Fusion of lipid vesicles with planar lipid bilayers induced by a combination of peptides

We studied the peptide-induced membrane fusion process between small unilamellar vesicles (SUVs) and supported planar bilayers (SPBs) with the aim of developing a method for incorporating membrane components into SPBs. As fusogenic peptides, two analogues of the N-terminal region of an influenza membrane fusion protein hemaggulutinin, anionic E5 and cationic K5, were synthesized, and the membrane fusion was investigated using SPB and SUVs composed of phosphatidylcholine from egg yolk (EggPC). We directly visualized the process of lipid transfer from SUVs to SPB by total internal reflection fluorescence (TIRF) microscopy. The transfer of fluorescent lipids was effectively induced only by the combination of two peptides. The TIRF microscopy observations of single SUV fusion events also revealed that lipid membranes from SUV could completely fuse into the SPB. However, the presence of single peptide (either E5 or K5) rather inhibited the lipid transfer, presumably due to the electrostatic repulsion between SUVs and SPB. The opposite effects induced by the peptides indicate the possibility for a designed application of two peptides as a means to control the membrane fusion spatially and temporally.     

Cholesterol modulated antibody binding in supported lipid membranes as determined by total internal reflectance microscopy on a microfabricated high-throughput glass chip

A high-throughput microfabricated all-glass microchip, lipid biochip, was created and used to measure fluorescently tagged antibody binding to dinitrophenol (DNP) haptens in planar supported phospholipid/cholesterol lipid bilayers as a function of cholesterol-to-lipid molar ratio (X(CHOL)). Multiple parallel microchannels etched in the lipid biochip allowed simultaneous measurement of antibody binding to hapten-containing and hapten-free lipid bilayers, for a range of aqueous antibody concentrations. Specific and nonspecific antibody binding to the supported lipid bilayers was determined from the internally calibrated intensity of the surface fluorescence using total internal reflectance fluorescence (TIRF) microscopy. The TIRF intensity data of the specific antibody binding were fitted to the Langmuir isotherm and Hill equation models to determine the apparent dissociation constant K(d), the maximum fluorescence parameter F(infinity), and binding cooperativity n. As X(CHOL) increased from 0 to 0.50, K(d) exhibited a minimum of approximately 4 microM and n reached a maximum of approximately 2.2 at X(CHOL) approximately 0.20. However, F(infinity) appeared to be insensitive to the cholesterol content. The nonspecific binding fraction (NS), defined as the ratio of the TIRF intensity for hapten-free bilayers to that with hapten, showed a minimum of approximately 0.08 also at X(CHOL) approximately 0.20. The results suggest that cholesterol regulates the specific binding affinity and cooperativity, as well as suppresses nonspecific binding of aqueous antibody to a planar supported lipid bilayer surface at an optimal cholesterol content of X(CHOL) approximately 0.20. Interestingly, for X(CHOL) approximately 0.40, NS reached a maximum of approximately 0.57, suggesting significant packing defects in the lipid bilayer surface, possibly as a result of lipid domain formation as predicted by the lipid superlattice model. We conclude that cholesterol plays a significant role in regulating both specific and nonspecific antibody/antigen binding events on the lipid bilayer surface and that our lipid biochip represents a new and useful high-resolution microfluidic device for measuring lipid/protein surface binding activities in a parallel and high-throughput fashion.     

A method for visualization of biomolecules labeled by a single quantum dot in living cells by a combination of total internal reflection fluorescence microscopy and intracellular fluorescence microscopy

