Updated on 2024/02/11

写真a

 
UCHIHASHI Takayuki
 
Organization
Graduate School of Science Professor
Graduate School
Graduate School of Science
Undergraduate School
School of Science Department of Physics
Title
Professor
Profile
1989 - 1993 B.Sc. Department of Physics, Hiroshima University (Supervisor: Prof. Seizo Morita)
1993 - 1995 M.Sc. Department of Physics, Hiroshima University (Supervisor: Prof. Seizo Morita)
1995 - 1997 PhD course, Department of Physics, Hiroshima University (Supervisor: Prof. Seizo Morita)
1997 - 1998 PhD. Department of Electronics, Osaka University (Supervisor: Prof. Seizo Morita)
1998 - 2000 Postdoctoral Research Fellow, Joint Research Center for Atom Technology, Tsukuba, Japan
2000 - 2002 Assistant Professor, Department of Electronic Engineering, Himeji Institute of Technology, Japan
2002 - 2004 Senior Researcher, SFI Nanoscience Laboratory, Trinity College, Dublin, Ireland
2004 - 2006 Assistant Professor, Department of Physics, Kanazawa University, Japan
2006 - 2015 Associate Professor, College of Science and Engineering, Kanazawa University, Japan
2015 - 2017 Professor, College of Science and Engineering, Kanazawa University, Japan
Director, Bio-AFM Frontier Research Center, Kanazawa University, Japan
2016-2017 Visiting Professor, Institute of Advance Energy, Kyoto University
2017 - Professor, Division of Material Science (Phyics), Graduate School of Science, Nagoya University, Nagoya University
2018 - 2019 Visiting Professor, Department of Life and Coordination-Complex Molecular Science, Institute for Molecular Science
2018 - Visiting Professor, Biomolecular Dynamics Observation Group (Collaborative Research Group), Exploratory Research Center on Life and Living Systems (ExCELLS)
External link

Degree 1

  1. 博士(工学) ( 1998.3   大阪大学 ) 

Research Interests 11

  1. Single-Molecule Imaging

  2. Protein

  3. Biophysics

  4. Scanning Probe Microscopy

  5. High-Speed Atomic Force Microscopy

  6. Atomic Force Microscopy

  7. Scanning Probe Microscopy

  8. Surface Science

  9. Biophysics

  10. Single-molecule measurement

  11. High-speed atomic force microscopy

Research Areas 3

  1. Others / Others  / Nanomaterials/Nanobioscience

  2. Life Science / Biophysics

  3. Life Science / Biophysics

Research History 14

  1. Institute for Glyco-core Research (iGCORE), Tokai National Higher Education and Research System

    2021

  2. Nagoya University   Division of Material Science(Physics), Graduate School of Science   Professor

    2017.4

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    Country:Japan

  3. Nagoya University   Division of Material Science(Physics), Graduate School of Science   Professor

    2017.4

  4. Nagoya University   Graduate School of Science, School of Science,   Professor

    2017.4

  5. Kanazawa University   Institute of Science and Engineering   Professor

    2015.4 - 2017.3

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    Country:Japan

  6. Kanazawa University   Bio-AFM Frontier Research Center, Institute of Science and Engineering   Director

    2015.4 - 2017.3

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    Country:Japan

  7. Kanazawa University   Institute of Science and Engineering   Professor

    2015.4 - 2017.3

  8. Kanazawa University   Bio-AFM Frontier Research Center, Institute of Science and Engineering   Director

    2015.4 - 2017.3

  9. Kanazawa University   Institute of Science and Engineering   Associate professor

    2008.4 - 2015.3

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    Country:Japan

  10. Kanazawa University   Graduate School of Natural Science & Technology   Associate Professor

    2006.4 - 2008.3

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    Country:Japan

  11. Kanazawa University   Graduate School of Natural Science & Technology   Associate Professor

    2004.4 - 2006.3

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    Country:Japan

  12. Trinity College, Dublin   SFI Nanoscience Institute   Senior Researcher

    2002.8 - 2004.3

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    Country:Japan

  13. Himeji Institute of Technology   Department of Electronic Engineering   Associate Professor

    2000.4 - 2002.7

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    Country:Japan

  14. Joint Research Center for Atom Technology   Postdoctoral Reseacher

    1998.4 - 2000.3

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    Country:Japan

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Education 5

  1. Osaka University   Graduate School, Division of Engineering

    - 1998

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    Country: Japan

  2. Osaka University   Graduate School, Division of Engineering

    - 1998

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    Country: Japan

  3. Hiroshima University   Graduate School, Division of Natural Science

    - 1995

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    Country: Japan

  4. Hiroshima University   Graduate School, Division of Natural Science

    - 1995

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    Country: Japan

  5. Hiroshima University   Faculty of Science

    - 1993

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    Country: Japan

Professional Memberships 11

  1. THE JAPAN SOCIETY OF APPLIED PHYSICS

  2. 日本生物物理学会

  3. The Japan Society of Vacuum and Surface Science

  4. 日本化学会

  5. 日本物理学会

  6. THE MOLECULAR BIOLOGY SOCIETY OF JAPAN

  7. Japanese Applied Physics Society

  8. Japanese Biophysical Society

  9. Japanese Surface Science Society

  10. THE CHEMICAL SOCIETY OF JAPAN

  11. THE PHYSICAL SOCIETY OF JAPAN

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Committee Memberships 5

  1. 日本生物物理学会   理事  

    2015   

  2. 日本生物物理学会 学会誌「生物物理」編集委員会   委員  

    2014 - 2015   

  3. 日本生物物理学会    代議員  

    2014 - 2015   

  4. 日本生物物理学会 分野別専門委員会   委員  

    2011   

  5. 日本生物物理学会 分野別専門委員会   委員  

    2011   

Awards 5

  1. IEEE 3M Nano 2019, Best Conference Paper Award

    2019.8   IEEE 3M Nano  

    Takayuki Uchihashi

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    Award type:Award from international society, conference, symposium, etc.  Country:China

  2. 平成25年度 科学技術分野の文部科学大臣表彰 科学技術賞(開発部門)

    2013.4   文部科学省   高速原子間力顕微鏡の開発

    安藤敏夫, 内橋貴之, 古寺哲幸

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    Country:Japan

  3. 平成21年度 ナノプローブテクノロジー賞

    2009.8   日本学術振興会ナノプローブテクノロジー第167委員会   液中AFMの高速化とタンパク質の動的撮影による研究

    内橋貴之

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    Country:Japan

  4. 第29回応用物理学会論文賞

    2007.9   日本応用物理学会   High-Speed Atomic Force Microscopy for Studying the Dynamic Behavior of Protein Molecules at Work

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    Country:Japan

  5. 2009 Nanoprobe Technology Award

    JSPS 167th Comittiee on Nano-Probe Technology   Improvement of High-Speed AFM and Its Application to Capturing Protein Dynamics

    Takayuki Uchihashi

 

Papers 187

  1. Product inhibition slow down the moving velocity of processive chitinase and sliding-intermediate state blocks re-binding of product

    Tanaka, Y; Uchihashi, T; Nakamura, A

    ARCHIVES OF BIOCHEMISTRY AND BIOPHYSICS   Vol. 752   page: 109854   2024.2

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    Processive movement is the key reaction for crystalline polymer degradation by enzyme. Product release is an important phenomenon in resetting the moving cycle, but how it affects chitinase kinetics was unknown. Therefore, we investigated the effect of diacetyl chitobiose (C2) on the biochemical activity and movement of chitinase A from Serratia marcescens (SmChiA). The apparent inhibition constant of C2 on crystalline chitin degradation of SmChiA was 159 μM. The binding position of C2 obtained by X-ray crystallography was at subsite +1, +2 and Trp275 interact with C2 at subsite +1. This binding state is consistent with the competitive inhibition obtained by biochemical analysis. The apparent inhibition constant of C2 on the moving velocity of high-speed (HS) AFM observations was 330 μM, which is close to the biochemical results, indicating that the main factor in crystalline chitin degradation is also the decrease in degradation activity due to inhibition of processive movement. The Trp275 is a key residue for making a sliding intermediate complex. SmChiA W275A showed weaker activity and affinity than WT against crystalline chitin because it is less processive than WT. In addition, biochemical apparent inhibition constant for C2 of SmChiA W275A was 45.6 μM. W275A mutant showed stronger C2 inhibition than WT even though the C2 binding affinity is weaker than WT. This result indicated that Trp275 is important for the interaction at subsite +1, but also important for making sliding intermediate complex and physically block the rebinding of C2 on the catalytic site for crystalline chitin degradation.

    DOI: 10.1016/j.abb.2023.109854

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  2. Quantitative Analysis of Therapeutic Antibody Interactions with Fcγ Receptors Using High-Speed Atomic Force Microscopy

    Yanaka Saeko, Watanabe Hiroki, Yogo Rina, Kongsema Mesayamas, Kondo Sachiko, Yagi Hirokazu, Uchihashi Takayuki, Kato Koichi

    Biological and Pharmaceutical Bulletin   Vol. 47 ( 1 ) page: 334 - 338   2024.1

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    Language:English   Publisher:The Pharmaceutical Society of Japan  

    <p>This study employed high-speed atomic force microscopy to quantitatively analyze the interactions between therapeutic antibodies and Fcγ receptors (FcγRs). Antibodies are essential components of the immune system and are integral to biopharmaceuticals. The focus of this study was on immunoglobulin G molecules, which are crucial for antigen binding <i>via</i> the Fab segments and cytotoxic functions through their Fc portions. We conducted real-time, label-free observations of the interactions of rituximab and mogamulizumab with the recombinant FcγRIIIa and FcγRIIa. The dwell times of FcγR binding were measured at the single-molecule level, which revealed an extended interaction duration of mogamulizumab with FcγRIIIa compared with that of rituximab. This is linked to enhanced antibody-dependent cellular cytotoxicity that is attributed to the absence of the core fucosylation of Fc-linked <i>N</i>-glycan. This study also emphasizes the crucial role of the Fab segments in the interaction with FcγRIIa as well as that with FcγRIIIa. This approach provided quantitative insight into therapeutic antibody interactions and exemplified kinetic proofreading, where cellular discrimination relies on ligand residence times. Observing the dwell times of antibodies on the effector molecules has emerged as a robust indicator of therapeutic antibody efficacy. Ultimately, these findings pave the way for the development of refined therapeutic antibodies with tailored interactions with specific FcγRs. This research contributes to the advancement of biopharmaceutical antibody design and optimizing antibody-based treatments for enhanced efficacy and precision.</p>

    DOI: 10.1248/bpb.b23-00751

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  3. The two‐step cargo recognition mechanism of heterotrimeric kinesin

    Xuguang Jiang, Tadayuki Ogawa, Kento Yonezawa, Nobutaka Shimizu, Sotaro Ichinose, Takayuki Uchihashi, Wataru Nagaike, Toshio Moriya, Naruhiko Adachi, Masato Kawasaki, Naoshi Dohmae, Toshiya Senda, Nobutaka Hirokawa

    EMBO reports   Vol. 24 ( 11 ) page: e56864   2023.11

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    Abstract

    Kinesin‐driven intracellular transport is essential for various cell biological events and thus plays a crucial role in many pathological processes. However, little is known about the molecular basis of the specific and dynamic cargo‐binding mechanism of kinesins. Here, an integrated structural analysis of the KIF3/KAP3 and KIF3/KAP3‐APC complexes unveils the mechanism by which KIF3/KAP3 can dynamically grasp APC in a two‐step manner, which suggests kinesin‐cargo recognition dynamics composed of cargo loading, locking, and release. Our finding is the first demonstration of the two‐step cargo recognition and stabilization mechanism of kinesins, which provides novel insights into the intracellular trafficking machinery.

    DOI: 10.15252/embr.202356864

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  4. Measuring mechanical properties with high-speed atomic force microscopy

    Ganser, C; Uchihashi, T

    MICROSCOPY   Vol. 73 ( 1 ) page: 14 - 21   2023.10

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  5. Elastic Polymer Coated Nanoparticles with Fast Clearance for <sup>19</sup>F MR Imaging

    Konishi Y., Minoshima M., Fujihara K., Uchihashi T., Kikuchi K.

    Angewandte Chemie - International Edition   Vol. 62 ( 40 ) page: e202308565   2023.10

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    19F magnetic resonance imaging (MRI) is a powerful molecular imaging technique that enables high-resolution imaging of deep tissues without background signal interference. However, the use of nanoparticles (NPs) as 19F MRI probes has been limited by the immediate trapping and accumulation of stiff NPs, typically of around 100 nm in size, in the mononuclear phagocyte system, particularly in the liver. To address this issue, elastic nanomaterials have emerged as promising candidates for improving delivery efficacy in vivo. Nevertheless, the impact of elasticity on NP elimination has remained unclear due to the lack of suitable probes for real-time and long-term monitoring. In this study, we present the development of perfluorocarbon-encapsulated polymer NPs as a novel 19F MRI contrast agent, with the aim of suppressing long-term accumulation. The polymer NPs have high elasticity and exhibit robust sensitivity in 19F MRI imaging. Importantly, our 19F MRI data demonstrate a gradual decline in the signal intensity of the polymer NPs after administration, which contrasts starkly with the behavior observed for stiff silica NPs. This innovative polymer-coated NP system represents a groundbreaking nanomaterial that successfully overcomes the challenges associated with long-term accumulation, while enabling tracking of biodistribution over extended periods.

    DOI: 10.1002/anie.202308565

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  6. Multiple roles of a conserved glutamate residue for unique biophysical properties in a new group of microbial rhodopsins homologous to TAT rhodopsin

    Kentaro Mannen, Takashi Nagata, Andrey Rozenberg, Masae Konno, María del Carmen Marín, Reza Bagherzadeh, Oded Béjà, Takayuki Uchihashi, Keiichi Inoue

    Journal of Molecular Biology     page: 168331 - 168331   2023.10

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    Language:English   Publishing type:Research paper (scientific journal)   Publisher:Elsevier BV  

    DOI: 10.1016/j.jmb.2023.168331

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  7. Structure of the human ATAD2 AAA+ histone chaperone reveals mechanism of regulation and inter-subunit communication. International journal

    Carol Cho, Christian Ganser, Takayuki Uchihashi, Koichi Kato, Ji-Joon Song

    Communications biology   Vol. 6 ( 1 ) page: 993 - 993   2023.9

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    ATAD2 is a non-canonical ATP-dependent histone chaperone and a major cancer target. Despite widespread efforts to design drugs targeting the ATAD2 bromodomain, little is known about the overall structural organization and regulation of ATAD2. Here, we present the 3.1 Å cryo-EM structure of human ATAD2 in the ATP state, showing a shallow hexameric spiral that binds a peptide substrate at the central pore. The spiral conformation is locked by an N-terminal linker domain (LD) that wedges between the seam subunits, thus limiting ATP-dependent symmetry breaking of the AAA+ ring. In contrast, structures of the ATAD2-histone H3/H4 complex show the LD undocked from the seam, suggesting that H3/H4 binding unlocks the AAA+ spiral by allosterically releasing the LD. These findings, together with the discovery of an inter-subunit signaling mechanism, reveal a unique regulatory mechanism for ATAD2 and lay the foundation for developing new ATAD2 inhibitors.

    DOI: 10.1038/s42003-023-05373-1

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  8. Visualizing the membrane disruption action of antimicrobial peptides by cryo-electron tomography. International journal

    Eric H-L Chen, Chun-Hsiung Wang, Yi-Ting Liao, Feng-Yueh Chan, Yui Kanaoka, Takayuki Uchihashi, Koichi Kato, Longsheng Lai, Yi-Wei Chang, Meng-Chiao Ho, Rita P-Y Chen

    Nature communications   Vol. 14 ( 1 ) page: 5464 - 5464   2023.9

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    The abuse of antibiotics has led to the emergence of multidrug-resistant microbial pathogens, presenting a pressing challenge in global healthcare. Membrane-disrupting antimicrobial peptides (AMPs) combat so-called superbugs via mechanisms different than conventional antibiotics and have good application prospects in medicine, agriculture, and the food industry. However, the mechanism-of-action of AMPs has not been fully characterized at the cellular level due to a lack of high-resolution imaging technologies that can capture cellular-membrane disruption events in the hydrated state. Previously, we reported PepD2M, a de novo-designed AMP with potent and wide-spectrum bactericidal and fungicidal activity. In this study, we use cryo-electron tomography (cryo-ET) and high-speed atomic force microscopy (HS-AFM) to directly visualize the pepD2M-induced disruption of the outer and inner membranes of the Gram-negative bacterium Escherichia coli, and compared with a well-known pore-forming peptide, melittin. Our high-resolution cryo-ET images reveal how pepD2M disrupts the E. coli membrane using a carpet/detergent-like mechanism. Our studies reveal the direct membrane-disrupting consequence of AMPs on the bacterial membrane by cryo-ET, and this information provides critical insights into the mechanisms of this class of antimicrobial agents.

    DOI: 10.1038/s41467-023-41156-2

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  9. Molecular Design of FRET Probes Based on Domain Rearrangement of Protein Disulfide Isomerase for Monitoring Intracellular Redox Status

    Maho Yagi-Utsumi, Haruko Miura, Christian Ganser, Hiroki Watanabe, Methanee Hiranyakorn, Tadashi Satoh, Takayuki Uchihashi, Koichi Kato, Kei-ichi Okazaki, Kazuhiro Aoki

    International Journal of Molecular Sciences   Vol. 24 ( 16 ) page: 12865 - 12865   2023.8

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    Language:English   Publishing type:Research paper (scientific journal)   Publisher:MDPI AG  

    Multidomain proteins can exhibit sophisticated functions based on cooperative interactions and allosteric regulation through spatial rearrangements of the multiple domains. This study explored the potential of using multidomain proteins as a basis for Förster resonance energy transfer (FRET) biosensors, focusing on protein disulfide isomerase (PDI) as a representative example. PDI, a well-studied multidomain protein, undergoes redox-dependent conformational changes, enabling the exposure of a hydrophobic surface extending across the b’ and a’ domains that serves as the primary binding site for substrates. Taking advantage of the dynamic domain rearrangements of PDI, we developed FRET-based biosensors by fusing the b’ and a’ domains of thermophilic fungal PDI with fluorescent proteins as the FRET acceptor and donor, respectively. Both experimental and computational approaches were used to characterize FRET efficiency in different redox states. In vitro and in vivo evaluations demonstrated higher FRET efficiency of this biosensor in the oxidized form, reflecting the domain rearrangement and its responsiveness to intracellular redox environments. This novel approach of exploiting redox-dependent domain dynamics in multidomain proteins offers promising opportunities for designing innovative FRET-based biosensors with potential applications in studying cellular redox regulation and beyond.

