Research at the Sato Lab

Research at the Sato Lab

We investigate the structures 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 cryo-TEM images using single particle analysis (SPA) technique, we determine high-resolution 3D structures of various proteins, especially membrane protein complexes using our newly-developed image analysis algorithms including artificial intelligence (AI). We have determined the 3D structures of signaling complexes, including ion channels, receptors, and oxidative stress sensors. We have also developed a new electron microscope, ASEM, for larger scale observation of samples in liquid. It is applied to study signaling in various biological phenomena, including  bacterial structures, microbe biofilm, virus activities, brain/neuronal differentiation, bone response to mechanical stress, embryogenesis, carcinogenesis and metastasis .


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 physiological functions.

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.

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.

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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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