Koji Sumitomo

The localisation of liquid-ordered (L-o) and liquid-crystalline (La) phase domains on a silicon substrate with a microwell array is investigated. Although the phase separation of the Lo and La phases on both a giant unilamellar vesicle (GUV) and a supported membrane remains stable for a long time, the lateral diffusion of lipids across each domain boundary occurs quickly. Since the phase separation and domain arrangement are governed by the stiffness and lateral tension of the lipid membrane, the phase separation is rearranged on a rnicropatterned substrate. Similar phase separation of the Lo and La phases is observed at a lipid membrane suspended over a microwell. However, the La phase is preferred at a suspended membrane, and saturated lipids and cholesterol are excluded toward the supporting membrane on the periphery. Since the Lo domain area is reduced by anisotropic diffusion through the boundary between the suspended and supported membranes, a very slow reduction rate with a La phase domain is observed at a membrane suspended over a microwell, which is membrane. linear functional relation is observed. Finally, a localized surrounded by an Lo phase supported membrane.

The fusion of proteoliposomes is a promising approach for incorporating membrane proteins in artificial lipid membranes. In this study, we employed an electrostatic interaction between vesicles and supported bilayer lipid membranes (s-BLMs) to control the fusion process. We combined large unilamellar vesicles (LUVs) containing anionic lipids, which we used instead of proteoliposomes, and s-BLMs containing cationic lipids to control electrostatic interaction. Anionic LUVs were never adsorbed or ruptured on the SiO2 substrate with a slight negative charge, and selectively fused with cationic s-BLMs. The LUVs can be fused effectively to the target position. Furthermore, as the vesicle fusion proceeds and some of the positive charges are neutralized, the attractive interaction weakens and finally the vesicle fusion saturates. In other words, we can control the number of LUVs fused with s-BLMs by controlling the concentration of the cationic lipids in the s-BLMs. The fluidity of the s-BLMs after vesicle fusion was confirmed to be sufficiently high. This indicates that the LUVs attached to the s-BLMs were almost completely fused, and there were few intermediate state vesicles in the fusion process. We could control the position and amount of vesicle fusion with the s-BLMs by employing an electrostatic interaction.

To provide a platform for biodevices designed to characterize membrane proteins, we fabricated a new type of microwell sealed with a lipid membrane on a SiO2 substrate. The microwell is surrounded by a Au ring with a self-assembled monolayer, on which a lipid membrane is formed to create a seal. Fluorescence and electrophysiological studies reveal that the structure prevents unfavorable ion leakage from/ into microwells. By separating the microwells and outer regions, ion diffusion through the water layer between the membrane and the substrate is reduced. This study offers a promising approach for the functional analysis of membrane proteins. (C) 2015 The Japan Society of Applied Physics

A membrane protein is one of the targets for understanding cellular function and for biosensor and drug screening applications. An artificial lipid membrane suspended over microwells can act as a platform when we attempt to realize a nanobio device based on a membrane protein function. With the aim of preparing a lipid membrane array on a substrate, we investigated lipid membrane formation on a hydrogel-confined micropatterned substrate because a hydrogel can support a lipid membrane both mechanically and functionally. The interaction between the positive charge of the hydrogel and the negative charge of the substrate surface allows the hydrogel to be confined in the microwells. When small unilamellar vesicles with a diameter smaller than the aperture of the microwells were added to the solution over the substrate, hydrogel-supported lipid membranes were formed. The vesicle fusion was facilitated by the electrostatic interaction between the vesicle charge and the opposite charge of the hydrogel. The continuous and fluid properties of the hydrogel-supported lipid membrane were confirmed by the fluorescent recovery after photobleaching method. Our results suggest that support provided by hydrogels in the microwells enables us to arrange membrane proteins on a nanobio device by the direct formation of a planar lipid membrane from proteoliposomes with a small diameter. (C) 2015 Elsevier B.V. All rights reserved.