    DOI: 10.3390/ijms241612865

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  10. Single microgel degradation governed by heterogeneous nanostructures

    Yuichiro Nishizawa, Hiroki Yokoi, Takayuki Uchihashi, Daisuke Suzuki

    Soft Matter   Vol. 19 ( 27 ) page: 5068 - 5075   2023.7

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    Language:English   Publishing type:Research paper (scientific journal)   Publisher:Royal Society of Chemistry (RSC)  

    The real-time visualization via high-speed atomic force microscopy revealed that single microgel exhibit heterogeneous degradation behavior.

    DOI: 10.1039/d3sm00216k

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  11. Multistep, site-selective noncovalent synthesis of two-dimensional block supramolecular polymers

    Norihiko Sasaki, Jun Kikkawa, Yoshiki Ishii, Takayuki Uchihashi, Hitomi Imamura, Masayuki Takeuchi, Kazunori Sugiyasu

    Nature Chemistry   Vol. 15 ( 7 ) page: 922 - +   2023.7

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    Language:English   Publishing type:Research paper (scientific journal)   Publisher:Springer Science and Business Media LLC  

    DOI: 10.1038/s41557-023-01216-y

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    Other Link: https://www.nature.com/articles/s41557-023-01216-y

  12. High Density of N- and O-Glycosylation Shields and Defines the Structural Dynamics of the Intrinsically Disordered Ectodomain of Receptor-type Protein Tyrosine Phosphatase Alpha

    Yu-Chun Chien, Yong-Sheng Wang, Deepa Sridharan, Chu-Wei Kuo, Chih-Ta Chien, Takayuki Uchihashi, Koichi Kato, Takashi Angata, Tzu-Ching Meng, Shang-Te Danny Hsu, Kay-Hooi Khoo

    JACS Au   Vol. 3 ( 7 ) page: 1864 - 1875   2023.6

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    Language:English   Publishing type:Research paper (scientific journal)   Publisher:American Chemical Society (ACS)  

    DOI: 10.1021/jacsau.3c00124

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  13. Closed-loop recycling of microparticle-based polymers

    Takumi Watanabe, Haruka Minato, Yuma Sasaki, Seina Hiroshige, Hayato Suzuki, Nahomi Matsuki, Koki Sano, Takeshi Wakiya, Yuichiro Nishizawa, Takayuki Uchihashi, Takuma Kureha, Mitsuhiro Shibayama, Toshikazu Takata, Daisuke Suzuki

    Green Chemistry   Vol. 25 ( 9 ) page: 3418 - 3424   2023.5

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    Publishing type:Research paper (scientific journal)   Publisher:Royal Society of Chemistry (RSC)  

    We propose a recycling strategy for tough polymers based on microparticles. The "microparticle-based" concept allows materials recycling without loss of their properties (‘closed-loop’ recycling).

    DOI: 10.1039/d3gc00090g

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  14. Antiparallel dimer structure of CELSR cadherin in solution revealed by high-speed atomic force microscopy

    Nishiguchi, S; Kasai, RS; Uchihashi, T

    PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA   Vol. 120 ( 18 ) page: e2302047120   2023.4

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    Language:English   Publisher:Proceedings of the National Academy of Sciences of the United States of America  

    Cadherin EGF LAG seven-pass G-type receptors (CELSR) cadherins, members of the cadherin superfamily, and adhesion G-protein-coupled receptors, play a vital role in cell–cell adhesion. The mutual binding of the extracellular domains (ectodomains) of CELSR cadherins between cells is crucial for tissue formation, including the establishment of planar cell polarity, which directs the proper patterning of cells. CELSR cadherins possess nine cadherin ectodomains (EC1–EC9) and noncadherin ectodomains. However, the structural and functional mechanisms of the binding mode of CELSR cadherins have not been determined. In this study, we investigated the binding mode of CELSR cadherins using single-molecule fluorescence microscopy, high-speed atomic force microscopy (HS-AFM), and bead aggregation assay. The fluorescence microscopy analysis results indicated that the trans-dimer of the CELSR cadherin constitutes the essential adhesive unit between cells. HS-AFM analysis and bead aggregation assay results demonstrated that EC1–EC8 entirely overlap and twist to form antiparallel dimer conformations and that the binding of EC1–EC4 is sufficient to sustain bead aggregation. The interaction mechanism of CELSR cadherin may elucidate the variation of the binding mechanism within the cadherin superfamily and physiological role of CELSR cadherins in relation to planar cell polarity.

    DOI: 10.1073/pnas.2302047120

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  15. Creation of single molecular conjugates of metal-organic cages and DNA. International journal

    Toshinobu Nakajo, Shinpei Kusaka, Haruka Hiraoka, Kohei Nomura, Noriaki Matsubara, Rintaro Baba, Yuki Yoshida, Kosuke Nakamoto, Masakazu Honma, Hiroaki Iguchi, Takayuki Uchihashi, Hiroshi Abe, Ryotaro Matsuda

    Chemical communications (Cambridge, England)   Vol. 59 ( 33 ) page: 4974 - 4977   2023.4

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    Here we report the development of an equimolar conjugate of a metal-organic cage (MOC) and DNA (MOC-DNA). Several MOC-DNA conjugates were assembled into a programmed structure by coordinating with a template DNA having a complementary base sequence. Moreover, conjugation with the MOC drastically enhanced the permeability of DNA through the lipid bilayer, presenting great potential as a drug delivery system.

    DOI: 10.1039/d3cc00460k

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  16. Dimerization Processes of Cell-cell Adhesion Molecule Cadherin Revealed by High-speed Atomic Force Microscopy Invited Reviewed

    Shigetaka Nishiguchi, Tadaomi Furuta, Takayuki Uchihashi

    Seibutu Butsuri   Vol. 63 ( 2 ) page: 104 - 106   2023.4

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    Language:Japanese   Publishing type:Research paper (scientific journal)   Publisher:The Biophysical Society of Japan General Incorporated Association  

    DOI: 10.2142/biophys.63.104

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  17. 3D Structure of Ring-shaped Microtubule Swarms Revealed by High-speed Atomic Force Microscopy

    Mst. Rubaya Rashid, Christian Ganser, Mousumi Akter, Syeda Rubaiya Nasrin, Arif Md. Rashedul Kabir, Kazuki Sada, Takayuki Uchihashi, Akira Kakugo

    Chemistry Letters   Vol. 52 ( 2 ) page: 100 - 104   2023.2

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    Language:English   Publishing type:Research paper (scientific journal)   Publisher:The Chemical Society of Japan  

    <p>Biomolecular motor, microtubule (MT) and kinesin based molecular swarm robots are important due to their applications in cooperative task achievements, while the structural details are still unknown. In this work, high-speed atomic force microscopy was used to observe the MT swarm ring structure at nanometer-level resolution. MTs were observed to pile up in multiple layers to form swarm rings. The number of MTs involved in force generation was estimated which will specifically impact the understanding of the force-generation capability of swarms.</p>

    DOI: 10.1246/cl.220491

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  18. Disulfide Bond-mediated Oligomerization of a Green Fluorescent Protein in Solution

    Julian Wong Soon, Koji Oohora, Takayuki Uchihashi, Takashi Hayashi

    Chemistry Letters   Vol. 52 ( 2 ) page: 105 - 109   2023.2

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    Language:English   Publishing type:Research paper (scientific journal)   Publisher:The Chemical Society of Japan  

    A green fluorescent protein (GFP) variant with two cysteine residues added to the protein surface was modified with 2,2′- dipyridyl disulfide and oligomerization of the modified protein and the unmodified variant via the disulfide bond was achieved in solution. The fluorescence anisotropy decay of the obtained GFP oligomer is capable of efficient homo-FRET.

    DOI: 10.1246/cl.220495

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  19. Biosynthesis of highly branched gold nanoparticles through structural engineering of fatty acids

    Yue Y., Yokota Y., Uchihashi T.

    iScience   Vol. 26 ( 1 ) page: 105864   2023.1

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    Biomimetic approaches have been used to develop inorganic nanomaterials with complex morphologies and functions. Fatty acids are among the most important and decomposable biomolecules in nature. However, the controlled synthesis of branched gold nanoparticles using these biomolecules has not been reported. Herein, we demonstrate a strategy to produce highly branched gold nanoparticles through structural engineering of fatty acids. Furthermore, we developed a method for tailoring fatty acid molecules by altering their aliphatic chains to facilitate the morphological evolution of gold nanoparticles from spherical to branched shape. It is found that the growth of the nanoparticles is sensitive to characteristics of fatty acids, such as saturation degrees. The growth of the nanoparticle is visualized by high-speed atomic force microscopy. The reaction mechanisms and growth processes of branched gold nanoparticles are proposed. This work may serve as a cornerstone to the design in a biomimetic fashion for the controllable synthesis of metallic nanomaterials.

    DOI: 10.1016/j.isci.2022.105864

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  20. Decoding of the ubiquitin code for clearance of colliding ribosomes by the RQT complex. International journal

    Yoshitaka Matsuo, Takayuki Uchihashi, Toshifumi Inada

    Nature communications   Vol. 14 ( 1 ) page: 79 - 79   2023.1

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    The collision sensor Hel2 specifically recognizes colliding ribosomes and ubiquitinates the ribosomal protein uS10, leading to noncanonical subunit dissociation by the ribosome-associated quality control trigger (RQT) complex. Although uS10 ubiquitination is essential for rescuing stalled ribosomes, its function and recognition steps are not fully understood. Here, we show that the RQT complex components Cue3 and Rqt4 interact with the K63-linked ubiquitin chain and accelerate the recruitment of the RQT complex to the ubiquitinated colliding ribosome. The CUE domain of Cue3 and the N-terminal domain of Rqt4 bind independently to the K63-linked ubiquitin chain. Their deletion abolishes ribosomal dissociation mediated by the RQT complex. High-speed atomic force microscopy (HS-AFM) reveals that the intrinsically disordered regions of Rqt4 enable the expansion of the searchable area for interaction with the ubiquitin chain. These findings provide mechanistic insight into the decoding of the ubiquitin code for clearance of colliding ribosomes by the RQT complex.

    DOI: 10.1038/s41467-022-35608-4

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  21. Introduction of Session 2, “Advanced methods for retinal proteins”

    Uchihashi Takayuki, Kandori Hideki

    Biophysics and Physicobiology   Vol. 20 ( Supplemental ) page: n/a   2023

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    Language:English   Publisher:The Biophysical Society of Japan  

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  22. Activity of Regional Branch

    Seibutsu Butsuri   Vol. 63 ( 5 ) page: 285 - 286   2023

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    DOI: 10.2142/biophys.63.285

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  23. Ring formation by Vibrio fusion protein composed of FliF and FliG, MS-ring and C-ring component of bacterial flagellar motor in membrane

    Kanji Takahashi, Tatsuro Nishikino, Hiroki Kajino, Seiji Kojima, Takayuki Uchihashi, Michio Homma

    BIOPHYSICS AND PHYSICOBIOLOGY   Vol. 20 ( 2 ) page: n/a   2023

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    The marine bacterium Vibrio alginolyticus has a single flagellum as a locomotory organ at the cell pole, which is rotated by the Na+-motive force to swim in a liquid. The base of the flagella has a motor composed of a stator and rotor, which serves as a power engine to generate torque through the rotor-stator interaction coupled to Na+ influx through the stator channel. The MS-ring, which is embedded in the membrane at the base of the flagella as part of the rotor, is the initial structure required for flagellum assembly. It comprises 34 molecules of the twotransmembrane protein FliF. FliG, FliM, and FliN form a C-ring just below the MS-ring. FliG is an important rotor protein that interacts with the stator PomA and directly contributes to force generation. We previously found that FliG promotes MS-ring formation in E. coli. In the present study, we constructed a fliF-fliG fusion gene, which encodes an approximately 100 kDa protein, and the successful production of this protein effectively formed the MS-ring in E. coli cells. We observed fuzzy structures around the ring using either electron microscopy or high-speed atomic force microscopy (HS-AFM), suggesting that FliM and FliN are necessary for the formation of a stable ring structure. The HS-AFM movies revealed flexible movements at the FliG region.

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  24. Clarification of Surface Deswelling of Thermoresponsive Microgels by Electrophoresis

    Yuichiro Nishizawa, Takumi Inui, Ryuji Namioka, Takayuki Uchihashi, Takumi Watanabe, Daisuke Suzuki

    Langmuir   Vol. 38 ( 51 ) page: 16084 - 16093   2022.12

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    DOI: 10.1021/acs.langmuir.2c02742

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  25. Lytic polysaccharide monooxygenase increases cellobiohydrolases activity by promoting decrystallization of cellulose surface

    Taku Uchiyama, Takayuki Uchihashi, Takuya Ishida, Akihiko Nakamura, Josh V. Vermaas, Michael F. Crowley, Masahiro Samejima, Gregg T. Beckham, Kiyohiko Igarashi

    Science Advances   Vol. 8 ( 51 ) page: eade5155   2022.12

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  26. Tip-scan high-speed atomic force microscopy with a uniaxial substrate stretching device for studying dynamics of biomolecules under mechanical stress

    Feng-Yueh Chan, Ryo Kurosaki, Christian Ganser, Tetsuya Takeda, Takayuki Uchihashi

    Review of Scientific Instruments   Vol. 93 ( 11 ) page: 113703 - 113703   2022.11

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    High-speed atomic force microscopy (HS-AFM) is a powerful tool for studying the dynamics of biomolecules in vitro because of its high temporal and spatial resolution. However, multi-functionalization, such as combination with complementary measurement methods, environment control, and large-scale mechanical manipulation of samples, is still a complex endeavor due to the inherent design and the compact sample scanning stage. Emerging tip-scan HS-AFM overcame this design hindrance and opened a door for additional functionalities. In this study, we designed a motor-driven stretching device to manipulate elastic substrates for HS-AFM imaging of biomolecules under controllable mechanical stimulation. To demonstrate the applicability of the substrate stretching device, we observed a microtubule buckling by straining the substrate and actin filaments linked by α-actinin on a curved surface. In addition, a BAR domain protein BIN1 that senses substrate curvature was observed while dynamically controlling the surface curvature. Our results clearly prove that large-scale mechanical manipulation can be coupled with nanometer-scale imaging to observe biophysical effects otherwise obscured.

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  27. Scanning Probe Microscopy

    Nakajima K., Sumitomo K., Nakayama T., Ken-Ichi Fukui , Kageshima M., Kawai S., Komeda T., Takahashi T., Uchihashi T.

    Japanese Journal of Applied Physics   Vol. 61 ( SL )   2022.9

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    DOI: 10.35848/1347-4065/ac7cc6

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  28. Mechanism of the Irreversible Transition from Pentamer to Monomer at pH 2 in a Blue Proteorhodopsin

    Sumikawa, M; Abe-Yoshizumi, R; Uchihashi, T; Kandori, H

    BIOCHEMISTRY   Vol. 61 ( 18 ) page: 1936 - 1944   2022.8

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    Proteorhodopsin (PR) is a light-driven proton pump found in marine bacteria, and thousands of PRs are classified as blue-absorbing PRs (BPR; λmax∼490 nm) and green-absorbing PRs (GPR; λmax∼525 nm). We previously converted BPR into GPR using an anomalous pH effect, which was achieved by an irreversible process at around pH 2. Recent size-exclusion chromatography (SEC) and atomic force microscopy (AFM) analyses of BPR from Vibrio califitulae (VcBPR) revealed the anomalous pH effect owing to the irreversible transition from pentamer to monomer. Different pKavalues of the Schiff base counterion between pentamer and monomer lead to different colors at the same pH. Here, we incorporate systematic mutation into VcBPR and examine the anomalous pH effect. The anomalous pH effect was observed for the mutants of key residues near the retinal chromophore such as D76N, D206N, and Q84L, indicating that the Schiff base counterions and the L/Q switch do not affect the irreversible transition from pentamer to monomer at pH ∼2. We then focus on the two specific interactions at the intermonomer interface in a pentamer, E29/R30/D31 and W13/H54. Single mutants such as E29Q, R30A, W13A, and H54A and the wild type (WT) exhibited an anomalous pH effect. In contrast, the anomalous pH effect was lost for E29Q/H54A, R30A/H54A, and W13A/E29Q. Size-exclusion chromatography (SEC) and atomic force microscopy (AFM) measurements showed monomer forms in the original states of the double mutants, being a clear contrast to the pentamer forms of all single mutants in the original states. It was concluded that the pentamer structure of VcBPR was stabilized by an electrostatic interaction in the E29/R30/D31 region and a hydrogen-bonding interaction in the W13/H54 region, which was disrupted at pH 2 and converted into monomers.

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  29. Multiple dimeric structures and strand-swap dimerization of E-cadherin in solution visualized by high-speed atomic force microscopy. International journal

    Shigetaka Nishiguchi, Tadaomi Furuta, Takayuki Uchihashi

    Proceedings of the National Academy of Sciences of the United States of America   Vol. 119 ( 30 ) page: e2208067119   2022.7

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    Classical cadherins play key roles in cell-cell adhesion. The adhesion process is thought to comprise mainly two steps: X-dimer and strand-swap (SS-) dimer formation of the extracellular domains (ectodomains) of cadherins. The dimerization mechanism of this two-step process has been investigated for type I cadherins, including E-cadherin, of classical cadherins, whereas other binding states also have been proposed, raising the possibility of additional binding processes required for the cadherin dimerization. However, technical limitations in observing single-molecule structures and their dynamics have precluded the investigation of the dynamic binding process of cadherin. Here, we used high-speed atomic force microscopy (HS-AFM) to observe full-length ectodomains of E-cadherin in solution and identified multiple dimeric structures that had not been reported previously. HS-AFM revealed that almost half of the cadherin dimers showed S- (or reverse S-) shaped conformations, which had more dynamic properties than the SS- and X-like dimers. The combined HS-AFM, mutational, and molecular modeling analyses showed that the S-shaped dimer was formed by membrane-distal ectodomains, while the binding interface was different from that of SS- and X-dimers. Furthermore, the formation of the SS-dimer from the S-shaped and X-like dimers was directly visualized, suggesting the processes of SS-dimer formation from S-shaped and X-dimers during cadherin dimerization.