We developed a simple fluorescence microscopy for acquisition of high-resolution images of single quantum dots (QDs) labeled to biomolecules on apical plasma membrane, in cell interior and on basal plasma membrane of living cells. The method was a combination of total internal reflection fluorescence microscopy (TIRFM) at apical cell surface and intracellular microscopy coupled with focusing objective. Insulin conjugated to single QD (insulin-QD) was chosen as the model system. In order to bind insulin-QDs to insulin receptors on the plasma membrane through the interaction between insulin and its receptor, as well as internalize them, the cells attached on a coverslip were incubated with biotinylated insulin and QD-streptavidin conjugate at 37 °C. Next, fluorescent molecules in the cells were photobleached by illuminating the cells using a 100-W mercury lamp with the wavelengths from 460 to 490 nm. Then, the incident angle of a laser beam was adjusted to produce total internal reflection at the apical surface of a single cell. In this case, the insulin-QDs in the whole cell were excited, and the fluorescent molecules outside the cell were not illuminated. Finally, the images of single insulin-QDs on the apical plasma membrane, in the cell interior and on the basal plasma membrane of the cell were taken by focusing the objective to different positions, respectively. The resolution and contrast of the fluorescent spots in the images were much higher than those obtained by using epi-fluorescence microscopy and comparable to those obtained by using the conventional TIRFM. The method improved the image acquisition speed for the images on the apical and basal plasma membrane using the conventional TIRFM, and could acquire the high-resolution images in the cell interior quickly.     

Lateral organization of a membrane protein in a supported binary lipid domain: direct observation of the organization of bacterial light-harvesting complex 2 by total internal reflection fluorescence microscopy

A unique method is described for directly observing the lateral organization of a membrane protein (bacterial light-harvesting complex LH2) in a supported lipid bilayer using total internal reflection fluorescence (TIRF) microscopy. The supported lipid bilayer consisted of anionic 1,2-dioleoyl-sn-glycero-3-[phospho-rac-(1'-glycerol)] (DOPG) and 1,2-distearoly-sn-3-[phospho-rac-(1'-glycerol)] (DSPG) and was formed through the rupture of a giant vesicle on a positively charged coverslip. TIRF microscopy revealed that the bilayer was composed of phase-separated domains. When a suspension of cationic phospholipid (1,2-dioleoyl-sn-glycero-3-ethylphosphocholine: EDOPC) vesicles (approximately 400 nm in diameter), containing LH2 complexes (EDOPC/LH2 = 1000/1), was put into contact with the supported lipid bilayer, the cationic vesicles immediately began to fuse and did so specifically with the fluid phase (DOPG-rich domain) of the supported bilayer. Fluorescence from the incorporated LH2 complexes gradually (over approximately 20 min) spread from the domain boundary into the gel domain (DSPG-rich domain). Similar diffusion into the domain-structured supported lipid membrane was observed when the fluorescent lipid (1,2-dioleoyl-sn-glycero-3-phosphoethanolamine-N-lissamine-rhodamine B sulfonyl: N-Rh-DOPE) was incorporated into the vesicles instead of LH2. These results indicate that vesicles containing LH2 and lipids preferentially fuse with the fluid domain, after which they laterally diffuse into the gel domain. This report describes for first time the lateral organization of a membrane protein, LH2, via vesicle fusion and subsequent lateral diffusion of the LH2 from the fluid to the gel domains in the supported lipid bilayer. The biological implications and applications of the present study are briefly discussed.     

Probing chemical induced cellular stress by non-Faradaic electrochemical impedance spectroscopy using an Escherichia coli capacitive biochip

A new capacitive biochip was developed using carboxy-CNT activated gold interdigitated (GID) capacitors immobilized with E. coli cells for the detection of cellular stress caused by chemicals. Here, acetic acid, H(2)O(2) and NaCl were employed as model chemicals to test the biochip and monitored the responses under AC electrical field by non-Faradaic electrochemical impedance spectroscopy (nFEIS). The electrical properties of E. coli cells under different stresses were studied based on the change in surface capacitance as a function of applied frequency (300-600 MHz) in a label-free and noninvasive manner. The capacitive response of the E. coli biochip under normal conditions exhibited characteristic dispersion peaks at 463 and 582 MHz frequencies. Deformation of these signature peaks determined the toxicity of chemicals to E. coli on the capacitive biochip. The E. coli cells were sensitive to, and severely affected by 166-498 mM (1-3%) acetic acid with declined capacitance responses. The E. coli biochip exposed to H(2)O(2) exhibited adaptive responses at lower concentrations (<2%), while at a higher level (882 mM, 3%), the capacitance response declined due to oxidative toxicity in cells. However, E. coli cells were not severely affected by high NaCl levels (513-684 mM, 3-4%) as the cells tend to resist the salt stress. Our results demonstrated that the biochip response at a particular frequency enabled the determination of the severity of the stress imposed by chemicals and it can be potentially applied for monitoring unknown chemicals as an indicator of cytotoxicity.     

