Surface-induced phase separation of a sphingomyelin/cholesterol/ganglioside GM1-planar bilayer on mica surfaces and microdomain molecular conformation that accelerates Abeta oligomerization.

Ganglioside GM1 mediates the amyloid beta (Abeta) aggregation that is the hallmark of Alzheimer's disease (AD). To investigate how ganglioside-containing lipid bilayers interact with Abeta, we examined the interaction between Abeta40 and supported planar lipid bilayers (SPBs) on mica and SiO(2) substrates by using atomic force microscopy, fluorescence microscopy, and molecular dynamics computer simulations. These SPBs contained several compositions of sphingomyelin, cholesterol, and GM1 and were treated at physiological salt concentrations. Surprisingly high-speed Abeta aggregation of fibril formations occurred at all GM1 concentrations examined on the mica surface, but on the SiO(2) surface, only globular agglomerates formed and they formed slowly. At a GM1 concentration of 20mol%, unique triangular regions formed on the mica surface and the rapidly formed Abeta aggregations were observed only outside these regions. We have found that some unique surface-induced phase separations are induced by the GM1 clustering effects and the strong interactions between the GM1 head group and the water layer adsorbed in the ditrigonal cavities on the mica surface. The speed of Abeta40 aggregation and the shape of the agglomerates depend on the molecular conformation of GM1, which varies depending on the substrate materials. We identified the conformation that significantly accelerates Abeta40 aggregation, and we think that the detailed knowledge about the GM1 molecular conformation obtained in this work will be useful to those investigating Abeta-GM1 interactions.

Anomalous diffusion in supported lipid bilayers induced by oxide surface nanostructures.

Hierarchic structure and anomalous diffusion on submicrometer scale were introduced into an artificial cell membrane, and the spatiotemporal dependence of lipid diffusion was visualized on nanostructured oxide surfaces. We observed the lipid diffusion in supported lipid bilayers (SLBs) on step-and-terrace TiO(2)(100) and amorphous SiO(2)/Si surfaces by single molecule tracking (SMT) method. The SMT at the time resolution of 500 µs to 30 ms achieved observation of the lipid diffusion over the spatial and temporal ranges of 100 nm/millisecond to 1 µm/second. The temporal dependence of the diffusion coefficient in the SLB on TiO(2)(100) showed that the crossover from anomalous diffusion to random diffusion occurred around 10 ms. The surface fine architecture on substrates will be applicable to induce hierarchic structures on the order of 100 nm or less, which correspond to the microcompartment size in vivo.

Construction and structural analysis of tethered lipid bilayer containing photosynthetic antenna proteins for functional analysis.

The construction and structural analysis of a tethered planar lipid bilayer containing bacterial photosynthetic membrane proteins, light-harvesting complex 2 (LH2), and light-harvesting core complex (LH1-RC) is described and establishes this system as an experimental platform for their functional analysis. The planar lipid bilayer containing LH2 and/or LH1-RC complexes was successfully formed on an avidin-immobilized coverglass via an avidin-biotin linkage. Atomic force microscopy (AFM) showed that a smooth continuous membrane was formed there. Lateral diffusion of these membrane proteins, observed by a fluorescence recovery after photobleaching (FRAP), is discussed in terms of the membrane architecture. Energy transfer from LH2 to LH1-RC within the tethered membrane was observed by steady-state fluorescence spectroscopy, indicating that the tethered membrane can mimic the natural situation.

Synchrotron-radiation-stimulated etching of polydimethylsiloxane using XeF(2) as a reaction gas.

The synchrotron radiation (SR) stimulated etching of silicon elastomer polydimethylsiloxane (PDMS) using XeF(2) as an etching gas has been demonstrated. An etching system with differential pumps and two parabolic focusing mirrors was constructed to perform the etching. The PDMS was found to be effectively etched by the SR irradiation under the XeF(2) gas flow, and the etching process was area-selective and anisotropic. An extremely high etching rate of 40-50 microm (10 min)(-1) was easily obtained at an XeF(2) gas pressure of 0.2-0.4 torr. This suggests that SR etching using XeF(2) gas provides a new microfabrication technology for thick PDMS membranes, which can open new applications such as the formation of three-dimensional microfluidic circuits.