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  30. Protein Needles Designed to Self‐Assemble through Needle Tip Engineering Reviewed

    Kosuke Kikuchi, Tatsuya Fukuyama, Takayuki Uchihashi, Tadaomi Furuta, Yusuke T. Maeda, Takafumi Ueno

    Small   Vol. 18 ( 10 ) page: 2106401 - 2106401   2022.3

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  31. Quantitative Visualization of the Interaction between Complement Component C1 and Immunoglobulin G: The Effect of C<sub>H</sub>1 Domain Deletion Reviewed International journal

    Yanaka, S; Nishiguchi, S; Yogo, R; Watanabe, H; Shen, JA; Yagi, H; Uchihashi, T; Kato, K

    INTERNATIONAL JOURNAL OF MOLECULAR SCIENCES   Vol. 23 ( 4 )   2022.2

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    Immunoglobulin G (IgG) adopts a modular multidomain structure that mediates antigen recognition and effector functions, such as complement-dependent cytotoxicity. IgG molecules are self-assembled into a hexameric ring on antigen-containing membranes, recruiting the complement component C1q. In order to provide deeper insights into the initial step of the complement pathway, we report a high-speed atomic force microscopy study for the quantitative visualization of the interaction between mouse IgG and the C1 complex composed of C1q, C1r, and C1s. The results showed that the C1q in the C1 complex is restricted regarding internal motion, and that it has a stronger binding affinity for on-membrane IgG2b assemblages than C1q alone, presumably because of the lower conformational entropy loss upon binding. Furthermore, we visualized a 1:1 stoichiometric interaction between C1/C1q and an IgG2a variant that lacks the entire CH 1 domain in the absence of an antigen. In addition to the canonical C1q-binding site on Fc, their interactions are mediated through a secondary site on the CL domain that is cryptic in the presence of the CH 1 domain. Our findings offer clues for novel-modality therapeutic antibodies.

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  32. Shape-selective one-step synthesis of branched gold nanoparticles on the crystal surface of redox-active Pd<SUP>II</SUP>-macrocycles Reviewed

    Yamashita, Y; Tashiro, S; Ishii, Y; Uchihashi, T; Matsushita, N; Kubota, R; Shionoya, M

    DALTON TRANSACTIONS   Vol. 51 ( 4 ) page: 1318 - 1324   2022.1

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    The synthesis of branched gold nanoparticles (AuNPs) with shape- and size-specific optical properties requires effective control of the particle formation mechanism using appropriate reducing agents and protective agents that prevent particle aggregation in solution. In this context, the heterogeneous synthesis of AuNPs using solid surfaces of graphene oxides and metal–organic frameworks has attracted much attention. These materials are characterized by their ability to immobilize and stabilize the particles grown on the surface without the need for additional protective agents. However, the shape- and size-selective synthesis of AuNPs using solid surfaces remains challenging. Herein, we report the shape-selective one-step synthesis of monodisperse branched AuNPs using a metal–macrocycle framework (MMF), a porous molecular crystal of PdII3-tris(phenylenediamine) macrocycle. Konpeito-Shaped branched AuNPs with uniform size were obtained on the surface of MMF by mixing HAuCl4·4H2O, l-ascorbic acid and MMF microcrystals. Spectroscopic and microscopic observations confirmed that MMF promoted the reduction of gold by its reductive activity as well as acted as a solid support to electrostatically immobilize the pseudo-seed particles for further growth on the crystal surface. In addition, the MMF also served as a substrate for in situ high-speed AFM imaging due to the effective immobilization of AuNPs on the surface, allowing direct visualization of the particle growth. Since the chemical structural features of MMF allow the growth of branched AuNPs via pseudo-seeding, this approach would provide new synthetic methods for obtaining a variety of gold nanostructures.

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  33. The Lipid-Binding Defective Dynamin 2 Mutant in Charcot-Marie-Tooth Disease Impairs Proper Actin Bundling and Actin Organization in Glomerular Podocytes. International journal

    Eriko Hamasaki, Natsuki Wakita, Hiroki Yasuoka, Hikaru Nagaoka, Masayuki Morita, Eizo Takashima, Takayuki Uchihashi, Tetsuya Takeda, Tadashi Abe, Ji-Won Lee, Tadahiro Iimura, Moin A Saleem, Naohisa Ogo, Akira Asai, Akihiro Narita, Kohji Takei, Hiroshi Yamada

    Frontiers in cell and developmental biology   Vol. 10   page: 884509 - 884509   2022

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    Dynamin is an endocytic protein that functions in vesicle formation by scission of invaginated membranes. Dynamin maintains the structure of foot processes in glomerular podocytes by directly and indirectly interacting with actin filaments. However, molecular mechanisms underlying dynamin-mediated actin regulation are largely unknown. Here, biochemical and cell biological experiments were conducted to uncover how dynamin modulates interactions between membranes and actin in human podocytes. Actin-bundling, membrane tubulating, and GTPase activities of dynamin were examined in vitro using recombinant dynamin 2-wild-type (WT) or dynamin 2-K562E, which is a mutant found in Charcot-Marie-Tooth patients. Dynamin 2-WT and dynamin 2-K562E led to the formation of prominent actin bundles with constant diameters. Whereas liposomes incubated with dynamin 2-WT resulted in tubule formation, dynamin 2-K562E reduced tubulation. Actin filaments and liposomes stimulated dynamin 2-WT GTPase activity by 6- and 20-fold, respectively. Actin-filaments, but not liposomes, stimulated dynamin 2-K562E GTPase activity by 4-fold. Self-assembly-dependent GTPase activity of dynamin 2-K562E was reduced to one-third compared to that of dynamin 2-WT. Incubation of liposomes and actin with dynamin 2-WT led to the formation of thick actin bundles, which often bound to liposomes. The interaction between lipid membranes and actin bundles by dynamin 2-K562E was lower than that by dynamin 2-WT. Dynamin 2-WT partially colocalized with stress fibers and actin bundles based on double immunofluorescence of human podocytes. Dynamin 2-K562E expression resulted in decreased stress fiber density and the formation of aberrant actin clusters. Dynamin 2-K562E colocalized with α-actinin-4 in aberrant actin clusters. Reformation of stress fibers after cytochalasin D-induced actin depolymerization and washout was less effective in dynamin 2-K562E-expressing cells than that in dynamin 2-WT. Bis-T-23, a dynamin self-assembly enhancer, was unable to rescue the decreased focal adhesion numbers and reduced stress fiber density induced by dynamin 2-K562E expression. These results suggest that the low affinity of the K562E mutant for lipid membranes, and atypical self-assembling properties, lead to actin disorganization in HPCs. Moreover, lipid-binding and self-assembly of dynamin 2 along actin filaments are required for podocyte morphology and functions. Finally, dynamin 2-mediated interactions between actin and membranes are critical for actin bundle formation in HPCs.

    DOI: 10.3389/fcell.2022.884509

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  34. Quantitative Visualization of the Interaction between Complement Component C1 and Immunoglobulin G: The Effect of CH1 Domain Deletion Reviewed

    Saeko Yanaka, Shigetaka Nishiguchi, Rina Yogo, Hiroki Watanabe, Jiana Shen, Hirokazu Yagi, Takayuki Uchihashi, and Koichi Kato

    International Journal of Molecular Sciences   Vol. 23 ( 4 ) page: 2090   2021.12

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    Immunoglobulin G (IgG) adopts a modular multidomain structure that mediates antigen recognition and effector functions, such as complement-dependent cytotoxicity. IgG molecules are self-assembled into a hexameric ring on antigen-containing membranes, recruiting the complement component, C1q. To provide deeper insights into the initial step of the complement pathway, we report a high-speed atomic force microscopy study for quantitative visualization of the interaction between IgG and the C1 complex composed of C1q, C1r, and C1s. Results showed that C1q in the C1 complex is restricted regarding internal motion and has a stronger binding affinity for on-membrane IgG assemblages than C1q alone, presumably because of smaller conformational entropy loss upon binding. Furthermore, we visualized a 1:1 stoichiometric interaction between C1/C1q and an IgG variant that lacks the entire CH1 domain in the absence of antigen. In addition to the canonical C1q-binding site on Fc, their interactions are mediated through a secondary site on the CL domain that is cryptic in the presence of the CH1 domain. Our findings offer clues for novel-modality therapeutic antibodies.

    DOI: 10.3390/ijms23042090.

  35. Molecular Origin of the Anomalous pH Effect in Blue Proteorhodopsin Reviewed

    Sumikawa, M; Abe-Yoshizumi, R; Uchihashi, T; Kandori, H

    JOURNAL OF PHYSICAL CHEMISTRY LETTERS   Vol. 12 ( 51 ) page: 12225 - 12229   2021.12

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    Proteorhodopsin (PR) is a light-driven proton pump found in marine bacteria, and thousands of PRs are classified into blue-absorbing PR (BPR; λmax ∼ 490 nm) and green-absorbing PR (GPR; λmax ∼ 525 nm). We previously presented conversion of BPR into GPR using the anomalous pH effect. When we lowered the pH of a BPR to pH 2 and returned to pH 7, the protein absorbs green light. This suggests the existence of the critical point of the irreversible process at around pH 2, but the mechanism of anomalous pH effect was fully unknown. The present size exclusion chromatography (SEC) and atomic force microscope (AFM) analysis of BPR from Vibrio califitulae (VcBPR) revealed the anomalous pH effect because of the conversion from pentamer to monomer. The different pKa of the Schiff base counterion between pentamer and monomer leads to different colors at the same pH.

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  36. Desiccation-induced fibrous condensation of CAHS protein from an anhydrobiotic tardigrade. Reviewed International journal

    Maho Yagi-Utsumi, Kazuhiro Aoki, Hiroki Watanabe, Chihong Song, Seiji Nishimura, Tadashi Satoh, Saeko Yanaka, Christian Ganser, Sae Tanaka, Vincent Schnapka, Ean Wai Goh, Yuji Furutani, Kazuyoshi Murata, Takayuki Uchihashi, Kazuharu Arakawa, Koichi Kato

    Scientific reports   Vol. 11 ( 1 ) page: 21328 - 21328   2021.11

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    Anhydrobiosis, one of the most extensively studied forms of cryptobiosis, is induced in certain organisms as a response to desiccation. Anhydrobiotic species has been hypothesized to produce substances that can protect their biological components and/or cell membranes without water. In extremotolerant tardigrades, highly hydrophilic and heat-soluble protein families, cytosolic abundant heat-soluble (CAHS) proteins, have been identified, which are postulated to be integral parts of the tardigrades' response to desiccation. In this study, to elucidate these protein functions, we performed in vitro and in vivo characterizations of the reversible self-assembling property of CAHS1 protein, a major isoform of CAHS proteins from Ramazzottius varieornatus, using a series of spectroscopic and microscopic techniques. We found that CAHS1 proteins homo-oligomerized via the C-terminal α-helical region and formed a hydrogel as their concentration increased. We also demonstrated that the overexpressed CAHS1 proteins formed condensates under desiccation-mimicking conditions. These data strongly suggested that, upon drying, the CAHS1 proteins form oligomers and eventually underwent sol-gel transition in tardigrade cytosols. Thus, it is proposed that the CAHS1 proteins form the cytosolic fibrous condensates, which presumably have variable mechanisms for the desiccation tolerance of tardigrades. These findings provide insights into molecular strategies of organisms to adapt to extreme environments.

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  37. Construction of ferritin hydrogels utilizing subunit–subunit interactions Reviewed

    Masaru Yamanaka, Tsuyoshi Mashima, Michio Ogihara, Mei Okamoto, Takayuki Uchihashi, Shun Hirota

    PLOS ONE   Vol. 16 ( 11 ) page: e0259052 - e0259052   2021.11

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    Various proteins form nanostructures exhibiting unique functions, making them attractive as next-generation materials. Ferritin is a hollow spherical protein that incorporates iron ions. Here, we found that hydrogels are simply formed from concentrated apoferritin solutions by acid denaturation and subsequent neutralization. The water content of the hydrogel was approximately 80%. The apoferritin hydrogel did not decompose in the presence of 1 M HCl, 2-mercaptoethanol, or methanol but was dissolved in the presence of 1 M NaOH, by heating at 80°C, or by treatment with trypsin or 6 M guanidine hydrochloride. The Young’s modulus of the hydrogel was 20.4 ± 12.1 kPa according to local indentation experimentes using atomic force microscopy, indicating that the hydrogel was relatively stiff. Transition electron microscopy measurements revealed that a fibrous network was constructed in the hydrogel. The color of the hydrogel became yellow-brown upon incubation in the presence of Fe<sup>3+</sup> ions, indicating that the hydrogel adsorbed the Fe<sup>3+</sup> ions. The yellow-brown color of the Fe<sup>3+</sup>-adsorbed hydrogel did not change upon incubation in pure water, whereas it became pale by incubating it in the presence of 100 mM ethylenediaminetetraacetic acid (EDTA). The apoferritin hydrogel also adsorbed Co<sup>2+</sup> and Cu<sup>2+</sup> ions and released them in the presence of EDTA, while it adsorbed less Ni<sup>2+</sup> ions; more Fe<sup>3+</sup> ions adsorbed to the apoferritin hydrogel than other metal ions, indicating that the hydrogel keeps the iron storage characteristic of ferritin. These results demonstrate a new property of ferritin: the ability to form a hydrogel that can adsorb/desorb metal ions, which may be useful in designing future biomaterials.

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  38. Deformation of microtubules regulates translocation dynamics of kinesin. Reviewed International coauthorship International journal

    Syeda Rubaiya Nasrin, Christian Ganser, Seiji Nishikawa, Arif Md Rashedul Kabir, Kazuki Sada, Takefumi Yamashita, Mitsunori Ikeguchi, Takayuki Uchihashi, Henry Hess, Akira Kakugo

    Science advances   Vol. 7 ( 42 ) page: eabf2211   2021.10

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    [Figure: see text].

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  39. Scanning Probe Microscopy FOREWORD

    Nakajima, K; Sumitomo, K; Nakayama, T; Fukui, K; Kageshima, M; Kawai, S; Komeda, T; Takahashi, T; Uchihashi, T

    JAPANESE JOURNAL OF APPLIED PHYSICS   Vol. 60 ( SE )   2021.9

  40. 高速原子間力顕微鏡による一分子動態計測:最近の応用研究 Invited

    内橋貴之

    生化学   Vol. 93   page: 526 - 561   2021.8

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  41. Tardigrade Secretory-Abundant Heat-Soluble Protein Has a Flexible β-Barrel Structure in Solution and Keeps This Structure in Dehydration. Reviewed International journal

    Kazuhisa Miyazawa, Satoru G Itoh, Hiroki Watanabe, Takayuki Uchihashi, Saeko Yanaka, Maho Yagi-Utsumi, Koichi Kato, Kazuharu Arakawa, Hisashi Okumura

    The journal of physical chemistry. B   Vol. 125 ( 32 ) page: 9145 - 9154   2021.8

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    Secretory-abundant heat-soluble (SAHS) proteins are unique heat-soluble proteins of Tardigrada and are believed to play an essential role in anhydrobiosis, a latent state of life induced by desiccation. To investigate the dynamic properties, molecular dynamics (MD) simulations of a SAHS protein, RvSAHS1, were performed in solution and under dehydrating conditions. For comparison purposes, MD simulations of a human liver-type fatty-acid binding protein (LFABP) were performed in solution. Furthermore, high-speed atomic force microscopy observations were conducted to ascertain the results of the MD simulations. Three properties of RvSAHS1 were found as follows. (1) The entrance region of RvSAHS1 is more flexible and can be more extensive in solutions compared with that of a human LFABP because there is no salt bridge between the βD and βE strands. (2) The intrinsically disordered domain in the N-terminal region significantly fluctuates and can form an amphiphilic α-helix. (3) The size of the entrance region gets smaller along with dehydration, keeping the β-barrel structure. Overall, the obtained results provide atomic-level dynamics of SAHS proteins.

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  42. Reconstruction of Three-Dimensional Conformations of Bacterial ClpB from High-Speed Atomic-Force-Microscopy Images. Reviewed International journal

    Bhaskar Dasgupta, Osamu Miyashita, Takayuki Uchihashi, Florence Tama

    Frontiers in molecular biosciences   Vol. 8   page: 704274 - 704274   2021.8

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    ClpB belongs to the cellular disaggretase machinery involved in rescuing misfolded or aggregated proteins during heat or other cellular shocks. The function of this protein relies on the interconversion between different conformations in its native condition. A recent high-speed-atomic-force-microscopy (HS-AFM) experiment on ClpB from Thermus thermophilus shows four predominant conformational classes, namely, open, closed, spiral, and half-spiral. Analyses of AFM images provide only partial structural information regarding the molecular surface, and thus computational modeling of three-dimensional (3D) structures of these conformations should help interpret dynamical events related to ClpB functions. In this study, we reconstruct 3D models of ClpB from HS-AFM images in different conformational classes. We have applied our recently developed computational method based on a low-resolution representation of 3D structure using a Gaussian mixture model, combined with a Monte-Carlo sampling algorithm to optimize the agreement with target AFM images. After conformational sampling, we obtained models that reflect conformational variety embedded within the AFM images. From these reconstructed 3D models, we described, in terms of relative domain arrangement, the different types of ClpB oligomeric conformations observed by HS-AFM experiments. In particular, we highlighted the slippage of the monomeric components around the seam. This study demonstrates that such details of information, necessary for annotating the different conformational states involved in the ClpB function, can be obtained by combining HS-AFM images, even with limited resolution, and computational modeling.

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  43. JRAB/MICAL-L2 undergoes liquid-liquid phase separation to form tubular recycling endosomes. Reviewed International journal

    Ayuko Sakane, Taka-Aki Yano, Takayuki Uchihashi, Kazuki Horikawa, Yusuke Hara, Issei Imoto, Shusaku Kurisu, Hiroshi Yamada, Kohji Takei, Takuya Sasaki

    Communications biology   Vol. 4 ( 1 ) page: 551 - 551   2021.5

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    Elongated tubular endosomes play essential roles in diverse cellular functions. Multiple molecules have been implicated in tubulation of recycling endosomes, but the mechanism of endosomal tubule biogenesis has remained unclear. In this study, we found that JRAB/MICAL-L2 induces endosomal tubulation via activated Rab8A. In association with Rab8A, JRAB/MICAL-L2 adopts its closed form, which functions in the tubulation of recycling endosomes. Moreover, JRAB/MICAL-L2 induces liquid-liquid phase separation, initiating the formation of tubular recycling endosomes upon overexpression. Between its N-terminal and C-terminal globular domains, JRAB/MICAL-L2 contains an intrinsically disordered region, which contributes to the formation of JRAB/MICAL-L2 condensates. Based on our findings, we propose that JRAB/MICAL-L2 plays two sequential roles in the biogenesis of tubular recycling endosomes: first, JRAB/MICAL-L2 organizes phase separation, and then the closed form of JRAB/MICAL-L2 formed by interaction with Rab8A promotes endosomal tubulation.