Loss of epithelial polarity is accompanied by differential association of proteins with intracellular membranes

Cellular membranes play an important role in the formation and maintenance of epithelial polarity, which is lost early during carcinogenesis. We set out to identify membrane proteins which are altered during loss of cell polarity in mammary epithelium. As a model system we used murine mammary epithelial cells expressing the conditional oncoprotein c-JunER, which induces a reversible loss of polarity upon beta-estradiol-driven activation [1]. When grown either in the absence or presence of hormone, these cells exhibit a polarized or unpolarized phenotype, respectively. Different membrane fractions of polarized or unpolarized cells were analyzed by two-dimensional electrophoresis (2-DE) and differentially expressed membrane proteins were identified. To distinguish between transmembrane orientation and peripheral attachment of these proteins, were performed extractions with carbonate at high pH or with Triton X-114. In addition, cytosolic proteins of both states were analyzed to investigate their differential association with distinct membrane fractions. We found ten protein spots preferentially or exclusively in polarized cells and 17 other proteins as being upregulated during loss of polarity. Some of the peripheral membrane proteins were identified by microsequencing. The resident Golgi protein nucleobindin and fructose-bisphosphate aldolase were preferentially associated with membranes of polarized cells, whereas alphaB crystallin was detected exclusively and in high amounts in unpolarized cells.     

Determination of berberine by measuring the enhanced total internal reflected fluorescence at water/tetrachloromethane interface in the presence of sodium dodecyl benzene sulfonate

A highly sensitive method for determination of berberine is proposed based on the measurements of total internal reflected fluorescence (TIRF) at water/ tetrachloromethane (H₂O/CCl₄) interface. In the pH range of 2.6–5.7, the co-adsorption of the berberine with the anionic surfactants such as sodium dodecyl benzene sulfonate (SDBS), sodium dodecylsulfonate (SDS), and sodium lauryl sulfate (SLS) occurs at the H₂O/CCl₄ interface, resulting in greatly enhanced TIRF signal characterized by the emission at 526 nm when excited with a 351 nm light beam. The enhanced TIRF intensity is in proportion to the berberine concentration in the range 0.2–10.0×10^-7 mol L^-1. The limit of detection is 1.7×10^-9 mol L^-1 (3). It was found that ions such as Ca(II), Cu(II), Fe(III), Cd(II), Mg(II), Zn(II), Pb(II), and Al(III) can be allowed larger than 1.0×10^-4 mol L^-1. Meanwhile, the organic compounds such as vitamin B, saccharine, and amino acid do not display any effect for the present TIRF method even if they are larger than 1.0×10^-2 mol L^-1in high concentration levels (larger than 1.0×10^-5 mol L^-1). The results of determination for synthetic samples were agreement with the desired values, and the ones for tablets were identical with those obtained according to the method of Chinese Pharmacopoeia.     

Combination of polarized TIRF and ATR spectroscopies for determination of the second and fourth order parameters of molecular orientation in thin films and construction of an orientation distribution based on the maximum entropy method

This article describes two mathematical formalisms for the determination of the second and fourth order parameters of molecular films using optical spectroscopy. Method A uses polarized total internal reflection fluorescence (TIRF) to calculate the second and fourth order parameters, {P2(cos theta)} and {P4(cos theta)}, using an independently determined value for the angle between the absorption and emission dipoles, gamma. Method B uses {P2(cos theta)} obtained from attenuated total reflectance (ATR) data, along with polarized TIRF measurements to calculate {P4(cos theta)} and {cos2 gamma}. The choice of a specific method should rely on experimental considerations. We also present a method to separate the contributions of substrate surface roughness and dipole orientation with respect to the molecular axis from the spectroscopically determined second and fourth order parameters. Finally, a maximum entropy approach for construction of an orientation distribution from order parameters is compared with the commonly used delta and Gaussian distributions.     

