texas-red and 1-2-dipalmitoyl-3-phosphatidylethanolamine

Binding of amphiphilic molecules to supported lipid bilayers (SLBs) often results in lipid fibril extension from the SLBs. Previous studies proposed that amphiphiles with large and flexible hydrophilic regions trigger lipid fibril formation in SLBs by inducing membrane curvature via their hydrophilic regions. However, no experimental studies have verified this mechanism of fibril formation. In this work, we investigated the binding of lipopolysaccharide (LPS) and cholesterol-modified gelatin to SLBs using fluorescence microscopy. SLBs with restricted and unrestricted bilayer areas were employed to identify the mechanism of fibril generation. We show that the main cause of lipid fibril formation is an approximately 20% expansion in the bilayer area rather than increased membrane curvature. The data indicate that bilayer area confinement plays a critical role in morphological changes of SLBs even when bound amphiphilic molecules have a large hydrophilic domain. We also show that bilayer area change after LPS insertion is dependent on the patch shape of the SLB. When an SLB patch consists of a broad bilayer segment connected to a long thin streak, bilayer area expansion mainly occurs within the bilayer streak. The results indicate that LPS insertion causes net lipid flow from the broad bilayer region to the streak area. The differential increase in area is explained by the instability of planar bilayer streaks that originate from the large energetic contribution of line tension arising along the bilayer edge.

Topics: Biomimetic Materials; Cholesterol; Gelatin; Lipid Bilayers; Lipopolysaccharides; Microscopy, Fluorescence; Phosphatidylcholines; Phosphatidylethanolamines; Surface Tension; Unilamellar Liposomes; Xanthenes

The fusion of lipid membranes is a key process in biology. It enables cells and organelles to exchange molecules with their surroundings, which otherwise could not cross the membrane barrier. To study such complex processes we use simplified artificial model systems, i.e., an optical fusion assay based on membrane-coated glass spheres. We present a technique to analyze membrane-membrane interactions in a large ensemble of particles. Detailed information on the geometry of the fusion stalk of fully fused membranes is obtained by studying the diffusional lipid dynamics with fluorescence recovery after photobleaching experiments. A small contact zone is a strong obstruction for the particle exchange across the fusion spot. With the aid of computer simulations, fluorescence-recovery-after-photobleaching recovery times of both fused and single-membrane-coated beads allow us to estimate the size of the contact zones between two membrane-coated beads. Minimizing delamination and bending energy leads to minimal angles close to those geometrically allowed.

Topics: Algorithms; Cell Fusion; Computer Simulation; Diffusion; Fluorescence Recovery After Photobleaching; Fluorescent Dyes; Glass; Heterocyclic Compounds, 4 or More Rings; Lipopeptides; Membrane Fusion; Membranes, Artificial; Microscopy, Confocal; Models, Theoretical; Phosphatidylcholines; Phosphatidylethanolamines; Silicon Dioxide; Xanthenes

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

DNA hydrogels are promising materials for various fields of research, such as in vitro protein production, drug carrier systems, and cell transplantation. For effective application and further utilization of DNA hydrogels, highly effective methods of nano- and microscale DNA hydrogel fabrication are needed. In this respect, the fundamental advantages of a core-shell structure can provide a simple remedy. An isolated reaction chamber and massive production platform can be provided by a core-shell structure, and lipids are one of the best shell precursor candidates because of their intrinsic biocompatibility and potential for easy modification. Here, we demonstrate a novel core-shell nanostructure made of gene-knitted X-shaped DNA (X-DNA) origami-networked gel core-supported lipid strata. It was simply organized by cross-linking DNA molecules via T4 enzymatic ligation and enclosing them in lipid strata. As a condensed core structure, the DNA gel shows Brownian behavior in a confined area. It has been speculated that they could, in the future, be utilized for in vitro protein synthesis, gene-integration transporters, and even new molecular bottom-up biological machineries.