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  44. Non-close-packed arrangement of soft elastomer microspheres on solid substrates Reviewed

    Yuma Sasaki, Seina Hiroshige, Masaya Takizawa, Yuichiro Nishizawa, Takayuki Uchihashi, Haruka Minato, Daisuke Suzuki

    RSC Advances   Vol. 11 ( 24 ) page: 14562 - 14567   2021.5

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  45. Nanostructure and thermoresponsiveness of poly(N-isopropyl methacrylamide)-based hydrogel microspheres prepared via aqueous free radical precipitation polymerization Reviewed

    Yuichiro Nishizawa, Haruka Minato, Takumi Inui, Ikuma Saito, Takuma Kureha, Mitsuhiro Shibayama, Takayuki Uchihashi, Daisuke Suzuki

    RSC Advances   Vol. 11 ( 22 ) page: 13130 - 13137   2021.4

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    <p>Nanostructure and thermoresponsiveness of single and packed poly(<italic>N</italic>-isopropyl methacrylamide)-based microgels observed by temperature-controllable high speed atomic force microscopy.</p>

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  46. Tuning the Fermi surface of In/Si(111)-(√7 x √3) by CuPc adsorption

    Sagehashi, R; Kobayashi, T; Uchihashi, T; Sakamoto, K

    SURFACE SCIENCE   Vol. 705   2021.3

  47. Dynamic Assembly/Disassembly of Staphylococcus aureus FtsZ Visualized by High-Speed Atomic Force Microscopy. Reviewed International journal

    Junso Fujita, Shogo Sugiyama, Haruna Terakado, Maho Miyazaki, Mayuki Ozawa, Nanami Ueda, Natsuko Kuroda, Shun-Ichi Tanaka, Takuya Yoshizawa, Takayuki Uchihashi, Hiroyoshi Matsumura

    International journal of molecular sciences   Vol. 22 ( 4 ) page: 1 - 10   2021.2

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    FtsZ is a key protein in bacterial cell division and is assembled into filamentous architectures. FtsZ filaments are thought to regulate bacterial cell division and have been investigated using many types of imaging techniques such as atomic force microscopy (AFM), but the time scale of the method was too long to trace the filament formation process. Development of high-speed AFM enables us to achieve sub-second time resolution and visualize the formation and dissociation process of FtsZ filaments. The analysis of the growth and dissociation rates of the C-terminal truncated FtsZ (FtsZt) filaments indicate the net growth and dissociation of FtsZt filaments in the growth and dissociation conditions, respectively. We also analyzed the curvatures of the full-length FtsZ (FtsZf) and FtsZt filaments, and the comparative analysis indicated the straight-shape preference of the FtsZt filaments than those of FtsZf. These findings provide insights into the fundamental dynamic behavior of FtsZ protofilaments and bacterial cell division.

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  48. Nanostructures, Thermoresponsiveness, and Assembly Mechanism of Hydrogel Microspheres during Aqueous Free-Radical Precipitation Polymerization. Reviewed International journal

    Yuichiro Nishizawa, Haruka Minato, Takumi Inui, Takayuki Uchihashi, Daisuke Suzuki

    Langmuir : the ACS journal of surfaces and colloids   Vol. 37 ( 1 ) page: 151 - 159   2021.1

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    Although techniques to produce uniformly sized hydrogel microspheres (microgels) by aqueous free-radical precipitation polymerization are well established, the details of the polymerization process remain mysterious. In the present study, the structural evolution and thermoresponsiveness of the developing microgels during the polymerization were evaluated by temperature-controlled high-speed atomic force microscopy. This analysis clarified that the swelling properties of the precursor microgels formed in the early stages of the polymerization are quite low due to the high incorporation of cross-linkers and that non-thermoresponsive deca-nanosized spherical domains are already present in the precursor microgels. Furthermore, we succeeded in tracking the formation of nuclei and their growth process, which has never been fully understood, in aqueous solution by real-time observations. These findings will help us to design functional microgels with the desired nanostructures via precipitation polymerization.

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  49. Single-molecule dynamics observed by high-speed atomic force microscopy

    Uchihashi Takayuki

      Vol. 93 ( 4 ) page: 526 - 531   2021

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  50. Structural insights into the mechanism of rhodopsin phosphodiesterase. Reviewed International journal

    Tatsuya Ikuta, Wataru Shihoya, Masahiro Sugiura, Kazuho Yoshida, Masahito Watari, Takaya Tokano, Keitaro Yamashita, Kota Katayama, Satoshi P Tsunoda, Takayuki Uchihashi, Hideki Kandori, Osamu Nureki

    Nature communications   Vol. 11 ( 1 ) page: 5605 - 5605   2020.11

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    Rhodopsin phosphodiesterase (Rh-PDE) is an enzyme rhodopsin belonging to a recently discovered class of microbial rhodopsins with light-dependent enzymatic activity. Rh-PDE consists of the N-terminal rhodopsin domain and C-terminal phosphodiesterase (PDE) domain, connected by 76-residue linker, and hydrolyzes both cAMP and cGMP in a light-dependent manner. Thus, Rh-PDE has potential for the optogenetic manipulation of cyclic nucleotide concentrations, as a complementary tool to rhodopsin guanylyl cyclase and photosensitive adenylyl cyclase. Here we present structural and functional analyses of the Rh-PDE derived from Salpingoeca rosetta. The crystal structure of the rhodopsin domain at 2.6 Å resolution revealed a new topology of rhodopsins, with 8 TMs including the N-terminal extra TM, TM0. Mutational analyses demonstrated that TM0 plays a crucial role in the enzymatic photoactivity. We further solved the crystal structures of the rhodopsin domain (3.5 Å) and PDE domain (2.1 Å) with their connecting linkers, which showed a rough sketch of the full-length Rh-PDE. Integrating these structures, we proposed a model of full-length Rh-PDE, based on the HS-AFM observations and computational modeling of the linker region. These findings provide insight into the photoactivation mechanisms of other 8-TM enzyme rhodopsins and expand the definition of rhodopsins.

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  51. Novel Babesia bovis exported proteins that modify properties of infected red blood cells. Reviewed International journal

    Hassan Hakimi, Thomas J Templeton, Miako Sakaguchi, Junya Yamagishi, Shinya Miyazaki, Kazuhide Yahata, Takayuki Uchihashi, Shin-Ichiro Kawazu, Osamu Kaneko, Masahito Asada

    PLoS pathogens   Vol. 16 ( 10 ) page: e1008917   2020.10

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    Babesia bovis causes a pathogenic form of babesiosis in cattle. Following invasion of red blood cells (RBCs) the parasite extensively modifies host cell structural and mechanical properties via the export of numerous proteins. Despite their crucial role in virulence and pathogenesis, such proteins have not been comprehensively characterized in B. bovis. Here we describe the surface biotinylation of infected RBCs (iRBCs), followed by proteomic analysis. We describe a multigene family (mtm) that encodes predicted multi-transmembrane integral membrane proteins which are exported and expressed on the surface of iRBCs. One mtm gene was downregulated in blasticidin-S (BS) resistant parasites, suggesting an association with BS uptake. Induced knockdown of a novel exported protein encoded by BBOV_III004280, named VESA export-associated protein (BbVEAP), resulted in a decreased growth rate, reduced RBC surface ridge numbers, mis-localized VESA1, and abrogated cytoadhesion to endothelial cells, suggesting that BbVEAP is a novel virulence factor for B. bovis.

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  52. Single-molecule level dynamic observation of disassembly of the apo-ferritin cage in solution. Reviewed International journal

    Basudev Maity, Zhipeng Li, Kento Niwase, Christian Ganser, Tadaomi Furuta, Takayuki Uchihashi, Diannan Lu, Takafumi Ueno

    Physical chemistry chemical physics : PCCP   Vol. 22 ( 33 ) page: 18562 - 18572   2020.9

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    The ferritin cage iron-storage protein assembly has been widely used as a template for preparing nanomaterials. This assembly has a unique pH-induced disassembly/reassembly mechanism that provides a means for encapsulating molecules such as nanoparticles and small enzymes for catalytic and biomaterial applications. Although several researchers have investigated the disassembly process of ferritin, the dynamics involved in the initiation of the process and its intermediate states have not been elucidated due to a lack of suitable methodology to track the process in real-time. We describe the use of high-speed atomic force microscopy (HS-AFM) to image the dynamic event in real-time with single-molecule level resolution. The HS-AFM movies produced in the present work enable direct visualization of the movements of single ferritin cages in solution and formation of a hole prior to disassembly into subunit fragments. Additional support for these observations was confirmed at the atomic level by the results of all-atom molecular dynamics (MD) simulations, which revealed that the initiation process includes the opening of 3-fold symmetric channels. Our findings provide an essential contribution to a fundamental understanding of the dynamics of protein assembly and disassembly, as well as efforts to redesign the apo-ferritin cage for extended applications.

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  53. Thermoresponsive structural changes of single poly(N-isopropyl acrylamide) hydrogel microspheres under densely packed conditions on a solid substrate Reviewed

    Haruka Minato, Yuichiro Nishizawa, Takayuki Uchihashi, Daisuke Suzuki

    Polymer Journal   Vol. 52 ( 9 ) page: 1137 - 1141   2020.9

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  54. Convergent evolution of processivity in bacterial and fungal cellulases. Reviewed International journal

    Taku Uchiyama, Takayuki Uchihashi, Akihiko Nakamura, Hiroki Watanabe, Satoshi Kaneko, Masahiro Samejima, Kiyohiko Igarashi

    Proceedings of the National Academy of Sciences of the United States of America   Vol. 117 ( 33 ) page: 19896 - 19903   2020.8

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    Cellulose is the most abundant biomass on Earth, and many microorganisms depend on it as a source of energy. It consists mainly of crystalline and amorphous regions, and natural degradation of the crystalline part is highly dependent on the degree of processivity of the degrading enzymes (i.e., the extent of continuous hydrolysis without detachment from the substrate cellulose). Here, we report high-speed atomic force microscopic (HS-AFM) observations of the movement of four types of cellulases derived from the cellulolytic bacteria Cellulomonas fimi on various insoluble cellulose substrates. The HS-AFM images clearly demonstrated that two of them (CfCel6B and CfCel48A) slide on crystalline cellulose. The direction of processive movement of CfCel6B is from the nonreducing to the reducing end of the substrate, which is opposite that of processive cellulase Cel7A of the fungus Trichoderma reesei (TrCel7A), whose movement was first observed by this technique, while CfCel48A moves in the same direction as TrCel7A. When CfCel6B and TrCel7A were mixed on the same substrate, "traffic accidents" were observed, in which the two cellulases blocked each other's progress. The processivity of CfCel6B was similar to those of fungal family 7 cellulases but considerably higher than those of fungal family 6 cellulases. The results indicate that bacteria utilize family 6 cellulases as high-processivity enzymes for efficient degradation of crystalline cellulose, whereas family 7 enzymes have the same function in fungi. This is consistent with the idea of convergent evolution of processive cellulases in fungi and bacteria to achieve similar functionality using different protein foldings.

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  55. Direct visualization of the conformational change of FUS/TLS upon binding to promoter-associated non-coding RNA. Reviewed International journal

    Nesreen Hamad, Hiroki Watanabe, Takayuki Uchihashi, Riki Kurokawa, Takashi Nagata, Masato Katahira

    Chemical communications (Cambridge, England)   Vol. 56 ( 64 ) page: 9134 - 9137   2020.8

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    High-speed AFM revealed the conformational change of fused in sarcoma (FUS) from a compact to an extended structure upon binding of non-coding RNA, which is supposed to allow FUS to bind to CBP/p300 for transcriptional interference. Thus, a mechanistic insight into transcription regulation by FUS and non-coding RNA is provided.

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  56. Scanning Probe Microscopy FOREWORD

    Nakajima, K; Sumitomo, K; Nakayama, T; Fukui, K; Kageshima, M; Kawai, S; Komeda, T; Takahashi, T; Uchihashi, T

    JAPANESE JOURNAL OF APPLIED PHYSICS   Vol. 59   2020.8

  57. Assembly mechanism of a supramolecular MS-ring complex to initiate bacterial flagellar biogenesis in Vibrio species. Reviewed International journal

    Hiroyuki Terashima, Keiichi Hirano, Yuna Inoue, Takaya Tokano, Akihiro Kawamoto, Takayuki Kato, Erika Yamaguchi, Keiichi Namba, Takayuki Uchihashi, Seiji Kojima, Michio Homma

    Journal of bacteriology   Vol. 202 ( 16 )   2020.8

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    The bacterial flagellum is an organelle responsible for motility and has a rotary motor comprising the rotor and the stator. Flagellar biogenesis is initiated by the assembly of the MS-ring, a supramolecular complex embedded in the cytoplasmic membrane. The MS-ring consists of a few dozen copies of the transmembrane FliF protein, and is an essential core structure which is a part of the rotor. The number and location of the flagella are controlled by the FlhF and FlhG proteins in some species. However, there is no clarity on the factors initiating MS-ring assembly, and contribution of FlhF/FlhG to this process. Here, we show that FlhF and a C-ring component FliG facilitate Vibrio MS-ring formation. When Vibrio FliF alone was expressed in Escherichia coli cells, MS-ring formation rarely occurred, indicating the requirement of other factors for MS-ring assembly. Consequently, we investigated if FlhF aided FliF in MS-ring assembly. We found that FlhF allowed GFP-fused FliF to localize at the cell pole in a Vibrio cell, suggesting that it increases local concentration of FliF at the pole. When FliF was co-expressed with FlhF in E. coli cells, the MS-ring was effectively formed, indicating that FlhF somehow contributes to MS-ring formation. The isolated MS-ring structure was similar to the MS-ring formed by Salmonella FliF. Interestingly, FliG facilitates MS-ring formation, suggesting that FliF and FliG assist in each other's assembly into the MS-ring and C-ring. This study aids in understanding the mechanism behind MS-ring assembly using appropriate spatial/temporal regulations.Importance Flagellar formation is initiated by the assembly of the FliF protein into the MS-ring complex, embedded in the cytoplasmic membrane. The appropriate spatial/temporal control of MS-ring formation is important for the morphogenesis of the bacterial flagellum. Here, we focus on the assembly mechanism of Vibrio FliF into the MS-ring. FlhF, a positive regulator of the number and location of flagella, recruits the FliF molecules at the cell pole and facilitates MS-ring formation. FliG also facilitates MS-ring formation. Our study showed that these factors control flagellar biogenesis in Vibrio, by initiating the MS-ring assembly. Furthermore, it also implies that flagellar biogenesis is a sophisticated system linked with the expression of certain genes, protein localization and a supramolecular complex assembly.

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  58. ヘリオロドプシンおよびシゾロドプシンの構造から明らかになった微生物型ロドプシンの多様性(Structures of heliorhodopsin and schizorhodopsin elucidate the structural diversity of microbial rhodopsins)

    Shihoya Wataru, Inoue Keiichi, Manish Singh, Higuchi Akimitsu, Konno Masae, Yoshizumi Rei, Uchihashi Takayuki, Kandori Hideki, Nureki Osamu

    生物物理   Vol. 60 ( Suppl.1-2 ) page: S154 - S154   2020.8

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  59. Structural Dynamics of a Protein Domain Relevant to the Water-Oxidizing Complex in Photosystem II as Visualized by High-Speed Atomic Force Microscopy. Reviewed International journal

    Takaya Tokano, Yuki Kato, Shogo Sugiyama, Takayuki Uchihashi, Takumi Noguchi

    The journal of physical chemistry. B   Vol. 124 ( 28 ) page: 5847 - 5857   2020.7

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    Photosystem II (PSII) is a multiprotein complex that has a function of light-driven water oxidation. The catalytic site of water oxidation is the Mn4CaO5 cluster, which is bound to the lumenal side of PSII through amino acid residues from the D1 and CP43 proteins and is further surrounded by the extrinsic proteins. In this study, we have for the first time visualized the structural dynamics of the lumenal region of a PSII core complex using high-speed atomic force microscopy (HS-AFM). The HS-AFM images of a PSII membrane fragment showed stepwise dissociation of the PsbP and PsbO extrinsic proteins. Upon subsequent destruction of the Mn4CaO5 cluster, the lumenal domain of CP43 was found to undergo a conformational fluctuation. The observed structural flexibility and conformational fluctuation of the CP43 lumenal domain are suggested to play important roles in the biogenesis of PSII and the photoassembly of the Mn4CaO5 cluster.

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  60. Dynamic behavior of an artificial protein needle contacting a membrane observed by high-speed atomic force microscopy. Reviewed International journal

    Takafumi Ueno, Kento Niwase, Daisho Tsubokawa, Kosuke Kikuchi, Natsumi Takai, Tadaomi Furuta, Ryuji Kawano, Takayuki Uchihashi

    Nanoscale   Vol. 12 ( 15 ) page: 8166 - 8173   2020.4

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    Bacteriophage T4 and other bacteriophages have a protein component known as a molecular needle which is used for the transmembrane reaction in the infection process. In this paper, the transmembrane reaction mechanisms of artificial protein needles (PNs) constructed by protein engineering of the component protein of bacteriophage T4 are elucidated by observation of single-molecules by high-speed atomic force microscopy (HS-AFM) and molecular dynamics (MD) simulations. The HS-AFM images indicate that the tip of the needle structure stabilizes the interaction of the needle with the membrane surface and is involved in controlling the contact angle and angular velocity with respect to the membrane. The MD simulations indicate that the dynamic behavior of PN is governed by hydrogen bonds between the membrane phosphate fragments and the tip. Moreover, quartz crystal microbalance (QCM) and electrophysiological experiments indicate that the tip structure of PN affects its kinetic behavior and membrane potential. These results demonstrate that protein assemblies derived from natural biosupramolecules can be used to create nanomaterials with rationally-designed functionality.

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  61. Dynamics of oligomer and amyloid fibril formation by yeast prion Sup35 observed by high-speed atomic force microscopy. Reviewed International journal

    Hiroki Konno, Takahiro Watanabe-Nakayama, Takayuki Uchihashi, Momoko Okuda, Liwen Zhu, Noriyuki Kodera, Yousuke Kikuchi, Toshio Ando, Hideki Taguchi

    Proceedings of the National Academy of Sciences of the United States of America   Vol. 117 ( 14 ) page: 7831 - 7836   2020.4

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    The yeast prion protein Sup35, which contains intrinsically disordered regions, forms amyloid fibrils responsible for a prion phenotype [PSI+]. Using high-speed atomic force microscopy (HS-AFM), we directly visualized the prion determinant domain (Sup35NM) and the formation of its oligomers and fibrils at subsecond and submolecular resolutions. Monomers with freely moving tail-like regions initially appeared in the images, and subsequently oligomers with distinct sizes of ∼1.7 and 3 to 4 nm progressively accumulated. Nevertheless, these oligomers did not form fibrils, even after an incubation for 2 h in the presence of monomers. Fibrils appeared after much longer monomer incubation. The fibril elongation occurred smoothly without discrete steps, suggesting gradual conversions of the incorporated monomers into cross-β structures. The individual oligomers were separated from each other and also from the fibrils by respective, identical lengths on the mica surface, probably due to repulsion caused by the freely moving disordered regions. Based on these HS-AFM observations, we propose that the freely moving tails of the monomers are incorporated into the fibril ends, and then the structural conversions to cross-β structures gradually occur.