A Spectrometric Setup for Synchronous Total Internal Reflection Fluorescence Measurement at the Solid／Liquid Interface

A spectrometric setup of perform total internal reflection fluorescence(TIRF) and synchronous TIRF measurements at solid/liquid interfaces is presented. The combination of TIRF and synchronous fluorescence was proposed to analyze simultaneously different components at interfaces. The TIRF excitation,emission and synchronous spectra of a water-soluble porphyrin were obtained from water/glass interface using this setup without the existence of a surfactant.     

A high-throughput microfluidic biochip to quantify bacterial adhesion to single host cells by real-time PCR assay

A high-throughput microfluidic poly-(dimethylsiloxane) biochip was developed to quantify bacterial adhesion to single host cells by real-time PCR assay. The biochip is simply structured with a two-dimensional array of 900 micro-wells, one inlet, and one outlet micro-channels. Isolation of single infected host cells into the individual micro-wells of the biochip was achieved by one-step vacuum-driven microfluidics. The adhered bacterial cells were then quantified by direct on-chip real-time PCR assay with single-bacterium-detection sensitivity. The performance of this microfluidic platform was demonstrated through profiling of the association of a common bacterial pathogen, Pseudomonas aeruginosa, to single host human lung epithelial A549 cells, revealing an adherence distribution that has not been previously reported. This microfluidic platform offers a simple and effective tool for biologists to analyze pathogen–host interaction at the single-cell level without the necessities of fluorescence labeling. The chip can similarly be used for other PCR-based applications requiring single-cell analysis.     

Detection of single-molecule DNA hybridization by using dual-color total internal reflection fluorescence microscopy

We examined the use of prism-type simultaneous dual-color total internal reflection fluorescence microscopy (TIRFM) to probe DNA molecules at the single-molecule level. The system allowed the direct detection of the complementary interactions between single-stranded probe DNA molecules (16-mer) and various lengths of single-stranded target DNA molecules (16-mer and 55-mer) that had been labeled with different fluorescent dyes (Cy3, Cy5, and fluorescein). The polymer-modified glass substrate and the extent of DNA probe immobilization were easily characterized either with standard TIRFM or with atomic force microscopy. However, only dual-color TIRFM could provide unambiguous images of individual single-stranded target DNA molecules hybridized with the correct sequence in the range of fM–aM. Succinic anhydride showed low RMS roughness and was found to be an optimal blocking reagent against non-specific adsorption, with an efficiency of 92%. This study provides a benchmark for directly monitoring the interactions and the detection of co-localization of two different DNA molecules and can be applied to the development of a nanoarray biochip at the single-molecule level.     

TIRF microscopy as a screening method for non-specific binding on surfaces

We report a method for studying nanoparticle-biosensor surface interactions based on total internal reflection fluorescence (TIRF) microscopy. We demonstrate that this simple technique allows for high throughput screening of non-specific adsorption (NSA) of nanoparticles on surfaces of different chemical composition. Binding events between fluorescent nanoparticles and functionalized Zeonor? surfaces are observed in real-time, giving a measure of the attractive or repulsive properties of the surface and the kinetics of the interaction. Three types of coatings have been studied: one containing a polymerized aminosilane network with terminal -NH(2) groups, a second film with a high density of -COOH surface groups and the third with sterically restraining branched poly(ethylene)glycol (PEG) functionality. TIRF microscopy revealed that the NSA of nanoparticles with negative surface charge on such modified coatings decreased in the following order -NH(2)>-branched PEG>-COOH. The surface specificity of the technique also allows discrimination of the degree of NSA of the same surface at different pH.     

