Membrane transport in cells is a fundamental biological process that is mediated by various channels, pumps and transporter proteins. AcrB is a major multidrug efflux transporter in gram-negative bacteria, which confer multidrug resistance. AcrB transports a wide variety of drugs or toxic compounds directly out of the cells driven by proton motive force. Now we solve the crystal structures of AcrB with and without substrates. The AcrB-drug complex consists of asymmetric three protomers, each of which has different conformation corresponding to one of the three functional states of the transport cycle. Bound substrate was found in the periplasmic domain of one of the three protomers. The voluminous binding pocket is aromatic and allows multi-site binding. The structures show that drugs are presumably exported by a three-step functionally rotating mechanism in which drugs undergo ordered binding change.
The membrane of each organelle has its own characteristic/specific lipid composition. The integrity of the lipid composition is essential for the organelles to maintain their surface properties, structure and functions. Late endosomes are the organelles which have roles in the intracellular macromolecule sorting as well as the degradation and metabolism of materials in the cell, and are rich in the negatively-charged unusual phospholipid, bis (monoacylglycero) phosphate (BMP). The structure of the late endosome is altered from multivesicular to multilamellar membranes under different pathological conditions, such as glycolipid-storage diseases, and by treatment with certain drugs, resulting in a disruption of the exit of cholesterol from the organelle. The molecular mechanisms of these phenomena have not been revealed. In this report, we show that the structural change of these organelles can be reproduced in vitro by incubating BMP membrane with glycolipids as well as a certain drug. It is also shown that cholesterol is trapped in such an in vitro system.
Mycoplasma, a group of parasitic bacteria, forms a membrane protrusion at a cell pole. They bind to solid surfaces such as animal cell and glass, and glide. The mycoplasma genomes do not have any genes involved in the bio-motility systems ever known, suggesting that they move with a mysterious mechanism. We have studied about the properties of motility, structures of machinery, and enzymatic reaction coupled to gliding, and suggested a putative molecular mechanism. “Many filamentous legs on the base of membrane protrusion repeat binding and release from sialic acid fixed on the solid surface and pull the cell body.”
Mechanobiology is indispensable to all living creatures ranging from bacteria to human beings because every living thing continually experiences mechanical stimuli, not only exogenous but also endogenous, and responds appropriately to these stimuli. At the cellular level, there are three major mechanical stresses such as, stretch, shear stress and pressure. We have been intensively investigating systems to apply these mechanical environments to cells. Analysis of these responses is critical to understand the mechanisms and regulation of diverse physiological processes such as arterial and regulation, sensory perception, muscle development, and so on. Therefore, to study mechanobiology, we employed the polydimethysiloxane (PDMS) based technology, which is called soft lithography. In this paper, I am going to focus on biomedical and clinical applications of soft lithography.
Analysis of intra- or inter-cellular responses plays an important role in understanding the bio-functional mechanisms that are undergoing in a single cell or cell groups or tissue or organ, which led to advanced disease diagnosis and improved treatments. In nature, cells possess different functional activities and these activities control gene and protein expressions and cell function altogether. To understand the different functions of each cell, there is a need to develop microchip platforms, which can perform high-throughput screening and analysis of genes and proteins at individual cell level and single-molecule level. For achieving these goals, we developed in our laboratory different kinds of chip devices by employing micro and nano technology and applied these devices in single molecule DNA detection, in vitro protein synthesis and single-cell analysis. In this review, we mainly focus on the analysis of cellular signaling using cell chip device systems. This review has been divided mainly into three topics; high-throughput single-cell analysis system, drug screening system using neural cell chip and nanobiodevice for cell signaling detection.
The label-free biosensing technique based on anomalous reflection of gold (AR) is reviewed. The AR method is a phenomenon that a large decrease in reflectivity occurs for blue and purple light upon adsorption of an ultrathin transparent film on a gold surface. This originates from the fact that gold has less metallic character for light with wavelengths shorter than 500 nm. Advantages of AR method for biosensing are (i) the optical setup is simple, (ii) the optical geometry is elastic, (iii) thickness of gold thin films is not limited, and (iv) the method provides us with high spatial resolution compared with conventional surface plasmon resonance imaging. The AR imaging of a protein microarray is also shown to demonstrate the capability of this method.
Here we introduce a scanning reflection high-energy electron diffraction technique (Scanning RHEED), that is used to measure simultaneously multiple RHEED intensity variation curves as a function of position, azimuth angle, incident angle or combinations of all three, during epitaxial film growth on a substrate. In this paper, the incident angle dependence of RHEED intensity oscillations is discussed in terms of oscillation amplitude, decay, and phase during SrTiO3 homoepitaxy on a (100) substrate. A simple way to adjust the incident angle of the electron beam in conventional RHEED systems is also introduced.