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  62. Schizorhodopsins: A family of rhodopsins from Asgard archaea that function as light-driven inward H+ pumps. Reviewed International journal

    Keiichi Inoue, Satoshi P Tsunoda, Manish Singh, Sahoko Tomida, Shoko Hososhima, Masae Konno, Ryoko Nakamura, Hiroki Watanabe, Paul-Adrian Bulzu, Horia L Banciu, Adrian-Ştefan Andrei, Takayuki Uchihashi, Rohit Ghai, Oded Béjà, Hideki Kandori

    Science advances   Vol. 6 ( 15 ) page: eaaz2441   2020.4

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    Schizorhodopsins (SzRs), a rhodopsin family first identified in Asgard archaea, the archaeal group closest to eukaryotes, are present at a phylogenetically intermediate position between typical microbial rhodopsins and heliorhodopsins. However, the biological function and molecular properties of SzRs have not been reported. Here, SzRs from Asgardarchaeota and from a yet unknown microorganism are expressed in Escherichia coli and mammalian cells, and ion transport assays and patch clamp analyses are used to demonstrate SzR as a novel type of light-driven inward H+ pump. The mutation of a cytoplasmic glutamate inhibited inward H+ transport, suggesting that it functions as a cytoplasmic H+ acceptor. The function, trimeric structure, and H+ transport mechanism of SzR are similar to that of xenorhodopsin (XeR), a light-driven inward H+ pumping microbial rhodopsins, implying that they evolved convergently. The inward H+ pump function of SzR provides new insight into the photobiological life cycle of the Asgardarchaeota.

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  63. Recent advances in bioimaging with high-speed atomic force microscopy. Reviewed International journal

    Takayuki Uchihashi, Christian Ganser

    Biophysical reviews   Vol. 12 ( 2 ) page: 363 - 369   2020.4

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    Among various microscopic techniques for characterizing protein structures and functions, high-speed atomic force microscopy (HS-AFM) is a unique technique in that it allows direct visualization of structural changes and molecular interactions of proteins without any labeling in a liquid environment. Since the development of the HS-AFM was first reported in 2001, it has been applied to analyze the dynamics of various types of proteins, including motor proteins, membrane proteins, DNA-binding proteins, amyloid proteins, and artificial proteins. This method has now become a versatile tool indispensable for biophysical research. This short review summarizes some bioimaging applications of HS-AFM reported in the last few years and novel applications of HS-AFM utilizing the unique ability of AFM to gain mechanical properties of samples in addition to structural information.

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  64. Single-molecule imaging analysis reveals the mechanism of a high-catalytic-activity mutant of chitinase A from Serratia marcescens Reviewed International journal

    Akasit Visootsat, Akihiko Nakamura, Paul Vignon, Hiroki Watanabe, Takayuki Uchihashi, Ryota Iino

    JOURNAL OF BIOLOGICAL CHEMISTRY   Vol. 295 ( 7 ) page: 1915 - 1925   2020.2

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    Chitin degradation is important for biomass conversion and has potential applications for agriculture, biotechnology, and the pharmaceutical industry. Chitinase A from the Gram-negative bacterium Serratia marcescens (SmChiA) is a processive enzyme that hydrolyzes crystalline chitin as it moves linearly along the substrate surface. In a previous study, the catalytic activity of SmChiA against crystalline chitin was found to increase after the tryptophan substitution of two phenylalanine residues (F232W and F396W), located at the entrance and exit of the substrate binding cleft of the catalytic domain, respectively. However, the mechanism underlying this high catalytic activity remains elusive. In this study, single-molecule fluorescence imaging and high-speed atomic force microscopy were applied to understand the mechanism of this high-catalytic-activity mutant. A reaction scheme including processive catalysis was used to reproduce the properties of SmChiA WT and F232W/F396W, in which all of the kinetic parameters were experimentally determined. High activity of F232W/F396W mutant was caused by a high processivity and a low dissociation rate constant after productive binding. The turnover numbers for both WT and F232W/F396W, determined by the biochemical analysis, were well-replicated using the kinetic parameters obtained from single-molecule imaging analysis, indicating the validity of the reaction scheme. Furthermore, alignment of amino acid sequences of 258 SmChiA-like proteins revealed that tryptophan, not phenylalanine, is the predominant amino acid at the corresponding positions (Phe-232 and Phe-396 for SmChiA). Our study will be helpful for understanding the kinetic mechanisms and further improvement of crystalline chitin hydrolytic activity of SmChiA mutants.

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  65. Induced-Fit Pathway Accelerated Binding of Agitoxin-2 to A K<SUP>+</SUP> Channel Imaged by HS-AFM

    Sumino, A; Sumikama, T; Uchihashi, T; Oiki, S

    BIOPHYSICAL JOURNAL   Vol. 118 ( 3 ) page: 236A - 236A   2020.2

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  66. Construction of a Hexameric Hemoprotein Sheet and Direct Observation of Dynamic Process of its Formation Reviewed

    Koji Oohora, Shota Hirayama, Takayuki Uchihashi, Takashi Hayashi

    Chemistry Letters   Vol. 49 ( 2 ) page: 186 - 190   2020.2

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    <p>A two-dimensional sheet assembly of a hexameric hemoprotein was constructed. A single cysteine residue was introduced onto each subunit surface in the hexameric hemoprotein and modified with a maleimide-tethering tripeptide FGG tag to provide a host–guest interaction between one cucurbit[8]uril molecule (CB8) and two FGG tags. The addition of CB8 into the engineered protein forms the assembly as a sheet-like precipitate. High-speed atomic force microscopic measurements directly reveal the detailed structure and the dynamic process of formation.</p>

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  67. High-speed near-field fluorescence microscopy combined with high-speed atomic force microscopy for biological studies Reviewed International journal

    Takayuki Umakoshi, Shingo Fukuda, Ryota Iino, Takayuki Uchihashi, Toshio Ando

    BIOCHIMICA ET BIOPHYSICA ACTA-GENERAL SUBJECTS   Vol. 1864 ( 2 ) page: 129325   2020.2

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    Background: High-speed atomic force microscopy (HS-AFM) has successfully visualized a variety of protein molecules during their functional activity. However, it cannot visualize small molecules interacting with proteins and even protein molecules when they are encapsulated. Thus, it has been desired to achieve techniques enabling simultaneous optical/AFM imaging at high spatiotemporal resolution with high correlation accuracy.Methods: Scanning near-field optical microscopy (SNOM) is a candidate for the combination with HS-AFM. However, the imaging rate of SNOM has been far below that of HS-AFM. We here developed HS-SNOM and metal tip-enhanced total internal reflection fluorescence microscopy (TIRFM) by exploiting tip-scan HS-AFM and exploring methods to fabricate a metallic tip on a tiny HS-AFM cantilever.Results: In tip-enhanced TIRFM/HS-AFM, simultaneous video recording of the two modalities of images was demonstrated in the presence of fluorescent molecules in the bulk solution at relatively high concentration. By using fabricated metal-tip cantilevers together with our tip-scan HS-AFM setup equipped with SNOM optics, we could perform simultaneous HS-SNOM/HS-AFM imaging, with correlation analysis between the two overlaid images being facilitated.Conclusions: This study materialized simultaneous tip-enhanced TIRFM/HS-AFM and HS-SNOM/HS-AFM imaging at high spatiotemporal resolution. Although some issues remain to be solved in the future, these correlative microscopy methods have a potential to increase the versatility of HS-AFM in biological research.General significance: We achieved an imaging rate of similar to 3 s/frame for SNOM imaging, more than 100-times higher than the typical SNOM imaging rate. We also demonstrated similar to 39 nm resolution in HS-SNOM imaging of fluorescently labeled DNA in solution.

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  68. Supramolecular tholos-like architecture constituted by archaeal proteins without functional annotation Reviewed International journal

    Maho Yagi-Utsumi, Arunima Sikdar, Chihong Song, Jimin Park, Rintaro Inoue, Hiroki Watanabe, Raymond N. Burton-Smith, Toshiya Kozai, Tatsuya Suzuki, Atsuji Kodama, Kentaro Ishii, Hirokazu Yagi, Tadashi Satoh, Susumu Uchiyama, Takayuki Uchihashi, Keehyoung Joo, Jooyoung Lee, Masaaki Sugiyama, Kazuyoshi Murata, Koichi Kato

    SCIENTIFIC REPORTS   Vol. 10 ( 1 ) page: 1540 - 1540   2020.1

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    Euryarchaeal genomes encode proteasome-assembling chaperone homologs, PbaA and PbaB, although archaeal proteasome formation is a chaperone-independent process. Homotetrameric PbaB functions as a proteasome activator, while PbaA forms a homopentamer that does not interact with the proteasome. Notably, PbaA forms a complex with PF0014, an archaeal protein without functional annotation. In this study, based on our previous research on PbaA crystal structure, we performed an integrative analysis of the supramolecular structure of the PbaA/PF0014 complex using native mass spectrometry, solution scattering, high-speed atomic force microscopy, and electron microscopy. The results indicated that this highly thermostable complex constitutes ten PbaA and ten PF0014 molecules, which are assembled into a dumbbell-shaped structure. Two PbaA homopentameric rings correspond to the dumbbell plates, with their N-termini located outside of the plates and C-terminal segments left mobile. Furthermore, mutant PbaA lacking the mobile C-terminal segment retained the ability to form a complex with PF0014, allowing 3D modeling of the complex. The complex shows a five-column tholos-like architecture, in which each column comprises homodimeric PF0014, harboring a central cavity, which can potentially accommodate biomacromolecules including proteins. Our findings provide insight into the functional roles of Pba family proteins, offering a novel framework for designing functional protein cages.

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  69. Thermoresponsive Micellar Assembly Constructed from a Hexameric Hemoprotein Modified with Poly(N-isopropylacrylamide) toward an Artificial Light-Harvesting System. Reviewed International journal

    Shota Hirayama, Koji Oohora, Takayuki Uchihashi, Takashi Hayashi

    Journal of the American Chemical Society   Vol. 142 ( 4 ) page: 1822 - 1831   2020.1

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    Artificial protein assemblies inspired by nature have significant potential in development of emergent functional materials. In order to construct an artificial protein assembly, we employed a mutant of a thermostable hemoprotein, hexameric tyrosine-coordinated heme protein (HTHP), as a building block. The HTHP mutant which has cysteine residues introduced on the bottom surface of its columnar structure was reacted with maleimide-tethering thermoresponsive poly(N-isopropylacrylamide), PNIPAAm, to generate the protein assembly upon heating. The site-specific modification of the cysteine residues with PNIPAAm on the protein surface was confirmed by SDS-PAGE and analytical size exclusion chromatography (SEC). The PNIPAAm-modified HTHP (PNIPAAm-HTHP) is found to provide a 43 nm spherical structure at 60 °C, and the structural changes observed between the assembled and the disassembled forms were duplicated at least five times. High-speed atomic force microscopic measurements of the micellar assembly supported by cross-linkage with glutaraldehyde indicate that the protein matrices are located on the surface of the sphere and cover the inner PNIPAAm core. Furthermore, substitution of heme with a photosensitizer, Zn protoporphyrin IX (ZnPP), in the micellar assembly provides an artificial light-harvesting system. Photochemical measurements of the ZnPP-substituted micellar assembly demonstrate that energy migration among the arrayed ZnPP molecules occurs within the range of several tens of picoseconds. Our present work represents the first example of an artificial light-harvesting system based on an assembled hemoprotein oligomer structure to replicate natural light-harvesting systems.

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  70. Corrigendum to "Dynamics of Inter-Molecular Interactions Between Single Aβ42 Oligomeric and Aggregate Species by High-Speed Atomic Force Microscopy". Reviewed International journal

    Lei Feng, Hiroki Watanabe, Paul Molino, Gordon G Wallace, Son L Phung, Takayuki Uchihashi, Michael J Higgins

    Journal of molecular biology   Vol. 432 ( 2 ) page: 633 - 633   2020.1

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  71. Rad50 zinc hook functions as a constitutive dimerization module interchangeable with SMC hinge. Reviewed International journal

    Hisashi Tatebe, Chew Theng Lim, Hiroki Konno, Kazuhiro Shiozaki, Akira Shinohara, Takayuki Uchihashi, Asako Furukohri

    Nature communications   Vol. 11 ( 1 ) page: 370 - 370   2020.1

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    The human Mre11/Rad50 complex is one of the key factors in genome maintenance pathways. Previous nanoscale imaging by atomic force microscopy (AFM) showed that the ring-like structure of the human Mre11/Rad50 complex transiently opens at the zinc hook of Rad50. However, imaging of the human Mre11/Rad50 complex by high-speed AFM shows that the Rad50 coiled-coil arms are consistently bridged by the dimerized hooks while the Mre11/Rad50 ring opens by disconnecting the head domains; resembling other SMC proteins such as cohesin or condensin. These architectural features are conserved in the yeast and bacterial Mre11/Rad50 complexes. Yeast strains harboring the chimeric Mre11/Rad50 complex containing the SMC hinge of bacterial condensin MukB instead of the RAD50 hook properly functions in DNA repair. We propose that the basic role of the Rad50 hook is similar to that of the SMC hinge, which serves as rather stable dimerization interface.

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  72. On-Membrane Dynamic Interplay between Anti-GM1 IgG Antibodies and Complement Component C1q Reviewed International journal

    Saeko Yanaka, Rina Yogo, Hiroki Watanabe, Yuki Taniguchi, Tadashi Satoh, Naoko Komura, Hiromune Ando, Hirokazu Yagi, Nobuhiro Yuki, Takayuki Uchihashi, Koichi Kato

    INTERNATIONAL JOURNAL OF MOLECULAR SCIENCES   Vol. 21 ( 1 )   2020.1

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    Guillain-Barre syndrome, an autoimmune neuropathy characterized by acute limb weakness, is often preceded by Campylobacter jejuni infection. Molecular mimicry exists between the bacterial lipo-oligosaccharide and human ganglioside. Such C. jejuni infection induces production of immunoglobulin G1 (IgG1) autoantibodies against GM1 and causes complement-mediated motor nerve injury. For elucidating the molecular mechanisms linking autoantigen recognition and complement activation, we characterized the dynamic interactions of anti-GM1 IgG autoantibodies on ganglioside-incorporated membranes. Using high-speed atomic force microscopy, we found that the IgG molecules assemble into a hexameric ring structure on the membranes depending on their specific interactions with GM1. Complement component C1q was specifically recruited onto these IgG rings. The ring formation was inhibited by an IgG-binding domain of staphylococcal protein A bound at the cleft between the C(H)2 and C(H)3 domains. These data indicate that the IgG assembly is mediated through Fc-Fc interactions, which are promoted under on-membrane conditions due to restricted translational diffusion of IgG molecules. Reduction and alkylation of the hinge disulfide impaired IgG ring formation, presumably because of an increase in conformational entropic penalty. Our findings provide mechanistic insights into the molecular processes involved in Guillain-Barre syndrome and, more generally, into antigen-dependent interplay between antibodies and complement components on membranes.

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  73. Structural basis of nucleosome assembly by the Abo1 AAA+ ATPase histone chaperone. Reviewed International journal

    Carol Cho, Juwon Jang, Yujin Kang, Hiroki Watanabe, Takayuki Uchihashi, Seung Joong Kim, Koichi Kato, Ja Yil Lee, Ji-Joon Song

    Nature communications   Vol. 10 ( 1 ) page: 5764 - 5764   2019.12

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    The fundamental unit of chromatin, the nucleosome, is an intricate structure that requires histone chaperones for assembly. ATAD2 AAA+ ATPases are a family of histone chaperones that regulate nucleosome density and chromatin dynamics. Here, we demonstrate that the fission yeast ATAD2 homolog, Abo1, deposits histone H3-H4 onto DNA in an ATP-hydrolysis-dependent manner by in vitro reconstitution and single-tethered DNA curtain assays. We present cryo-EM structures of an ATAD2 family ATPase to atomic resolution in three different nucleotide states, revealing unique structural features required for histone loading on DNA, and directly visualize the transitions of Abo1 from an asymmetric spiral (ATP-state) to a symmetric ring (ADP- and apo-states) using high-speed atomic force microscopy (HS-AFM). Furthermore, we find that the acidic pore of ATP-Abo1 binds a peptide substrate which is suggestive of a histone tail. Based on these results, we propose a model whereby Abo1 facilitates H3-H4 loading by utilizing ATP.

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  74. Crystal structure of heliorhodopsin. Reviewed International journal

    Wataru Shihoya, Keiichi Inoue, Manish Singh, Masae Konno, Shoko Hososhima, Keitaro Yamashita, Kento Ikeda, Akimitsu Higuchi, Tamaki Izume, Sae Okazaki, Masanori Hashimoto, Ritsu Mizutori, Sahoko Tomida, Yumeka Yamauchi, Rei Abe-Yoshizumi, Kota Katayama, Satoshi P Tsunoda, Mikihiro Shibata, Yuji Furutani, Alina Pushkarev, Oded Béjà, Takayuki Uchihashi, Hideki Kandori, Osamu Nureki

    Nature   Vol. 574 ( 7776 ) page: 132 - +   2019.10

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    Heliorhodopsins (HeRs) are a family of rhodopsins that was recently discovered using functional metagenomics1. They are widely present in bacteria, archaea, algae and algal viruses2,3. Although HeRs have seven predicted transmembrane helices and an all-trans retinal chromophore as in the type-1 (microbial) rhodopsin, they display less than 15% sequence identity with type-1 and type-2 (animal) rhodopsins. HeRs also exhibit the reverse orientation in the membrane compared with the other rhodopsins. Owing to the lack of structural information, little is known about the overall fold and the photoactivation mechanism of HeRs. Here we present the 2.4-Å-resolution structure of HeR from an uncultured Thermoplasmatales archaeon SG8-52-1 (GenBank sequence ID LSSD01000000). Structural and biophysical analyses reveal the similarities and differences between HeRs and type-1 microbial rhodopsins. The overall fold of HeR is similar to that of bacteriorhodopsin. A linear hydrophobic pocket in HeR accommodates a retinal configuration and isomerization as in the type-1 rhodopsin, although most of the residues constituting the pocket are divergent. Hydrophobic residues fill the space in the extracellular half of HeR, preventing the permeation of protons and ions. The structure reveals an unexpected lateral fenestration above the β-ionone ring of the retinal chromophore, which has a critical role in capturing retinal from environment sources. Our study increases the understanding of the functions of HeRs, and the structural similarity and diversity among the microbial rhodopsins.

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  75. 高速原子間力顕微鏡による生体・合成高分子の動態イメージング Invited Reviewed

    高分子   Vol. 68 ( 10 ) page: 564 - 568   2019.10

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  76. Protein uptake into individual hydrogel microspheres visualized by high-speed atomic force microscopy. Reviewed International journal

    Shusuke Matsui, Kensuke Hosho, Haruka Minato, Takayuki Uchihashi, Daisuke Suzuki

    Chemical communications (Cambridge, England)   Vol. 55 ( 68 ) page: 10064 - 10067   2019.9

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    The moment of protein uptake into hydrogel microspheres (microgels) was directly monitored at the nanoscale by high-speed atomic force microscopy. The dynamic morphological changes in the microgels during protein uptake are largely different depending on the type of protein, and the properties and structure of the microgels, which would affect the colloidal stability of protein-microgel hybrids both in vitro and in vivo.