Topics: Bacteriophage T4; Benzothiazoles; Cholesterol; Diamines; DNA, Single-Stranded; Fluorescent Dyes; Hydrogels; Ligases; Microscopy, Electron, Transmission; Nanostructures; Nucleic Acid Conformation; Organic Chemicals; Phosphatidylcholines; Phosphatidylethanolamines; Phosphatidylglycerols; Quinolines; Viral Proteins; Xanthenes

A pH controlled flow cell device was constructed to allow electrophoretic movement of charged lipids and membrane associated proteins in supported phospholipid bilayers. The device isolated electrolysis products near the electrodes from the electrophoresis process within the bilayer. This allowed the pH over the bilayer region to remain within ±0.2 pH units or better over many hours at salt concentrations up to 10 mM. Using this setup, it was found that the electrophoretic mobility of a dye conjugated lipid (Texas Red 1,2-dihexadecanoyl-sn-glycero-3-phosphoethanolamine (TR-DHPE)) was essentially constant between pH 3.3 and 9.3. In contrast, streptavidin, which was bound to biotinylated lipids, shifted from migrating cathodically at acidic pH values to migrating anodically under basic conditions. This shift was due to the modulation of the net charge on the protein, which changed the electrophoretic forces experienced by the macromolecule. The addition of a polyethylene glycol (PEG) cushion beneath the bilayer or the increase in the ionic strength of the buffer solution resulted in a decrease of the electroosmotic force experienced by the streptavidin with little effect on the Texas Red-DHPE. As such, it was possible in part to control the electrophoretic and electroosmotic contributions to streptavidin independently of one another.

Topics: Biotin; Buffers; Electroosmosis; Electrophoresis; Ethanolamines; Hydrogen-Ion Concentration; Lipid Bilayers; Phosphatidylethanolamines; Streptavidin; Xanthenes

Total internal reflection fluorescence microscopy is widely used to confine the excitation of a complex fluorescent sample very close to the material on which it is supported. By working with high refractive index solid supports, it is possible to confine even further the evanescent field, and by varying the angle of incidence, to obtain quantitative information on the distance of the fluorescent object from the surface. We report the fabrication of hybrid surfaces consisting of nm layers of SiO(2) on lithium niobate (LiNbO(3), n = 2.3). Supported lipid bilayer membranes can be assembled and patterned on these hybrid surfaces as on conventional glass. By varying the angle of incidence of the excitation light, we are able to obtain fluorescent contrast between 40-nm fluorescent beads tethered to a supported bilayer and fluorescently labeled protein printed on the surface, which differ in vertical position by only tens of nm. Preliminary experiments that test theoretical models for the fluorescence-collection factor near a high refractive index surface are presented, and this factor is incorporated into a semiquantitative model used to predict the contrast of the 40-nm bead/protein system. These results demonstrate that it should be possible to profile the vertical location of fluorophores on the nm distance scale in real time, opening the possibility of many experiments at the interface between supported membranes and living cells. Improvements in materials and optical techniques are outlined.

Topics: Contrast Sensitivity; Fluorescent Dyes; Image Enhancement; Lipid Bilayers; Mathematical Computing; Microscopy, Fluorescence; Niobium; Oxides; Phosphatidylcholines; Phosphatidylethanolamines; Silicon Dioxide; Xanthenes

Monolayer mixtures of dihydrocholesterol and phospholipids at the air-water interface are used to model membranes containing cholesterol and phospholipids. Specific, stoichiometric interactions between cholesterol and some but not all phospholipids have been proposed to lead to the formation of condensed complexes. It is reported here that an externally applied electric field of the appropriate sign can destabilize these complexes, resulting in their dissociation. This is demonstrated through the application of an electric field gradient that leads to phase separations in otherwise homogeneous monolayers. This is observed only when the monolayer composition is close to the stoichiometry of the complex. The electric field effect is analyzed with the same mean field thermodynamic model as that used previously to account for pairs of upper miscibility critical points in these mixtures. The concentrations of dihydrocholesterol, phospholipid, and complex vary strongly and sometimes discontinuously in the monolayer membrane in the field gradient. The model is an approximation to a two-dimensional liquid in which molecules freely exchange between free and complexed form so that the chemical potentials are constant throughout the membrane. The calculations are illustrated for a complex of about 15 molecules, composed of 5 cholesterol molecules and 10 phospholipid molecules.