pH对磷脂膜中Slc11a1跨膜区二级结构和取向的影响

采用圆二色光谱、 荧光光谱、 红外衰减全反射光谱和差示扫描量热分析等方法对不同pH条件下膜蛋白Slc11a1(溶质转运蛋白家族11成员1)的第二、 第三和第四跨膜区(TMD2~TMD4)在磷脂膜[二肉豆蔻酰磷脂酰胆碱(DMPC)和二肉豆蔻酰磷脂酰甘油(DMPG)的摩尔比为2∶1]中的二级结构和取向进行了研究. 结果表明, TMD3的二级结构及在磷脂膜内的位置与pH密切相关, 在pH=7时TMD3主要为β股结构, 在膜中埋入较浅; 而在pH=5.5时TMD3形成部分α螺旋结构, 并较深地埋入膜中. 对TMD3进行E139A突变后的结果证明, TMD3的这些性质与位于中间的谷氨酸的质子化性质密切相关. 实验结果还表明, TMD2和TMD4在不同pH条件下都形成α螺旋结构并分别以26°和35°的倾斜角插入磷脂膜内, 它们在磷脂膜内的位置基本不受pH影响.     

A High‐Affinity Fluorescent Sensor for Catecholamine: Application to Monitoring Norepinephrine Exocytosis

A fluorescent sensor for catecholamines, NS510, is presented. The sensor is based on a quinolone fluorophore incorporating a boronic acid recognition element that gives it high affinity for catecholamines and a turn‐on response to norepinephrine. The sensor results in punctate staining of norepinephrine‐enriched chromaffin cells visualized using confocal microscopy indicating that it stains the norepinephrine in secretory vesicles. Amperometry in conjunction with total internal reflection fluorescence (TIRF) microscopy demonstrates that the sensor can be used to observe destaining of individual chromaffin granules upon exocytosis. NS510 is the highest affinity fluorescent norepinephrine sensor currently available and can be used for measuring catecholamines in live‐cell assays.     

High-resolution analyses of cell fusion dynamics in a biochip

In this study, we analyzed the electrofusion of two cells in a biochip that has been developed to perform the capture by dielectrophoresis and the electrofusion of pairs of cells. The good transparency of the microsystem allowed analyzing the details of the fusion events. By staining one of the cells, the mixing of the two cytosols could be observed during the electrofusion experiment. We show for the first time the rapidity of the mixing of the two cytosols: less than 5 s under our experimental conditions. By comparing these experimental results to a numerical simulation, we found that the rate of this phenomenon is compatible with a diffusion-only mechanism, showing that during the fusion, the two cell membranes in contact are affected by very rapid structural changes and do not limit the exchange of the cytosols between the two cells. A point of interest is the use of dielectric structures to concentrate the electric field and of positive dielectrophoresis to capture cells in the area where the electric field is more intense. This technique allows the increase of the cell-to-cell contact and limits cell cytosol leakages during the fusion process.     

用于细胞破裂的微流控生物芯片的研制

基于微电子机械系统(MEMS)技术,研制成一种夹流式血细胞破裂微流控生物芯片。细胞样品在破胞试剂夹流作用下导入芯片并在微沟道中流动,两种液体在流动过程中充分混合,导致细胞破裂。采用抗凝全血为细胞样品,比较胍盐和曲拉通的破胞效果;并分析在胍盐破裂细胞条件下,细胞浓度和流速对破胞效果的影响。控制破胞试剂流速远大于样品流速,可在几秒钟内完成细胞的破裂;保持破胞试剂与样品流速的比例,同时提高流速可在芯片上实现细胞的快速破裂。夹流式细胞破裂芯片具有与细胞分离芯片和脱氧核糖核酸(DNA)提取芯片相集成的潜力,可实现对复杂生物样品预处理操作,为实现微全分析系统打下良好基础。     

Membrane microextension: a possible mechanism for establishing molecular contact in electrofusion.