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  77. Evaluation of thermoresponsive structural changes in hydrogel microspheres by high-speed atomic force microscopy

    Nishizawa, Y; Matsui, S; Urayama, K; Kureha, T; Sibayama, M; Uchihashi, T; Suzuki, D

    ABSTRACTS OF PAPERS OF THE AMERICAN CHEMICAL SOCIETY   Vol. 258   2019.8

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  78. Assembly of rod-shaped hydrogel microspheres at the air/water interface

    Kenshiro Honda, Yuka Sazuka, Kojiro Iizuka, Shusuke Matsui, Takayuki Uchihashi, Takuma Kureha, Mitsuhiro Shibayama, Takumi Watanabe, Daisuke Suzuki

    ABSTRACTS OF PAPERS OF THE AMERICAN CHEMICAL SOCIETY   Vol. 258   2019.8

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  79. The Fab portion of immunoglobulin G contributes to its binding to Fc gamma receptor III Reviewed International journal

    Rina Yogo, Yuki Yamaguchi, Hiroki Watanabe, Hirokazu Yagi, Tadashi Satoh, Mahito Nakanishi, Masayoshi Onitsuka, Takeshi Omasa, Mari Shimada, Takahiro Maruno, Tetsuo Torisu, Shio Watanabe, Daisuke Higo, Takayuki Uchihashi, Saeko Yanaka, Susumu Uchiyama, Koichi Kato

    SCIENTIFIC REPORTS   Vol. 9 ( 1 ) page: 11957 - 11957   2019.8

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    Most cells active in the immune system express receptors for antibodies which mediate a variety of defensive mechanisms. These receptors interact with the Fc portion of the antibody and are therefore collectively called Fc receptors. Here, using high-speed atomic force microscopy, we observe interactions of human, humanized, and mouse/human-chimeric immunoglobulin G1 (IgG1) antibodies and their cognate Fc receptor, Fc gamma RIIIa. Our results demonstrate that not only Fc but also Fab positively contributes to the interaction with the receptor. Furthermore, hydrogen/deuterium exchange mass spectrometric analysis reveals that the Fab portion of IgG1 is directly involved in its interaction with Fc gamma RIIIa, in addition to the canonical Fc-mediated interaction. By targeting the previously unidentified receptor-interaction sites in IgG-Fab, our findings could inspire therapeutic antibody engineering.

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  80. Atomically Controlled Surfaces, Interfaces and Nanostructures/Scanning Probe Microscopy FOREWORD

    Takahashi, T; Fujita, D; Fukidome, H; Fukui, K; Hibino, H; Kageshima, M; Komeda, T; Nakajima, K; Nakamura, M; Nakayama, T; Nohira, H; Sasakawa, K; Sasaki, N; Sumitomo, K; Uchihashi, T; Watanabe, T; Yamada, Y

    JAPANESE JOURNAL OF APPLIED PHYSICS   Vol. 58 ( SI )   2019.8

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  81. Development of wide-area tip-scanning high-speed atomic force microscopy

    Watanabe H., Uchihashi T.

    2019 IEEE International Conference on Manipulation, Manufacturing and Measurement on the Nanoscale, 3M-NANO 2019 - Proceedings     page: 281 - 285   2019.8

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    Recently a tip-scanning type high-speed atomic force microscopy (HS-AFM), which is combined with total internal reflection fluorescence microscope (TIRFM), has been developed. Using this system, the topographic and fluorescence images of biological samples in action have been simultaneously visualized. However, the maximum scanning range of the combined system was limited to ∼20 μm and ∼3 μm in X- and Y-directions respectively. This limitation makes it impossible to visualize the dynamics of much larger samples, including bacteria or mammalian cells. Here, we improved a laser-tracking system and expand the tracking range in X- and Y-directions to ∼50 μm and ∼32 μm, respectively by increasing an angular variation of a mirror-tilter unit changing the arrangement and the adhesive area between piezoactuators and base plate of the unit. The Shift of the X-direction of the scanner with large displacement of the Z-scanner is eliminated by modifying an input triangular signal to the mirror-tilter unit. We also developed a scanner, which could cover in a wider imaging area, for the expanded mirror-tilter unit in the tip-scanning HS-AFM system. The capability of this expanded system is demonstrated by measuring the laser-spot displacement, large-area imaging of a square grating sample and eukaryotic cells.

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  82. Real-time nanoscale visualization of biological molecules at work with high-speed atomic force microscopy

    Uchihashi T.

    2019 IEEE International Conference on Manipulation, Manufacturing and Measurement on the Nanoscale, 3M-NANO 2019 - Proceedings     page: 253 - 256   2019.8

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    Atomic force microscopy (AFM) is a powerful tool to visualize biological molecules at nm resolution in liquid conditions and thus has been applied to a wide variety of biological molecules. One of the most coveted functions of AFM is 'fast recording' because it should allow us to directly visualize dynamic processes of biological molecules at work. We have been challenging to break this limitation over 15 years and established fast imaging of dynamic molecular processes. The most advanced high-speed AFM can now capture successive images at 30-60 ms/frame. Further, the interaction force between the tip and the sample, which significantly influences on soft biological molecules, is greatly reduced without deterioration of the scanning performance. In this short paper, typical applications of HS-AFM on biological and artificial molecules will be demonstrated.

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  83. Publisher Correction: Dynamic structural states of ClpB involved in its disaggregation function. Reviewed International journal

    Takayuki Uchihashi, Yo-Hei Watanabe, Yosuke Nakazaki, Takashi Yamasaki, Hiroki Watanabe, Takahiro Maruno, Kentaro Ishii, Susumu Uchiyama, Chihong Song, Kazuyoshi Murata, Ryota Iino, Toshio Ando

    Nature communications   Vol. 10 ( 1 ) page: 3079 - 3079   2019.7

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    An amendment to this paper has been published and can be accessed via a link at the top of the paper.

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  84. Dynamics of Inter-Molecular Interactions Between Single Aβ42 Oligomeric and Aggregate Species by High-Speed Atomic Force Microscopy. Reviewed International journal

    Lei Feng, Hiroki Watanabe, Paul Molino, Gordon G Wallace, Son L Phung, Takayuki Uchihashi, Michael J Higgins

    Journal of molecular biology   Vol. 431 ( 15 ) page: 2687 - 2699   2019.7

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    Within the amyloid hypothesis in Alzheimer's disease, current focus has shifted to earlier stages of amyloid beta (Aβ) peptide assembly, involving soluble oligomers and smaller aggregates, which are more toxic to cells compared to their morphological distinct fibril forms. Critical to the Aβ field is unlocking the molecular-level kinetic pathways of oligomerization, leading to the culprit subset or specific species of Aβ oligomer populations responsible for the disease etiology. Here, we apply high-speed atomic force microscopy to enable direct visualization of dynamic interactions between single Aβ42 oligomers and aggregate forms, with combined nanometre structural and millisecond temporal resolution in liquid. Analysis of dimensions revealed up to three main Aβ42 species distributions, in addition to the appearance of monomers that showed fast surface diffusion compared to the larger Aβ42 species. Significantly, we devised a new single-molecule analysis based on image contrast in high-speed atomic force microscopy movies to quantify rate determining kinetic constants for interactions between the different Aβ42 species. The findings revealed that smaller Aβ42 species show an exponential decay of lifetime distribution, indicating that all molecules undergo the same process with a single well-defined energy barrier. In contrast, larger aggregates show randomized lifetimes, indicating a distribution of interactions energies/barriers that must be overcome in order to dissociate. We interpret the latter as being due to "permissive" binding, arising from different conformation states of the aggregates, along with a variety of accessible interacting groups. Inevitably, this may lead to the formation of different complexes or alloforms, which is known to contribute to difficulties in identifying Aβ oligomer toxicity and has implications for mechanisms underlying neuronal death accompanying Alzheimer's disease.

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  85. An Assessment of the Ability of Submicron- and Micron-Size Silicone Oil Droplets in Dropped Prefillable Syringes to Invoke Early- and Late-Stage Immune Responses. Reviewed International journal

    Elena Krayukhina, Masami Yokoyama, Kayoko Kakuhou Hayashihara, Takahiro Maruno, Masanori Noda, Hiroki Watanabe, Takayuki Uchihashi, Susumu Uchiyama

    Journal of pharmaceutical sciences   Vol. 108 ( 7 ) page: 2278 - 2287   2019.7

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    A number of biopharmaceuticals are available as lyophilized formulations along with a prefilled syringe (PFS) containing water for injection (WFI). Submicron- and micron-size droplets of lubricating silicone oil (SO) applied to the inner surface of the PFS barrel might migrate into the WFI, to which protein pharmaceuticals can adsorb, potentially inducing an immune response. In the present study, we subjected siliconized cyclo-olefin polymer PFSs filled with WFI to dropping stress to simulate actual shipping conditions as well as evaluated the risk associated with the released SO droplets. The results confirmed the undesirable effects of SO on therapeutic proteins, including adsorption to SO droplets and increased secretion of several innate cytokines from human peripheral blood mononuclear cells of a small donor panel. Assessment of immunogenicity in vivo using BALB/c mice revealed a slight increase in the plasma concentrations of antidrug antibodies over 21 days in response to SO-containing antibody samples compared to the absence of SO. These results indicate that SO droplets form complexes with pharmaceutical proteins that can potentially invoke early- and late-stage immune responses. Therefore, the use of SO-free cyclo-olefin polymer PFSs as primary containers for WFI could contribute to the enhanced safety of reconstituted biopharmaceuticals.

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  86. High-speed AFM reveals accelerated binding of agitoxin-2 to a K+ channel by induced fit

    Sumino A., Sumikama T., Uchihashi T., Oiki S.

    SCIENCE ADVANCES   Vol. 5 ( 7 )   2019.7

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    Agitoxin-2 (AgTx2) from scorpion venom is a potent blocker of K+ channels. The docking model has been elucidated, but it remains unclear whether binding dynamics are described by a two-state model (AgTx2-bound and AgTx2-unbound) or a more complicated mechanism, such as induced fit or conformational selection. Here, we observed the binding dynamics of AgTx2 to the KcsA channel using high-speed atomic force microscopy. From images of repeated binding and dissociation of AgTx2 to the channel, single-molecule kinetic analyses revealed that the affinity of the channel for AgTx2 increased during persistent binding and decreased during persistent dissociation. We propose a four-state model, including high- and low-affinity states of the channel, with relevant rate constants. An induced-fit pathway was dominant and accelerated binding by 400 times. This is the first analytical imaging of scorpion toxin binding in real time, which is applicable to various biological dynamics including channel ligands, DNA-modifier proteins, and antigen-antibody complexes.

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  87. High-speed AFM reveals accelerated binding of agitoxin-2 to a K+ channel by induced fit Reviewed

    A. Sumino, T. Sumikama, T. Uchihashi, S. Oiki

    Science Advances   Vol. 5 ( 7 ) page: 1 - 10   2019.7

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  88. Non-Thermoresponsive Decanano-sized Domains in Thermoresponsive Hydrogel Microspheres Revealed by Temperature-Controlled High-Speed Atomic Force Microscopy. Reviewed International journal

    Yuichiro Nishizawa, Shusuke Matsui, Kenji Urayama, Takuma Kureha, Mitsuhiro Shibayama, Takayuki Uchihashi, Daisuke Suzuki

    Angewandte Chemie (International ed. in English)   Vol. 58 ( 26 ) page: 8809 - 8813   2019.6

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    Despite the tremendous efforts devoted to the structural analysis of hydrogel microspheres (microgels), many details of their structures remain unclear. Reported in this study is that thermoresponsive poly(N-isopropyl acrylamide) (pNIPAm)-based microgels exhibit not only the widely accepted core-shell structures, but also inhomogeneous decanano-sized non-thermoresponsive spherical domains within their dense cores, which was revealed by temperature-controlled high-speed atomic force microscopy (TC-HS-AFM). Based on a series of experiments, it is concluded that the non-thermoresponsive domains are characteristic for pNIPAm microgels synthesized by precipitation polymerization, and plausible structures for microgels prepared by other polymerization techniques are proposed.

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  89. Non‐Thermoresponsive Decanano‐sized Domains in Thermoresponsive Hydrogel Microspheres Revealed by Temperature‐Controlled High‐Speed Atomic Force Microscopy Reviewed

    Yuichiro Nishizawa, Shusuke Matsui, Kenji Urayama, Takuma Kureha, Mitsuhiro Shibayama, Takayuki Uchihashi, Daisuke Suzuki

    Angewandte Chemie   Vol. 131 ( 26 ) page: 8901 - 8905   2019.6

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  90. Hydrogel Microellipsoids that Form Robust String-Like Assemblies at the Air/Water Interface. Reviewed International journal

    Kenshiro Honda, Yuka Sazuka, Kojiro Iizuka, Shusuke Matsui, Takayuki Uchihashi, Takuma Kureha, Mitsuhiro Shibayama, Takumi Watanabe, Daisuke Suzuki

    Angewandte Chemie (International ed. in English)   Vol. 58 ( 22 ) page: 7294 - 7298   2019.5

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    Soft colloidal particles such as hydrogel microspheres assemble at air/water or oil/water interfaces, where the soft colloids are highly deformed and their surface polymer chains are highly entangled with each other. Herein, we report the formation of robust one-dimensional, string-like colloidal assemblies through self-organization of hydrogel microspheres with shape anisotropy at the air/water interface of sessile droplets. Shape-anisotropic hydrogel microspheres were synthesized via two-step polymerization, whereby a hydrogel shell was formed onto preformed rigid microellipsoids. The shape anisotropy of the hydrogel microspheres was confirmed by transmission electron microscopy and high-speed atomic force microscopy as well as by light-scattering measurements. The present findings are crucial for the understanding of natural self-organization phenomena, where "softness" influences microscopic assembled structures such as those of Nostoc bacteria.

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  91. Mutational and Combinatorial Control of Self-Assembling and Disassembling of Human Proteasome α Subunits. Reviewed International journal

    Taichiro Sekiguchi, Tadashi Satoh, Eiji Kurimoto, Chihong Song, Toshiya Kozai, Hiroki Watanabe, Kentaro Ishii, Hirokazu Yagi, Saeko Yanaka, Susumu Uchiyama, Takayuki Uchihashi, Kazuyoshi Murata, Koichi Kato

    International journal of molecular sciences   Vol. 20 ( 9 )   2019.5

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    Eukaryotic proteasomes harbor heteroheptameric α-rings, each composed of seven different but homologous subunits α1-α7, which are correctly assembled via interactions with assembly chaperones. The human proteasome α7 subunit is reportedly spontaneously assembled into a homotetradecameric double ring, which can be disassembled into single rings via interaction with monomeric α6. We comprehensively characterized the oligomeric state of human proteasome α subunits and demonstrated that only the α7 subunit exhibits this unique, self-assembling property and that not only α6 but also α4 can disrupt the α7 double ring. We also demonstrated that mutationally monomerized α7 subunits can interact with the intrinsically monomeric α4 and α6 subunits, thereby forming heterotetradecameric complexes with a double-ring structure. The results of this study provide additional insights into the mechanisms underlying the assembly and disassembly of proteasomal subunits, thereby offering clues for the design and creation of circularly assembled hetero-oligomers based on homo-oligomeric structural frameworks.

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  92. Construction of a Quadrangular Tetramer and a Cage-Like Hexamer from Three-Helix Bundle-Linked Fusion Proteins. Reviewed International journal

    Takaaki Miyamoto, Yugo Hayashi, Keito Yoshida, Hiroki Watanabe, Takayuki Uchihashi, Kento Yonezawa, Nobutaka Shimizu, Hironari Kamikubo, Shun Hirota

    ACS synthetic biology   Vol. 8 ( 5 ) page: 1112 - 1120   2019.5

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    Self-assembled protein nanostructures have gained interest, owing to their potential applications in biomaterials; however, successful design and construction of protein nanostructures are limited. Herein, we constructed fusion protein 1 by linking the C-terminus of a dimerization domain and the N-terminus of another dimerization domain with a three-helix bundle protein, where it self-assembled mainly into tetramers. By replacing the C-terminal dimerization domain of 1 with a trimerization domain (fusion protein 2), hexamers were mainly obtained. According to ab initio structural models reconstructed from the small-angle X-ray scattering data, the tetramer of 1 and hexamer of 2 adopted quadrangle and cage-like structures, respectively, although they were combinations of different conformations. High-speed atomic force microscopy observations indicated that the tetramer and hexamer exhibit conformational dynamics. These results show that the present method utilizing three-helix bundle-linked fusion proteins is useful in the construction of protein nanostructures.

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  93. Inner lumen proteins stabilize doublet microtubules in cilia and flagella. Reviewed International journal

    Mikito Owa, Takayuki Uchihashi, Haru-Aki Yanagisawa, Takashi Yamano, Hiro Iguchi, Hideya Fukuzawa, Ken-Ichi Wakabayashi, Toshio Ando, Masahide Kikkawa

    Nature communications   Vol. 10 ( 1 ) page: 1143 - 1143   2019.3

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    Motile cilia are microtubule-based organelles that play important roles in most eukaryotes. Although axonemal microtubules are sufficiently stable to withstand their beating motion, it remains unknown how they are stabilized while serving as tracks for axonemal dyneins. To address this question, we have identified two uncharacterized proteins, FAP45 and FAP52, as microtubule inner proteins (MIPs) in Chlamydomonas. These proteins are conserved among eukaryotes with motile cilia. Using cryo-electron tomography (cryo-ET) and high-speed atomic force microscopy (HS-AFM), we show that lack of these proteins leads to a loss of inner protrusions in B-tubules and less stable microtubules. These protrusions are located near the inner junctions of doublet microtubules and lack of both FAP52 and a known inner junction protein FAP20 results in detachment of the B-tubule from the A-tubule, as well as flagellar shortening. These results demonstrate that FAP45 and FAP52 bind to the inside of microtubules and stabilize ciliary axonemes.

