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| 1.
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Watanabe, Shinji ; Ando, Toshio ; 渡邉, 信嗣 ; 安藤, 敏夫
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金沢大学理工研究域バイオＡＦＭ先端研究センター<br />We describe a tip-scan-type high-speed XYZ-nanopositioner designed for scanning ion conduct
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ance microscopy (SICM), with a special care being devoted to the way of nanopipette holding. The nanopipette probe is mounted in the center of a hollow piezoactuator, both ends of which are attached to identical diaphragm flexures, for Z-positioning. This design minimizes the generation of undesirable mechanical vibrations. Mechanical amplification is used to increase the XY-travel range of the nanopositioner. The first resonance frequencies of the nanopositioner are measured as ∼100 kHz and ∼2.3 kHz for the Z- and XY-displacements, respectively. The travel ranges are ∼6 μm and ∼34 μm for Z and XY, respectively. When this nanopositioner is used for hopping mode imaging of SICM with a ∼10-nm radius tip, the vertical tip velocity can be increased to 400 nm/ms; hence, the one-pixel acquisition time can be minimized to ∼1 ms. © 2017 Author(s).<br />Embargo Period 12 months
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2. Real-space and real-Time dynamics of CRISPR-Cas9 visualized by high-speed atomic force microscopy
Shibata, Mikihiro ; Nishimasu, Hiroshi ; Kodera, Noriyuki ; Hirano, Seiichi ; Ando, Toshio ; Uchihashi, Takayuki ; Nureki, Osamu ; 柴田, 幹大 ; 古寺, 哲幸 ; 安藤, 敏夫 ; 内橋, 貴之
概要:
金沢大学新学術創成研究機構<br />The CRISPR-Associated endonuclease Cas9 binds to a guide RNA and cleaves double-stranded DNA with a s
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equence complementary to the RNA guide. The Cas9-RNA system has been harnessed for numerous applications, such as genome editing. Here we use high-speed atomic force microscopy (HS-AFM) to visualize the real-space and real-Time dynamics of CRISPR-Cas9 in action. HS-AFM movies indicate that, whereas apo-Cas9 adopts unexpected flexible conformations, Cas9-RNA forms a stable bilobed structure and interrogates target sites on the DNA by three-dimensional diffusion. These movies also provide real-Time visualization of the Cas9-mediated DNA cleavage process. Notably, the Cas9 HNH nuclease domain fluctuates upon DNA binding, and subsequently adopts an active conformation, where the HNH active site is docked at the cleavage site in the target DNA. Collectively, our HS-AFM data extend our understanding of the action mechanism of CRISPR-Cas9. © 2017 The Author(s).<br />出版社版
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Ando, Toshio ; 安藤, 敏夫
概要:
金沢大学理工研究域バイオＡＦＭ先端研究センター<br />Proteins are dynamic in nature and work at the single molecule level. Therefore, directly w
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atching protein molecules in dynamic action at high spatiotemporal resolution must be the most straightforward approach to understanding how they function. To make this observation possible, high-speed atomic force microscopy (HS-AFM) has been developed. Its current performance allows us to film biological molecules at 10–16 frames/s, without disturbing their function. In fact, dynamic structures and processes of various proteins have been successfully visualized, including bacteriorhodopsin responding to light, myosin V walking on actin filaments, and even intrinsically disordered proteins undergoing order/disorder transitions. The molecular movies have provided insights that could not have been reached in other ways. Moreover, the cantilever tip can be used to manipulate molecules during successive imaging. This capability allows us to observe changes in molecules resulting from dissection or perturbation. This mode of imaging has been successfully applied to myosin V, peroxiredoxin and doublet microtubules, leading to new discoveries. Since HS-AFM can be combined with other techniques, such as super-resolution optical microscopy and optical tweezers, the usefulness of HS-AFM will be further expanded in the near future. © 2017, International Union for Pure and Applied Biophysics (IUPAB) and Springer-Verlag GmbH Germany.<br />Embargo Period 12 months
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Uchihashi, Takayuki ; Kodera, Noriyuki ; Ando, Toshio ; 内橋, 貴之 ; 古寺, 哲幸 ; 安藤, 敏夫
概要:
金沢大学理工研究域バイオＡＦＭ先端研究センター<br />The dynamics of proteins, such as their conformational change and dynamic interplay with in
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teraction partners, is crucial to their biological functions. To reveal the dynamic behavior of proteins, single-molecule approaches are indispensable because the molecules behave in an unsynchronized manner, which makes it difficult to monitor their dynamics by ensemble average experiments. Fluorescence microscopy has been widely used to study the functional behavior of single-protein molecules (Peterman et al. 2004; Joo et al. 2008; Roy et al. 2008; Yanagida and Ishii 2008). However, it only visualizes featureless fluorescent spots, meaning that protein molecules themselves are invisible. Moreover, what we can analyze using fluorescence techniques is limited to the behavior of a selected portion where a fluorophore is attached. Therefore, inferences have to be made to bridge the gap between the observed behavior of fluorescent spots and the actual behavior of labeled protein molecules. Consequently, it takes a considerably long time to reach a persuasive conclusion on how a protein dynamically behaves to function. © Springer Science+Business Media New York 2013.<br />Embargo Period 12 months<br />Book Chapter
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Fukuma, Takeshi ; Okazaki, Yasutaka ; Kodera, Noriyuki ; Uchihashi, Takayuki ; Ando, Toshio
概要:
金沢大学 理工研究域 数物科学系<br />We have developed the atomic force microscope scanner with the high resonance frequency of 540 kHz
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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.