Apoptosis plays an important role in many physiologic and pathologic conditions. The biochemical and morphological characteristics of apoptosis including cellular volume decrease, cell membrane blebbing, and phosphatidylserine translocation from the inner to the outer leaflet of the cell membrane are considered important events for phagocyte detection. Despite its importance, the relationship between the biological and morphological changes in a living cell has remained controversial. Scanning ion conductance microscopy is a suitable technique for investigating a series of these changes, because it allows us to observe the morphology of living cells without any mechanical interactions between the probe and the sample surface with a high resolution. Here, we investigated the biochemical and morphological changes in single neurons during the early stages of apoptosis, including apoptotic volume decrease, membrane blebbing and phosphatidylserine translocation, by using scanning ion conductance microscopy. Time-course imaging of apoptotic neurons showed there was a reduction in apoptotic volume after exposure to staurosporine and subsequent membrane bleb formation, which has a similar onset time to phosphatidylserine translocation. Our results show that a reduction in cellular volume is one of the earliest morphological changes in apoptosis, and membrane blebbing and phosphatidylserine translocation occur as subsequent biological and morphological changes. This is the first report to describe this series of morphological and biochemical changes ranging from an apoptotic volume decrease to membrane blebbing and PS translocation by scanning ion conductance microscopy (SICM). This new and direct imaging technique will provide new insight into the relationship between biochemical events inside a cell and cellular morphological changes. (C) 2015 Elsevier Inc. All rights reserved.

We investigated structural changes in ligand-gated ion channel proteins reconstituted into supported lipid bilayers. The ion channels' extracellular parts were highly dynamic and exhibited agonist-induced changes, which were inhibited by antagonists. Our data demonstrate that, by using small exogenously applied compounds (i.e., ligands), ion channel proteins reconstituted into supported lipid bilayers on a substrate can be controlled. (C) 2014 The Japan Society of Applied Physics

To provide a platform for a nanobiodevice, we fabricated microcavities on a Si/SiO2 substrate covered by a thin SiO2 layer with nanohole arrays that we call a pepper shaker substrate. Fluorescence and atomic force microscopy images showed that the structure of the pepper shaker substrate improved both the probability of membrane sealing over the microcavities by rupturing giant unilameller vesicles and the lifetime of the lipid membrane suspended over the microcavities. The success of this study reveals the potential for fabricating an artificial cell array as a tool for the functional and high throughput analysis of membrane proteins. (C) 2014 The Japan Society of Applied Physics

We successfully sealed a gel-confined microwell array on a Si substrate with a lipid membrane by rupturing giant unilamellar vesicles. Atomic force microscope measurements suggested that gel with a hillock structure supported lipid membranes gently because of its soft and elastic properties and improved the stability of the lipid membrane over the microwell array. We found that the lipid membrane sealed the gel-confined microwells and no Ca2+ leakage through the lipid membrane was observed within the detection range of our calcium indicator. We expect that gel with properties similar to cytoplasm consisting of a cytoskeletal network is a potential candidate for providing lipid membranes with mechanical support. This study proposes an artificial cell array system for fluorescence and atomic force microscope observations of functional membrane proteins on silicon-based nanobiodevices. (C) 2014 The Japan Society of Applied Physics

For the functional analysis of ion channel activity, an artificial lipid bilayer suspended over microwells was formed that ruptured giant unilamellar vesicles on a Si substrate. Ca2+ ion indicators (fluo-4) were confined in the microwells by sealing the microwells with a lipid bilayer. An overhang formed at the microwells prevented the lipid membrane from falling into them and allowed the stable confinement of the fluorescent probes. The transport of Ca2+ ions through the channels formed by a-hemolysin inserted in a lipid membrane was analyzed by employing the fluorescence intensity change of fluo-4 in the microwells. The microwell volume was very small (1-100 fl), so a highly sensitive monitor could be realized. The detection limit is several tens of ions/s/mu m(2), and this is much smaller than the ion current in a standard electrophysiological measurement. Smaller microwells will make it possible to mimic a local ion concentration change in the cells, although the signal to noise ratio must be further improved for the functional analysis of a single channel. We demonstrated that a microwell array with confined fluorescent probes sealed by a lipid bilayer could constitute a basic component of a highly sensitive biosensor array that works with functional membrane proteins. This array will allow us to realize high throughput and parallel testing devices. (C) 2011 Elsevier B.V. All rights reserved.

