Research

Our research aims and results

7-transmembrane proteins

画像:7回膜貫通型タンパク質

Structures of membrane proteins with 7-transmembrane helices including bacteriorhodopsin as well as G-protein-coupled receptors (GPCRs) are interested in and bacteriorhodopsin structure was analysed at 3 Å resolution by electron crystallography. While structural study of GPCRs is more difficult, we are continuously studying their structures and functions, because they are important drug targets and key molecules of signal transduction cascades in biological cells.

Water channel

画像:水チャネル

To understand both the fast conduction and the high selectivity of water molecules through water channel (aquaporin), we focused on determining the atomic structure of aquaporin buried in the lipid membrane close to in vivo situation. In particular, out of the 13 water channels in the human body, we choose aquaporin-4 and aquaporin-11, which is expressed in the brains and the kidney, respectively. We often use cryo-electron microscope and electron crystallography to solve the 3D structure. Furthermore, to elucidate the function in vivo including the higher functional relation between the brain and water channels, we also use freeze fracture technique, optical microscope, and stopped-flow analysis as well as molecular biology methods.

Voltage-gated sodium channels (Navs)

画像:膜電位感受性ナトリウムチャネル

Navs generate the rapid upstroke of action potentials in nerve cell axons.
We focus on elucidating the molecular mechanisms for sodium selectivity and activity regulation, utilizing both of crystallography and electrophysiology.

Gap Junction Channel

画像:ギャップ結合チャネル

Most multicellular organisms, such as vertebrates and invertebrates, possess gap junctions comprising clusters of channels that mediate the direct communication between adjacent cells. Three-dimensional structures of gap junction channels are investigated using cryo-electron microscopy (cryo-EM) to elucidate the molecular basis of the gating mechanism and the biological significance in vivo. We recently determined an atomic model of a gap junction channel by single particle cryo-EM. This method will essentially be used for structure determination of membrane proteins.
In addition to the chordate gap junction protein connexin, we also study the invertebrate gap junction protein innexin to investigate the diversity and shared function of gap junction channels in nature.

Gastric Proton pump

画像:胃プロトンポンプ

When we have food intake, pH of our stomach reaches around 1. This highly acidic environment is indispensable for digestion, and also acts as the first barrier against bacterial or viral infection. Conversely, too much acidification of the stomach induces gastric ulcer or gastroesophageal-reflux diseases. Gastric proton pump, H+,K+-ATPase is a membrane protein responsible for the gastric acid secretion. Its generated 6 units of pH difference, that is, more than a million-fold cation gradient, is hardly met by any other pump in nature. We would like to address its unique molecular mechanisms through functional and structural studies by electron crystallography.

Tight Junction

画像:IP39

The surface of tissue from skin, digestive system, organs and blood vessels etc., is covered with epithelial cells. It works not only to separate the internal environments from outside environments literally, but also to regulate molecular permeation for keeping homeostatic system of our body. Such functions can be achieved by the intercellular apparatus called as tight junction (TJ), where each cells come close to be closely aligned and to form intercellular pathway for the regulation of molecular permeations including ions and small molecule. The major membrane protein component of TJ is claudin, which forms branching network by its polymerization. We would like to elucidate the structure of the molecular arrangements and the mechanisms for the polymerization by using X-ray crystallography, electron crystallography and freeze-fracture electron microscopy.

Influenza virus

画像:インフルエンザウィルス

Envelope structures of influenza A virus particles were observed by high resolution cryo-electron microscopy and were ascribed to a combination of a thin outer lipid monolayer and a 72 Å thick protein-containing inner layer. The dense influenza virus particles with the asymmetric envelopes have high infectivity, while the large and the pale virus particles with phospholipid bilayer envelopes have less or no infectivity. We proposed a model for active influenza virus as shown in right side drawing with thin outer lipid monolayer and a 72 Å thick protein-containing inner layer.

molecular mechanisms of synapses

画像:シナプスの分子機構

We are investigating the molecular mechanisms of synapses, at which neuronal transmissions take place. We focus on the excitatory glutamatergic synapses, especially the post-synaptic density-95 (PSD-95) scaffold protein, which is believed to control the protein accumulation at post-synaptic membrane, and the LRRTM proteins, the post-synaptic cell adhesion molecules. Our works involves electrophysiology, biochemistry and fluorescence microscopy.

Multipotent stem cells

画像:Multipotent stem cells

We are analyzing the characterization of multipotent stem cells in the body. One of our goals is to differentiate these cells into many kinds of cells which make up adult tissues. We study cell migration as well as its mechanisms of these cells in vitro/vivo. We also investigate key molecules including receptors involved in the migrations.

Development of cryo-electron microscopes

画像:極低温電子顕微鏡

For high resolution structural study, the radiation sensitivity of biological molecules gave us a difficult challenge and forces us to develop an effective and stable cryo-electron microscope with a helium cooled specimen stage. We have therefore developed a super fluid helium stage that achieved a resolution of 2.0 Å. Thermal shield by liquid Nitrogen and Helium tank are gold plated to minimize radial heat. Small liquid Helium container, which was set at the center of the stage, was connected from the Helium tank by a capillary which could be cooled down to 1.5K by superfluid Helium. Recently we developed a new cryo-microscope, the seventh generation cryo-EM with an outer-control tilting device for electron tomography and some other analyses.

Electron crystallography

画像:電子線結晶学

Electron crystallography is a powerful tool for determining membrane proteins, because of the following reasons. 1) Keeping membrane proteins in the lipid bilayers similar to the natural environment. 2) Using a two-dimensional (2D) crystal rather than a three-dimensional (3D) crystal, which always has artificial 3D contacts due to crystal packing. 3) A 3D structure can be obtained from not so well-ordered crystals, since phase information is calculated from electron micrographs directly. On the other hand, the structural analysis program packages in EM field are not so matured in comparison to those in X-ray crystallography. Thus the structural analysis was very laborious and a time consuming step. We develop the graphics user interfaced programs based on the MRC package to overcome such problems.