True cell membrane contact is an essential condition for electro-pulsed cell fusion, but initial morphological perturbation leading to true contact is still not clear. Dielectrophoresis mediated compression and fusogenic pulse induced compaction of cells led to rapid merger of tight membranes, and deprived direct microscopic view of surface membrane perturbation. Freely suspending cells with large and different cell-cell gaps may proceed to electrofusion with perturbed membrane and initiates fusion events at different time. These pulsed exposed cells can be used for capturing changes in the membrane surface and early electrofusion events. Early stage of fusion of freely suspended intact human erythrocytes exposed to single exponential decay pulse was studied by scanning electron microscopy (SEM). Field pulse induces small membrane bumps. Interaction of bumps on adjacent membranes lead to true membrane contact and form bridges between the membranes as microextension, combining both membranes into a topologically single structure. Some fusion products showed expanded fusion zones, which suggest indication of open lumen at contact area.     

Fibrinogen adsorption on three silica-based surfaces: conformation and kinetics

Using AFM (atomic force microscopy) to probe protein conformation and arrangement, and TIRF (total internal reflectance fluorescence) to monitor kinetics, fibrinogen adsorption on three different silica-based surfaces was studied: the native oxide on silicon, acid-etched microscope slides, and acid-etched polished glass. The three are chemically similar, but the microscope slide is rougher and induces AFM tip instabilities that appear as high spots on the bare surface. Fibrinogen's conformation and transport-limited adsorption kinetics are found to be quantitatively similar on all three surfaces. Further, the number of adsorbed proteins in progressive AFM micrographs quantitatively match the coverages measured by TIRF during early adsorption. Surfaces appear full, via AFM, when adsorbed amounts are about an order of magnitude below their true saturation levels (via TIRF) because, above about 0.26 mg/m(2), individual proteins cannot be discerned. The results demonstrate how the appearance of AFM micrographs can be misleading regarding surface saturation. On all three surfaces, fibrinogen is, at most, slightly aggregated, showing limited, if any, surface mobility. The complexities of the microscope slide's surface landscape minimally impact adsorption.     

Magnetic, electronic, and structural characterization of nonstoichiometric iron oxides at the nanoscale

We have investigated the structural, magnetic, and electronic properties of nonstoichiometric iron oxide nanocrystals prepared by decomposition of iron(II) and iron(0) precursors in the presence of organic solvents and capping groups. The highly uniform, crystalline, and monodisperse nanocrystals that were produced enabled a full structural and compositional survey by electron microscopy and X-ray diffraction. The complex and metastable behavior of nonstoichiometric iron oxide (wüstite) at the nanoscale was studied by a combination of Mossbauer spectroscopy and magnetic characterization. Deposition from hydrocarbon solvents with subsequent self-assembly of iron oxide nanocrystals into superlattices allowed the preparation of continuous thin films suitable for electronic transport measurements.     

Long-range transport of giant vesicles along microtubule networks

We report on a minimal system to mimic intracellular transport of membrane-bounded, vesicular cargo. In a cell-free assay, purified kinesin-1 motor proteins were directly anchored to the membrane of giant unilamellar vesicles, and their movement studied along two-dimensional microtubule networks. Motion-tracking of vesicles with diameters of 1-3 μm revealed traveling distances up to the millimeter range. The transport velocities were identical to velocities of cargo-free motors. Using total internal reflection fluorescence (TIRF) microscopy, we were able to estimate the number of GFP-labeled motors involved in the transport of a single vesicle. We found that the vesicles were transported by the cooperative activity of typically 5-10 motor molecules. The presented assay is expected to open up further applications in the field of synthetic biology, aiming at the in vitro reconstitution of sub-cellular multi-motor transport systems. It may also find applications in bionanotechnology, where the controlled long-range transport of artificial cargo is a promising means to advance current lab-on-a-chip systems.