We investigated techniques for regulating the orientation of ion channel-type membrane proteins reconstituted in lipid bilayers. Free ion channel proteins aligned their long axis parallel to the substrate. In contrast, immunochemical and atomic force microscopy images revealed that ion channels reconstituted in supported lipid bilayers oriented upward, with their long axis perpendicular to the substrate. Our data demonstrates that the reconstitution of ion channels into planar lipid bilayers by rupturing small unilamellar proteoliposomes is a promising way of aligning ion channels upward in a membrane and of obtaining ion channels with controlled functions. (C) 2011 The Japan Society of Applied Physics

We investigated the optimum architecture for confining fluorescent probes in microwells on a Si substrate by covering it with a lipid bilayer. We modified the structure of the wells to prevent the lipid membrane from falling into them, and the overhang shape at the aperture improved the probability of confinement. The fluorescence intensity from the calcein confined in the wells remained unchanged for one hour or more, indicating that the probes remain stably in the wells without flowing out. An artificial cell sealed with the suspended membrane is a promising tool for the functional analysis of membrane proteins. (C) 2010 The Japan Society of Applied Physics

In this study, we observed the topology of a single protein in a stretched lipid bilayer (membrane) suspended over a nanoscale well using a fast-scanning atomic force microscope (AFM). The membrane was located stably enough on the well to prevent the leakage of a liquid placed in the well, and it allowed us to observe membrane stretching using an AFM. We successfully observed the gradual stretching of the suspended membrane. We also observed single bacteriorhodopsin proteins in the stretched membrane, and found that they maintained their trimeric structure, but that the distances between the trimers increased. (C) 2010 The Japan Society of Applied Physics

Atomic force microscopy (AFM) imaging of membrane proteins suspended over a nanostructure in liquid is a promising way to understand the structure and function of working proteins, although the membrane deformation that occurs during scanning makes it difficult to obtain a high resolution image. This study proposes an artificial cell system for the AFM observation of functional membrane proteins that consists of a sub-micron well on Si, a biological membrane, and a carbon nanotube (CNT) network. We successfully observed molecular-scale images of a purple membrane suspended over sub-micron well patterns. By using a CNT network to hold the suspended membrane, we suppressed the membrane deformation caused by the "imaging force". The CNT network takes the place of a cytoskeleton in supporting the cell membrane suspended over the well thus improving the spatial resolution of AFM measurement. (C) 2009 The Japan Society of Applied Physics

The ATP-gated P2X4 receptor is a cation channel, which is important in various pathophysiological events. The architecture of the P2X 4 receptor in the activated state and how to change its structure in response to ATP binding are not fully understood. Here, we analyze the architecture and ATP-induced structural changes in P2X4 receptors using fast-scanning atomic force microscopy (AFM). AFM images of the membrane-dissociated and membrane-inserted forms of P2X4 receptors and a functional analysis revealed that P2X4 receptors have an upward orientation on mica but lean to one side. Time-lapse imaging of the ATP-induced structural changes in P2X4 receptors revealed two different forms of activated structures under 0 Ca2+ conditions, namely a trimer structure and a pore dilation-like tripartite structure. A dye uptake measurement demonstrated that ATP-activated P2X4 receptors display pore dilation in the absence of Ca2+. With Ca2+, the P2X4 receptors exhibited only a disengaged trimer and no dye uptake was observed. Thus our data provide a new insight into ATP-induced structural changes in P2X4 receptors that correlate with pore dynamics. © 2009 Shinozaki et al.

We have probed the mechanical properties of purple membrane (PM) in a physiological environment using the atomic force microscope (AFM). By suspending PM over nano-trenches, the elastic properties of PM can be evaluated free from the interaction with the substrate. Force-displacement curves were obtained on the suspended membrane and the data was compared to that of a simple model of a thin film over a trench. By fitting the data to the model, the elastic modulus of PM was estimated to be 8 MPa. When the membrane is repeatedly indented, we observed a change in the force-distance data consistent with damage to the two-dimensional crystal of PM. In this paper we demonstrate that the AFM allows us to evaluate the mechanics of biological membranes in their native conditions. (C) 2008 Elsevier B.V. All rights reserved.