The PDMS-based microfluidic channel fabricated by synchrotron radiation stimulated etching.

Micro pattern on PDMS surface has been achieved by using synchrotron radiation (SR) stimulated etching. The experimental results indicated that SR stimulated etching has many advantages, such as extremely high etching rate (as large as 40-50 mum per 10 min), area-selectivity and anisotropy at room temperature, high spatial resolution. Combining the SR stimulated etching with photolithography, a PDMS-based microfluidic channel was obtained. The aim of this work is to develop a three-dimensional microfluidic channel with a special through hole, which is beneficial for cell differentiation, functionality and longevity and cannot be fabricated by conventional direct tooling techniques.

Incubation type Si-based planar ion channel biosensor.

A new planar-type ion channel biosensor with the function of cell culture has been fabricated using silicon on an insulator substrate as the sensor chip material. Coating of the sensor chip with fibronectin was essentially important for cell incubation on the chip. Although the seal resistance was quite low (approximately 7 Mohms) compared with the pipette patch-clamp gigaohm seal, the whole-cell channel current of the transient receptor potential vanilloid type 1 (TRPV1) channel expressing HEK293 cells was successfully observed, with a good signal-to-noise ratio, using capsaicin as a ligand molecule.

Formation of high-resistance supported lipid bilayer on the surface of a silicon substrate with microelectrodes.

We have developed two basic technologies for fabrication of supported planar lipid bilayer membrane ion channel biosensors: a defect-free lipid bilayer formation on the substrate surface with electrode pores and a patterning technique for the hydrophobic self-assembled-monolayer to form the guard ring that reduces the lipid bilayer edge-leak current. The importance of the supported-membrane structure to achieve low noise and high-speed performance is suggested on the basis of the observed relation between the single-ion-channel current noise and the pore size.

Surface characteristics and electrical properties of PMMA chips for incubation-type planar-patch-clamp biosensors.

Polymethylmethacrylate (PMMA) plates were successfully applied as sensor chips in an incubation-type planar patch clamp (IPPC). Hot embossing both sides formed the PMMA plates, and a focused ion beam realized micropores. The low seal resistance of the IPPC was investigated by analyzing the surface roughness of the chips. Atomic force microscopy (AFM) showed that the chip surface had a roughness of several nanometers due to the molding process. Coating the molded surface with an anti-adhesive compound further increased the surface roughness of the PMMA chip because the anti-adhesive compound itself had a large roughness and in some case, the compound partially peeled off while detaching the mold. Similarly, coating a chip with extracellular matrix (ECM) poly-l-lysine (PLL) also increased the surface roughness. The measured seal resistance of the PMMA chip for an HEK293 cell was in the range of 4-15 MΩ. The low seal resistance was attributed to the sharp-edge structure of the micropore and the surface roughness of the chip. Nevertheless, the whole cell current was successfully recorded from HEK293 cells expressing channel rhodopsin wide receiver (ChRWR) using salt-bridge-type stable Ag/AgCl electrodes. Another advantage of the PMMA sensor chip was the small parasitic capacitance.

Positioning of the sensor cell on the sensing area using cell trapping pattern in incubation type planar patch clamp biosensor.

Positioning the sensor cell on the micropore of the sensor chip and keeping it there during incubation are problematic tasks for incubation type planar patch clamp biosensors. To solve these problems, we formed on the Si sensor chip's surface a cell trapping pattern consisting of a lattice pattern with a round area 5 µm deep and with the micropore at the center of the round area. The surface of the sensor chip was coated with extra cellular matrix collagen IV, and HEK293 cells on which a chimera molecule of channel-rhodopsin-wide-receiver (ChR-WR) was expressed, were then seeded. We examined the effects of this cell trapping pattern on the biosensor's operation. In the case of a flat sensor chip without a cell trapping pattern, it took several days before the sensor cell covered the micropore and formed an almost confluent state. As a result, multi-cell layers easily formed and made channel current measurements impossible. On the other hand, the sensor chip with cell trapping pattern easily trapped cells in the round area, and formed the colony consisted of the cell monolayer covering the micropore. A laser (473 nm wavelength) induced channel current was observed from the whole cell arrangement formed using the nystatin perforation technique. The observed channel current characteristics matched measurements made by using a pipette patch clamp.

