High speed atomic force microscopy: In-situ and real-time visualization of biomolecular dynamics of myosin
High speed AFM imaging of myosin V yielded unprecedented insights into the dynamics of two-headed molecular cargo transporter in cells.
The high-speed atomic force microscopy (HS-AFM) technology at Kanazawa University exhibits the highest performance in the world with a frame rate of 30 ms/frame, a tapping force of ~30 pN, and spatial resolution of 1 nm.
Noriyuki Kodera has been one of the key players in the development of the HS-AFM and was awarded the prestigious 14th Japan Society for the Promotion of Science (JSPS) Prize for his contributions to AFM imaging . “My expertise is in visualizing biological samples with the HS-AFM system developed by Professor Toshio Ando of Kanazawa University,” says Kodera. “I have been working with Professor Ando since my Bachelor’s degree. The research on visualizing myosin was extremely challenging, taking many years of perseverance before we succeeded in obtaining images in 2010. Our results were published in Nature”. The images showed that myosin-V has two heads with actin binding and ATP hydrolysis sites, each with long neck domains, and a coiled-coil domain followed by a globular cargo binding domain .
The Kanazawa HS-AFM is a tapping mode system that is now commercially available and used worldwide for measuring biological samples. Importantly, a special feedback operation maintains a constant interactive force between the cantilever tip and sample surface. Furthermore, the short 7 micrometer long cantilevers of the HS-AFM (compared with approximately 200 micrometers in conventional AFMs) enables much faster and more accurate scans (Fig. 1).
“The high-speed AFM enabled us to perform real time imaging of functioning biomolecules at nano-meter and sub-second spatiotemporal resolution, as shown in movies posted on our website,” says Kodera. “The performance of our high-speed AFM is the best in the world for studying the structural dynamics of biomolecules. In addition to myosin V walking along actin filaments we have also directly visualized bacteriorhodopsin in response to light illumination, rotary motion by rotorless F1-ATPase, and more recently of real-time dynamics of CRISPR-Cas9 for genome editing reported by Mikihiro Shibata.” 
Kodera and his colleagues have also demonstrated high-speed AFM visualization of the thin and flexible structure of “intrinsically disordered proteins”. “This means high-speed AFM can visualize a moving single polypeptide chain,” explains Kodera. “These direct observations yield both visual insights into the structural dynamics of functioning proteins and importantly, shed light on their functional mechanisms.”
Fig. 1: Cantilever and tip of HS-AFM system at Kanazawa University
Noriyuki Kodera put HS-AFM on the world stage with the publication of the first direct visualization of myosin V molecules translocating along actin filaments. This report gave direct visual confirmation of molecular dynamics such as lever-arm swing, foot stomping, unfolding of the coiled-coil tail, and so on. This paper opened up many new interdisciplinary areas of research based on HS-AFM imaging of ‘functional biomolecules’.
Myosin V walking along actin filament
- N. Kodera, D. Yamamoto, R. Ishikawa & T. Ando, “Video imaging of walking myosin V by high-speed atomic force microscopy”, Nature 468, 72–76, (2010).
- M. Shibata, H. Nishimasu, N. Kodera, S. Hirano, T. Ando, T. Uchihashi & O. Nureki, “Real-space and real-time dynamics of CRISPR-Cas9 visualized by high-speed atomic force microscopy”, Nature Communications 8, 1430, (2017).
http://biophys.w3.kanazawa-u.ac.jp/ (Lab. Website)