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Fukuma, Takeshi ; Okazaki, Yasutaka ; Kodera, Noriyuki ; Uchihashi, Takayuki ; Ando, Toshio
概要:
金沢大学フロンティアサイエンス機構<br />金沢大学理工研究域数物科学系<br />We have developed the atomic force microscope scanner with the high resonance
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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.
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Kodera, Noriyuki ; Yamashita, Hayato ; Ando, Toshio
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金沢大学大学院自然科学研究科物理学<br />金沢大学理学部<br />The scanner that moves the sample stage in three dimensions is a crucial device that
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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's 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.
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Kodera, Noriyuki ; Kinoshita, Tatsuya ; Ito, Takahiro ; Ando, Toshio
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金沢大学理学部<br />The atomic force microscope (AFM) is a powerful tool for imaging biological molecules on a substrate, in so
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lution. 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.
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Amitani, Ichiro ; Sakamoto, Takeshi ; Ando, Toshio
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金沢大学大学院自然科学研究科物理学<br />金沢大学理学部<br />We have attempted to link the solution actomyosin ATPase with the mechanical propert
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ies of in vitro actin filament sliding over heavy meromyosin. To accomplish this we perturbed the system by altering the substrate with various NTPs and divalent cations, and by altering ionic strength. A wide variety of enzymatic and mechanical measurements were made under very similar solution conditions. Excellent correlations between the mechanical and enzymatic quantities were revealed. Analysis of these correlations based on a force-balance model led us to two fundamental equations, which can be described approximately as follows: the maximum sliding velocity is proportional to √VmaxKm/A, where Km/A is the actin concentration at which the substrate turnover rate is half of its maximum (Vmax). The active force generated by a cross-bridge under no external load or under a small external load is proportional to √Vmax/Km/A. The equations successfully accounted for the correlations observed in the present study and observations in other laboratories.
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Milhiet, Pierre-Emmanuel ; Yamamoto, Daisuke ; Berthoumieu, Olivia ; Dosset, Patrice ; Grimellec, Christian Le ; Verdier, Jean-Michel ; Marchal, Stéphane ; Ando, Toshio
概要:
金沢大学理工研究域数物科学系<br />Formation of fibrillar structures of proteins that deposit into aggregates has been suggested to pla
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y a key role in various neurodegenerative diseases. However mechanisms and dynamics of fibrillization remains to be elucidated. We have previously established that lithostathine, a protein overexpressed in the pre-clinical stages of Alzheimer's disease and present in the pathognomonic lesions associated with this disease, form fibrillar aggregates after its N-terminal truncation. In this paper we visualized, using high-speed atomic force microscopy (HS-AFM), growth and assembly of lithostathine protofibrils under physiological conditions with a time resolution of one image/s. Real-time imaging highlighted a very high velocity of elongation. Formation of fibrils via protofibril lateral association and stacking was also monitored revealing a zipper-like mechanism of association. We also demonstrate that, like other amyloid ß peptides, two lithostathine protofibrils can associate to form helical fibrils. Another striking finding is the propensity of the end of a growing protofibril or fibril to associate with the edge of a second fibril, forming false branching point. Taken together this study provides new clues about fibrillization mechanism of amyloid proteins. © 2010 Milhiet et al.
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