilayer. It was also found that residues 348 and 349, which are in the loop connecting TH8 and TH9, reached the opposite trans side of the bilayer, but did not protrude fully into the aqueous environment. These boundaries suggest the membrane-inserted segments of TH8 and TH9 form transmembrane helices about 25 residues in length, and suggest that they are connected by a tight turn. It is concluded that this combination of fluorescent techniques can be combined to obtain transmembrane helix topography.

Topics: Boron Compounds; Bridged Bicyclo Compounds; Diphtheria Toxin; Fluorescent Dyes; Membrane Proteins; Models, Molecular; Peptide Fragments; Phosphatidylcholines; Protein Structure, Secondary

A fluorometric method for evaluation of activities of lipophilic antioxidants toward peroxyl radicals in a lipophilic medium (octane:butyronitrile; 9:1, v/v) and dioleoylphosphatidyl choline (DOPC) liposomal suspension in Tris-HCl buffer (pH 7.4 at 40 degrees C) has been developed. This fluorometric method is based on the use of the fatty acid 4,4-difluoro-5-(4-phenyl-1, 3-butadienyl)-4-bora-3a,4a-diaza-s-indacene-3-undec anoic acid (BODIPY 581/591 C11) as an indicator, 2,2'-azobis-2,4-dimethyl valeronitrile (AMVN) as a peroxyl radical generator, and 6-hydroxy-2, 5,7,8-tetramethyl chroman-2-carboxylic acid (Trolox) as a calibrator. In the absence of antioxidants, a linear correlation was found between the fluorescence decay of BODIPY 581/591 C11 and the concentration of AMVN. Trolox was used as a standard reference material for validating this newly developed method. A linear correlation was found between the net protection of the fluorescence of BODIPY 581/591 C11 and the amounts of added Trolox. Employing this method, the relative peroxyl radical scavenging activities of Trolox, alpha-tocopherol, alpha-carotene, beta-carotene, lutein, and probucol in octane:butyronitrile (9:1, v/v) were determined to be 1. 0:0.7:0.5:0.3:0.4:0.3. In the DOPC system, beta-carotene showed an antioxidant activity which is a factor of two greater than that of alpha-tocopherol. This assay is suitable for measurement of activities of antioxidants in an organic lipid environment and a liposomal medium.

Topics: Antioxidants; Boron Compounds; Fluorescent Dyes; Free Radical Scavengers; Liposomes; Peroxides; Phosphatidylcholines; Spectrometry, Fluorescence