Extracellular and intraneuronal HMW-AbetaOs represent a molecular basis of memory loss in Alzheimer's disease model mouse.

BACKGROUND: Several lines of evidence indicate that memory loss represents a synaptic failure caused by soluble amyloid ß (Aß) oligomers. However, the pathological relevance of Aß oligomers (AßOs) as the trigger of synaptic or neuronal degeneration, and the possible mechanism underlying the neurotoxic action of endogenous AßOs remain to be determined. RESULTS: To specifically target toxic AßOs in vivo, monoclonal antibodies (1A9 and 2C3) specific to them were generated using a novel design method. 1A9 and 2C3 specifically recognize soluble AßOs larger than 35-mers and pentamers on Blue native polyacrylamide gel electrophoresis, respectively. Biophysical and structural analysis by atomic force microscopy (AFM) revealed that neurotoxic 1A9 and 2C3 oligomeric conformers displayed non-fibrilar, relatively spherical structure. Of note, such AßOs were taken up by neuroblastoma (SH-SY5Y) cell, resulted in neuronal death. In humans, immunohistochemical analysis employing 1A9 or 2C3 revealed that 1A9 and 2C3 stain intraneuronal granules accumulated in the perikaryon of pyramidal neurons and some diffuse plaques. Fluoro Jade-B binding assay also revealed 1A9- or 2C3-stained neurons, indicating their impending degeneration. In a long-term low-dose prophylactic trial using active 1A9 or 2C3 antibody, we found that passive immunization protected a mouse model of Alzheimer's disease (AD) from memory deficits, synaptic degeneration, promotion of intraneuronal AßOs, and neuronal degeneration. Because the primary antitoxic action of 1A9 and 2C3 occurs outside neurons, our results suggest that extracellular AßOs initiate the AD toxic process and intraneuronal AßOs may worsen neuronal degeneration and memory loss. CONCLUSION: Now, we have evidence that HMW-AßOs are among the earliest manifestation of the AD toxic process in mice and humans. We are certain that our studies move us closer to our goal of finding a therapeutic target and/or confirming the relevance of our therapeutic strategy.

Polymerized lipid bilayers on a solid substrate: morphologies and obstruction of lateral diffusion.

Substrate supported planar lipid bilayers (SPBs) are versatile models of the biological membrane in biophysical studies and biomedical applications. We previously developed a methodology for generating SPBs composed of polymeric and fluid phospholipid bilayers by using a photopolymerizable diacetylene phospholipid (DiynePC). Polymeric bilayers could be generated with micropatterns by conventional photolithography, and the degree of polymerization could be controlled by modulating UV irradiation doses. After removing nonreacted monomers, fluid lipid membranes could be integrated with polymeric bilayers. Herein, we report on a quantitative study of the morphology of polymeric bilayer domains and their obstruction toward lateral diffusion of membrane-associated molecules. Atomic force microscopy (AFM) observations revealed that polymerized DiynePC bilayers were formed as nanometer-sized domains. The ratio of polymeric and fluid bilayers could be modulated quantitatively by changing the UV irradiation dose for photopolymerization. Lateral diffusion coefficients of lipid molecules in fluid bilayers were measured by fluorescence recovery after photobleaching (FRAP) and correlated with the amount of polymeric bilayer domains on the substrate. Controlled domain structures, lipid compositions, and lateral mobility in the model membranes should allow us to fabricate model membranes that mimic complex features of biological membranes with well-defined structures and physicochemical properties.

Lipid bilayer membrane with atomic step structure: supported bilayer on a step-and-terrace TiO2(100) surface.