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  94. A ring-shaped hemoprotein trimer thermodynamically controlled by the supramolecular heme-heme pocket interaction

    Oohora Koji, Kajihara Ryota, Fujimaki Nishiki, Uchihashi Takayuki, Hayashi Takashi

    CHEMICAL COMMUNICATIONS   Vol. 55 ( 11 ) page: 1544 - 1547   2019.2

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  95. Microtubule self-healing and defect creation investigated by in-line force measurements during high-speed atomic force microscopy imaging Reviewed

    Christian Ganser, Takayuki Uchihashi

    Nanoscale   Vol. 11 ( 1 ) page: 125 - 135   2019.1

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    DOI: 10.1039/c8nr07392a

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  96. Single-unit imaging of membrane protein-embedded nanodiscs from two oriented sides by high-speed atomic force microscopy Reviewed

    T. Haruyama, Y. Sugano, N. Kodera, T. Uchihashi, T. Ando, Y. Tanaka, H. Konno, T. Tsukazaki

    Structure   Vol. 27 ( 1 ) page: 152 - +   2019.1

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    DOI: 10.1016/j.str.2018.09.005

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  97. Metastable asymmetrical structure of a shaftless V1 motor Reviewed

    Shintaro Maruyama, Kano Suzuki, Motonori Imamura, Hikaru Sasaki, Hideyuki Matsunami, Kenji Mizutani, Yasuko Saito, Fabiana L. Imai, Yoshiko Ishizuka-Katsura, Tomomi Kimura-Someya, Mikako Shirouzu, Takayuki Uchihashi, Toshio Ando, Ichiro Yamato, Takeshi Murata

    Science Advances   Vol. 5 ( 1 ) page: eaau8149 - eaau8149   2019.1

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    V<sub>1</sub>-ATPase is an ATP-driven rotary motor that is composed of a ring-shaped A<sub>3</sub>B<sub>3</sub> complex and a central DF shaft. The nucleotide-free A<sub>3</sub>B<sub>3</sub> complex of <italic>Enterococcus hirae</italic>, composed of three identical A<sub>1</sub>B<sub>1</sub> heterodimers, showed a unique asymmetrical structure, probably due to the strong binding of the N-terminal barrel domain, which forms a crown structure. Here, we mutated the barrel region to weaken the crown, and performed structural analyses using high-speed atomic force microscopy and x-ray crystallography of the mutant A<sub>3</sub>B<sub>3</sub>. The nucleotide-free mutant A<sub>3</sub>B<sub>3</sub> complex had a more symmetrical open structure than the wild type. Binding of nucleotides produced a closely packed spiral-like structure with a disrupted crown. These findings suggest that wild-type A<sub>3</sub>B<sub>3</sub> forms a metastable (stressed) asymmetric structure composed of unstable A<sub>1</sub>B<sub>1</sub> conformers due to the strong constraint of the crown. The results further the understanding of the principle of the cooperative transition mechanism of rotary motors.

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  98. 高速原子間力顕微鏡による生体・合成高分子の動態イメージング Invited

    内橋貴之

    高分子   Vol. 68 ( 01 ) page: 564 - 568   2019

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  99. Development of Wide-area Tip-scanning High-speed Atomic Force Microscopy

    Watanabe Hiroki, Uchihashi Takayuki

    2019 IEEE INTERNATIONAL CONFERENCE ON MANIPULATION, MANUFACTURING AND MEASUREMENT ON THE NANOSCALE (IEEE 3M-NANO)     page: 281 - 285   2019

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  100. Real-time Nanoscale Visualization of Biological Molecules at Work with High-speed Atomic Force Microscopy

    Uchihashi Takayuki

    2019 IEEE INTERNATIONAL CONFERENCE ON MANIPULATION, MANUFACTURING AND MEASUREMENT ON THE NANOSCALE (IEEE 3M-NANO)     page: 253 - 256   2019

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  101. Development of Wide-area Tip-scanning High-speed Atomic Force Microscopy

    Hiroki Watanabe, Takayuki Uchihashi

    2019 IEEE INTERNATIONAL CONFERENCE ON MANIPULATION, MANUFACTURING AND MEASUREMENT ON THE NANOSCALE (IEEE 3M-NANO)     page: 281 - 285   2019

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    Recently a tip-scanning type high-speed atomic force microscopy (HS-AFM), which is combined with total internal reflection fluorescence microscope (TIRFM), has been developed. Using this system, the topographic and fluorescence images of biological samples in action have been simultaneously visualized. However, the maximum scanning range of the combined system was limited to similar to 20 mu m and similar to 3 mu m in X- and Y-directions respectively. This limitation makes it impossible to visualize the dynamics of much larger samples, including bacteria or mammalian cells. Here, we improved a laser-tracking system and expand the tracking range in X- and Y-directions to similar to 50 mu m and similar to 32 mu m, respectively by increasing an angular variation of a mirror-tilter unit changing the arrangement and the adhesive area between piezoactuators and base plate of the unit. The Shift of the X-direction of the scanner with large displacement of the Z-scanner is eliminated by modifying an input triangular signal to the mirror-tilter unit. We also developed a scanner, which could cover in a wider imaging area, for the expanded mirror-tilter unit in the tip-scanning HS-AFM system. The capability of this expanded system is demonstrated by measuring the laser-spot displacement, large-area imaging of a square grating sample and eukaryotic cells.

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  102. Versatility of RNA-Binding Proteins in Living Cells Reviewed

    Riki Kurokawa, Akihide Takeuchi, Nobuyuki Shiina, Masato Katahira, Takefumi Yamashita, Yoko Matsuno, Keisuke Hitachi, Shinsuke Ishigaki, Nesreen Haamad, Ryoma Yoneda, Naomi Ueda, Kei Iida, Motoyasu Hosokawa, Masatoshi Hagiwara, Mamiko Iida, Tsukasa Mashima, Yudai Yamaoki, Masatomo So, Takashi Nagata, Gen Sobue, Keiko Kondo, Hiroki Watanabe, Takayuki Uchihashi

    Biomedical Sciences   Vol. 5 ( 1 ) page: 7 - 7   2019

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    DOI: 10.11648/j.bs.20190501.12

  103. Direct Observation and Manipulation of Supramolecular Polymerization by High-Speed Atomic Force Microscopy

    Fukui Tomoya, Uchihashi Takayuki, Sasaki Norihiko, Watanabe Hiroki, Takeuchi Masayuki, Sugiyasu Kazunori

    ANGEWANDTE CHEMIE-INTERNATIONAL EDITION   Vol. 57 ( 47 ) page: 15465 - 15470   2018.11

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    Despite recent advances in mechanistic understanding and controlled-synthesis methodologies regarding synthetic supramolecular assemblies, it has remained challenging to capture the molecular-level phenomena in real time, thus hindering further progress in this research field. In this study, we applied high-speed atomic-force microscopy (AFM), which has extraordinary spatiotemporal resolution (1 nm and sub-100 ms), to capture dynamic events occurring during synthetic molecular self-assembly. High-speed AFM permitted the visualization of unique dynamic behavior, such as seeded growth and self-repair in real time. Furthermore, scanning-probe AFM permitted the site-specific manipulation and functionalization of a molecular self-assembly. This powerful combination of bottom-up and top-down approaches at the molecular level should enable targeted syntheses of unprecedented functional nanoarchitectures.

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  104. Monitoring Thermoresponsive Morphological Changes in Individual Hydrogel Microspheres. Reviewed International journal

    Shusuke Matsui, Yuichiro Nishizawa, Takayuki Uchihashi, Daisuke Suzuki

    ACS omega   Vol. 3 ( 9 ) page: 10836 - 10842   2018.9

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    Real-time morphology/structure changes in individual hydrogel microspheres (microgels) were directly visualized at high spatiotemporal resolution using high-speed atomic force microscopy (HS-AFM) under temperature control ranging from room temperature to ∼40 °C. The recorded HS-AFM movies demonstrate that the size and morphology of thermoresponsive poly(N-isopropyl acrylamide)-based microgels change with increasing temperature at the individual microgel level. Specifically, the height of the microgels gradually decreases and domain structures appeared even below the volume phase transition temperature. Moreover, the domain structure is retained, even after the microgels have fully collapsed. The present study thus demonstrates that temperature-controlled HS-AFM is a useful tool for monitoring stimulus-responsiveness of microgels. In the near future, it should furthermore be possible to extend this temperature-controlled HS-AFM to other stimulus-responsive materials, including autonomously oscillating microgels.

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  105. Supramolecular Hemoprotein Assembly with a Periodic Structure Showing Heme-Heme Exciton Coupling

    Oohora Koji, Fujimaki Nishiki, Kajihara Ryota, Watanabe Hiroki, Uchihashi Takayuki, Hayashi Takashi

    JOURNAL OF THE AMERICAN CHEMICAL SOCIETY   Vol. 140 ( 32 ) page: 10145 - 10148   2018.8

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  106. Revealing circadian mechanisms of integration and resilience by visualizing clock proteins working in real time

    Mori Tetsuya, Sugiyama Shogo, Byrne Mark, Johnson Carl Hirschie, Uchihashi Takayuki, Ando Toshio

    NATURE COMMUNICATIONS   Vol. 9 ( 1 ) page: 3245   2018.8

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    DOI: 10.1038/s41467-018-05438-4

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  107. Scanning probe microscopy

    Takahashi T., Fukui K.I., Kageshima M., Komeda T., Nakajima K., Nakayama T., Sumitomo K., Uchihashi T.

    Japanese Journal of Applied Physics   Vol. 57 ( 8 )   2018.8

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  108. Quantum-dot antibody conjugation visualized at the single-molecule scale with high-speed atomic force microscopy

    Takayuki Umakoshi, Hikari Udaka, Takayuki Uchihashi, Toshio Ando, Miho Suzuki, Takeshi Fukuda

    COLLOIDS AND SURFACES B-BIOINTERFACES   Vol. 167   page: 267 - 274   2018.7

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    Conjugates of semiconductor quantum dots (QDs) and antibodies have emerged as a promising bioprobes due to their great combination of QD's efficient fluorescence and the high specificity of antigen-antibody reactions. For further developments in this field, it is essential to understand the molecular conformation of the QD-antibody conjugates at the single-molecule scale. Here, we report on the direct imaging of QD-antibody conjugates at the single-molecule scale by using high-speed atomic force microscopy (HS-AFM). Owing to the high spatiotemporal resolution of HS-AFM, we observed the dynamic splitting of individual antibodies during the conjugation process. QD-antibody conjugates were also clearly visualized at the single-molecule scale details. Several important features were even discovered through dynamic observation of the QD-antibody conjugates. We observed an intermediate state of conjugation, where the antibodies attached and detached to QDs repeatedly. We also revealed that the attached antibodies were not steady but drastically fluctuated in their recognition areas due to the Brownian motion. We also demonstrated that HS-AFM observation is useful for the quantitative analysis of fabricated conjugates. (C) Elsevier B.V. All rights reserved.

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  109. Translating MOF chemistry into supramolecular chemistry: Soluble coordination nanofibers showing efficient photon upconversion

    Masanori Hosoyamada, Nobuhiro Yanai, Keisuke Okumura, Takayuki Uchihashi, Nobuo Kimizuka

    Chemical Communications   Vol. 54 ( 50 ) page: 6828 - 6831   2018.6

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    A method for synthesizing coordination nanofibers by extracting the structural motifs of metal-organic frameworks (MOFs) is demonstrated. In these soluble nanofibers, multiple chromophores with largely different sizes and shapes can be arranged at desired compositions, and excited triplet energy migrates among the densely assembled chromophore arrays, showing an efficient photon upconversion even at very low concentration.

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  110. Dynamic structural states of CIpB involved in its disaggregation function

    Takayuki Uchihashi, Yo-hei Watanabe, Yosuke Nakazaki, Takashi Yamasaki, Hiroki Watanabe, Takahiro Maruno, Kentaro Ishii, Susumu Uchiyama, Chihong Song, Kazuyoshi Murata, Ryota Iino, Toshio Ando

    NATURE COMMUNICATIONS   Vol. 9 ( 1 ) page: 2147   2018.6

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    The ATP-dependent bacterial protein disaggregation machine, CIpB belonging to the AAA+ superfamily, refolds toxic protein aggregates into the native state in cooperation with the cognate Hsp70 partner. The ring-shaped hexamers of CIpB unfold and thread its protein substrate through the central pore. However, their function-related structural dynamics has remained elusive. Here we directly visualize CIpB using high-speed atomic force microscopy (HS-AFM) to gain a mechanistic insight into its disaggregation function. The HS-AFM movies demonstrate massive conformational changes of the hexameric ring during ATP hydrolysis, from a round ring to a spiral and even to a pair of twisted half-spirals. HS-AFM observations of Walker-motif mutants unveil crucial roles of ATP binding and hydrolysis in the oligomer formation and structural dynamics. Furthermore, repressed and hyperactive mutations result in significantly different oligomeric forms. These results provide a comprehensive view for the ATP-driven oligomeric-state transitions that enable CIpB to disentangle protein aggregates.

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  111. Sweeping of Adsorbed Therapeutic Protein on Prefillable Syringes Promotes Micron Aggregate Generation Reviewed

    Takahiro Maruno, Hiroki Watanabe, Saki Yoneda, Takayuki Uchihashi, Satoru Adachi, Kunihito Arai, Taichi Sawaguchi, Susumu Uchiyama

    Journal of Pharmaceutical Sciences   Vol. 107 ( 6 ) page: 1521 - 1529   2018.6

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    This study evaluated how differences in the surface properties of prefillable syringe barrels and in-solution sampling methods affect micron aggregates and protein adsorption levels. Three syringe types (glass barrel with silicone oil coating [GLS/SO+], glass barrel without silicone oil coating [GLS/SO−], and cyclo-olefin polymer [COP] barrel syringes) were tested with 3 therapeutic proteins (adalimumab, etanercept, and infliximab) using 2 sampling methods (aspiration or ejection). After quiescent incubation, solutions sampled by aspiration exhibited no significant change in micron aggregate concentration in any syringes, whereas those sampled by ejection exhibited increased micron aggregates in both GLS syringe types. Micron aggregate concentration in ejected solutions generally increased with increasing density of adsorbed proteins. Notably, COP syringes contained the lowest micron aggregate concentrations, which were independent of the sampling method. Correspondingly, the adsorbed protein density on COP syringes was the lowest at 1-2 mg/m2, which was much less compared with that on GLS syringes and was calculated to be equivalent to only 1–2 protein layers, as visually confirmed by high-speed atomic force microscopy. These data indicate that low-adsorption prefillable syringes should be used for therapeutic proteins because protein aggregate concentration in the ejected solution is elevated by increased protein adsorption to the syringe surface.

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  112. Oligomeric states of microbial rhodopsins determined by high-speed atomic force microscopy and circular dichroic spectroscopy

    Mikihiro Shibata, Keiichi Inoue, Kento Ikeda, Masae Konno, Manish Singh, Chihiro Kataoka, Rei Abe-Yoshizumi, Hideki Kandori, Takayuki Uchihashi

    Scientific Reports   Vol. 8 ( 1 ) page: 8262   2018.5

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    Oligomeric assembly is a common feature of membrane proteins and often relevant to their physiological functions. Determining the stoichiometry and the oligomeric state of membrane proteins in a lipid bilayer is generally challenging because of their large size, complexity, and structural alterations under experimental conditions. Here, we use high-speed atomic force microscopy (HS-AFM) to directly observe the oligomeric states in the lipid membrane of various microbial rhodopsins found within eubacteria to archaea. HS-AFM images show that eubacterial rhodopsins predominantly exist as pentamer forms, while archaeal rhodopsins are trimers in the lipid membrane. In addition, circular dichroism (CD) spectroscopy reveals that pentameric rhodopsins display inverted CD couplets compared to those of trimeric rhodopsins, indicating different types of exciton coupling of the retinal chromophore in each oligomer. The results clearly demonstrate that the stoichiometry of the fundamental oligomer of microbial rhodopsins strongly correlate with the phylogenetic tree, providing a new insight into the relationship between the oligomeric structure and function-structural evolution of microbial rhodopsins.

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  113. Structural properties determining low K affinity of the selectivity filter in the TWIK1 K channel

    Hisao Tsukamoto, Masahiro Higashi, Hideyoshi Motoki, Hiroki Watanabe, Christian Ganser, Koichi Nakajo, Yoshihiro Kubo, Takayuki Uchihashi, Yuji Furutani

    Journal of Biological Chemistry   Vol. 293 ( 18 ) page: 6969 - 6984   2018.5

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    Canonical K channels are tetrameric and highly K-selective, whereas two-pore– domain K (K2P) channels form dimers, but with a similar pore architecture. A two-pore– domain potassium channel TWIK1 (KCNK1 or K2P1) allows permeation of Na and other monovalent ions, resulting mainly from the presence of Thr-118 in the P1 domain. However, the mechanistic basis for this reduced selectivity is unclear. Using ion-exchange–induced difference IR spectroscopy, we analyzed WT TWIK1 and T118I (highly K-selective) and L228F (substitution in the P2 domain) TWIK1 variants and found that in the presence of K ions, WT and both variants exhibit an amide-I band at 1680 cm1. This band corresponds to interactions of the backbone carbonyls in the selectivity filter with K, a feature very similar to that of the canonical K channel KcsA. Computational analysis indicated that the relatively high frequency for the amide-I band is well explained by impairment of hydrogen bond formation with water molecules. Moreover, concentration-dependent spectral changes indicated that the K affinity of the WT selectivity filter was much lower than those of the variants. Furthermore, only the variants displayed a higher frequency shift of the 1680-cm1 band upon changes from K to Rb or Cs conditions. High-speed atomic force microscopy disclosed that TWIK1’s surface morphology largely does not change in K and Na solutions. Our results reveal the local conformational changes of the TWIK1 selectivity filter and suggest that the amide-I bands may be useful “molecular fingerprints” for assessing the properties of other K channels.

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  114. Construction of a Triangle-Shaped Trimer and a Tetrahedron Using an α-Helix-Inserted Circular Permutant of Cytochrome c 555

    Akiya Oda, Satoshi Nagao, Masaru Yamanaka, Ikki Ueda, Hiroki Watanabe, Takayuki Uchihashi, Naoki Shibata, Yoshiki Higuchi, Shun Hirota

    Chemistry - An Asian Journal   Vol. 13 ( 8 ) page: 964 - 967   2018.4

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  115. Insight into structural remodeling of the FlhA ring responsible for bacterial flagellar type III protein export

    Naoya Terahara, Yumi Inoue, Noriyuki Kodera, Yusuke V. Morimoto, Takayuki Uchihashi, Katsumi Imada, Toshio Ando, Keiichi Namba, Tohru Minamino

    Science Advances   Vol. 4 ( 4 ) page: eaao7054   2018.4

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    The bacterial flagellum is a supramolecular motility machine. Flagellar assembly begins with the basal body, followed by the hook and finally the filament. A carboxyl-terminal cytoplasmic domain of FlhA (FlhAC) forms a nonameric ring structure in the flagellar type III protein export apparatus and coordinates flagellar protein export with assembly. However, the mechanism of this process remains unknown. We report that a flexible linker of FlhAC (FlhAL) is required not only for FlhAC ring formation but also for substrate specificity switching of the protein export apparatus from the hook protein to the filament protein upon completion of the hook structure. FlhAL was required for cooperative ring formation of FlhAC. Alanine substitutions of residues involved in FlhAC ring formation interfered with the substrate specificity switching, thereby inhibiting filament assembly at the hook tip. These observations lead us to propose a mechanistic model for export switching involving structural remodeling of FlhAC.

