Research at the Sato Lab

  We investigate the structure of protein complexes, cells and tissues using a combination of Cryo-Transmission Electron Microscopy (TEM) and our original Atmospheric Scanning Electron Microscopy (ASEM). By combining large numbers of TEM images using single particle analysis (SPA) technique, we determine a high-resolution 3D structure of various membrane proteins complexes using our newly-developed image analysis algorithms. We have determined the 3D structures of signaling complexes, including ion channels, receptors, and oxidative stress sensors (Fig.1). We have also developed an in-solution observation microscope, ASEM. It is applied to study the mechanisms of signaling protein complexes, including ion channels, receptors, and antibodies. Cell and tissue level observation are also developed to study molecular complex in cells in various phenomena, including neuronal differentiation and processing, immunity, embryogenesis, carcinogenesis and metastasis.


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Fig.1 Cryo-TEM and Sodium channel structure              ( nature 2001)

For example, structure determination of membrane proteins is important to understand the signaling systems in our cells and also for drug design. However, their crystallization is generally hard. We have been developing the structure determination method using cryo-TEM without protein crystal, that is SPA, for more than ten years. This methodology recently reached atomic resolution and is expected to be widely applied to determine the structure of membrane proteins and molecular complexes. We focus on solving the mechanisms of the signaling proteins and their complexes, including channels, receptors and sensors (Fig. 2), to understand the molecular machinery realizing our physiological functions.


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Fig.2 Structure of signal processing proteins, IP3 receptor (left ; JMB 2004) and oxidative stress sensor KEAP1 preventing cancer (right ; PNAS 2010), revealed by single particle reconstruction using TEM

  ASEM has been developed to realize high resolution observation of a sample immersed in aqueous liquid in a readily accessible, open ASEM dish. Fixed cells or tissues in radical scavenger solution (10 mg/ml glucose) can be directly observed by SEM through a thin silicon nitride (SiN) film in the base of the ASEM dish. The observable depth of ASEM is 2-3 micro meter and the resolution is 8 nm. Using ASEM, we have successfully observed protein complex formations in cells or in small synaptic connections, which can be adopted for basic biology and medicine. It could be also applied for quicker intra-operative cancer diagnosis or diagnosis of infectious diseases.
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Fig. 3  ASEM (left ; JSB 2012) and its image of fine synapses between neurons (middle) and quick visualization of normal spinal cord (Right: 1st column) and breast-cancer metastasized spinal cord (Right: 2nd column; IJO 2015).

Rapid imaging of mycoplasma in solution using Atmospheric Scanning Electron Microscopy (ASEM)

We directly observed the model species Mycoplasma Mobile in buffer with the newly developed Atmospheric Scanning Electron Microscope (ASEM). Quick, in-solution EM observation of mycoplasma should aid in the early diagnosis of various mycoplasma-induced diseases.
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Under non-stress conditions, Keap1 grabs the cytoprotective transcription factor Nrf2 and represses its activity.

keapRH
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Research at the Sato Lab

We investigate the structure of nerve cells, proteins and molecular complexes at the macromolecular level, using a combination of Scanning Electron Microscopy (SEM), Transmission Electron Microscopy (TEM), and Optical Microscopy (OM). Using OM, whole cells can be observed at resolutions of up to 200 nm. By combining large numbers of EM images using Single Particle Analysis (SPA) techniques, we can determinate a high-resolution 3D structure. We have determined the structure of various membrane proteins using SPA with newly-developed image analysis methods. This will facilitate the study of nerves, calcium signaling mechanisms, and the transmission of pain and other sensations, pertinent to Alzheimer’s and many other diseases.

In collaboration with JEOL, we have developed a new electron microscope that allows direct observation of specimens in open atmosphere. Using SiN film developed for the semiconductor industry, the new ASEM can image the same specimen with both OM and EM, in liquid buffer.

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Sodium Channel

sodium channel

The voltage-sensitive sodium channels are responsible for initiating action potentials in electrically excitable cells such as nerves and muscles. This means they are a critical link in the chain of cellular-level events that produce movement and sensation, and even thought and emotion. This seminal article presented the first three-dimensional view of the sodium-channel protein.

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Keap1

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Under non-stress conditions, Keap1 grabs cytoprotective transcription factor Nrf2 and represses its activity. Oxidative stresses, sensed by regions inside the large sphere and/or inside the forked stem, allow the translocation of Nrf2 to the nucleus. Nrf2 activates many cytoprotective genes, which protect the cell from damage caused by oxidative stress.

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Orai1

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Our immune system consists of white blood cells, including lymphocytes. Lymphocytes are regulated by calcium permeable ion channels on their plasma membrane. These ion channels are believed to be composed of two major proteins, STIM1 and Orai1. Mutations in Orai1 cause severe immune deficiency syndrome.

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TRPC3

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TRPC3 is a key player in many cellular functions, controlling growth cone guidance and synaptic plasticity in the central nervous system, blood vessel constriction , and the differentiation of immune cells. Mutations of this protein are implicated in various cardiovascular and kidney diseases.

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IP3R

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The inositol trisphosphate receptor is a membrane glycoprotein complex acting as a calcium regulator for the cell. This is important for various cell activities and is implicated in neural plasticity. Using single-particle analysis and a helium-stage-equipped electron microscope, we created a three-dimensional model which illuminates the mechanism for the regulation of calcium release by the combined action of Ca2+ and inositol 1,4,5-triphosphate.

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TRPM2

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The TRPM2 protein is a heat-activated sensor of oxidative stress which has been implicated in diseases such as diabetes. Using single-particle analysis of electron microscope images, we are able to present a three-dimensional structure to further the understanding of its physiological role.

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Gamma-Secretase

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To gain insight into the structure of this complex enzyme, we purified functional c-secretase complex reconstituted in Sf9 cells and analyzed it using negative stain electron microscopy and 3D reconstruction techniques. Analysis of 2341 negatively stained particle images resulted in the three-dimensional representation of c-secretase at a resolution of 48 Å.

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SPP

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Signal peptide peptidase is an atypical aspartic protease that hydrolyzes peptide bonds within the transmembrane domain of substrates and is implicated in several biolgical and pathological functions.

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MG23

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Mitsugumin 23 (MG23) is a large transmembrane protein found in the SR/ER and nuclear membranes of many cell types, but its physiological role is unknown. Here we report the biochemical and biophysical characterization of MG23.

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M. mobile

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We directly observed the model species Mycoplasma Mobile in buffer with the newly developed Atmospheric Scanning Electron Microscope (ASEM). Quick, in-solution EM observation of mycoplasma should aid in the early diagnosis of various mycoplasma-induced diseases.

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Materials Science

ASEM dishes

We observed various physical phenomena in real time at EM resolutions--in open atmosphere.

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We investigate the structure of proteins and molecular complexes at the macromolecular level, using a combination of Scanning Electron Microscopy (SEM), Transmission Electron Microscopy (TEM), and Optical Microscopy (OM).