Topics: 1,2-Dipalmitoylphosphatidylcholine; Air; Cholestanol; Dimyristoylphosphatidylcholine; Electromagnetic Fields; Fluorescent Dyes; Macromolecular Substances; Membranes, Artificial; Models, Chemical; Phosphatidylethanolamines; Phospholipids; Solubility; Sphingomyelins; Thermodynamics; Unithiol; Water; Xanthenes

The formation of individually addressable micropatterned solid-supported lipid bilayers has been accomplished by means of micromolding in capillaries. Small unilamellar vesicles were spread on glass slides to form planar supported membranes along microscopic capillaries molded as trenches into a polydimethylsiloxane (PDMS) elastomer. PDMS provides an elastic and transparent carrier for microcapillaries molded from silicon wafers displaying the desired inverse trenches. The so-called master structure has been conventionally etched into silicon by photolithography. The cured PDMS elastomer was briefly exposed to an oxygen plasma, rendering the surface hydrophilic, and subsequently attached to a glass surface in order to form hydrophilic capillaries equipped with flow-promoting pads on either side. One flowpad acts as a reservoir to be filled with the vesicle suspension, while the other one serves as a collector to ensure a sufficient capillary flow to cover the substrate completely. Formation of planar lipid bilayers on the glass slide along the capillaries was followed by imaging the flow and spreading of fluorescently labeled DMPC liposomes with confocal laser scanning microscopy. By means of scanning force microscopy in aqueous solution the formed lipid structures were identified and the height of the lipid bilayers was accurately determined. With both techniques, it was shown that the patterned bilayers remain separated and persist for several hours on the substrate in aqueous solution.

Topics: Biocompatible Materials; Capillary Action; Dimethylpolysiloxanes; Dimyristoylphosphatidylcholine; Elasticity; Lipid Bilayers; Liposomes; Microscopy, Atomic Force; Microscopy, Confocal; Models, Biological; Nylons; Phosphatidylethanolamines; Xanthenes

Brownian ratchets use a time-varying asymmetric potential that can be applied to separate diffusing particles or molecules. A new type of Brownian ratchet, a geometrical Brownian ratchet, has been realized. Charged, fluorescently labeled phospholipids in a two-dimensional fluid bilayer were driven in one direction by an electric field through a two-dimensional periodic array of asymmetric barriers to lateral diffusion fabricated from titanium oxide on silica. Diffusion spreads the phospholipid molecules in the orthogonal direction, and the asymmetric barriers rectify the Brownian motion, causing a directional transport of molecules. The geometrical ratchet can be used as a continuous molecular sieve to separate mixtures of membrane-associated molecules that differ in electrophoretic mobility and diffusion coefficient.

Topics: 4-Chloro-7-nitrobenzofurazan; Chemical Phenomena; Chemistry, Physical; Diffusion; Electrophoresis; Fluorescence; Fluorescent Dyes; Lipid Bilayers; Membrane Fluidity; Membrane Proteins; Phosphatidylethanolamines; Phosphatidylserines; Phospholipids; Temperature; Xanthenes

Individual synthetic vesicles 0.1-1.0 micron in diameter are sized by using single-particle fluorescence emission spectra obtained in a custom sheath flow cell with an imaging spectrograph and a charge-coupled device. Data are acquired at 1 Hz, with limits of detection (3 sigma) less than 6.0 x 10(3) and 1.0 x 10(4) molecules of free sulforhodamine 101 and fluorescein, respectively, and with a spectral range for fluorescence emission collection from 350 to 800 nm (0.45 nm/pixel resolution). The system is used for small-particle population analysis by analyzing a suspension of submicron, unilamellar, synthetic vesicles prepared by standard procedures with phosphatidylserine and Texas red- or fluorescein head-group-conjugated dihydropalmitoylphosphatidylethanolamine. The submicron particles are individually identified, sized, and discriminated based on single-particle fluorescence emission spectra. Excellent agreement is found between fluorescence sizing data and transmission electron microscopic measurements.

Topics: Animals; Aplysia; Calibration; Flow Cytometry; Fluorescein; Fluoresceins; Fluorescent Dyes; Liposomes; Microscopy, Electron; Particle Size; Phosphatidylethanolamines; Spectrometry, Fluorescence; Xanthenes