Visualization of mobility by atomic force microscopy

Intrinsically disordered regions (IDRs) of proteins are very thin and hence hard to be visualized by electron microscopy. Thus far, only high-speed atomic force microscopy (HS-AFM) can visualize them. The molecular movies identify the alignment of IDRs and ordered regions in an intrinsically disordered protein (IDP) and show undulation motion of the IDRs. The visualized tail-like structures contain the information of mechanical properties of the IDRs. Here, we describe methods of HS-AFM visualization of IDPs and methods of analyzing the obtained images to characterize IDRs. © 2012 Springer Science+Business Media New York

Modeling of DNA binding to the condensin hinge domain using molecular dynamics simulations guided by atomic force microscopy

The condensin protein complex compacts chromatin during mitosis using its DNA-loop extrusion activity. Previous studies proposed scrunching and loop-capture models as molecular mechanisms for the loop extrusion process, both of which assume the binding of double-strand (ds) DNA to the hinge domain formed at the interface of the condensin subunits Smc2 and Smc4. However, how the hinge domain contacts dsDNA has remained unknown. Here, we conducted atomic force microscopy imaging of the budding yeast condensin holo-complex and used this data as basis for coarse-grained molecular dynamics simulations to model the hinge structure in a transient open conformation. We then simulated the dsDNA binding to open and closed hinge conformations, predicting that dsDNA binds to the outside surface when closed and to the outside and inside surfaces when open. Our simulations also suggested that the hinge can close around dsDNA bound to the inside surface. Based on these simulation results, we speculate that the conformational change of the hinge domain might be essential for the dsDNA binding regulation and play roles in condensin-mediated DNA-loop extrusion

Video-rate high-speed atomic force microscopy for biological sciences

金沢大学理工研究域数物科学系The atomic force microscope (AFM) is unique in its capability to capture high-resolution images of biological samples in liquids. This capability becomes more valuable to biological sciences if AFM additionally acquires an ability of high-speed imaging. "Direct and real-time visualization" is a straightforward and powerful means of understanding biomolecular processes. With conventional AFM, it takes more than a minute to capture an image, while biomolecular processes generally occur on a millisecond timescale. In order to fill this large gap,various efforts have been carried out in the past decade. Here, we review these past efforts, describe the current state of the capability and limitations of our high-speed AFM, and discuss possibilities that may break the limitations, leading to an innovative high-speed bioAFM

High-speed atomic force microscopy

The technology of high-speed atomic force microscopy (HS-AFM) has reached maturity. HS-AFM enables us to directly visualize the structure and dynamics of biological molecules in physiological solutions at subsecond to sub-100 ms temporal resolution. By this microscopy, dynamically acting molecules such as myosin V walking on an actin filament and bacteriorhodopsin in response to light are successfully visualized. Highresolution molecular movies reveal the dynamic behavior of molecules in action in great detail. Inferences no longer have to be made from static snapshots of molecular structures and from the dynamic behavior of optical markers attached to biomolecules. In this review, we first describe theoretical considerations for the highest possible imaging rate, then summarize techniques involved in HS-AFM and highlight recent imaging studies. Finally, we briefly discuss future challenges to explore. © 2012 The Japan Society of Applied Physics

Active damping of the scanner for high-speed atomic force microscopy

金沢大学大学院自然科学研究科物理学金沢大学理学部The scanner that moves the sample stage in three dimensions is a crucial device that limits the imaging rate of atomic force microscopy. This limitation derives mainly from the resonant vibrations of the scanner in the z direction (the most frequent scanning direction). Resonance originates in the scanner\u27s mechanical structure as well as in the z piezoactuator itself. We previously demonstrated that the resonance originating in the structure can be minimized by a counterbalancing method. Here we report that the latter resonance from the actuator can be eliminated by an active damping method, with the result the bandwidth of the z scanner nearly reaches the first resonant frequency (150 kHz) of the z piezoactuator. © 2005 American Institute of Physics

高速原子間力顕微鏡の開発

取得学位：博士(理学)，学位授与番号：博甲第754号，学位授与年月日：平成17年9月30日,学位授与年：200

Video imaging of walking myosin V by high-speed atomic force microscopy

金沢大学理工研究域数物科学系The dynamic behaviour of myosin V molecules translocating along actin filaments has been mainly studied by optical microscopy. The processive hand-over-hand movement coupled with hydrolysis of adenosine triphosphate was thereby demonstrated. However, the protein molecules themselves are invisible in the observations and have therefore been visualized by electron microscopy in the stationary states. The concomitant assessment of structure and dynamics has been unfeasible, a situation prevailing throughout biological research. Here we directly visualize myosin V molecules walking along actin tracks, using high-speed atomic force microscopy. The high-resolution movies not only provide corroborative \u27visual evidence\u27 for previously speculated or demonstrated molecular behaviours, including lever-arm swing, but also reveal more detailed behaviours of the molecules, leading to a comprehensive understanding of the motor mechanism. Our direct and dynamic high-resolution visualization is a powerful new approach to studying the structure and dynamics of biomolecules in action

High-resolution imaging of myosin motor in action by a high-speed atomic force microscope

金沢大学理学部The atomic force microscope (AFM) is a powerful tool for imaging biological molecules on a substrate, in solution. However, there is no effective time axis with AFM; commercially available AFMs require minutes to capture an image, but many interesting biological processes occur at much higher rate. Hence, what we can observe using the AFM is limited to stationary molecules, or those moving very slowly. We sought to increase markedly the scan speed of the AFM, so that in the future it can be used to study the dynamic behaviour of biomolecules. For this purpose, we have developed various devices optimised for high-speed scanning. Combining these devices has produced an AFM that can capture a 100 x 100 pixel image within 80 ms, thus generating a movie consisting of many successive images of a sample in aqueous solution. This is demonstrated by imaging myosin V molecules moving on mica, in solution

High resonance frequency force microscope scanner using inertia balance support.

金沢大学 理工研究域 数物科学系We have developed the atomic force microscope scanner with the high resonance frequency of 540 kHz in the z axis using a piezosupport mechanism "inertia balance support." In the method, a cubic piezoactuator is supported at the four sides perpendicular to the extension axis, by which the resonance frequency of the scanner remains as high as that of the actuator in the free vibration. The scanner allows driving at low voltage ±15 V for the practical z scan range of 330 nm. We demonstrate the applicability of the scanner to the true-atomic-resolution imaging of mica in liquid. © 2008 American Institute of Physics