The formation of a supported planar lipid bilayer (SPLB) and its morphology on step-and-terrace rutile TiO 2(100) surfaces were investigated by fluorescence microscopy and atomic force microscopy. The TiO 2(100) surfaces consisting of atomic steps and flat terraces were formed on a rutile TiO 2 single-crystal wafer by a wet treatment and annealing under a flow of oxygen. An intact vesicular layer formed on the TiO 2(100) surface when the surface was incubated in a sonicated vesicle suspension under the condition that a full-coverage SPLB forms on SiO 2, as reported in previous studies. However, a full-coverage, continuous, fluid SPLB was obtained on the step-and-terrace TiO 2(100) depending on the lipid concentration, incubation time, and vesicle size. The SPLB on the TiO 2(100) also has step-and-terrace morphology following the substrate structure precisely even though the SPLB is in the fluid phase and an approximately 1-nm-thick water layer exists between the SPLB and the substrate. This membrane distortion on the atomic scale affects the phase-separation structure of a binary bilayer of micrometer order. The interaction energy calculated including DLVO and non-DLVO factors shows that a lipid membrane on the TiO 2(100) gains 20 times more energy than on SiO 2. This specifically strong attraction on TiO 2 makes the fluid SPLB precisely follow the substrate structure of angstrom order.

Synchrotron-radiation-stimulated etching of SiO2 thin films with a tungsten nano-pillar mask.

A nano-pattern of SiO(2) on a Si (100) surface has been demonstrated by synchrotron-radiation-stimulated etching with a tungsten nano-pillar mask. The reaction gas was a mixture of SF(6) and O(2). The mask was fabricated using a focused ion beam with W(CO)(6) as the source gas. The width and height of the tungsten nano-pillar were approximately 80 nm and 160 nm, respectively. Synchrotron radiation irradiation with flowing SF(6) and O(2) effectively etches the silicon dioxide, and the etching process followed the surface photochemical reaction. The etched surface was very flat, and no undercutting occurred during the etching process.

Supported phospholipid bilayer formation on hydrophilicity-controlled silicon dioxide surfaces.

We investigated the influence of surface hydroxyl groups (-OHs) on the supported planar phospholipid bilayer (SPB) formation and characteristics. We prepared SiO2 surfaces with different hydrophilicity degree by annealing the SiO2 layer on Si(100) formed by wet chemical treatments. The hydrophilicity reduced with irreversible thermal desorption of -OHs. We formed SPB of dimyristoylphosphatidylcholine on the SiO2 surfaces by incubation at a 100-nm-filtered vesicle suspension. The formation rate was faster on less hydrophilic surfaces. We proposed that a stable hydrogen-bonded water layer on the SiO2 surface worked as a barrier to prevent vesicle adhesion on the surface. Theoretical calculation indicates that water molecules on vicinal surface -OHs take a stable surface-unique geometry, which disappears on an isolated -OH. The surface -OH density, however, affected little the fluidity of once formed SPBs, which was measured by the fluorescence recovery after the photobleaching method. We also describe the area-selective SPB deposition using surface patterning by the focused ion beam.

Lipid membrane formation by vesicle fusion on silicon dioxide surfaces modified with alkyl self-assembled monolayer islands.

Using atomic force microscopy, we have investigated the formation of the dipalmitoylphosphatidylcholine (DPPC) membrane by the vesicle fusion method on SiO2 surfaces modified with self-assembled monolayer (SAM) islands of octadecyltrichlorosilane (OTS) with sizes comparable to those of the vesicles. OTS-SAM islands with various sizes and coverages can be constructed on the SiO2 surfaces prepared by thermal oxidation followed by partial hydroxylation in a H2O2/H2SO4 solution. When vesicles are sufficiently smaller than the SiO2 domains, DPPC bilayers and DPPC/OTS layers form on the SiO2 and OTS domains, respectively. However, the adhesion of larger vesicles onto SiO2 is prevented by the OTS islands; therefore only DPPC/OTS layers form without formation of DPPC bilayers on the SiO2 domains. On surfaces with domains on the scale of tens to hundreds of nanometers, the relative size between the hydrophilic domains and the vesicles becomes an important factor in the membrane formation by the fusion of vesicles.