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  116. Front Cover: Construction of a Triangle-Shaped Trimer and a Tetrahedron Using an α-Helix-Inserted Circular Permutant of Cytochrome c 555 (Chem. Asian J. 10/2018) Reviewed

    Akiya Oda, Satoshi Nagao, Masaru Yamanaka, Ikki Ueda, Hiroki Watanabe, Takayuki Uchihashi, Naoki Shibata, Yoshiki Higuchi, Shun Hirota

    Chemistry - An Asian Journal   Vol. 13 ( 10 ) page: 1229   2018.4

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  117. High-speed AFM reveals detail understanding in adsorption of soft hydrogel microspheres onto solid substrate in aqueous solution

    Matsui Shusuke, Uchihashi Takayuki, Suzuki Daisuke

    ABSTRACTS OF PAPERS OF THE AMERICAN CHEMICAL SOCIETY   Vol. 255   page: .   2018.3

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  118. Real-time adsorption behavior of hydrogel microspheres onto solid/liquid interface

    Matsui Shusuke, Uchihashi Takayuki, Suzuki Daisuke

    ABSTRACTS OF PAPERS OF THE AMERICAN CHEMICAL SOCIETY   Vol. 255   page: .   2018.3

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  119. High-speed AFM reveals detail understanding in adsorption of soft hydrogel microspheres onto solid substrate in aqueous solution

    Matsui Shusuke, Uchihashi Takayuki, Suzuki Daisuke

    ABSTRACTS OF PAPERS OF THE AMERICAN CHEMICAL SOCIETY   Vol. 255   page: .   2018.3

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  120. Real-time adsorption behavior of hydrogel microspheres onto solid/liquid interface

    Matsui Shusuke, Uchihashi Takayuki, Suzuki Daisuke

    ABSTRACTS OF PAPERS OF THE AMERICAN CHEMICAL SOCIETY   Vol. 255   page: .   2018.3

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  121. Negatively Charged Lipids Are Essential for Functional and Structural Switch of Human 2-Cys Peroxiredoxin II Reviewed

    Takamitsu Haruyama, Takayuki Uchihashi, Yutaro Yamada, Noriyuki Kodera, Toshio Ando, Hiroki Konno

    Journal of Molecular Biology   Vol. 430 ( 5 ) page: 602 - 610   2018.3

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    The function of ubiquitous 2-Cys peroxiredoxins (Prxs) can be converted alternatively from peroxidases to molecular chaperones. This conversion has been reported to occur by the formation of high-molecular-weight (HMW) complexes upon overoxidation of or ATP/ADP binding to 2-Cys Prxs, but its mechanism is not well understood. Here, we show that upon binding to phosphatidylserine or phosphatidylglycerol dimeric human 2-Cys PrxII (hPrxII) is assembled to trefoil-shaped small oligomers (possibly hexamers) with full chaperone and null peroxidase activities. Spherical HMW complexes are formed, only when phosphatidylserine or phosphatidylglycerol is bound to overoxidized or ATP/ADP-bound hPrxII. The spherical HMW complexes are lipid vesicles covered with trefoil-shaped oligomers arranged in a hexagonal lattice pattern. Thus, these lipids with a net negative charge, which can be supplied by increased membrane trafficking under oxidative stress, are essential for the structural and functional switch of hPrxII and possibly most 2-Cys Prxs.

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  122. Rate constants, processivity, and productive binding ratio of chitinase A revealed by single-molecule analysis. International journal

    Akihiko Nakamura, Tomoyuki Tasaki, Yasuko Okuni, Chihong Song, Kazuyoshi Murata, Toshiya Kozai, Mayu Hara, Hayuki Sugimoto, Kazushi Suzuki, Takeshi Watanabe, Takayuki Uchihashi, Hiroyuki Noji, Ryota Iino

    Physical chemistry chemical physics : PCCP   Vol. 20 ( 5 ) page: 3010 - 3018   2018.2

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    Serratia marcescens chitinase A is a linear molecular motor that hydrolyses crystalline chitin in a processive manner. Here, we quantitatively determined the rate constants of elementary reaction steps, including binding (kon), translational movement (ktr), and dissociation (koff) with single-molecule fluorescence imaging. The kon for a single chitin microfibril was 2.1 × 109 M-1 μm-1 s-1. The koff showed two components, k (3.2 s-1, 78%) and k (0.38 s-1, 22%), corresponding to bindings to different crystal surfaces. From the kon, k, k and ratio of fast and slow dissociations, dissociation constants for low and high affinity sites were estimated as 2.0 × 10-9 M μm and 8.1 × 10-10 M μm, respectively. The ktr was 52.5 nm s-1, and processivity was estimated as 60.4. The apparent inconsistency between high turnover (52.5 s-1) calculated from ktr and biochemically determined low kcat (2.6 s-1) is explained by a low ratio (4.8%) of productive enzymes on the chitin surface (52.5 s-1 × 0.048 = 2.5 s-1). Our results highlight the importance of single-molecule analysis in understanding the mechanism of enzymes acting on a solid-liquid interface.

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  123. Correction: Rate constants, processivity, and productive binding ratio of chitinase A revealed by single-molecule analysis. International journal

    Akihiko Nakamura, Tomoyuki Tasaki, Yasuko Okuni, Chihong Song, Kazuyoshi Murata, Toshiya Kozai, Mayu Hara, Hayuki Sugimoto, Kazushi Suzuki, Takeshi Watanabe, Takayuki Uchihashi, Hiroyuki Noji, Ryota Iino

    Physical chemistry chemical physics : PCCP   Vol. 20 ( 5 ) page: 3844 - 3844   2018.2

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    Correction for 'Rate constants, processivity, and productive binding ratio of chitinase A revealed by single-molecule analysis' by Akihiko Nakamura et al., Phys. Chem. Chem. Phys., 2018, DOI: .

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  124. Dynamic Observation of Kai Proteins by HS-AFM Reveals a Mechanism of the Robustness in the Cyanobacterial Circadian Oscillator

    Sugiyama Shogo, Mori Tetsya, Byrne Mark, Uchihashi Takayuki, Johnson Carl H., Ando Toshio

    BIOPHYSICAL JOURNAL   Vol. 114 ( 3 ) page: 68A - 68A   2018.2

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  125. Dynamic Observation of Kai Proteins by HS-AFM Reveals a Mechanism of the Robustness in the Cyanobacterial Circadian Oscillator

    Sugiyama Shogo, Mori Tetsya, Byrne Mark, Uchihashi Takayuki, Johnson Carl H, Ando Toshio

    BIOPHYSICAL JOURNAL   Vol. 114 ( 3 ) page: 68A-68A   2018.2

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  126. Applications of high-speed atomic force microscopy to real-time visualization of dynamic biomolecular processes.

    Uchihashi T, Scheuring S

    Biochimica et biophysica acta   Vol. 1862 ( 2 ) page: 229 - 240   2018.2

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    DOI: 10.1016/j.bbagen.2017.07.010

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  127. Dynamic clustering of dynamin-amphiphysin helices regulates membrane constriction and fission coupled with GTP hydrolysis Reviewed

    Tetsuya Takeda, Toshiya Kozai, Huiran Yang, Daiki Ishikuro, Kaho Seyama, Yusuke Kumagai, Tadashi Abe, Hiroshi Yamada, Takayuki Uchihashi, Toshio Ando, Kohji Takei

    eLife   Vol. 7   2018.1

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    Dynamin is a mechanochemical GTPase essential for membrane fission during clathrin-mediated endocytosis. Dynamin forms helical complexes at the neck of clathrin-coated pits and their structural changes coupled with GTP hydrolysis drive membrane fission. Dynamin and its binding protein amphiphysin cooperatively regulate membrane remodeling during the fission, but its precise mechanism remains elusive. In this study, we analyzed structural changes of dynamin-amphiphysin complexes during the membrane fission using electron microscopy (EM) and high-speed atomic force microscopy (HS-AFM). Interestingly, HS-AFM analyses show that the dynamin-amphiphysin helices are rearranged to form clusters upon GTP hydrolysis and membrane constriction occurs at protein-uncoated regions flanking the clusters. We also show a novel function of amphiphysin in size control of the clusters to enhance biogenesis of endocytic vesicles. Our approaches using combination of EM and HS-AFM clearly demonstrate new mechanistic insights into the dynamics of dynamin-amphiphysin complexes during membrane fission.

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  128. Conversion of functionally undefined homopentameric protein PbaA into a proteasome activator by mutational modification of its C-terminal segment conformation Reviewed International journal

    Maho Yagi-Utsumi, Arunima Sikdar, Toshiya Kozai, Rintaro Inoue, Masaaki Sugiyama, Takayuki Uchihashi, Hirokazu Yagi, Tadashi Satoh, Koichi Kato

    PROTEIN ENGINEERING DESIGN & SELECTION   Vol. 31 ( 1 ) page: 29 - 36   2018.1

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    Recent bioinformatic analyses identified proteasome assembly chaperone-like proteins, PbaA and PbaB, in archaea. PbaB forms a homotetramer and functions as a proteasome activator, whereas PbaA does not interact with the proteasome despite the presence of an apparent C-terminal proteasome activation motif. We revealed that PbaA forms a homopentamer predominantly in the closed conformation with its C-terminal segments packed against the core domains, in contrast to the PbaB homotetramer with projecting C-terminal segments. This prompted us to create a novel proteasome activator based on a well-characterized structural framework. We constructed a panel of chimeric proteins comprising the homopentameric scaffold of PbaA and C-terminal segment of PbaB and subjected them to proteasome-activating assays as well as small-angle X-ray scattering and high-speed atomic force microscopy. The results indicated that the open conformation and consequent proteasome activation activity could be enhanced by replacement of the crystallo-graphically disordered C-terminal segment of PbaA with the corresponding disordered segment of PbaB. Moreover, these effects can be produced just by incorporating two glutamate residues into the disordered C-terminal segment of PbaA, probably due to electrostatic repulsion among the negatively charged segments. Thus, we successfully endowed a functionally undefined protein with proteasome-activating activity by modifying its C-terminal segment.

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  129. Visualization of Protein Dynamics using High-Speed Atomic Force Microscopy and Image Analysis

    Uchihashi Takayuki

    JOURNAL OF COMPUTER CHEMISTRY-JAPAN   Vol. 17 ( 1 ) page: 20-30 - 30   2018

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    <p>Recent progress of high-speed atomic force microscopy (HS-AFM) has enabled us to visualize dynamic conformational change and molecular interaction of proteins in realtime under a physiological condition. Molecular mechanisms through dynamic phenomena that have been deduced from the accumulation of experimental evidence by various analytical techniques are now able to be directly and intuitively understood from AFM movies. While a movie provides a great deal of information compared to a static image, extracting useful information based on objective criteria is essential for the next step. This review describes representative HS-AFM data for three typical dynamic biological phenomena; conformational dynamics of single proteins, linear movement of enzymes and velocity analysis and dynamic molecular interaction together with imaging processing techniques for interpreting AFM data and efficient analysis of successive images.</p>

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  130. Optimum Substrates for Imaging Biological Molecules with High-Speed Atomic Force Microscopy

    Uchihashi, T; Watanabe, H; Kodera, N

    NANOSCALE IMAGING: METHODS AND PROTOCOLS   Vol. 1814   page: 159 - 179   2018

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    Recent progresses in high-speed atomic force microscopy (HS-AFM) have enabled us to directly visualize dynamic processes of various proteins in liquid conditions. One of the key factors leading to successful HS-AFM observations is the selection of an appropriate substrate depending on molecules to be observed. For the HS-AFM imaging, a target molecule must be absorbed on a substrate by controlling its orientation without impairing the dynamics or physiological function of the molecule. In this chapter, we describe protocols for preparation of substrates that have been used for HS-AFM and then introduce observation examples on dynamic processes of biological molecules.

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  131. High-speed scanning near-field optical microscopy

    Umakoshi T., Fukuda S., Uchihashi T., Verma P., Ando T.

    Optics InfoBase Conference Papers   Vol. Part F125-JSAP 2018   2018

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    Scanning near-field optical microscopy (SNOM) has been recognized as a powerful technique for super-resolution optical imaging [1]. It has been even more unique compared with other super-resolution fluorescent imaging techniques because it utilizes near-field light at a metallic tip as a nano-light-source, which realizes super-resolution not only in fluorescence but also in any other optical signals such as Raman scattering, infrared absorption and photoluminescence. Such a strong advantage has been stimulating biological fields as a new class of analytical techniques. However, it has been yet highly challenging to apply SNOM for biological samples. Although there have been several issues for biological applications, one of the major problems is its low imaging speed. Because it requires to physically scan a metallic tip, the imaging rate was typically limited to several minutes per frame, which is too slow to capture dynamic biological samples.

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  132. Optimum Substrates for Imaging Biological Molecules with High-Speed Atomic Force Microscopy Reviewed

    Takayuki Uchihashi, Hiroki Watanabe, Noriyuki Kodera

    Methods in Molecular Biology   Vol. 1814   page: 159 - 179   2018

  133. エンドサイトーシス生物学の新展開 ダイナミンによる膜切断過程の動態イメージング

    竹田 哲也, 小財 稔矢, 楊 恵然, 石黒 大輝, 背山 佳穂, 熊谷 祐介, 阿部 匡史, 山田 浩司, 内橋 貴之, 安藤 敏夫, 竹居 孝二

    生命科学系学会合同年次大会   Vol. 2017年度   page: [4AW17 - 2]   2017.12

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  134. Two-step process for disassembly mechanism of proteasome α7 homo-tetradecamer by α6 revealed by high-speed atomic force microscopy. International journal

    Toshiya Kozai, Taichiro Sekiguchi, Tadashi Satoh, Hirokazu Yagi, Koichi Kato, Takayuki Uchihashi

    Scientific reports   Vol. 7 ( 1 ) page: 15373 - 15373   2017.11

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    The 20S proteasome is a core particle of the eukaryotic proteasome responsible for proteolysis and is composed of layered α and β hetero-heptameric rings. The α7 subunit, which is one of components of the α ring, is known to self-assemble into a double-ringed homo-tetradecamer composed of two layers of the α7 heptameric ring. The α7 tetradecamer is known to disassemble upon the addition of α6 subunit, producing a 1:7 hetero-octameric α6-α7 complex. However, the detailed disassembly mechanism remains unclear. Here, we applied high-speed atomic force microscopy (HS-AFM) to dissect the disassembly process of the α7 double ring caused by interaction with the α6. HS-AFM movies clearly demonstrated two different modes of interaction in which the α6 monomer initially cracks at the interface between the stacked two α7 single rings and the subsequent intercalation of the α6 monomer in the open pore of the α7 single ring blocks the re-association of the single rings into the double ring. This result provides a mechanistic insight about the disassembly process of non-native homo-oligomers formed by proteasome components which is crucial for the initial process for assembly of 20S proteasome.

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  135. Real-space and real-time dynamics of CRISPR-Cas9 visualized by high-speed atomic force microscopy

    Mikihiro Shibata, Hiroshi Nishimasu, Noriyuki Kodera, Seiichi Hirano, Toshio Ando, Takayuki Uchihashi, Osamu Nureki

    NATURE COMMUNICATIONS   Vol. 8 ( 1 ) page: 1430   2017.11

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    The CRISPR-associated endonuclease Cas9 binds to a guide RNA and cleaves double-stranded DNA with a sequence 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.

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  136. Na+-induced structural transition of MotPS for stator assembly of the Bacillus flagellar motor

    Naoya Terahara, Noriyuki Kodera, Takayuki Uchihashi, Toshio Ando, Keiichi Namba, Tohru Minamino

    SCIENCE ADVANCES   Vol. 3 ( 11 ) page: eaao4119   2017.11

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    The bacterial flagellar motor consists of a rotor and a dozen stator units and regulates the number of active stator units around the rotor in response to changes in the environment. The MotPS complex is a Na+-type stator unit in the Bacillus subtilis flagellar motor and binds to the peptidoglycan layer through the peptidoglycan-binding (PGB) domain of MotS to act as the stator. The MotPS complex is activated in response to an increase in the Na+ concentration in the environment, but the mechanism of this activation has remained unknown. We report that activation occurs by a Na+-induced folding and dimer formation of the PGB domain of MotS, as revealed in real-time imaging by high-speed atomic force microscopy. The MotPS complex showed two distinct ellipsoid domains connected by a flexible linker. A smaller domain, corresponding to the PGB domain, became structured and unstructured in the presence and absence of 150 mM NaCl, respectively. When the amino-terminal portion of the PGB domain adopted a partially stretched conformation in the presence of NaCl, the center-to-center distance between these two domains increased by up to 5 nm, allowing the PGB domain to reach and bind to the peptidoglycan layer. We propose that assembly of the MotPS complex into a motor proceeds by means of Na+-induced structural transitions of its PGB domain.

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  137. Dedifferentiated liposarcoma in the maxillary gingiva: A clinical report and review of the literature

    Enomoto Akifumi, Matsunaga Kazuhide, Shimoide Takeshi, Mukai Takao, Uchihashi Takayuki, Hamada Suguru

    JOURNAL OF ORAL AND MAXILLOFACIAL SURGERY MEDICINE AND PATHOLOGY   Vol. 29 ( 6 ) page: 542 - 545   2017.11

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  138. Fast Adsorption of Soft Hydrogel Microspheres on Solid Surfaces in Aqueous Solution

    Shusuke Matsui, Takuma Kureha, Seina Hiroshige, Mikihiro Shibata, Takayuki Uchihashi, Daisuke Suzuki

    ANGEWANDTE CHEMIE-INTERNATIONAL EDITION   Vol. 56 ( 40 ) page: 12146 - 12149   2017.9

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    The real-time adsorption behavior of polymeric colloidal microspheres onto solid surfaces in aqueous solution was visualized for the first time using high-speed atomic force microscopy (HS-AFM) to reveal how the softness of the microspheres affects their dynamic adsorption. Studies that focus on the deformability of microspheres upon dynamic adsorption have not yet been reported, most likely on account of a lack of techniques that appropriately depict the dynamic adsorption and deformation behavior of individual microspheres at the nanoscale in real time. In this study, the deformability of microspheres plays a crucial role on the adsorption kinetics, that is, soft hydrogel microspheres adsorb faster than harder elastomeric or rigid microspheres. These results should provide insight towards development of new colloidal nanomaterials that exhibit effective adsorption on specific sites in aqueous solution.

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  139. Visualisation of a flexible modular structure of the ER folding-sensor enzyme UGGT

    Tadashi Satoh, Chihong Song, Tong Zhu, Takayasu Toshimori, Kazuyoshi Murata, Yugo Hayashi, Hironari Kamikubo, Takayuki Uchihashi, Koichi Kato

    SCIENTIFIC REPORTS   Vol. 7 ( 1 ) page: 12142   2017.9

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    In the endoplasmic reticulum (ER), a protein quality control system facilitates the efficient folding of newly synthesised proteins. In this system, a series of N-linked glycan intermediates displayed on the protein surface serve as quality tags. The ER folding-sensor enzyme UDP-glucose: glycoprotein glucosyltransferase (UGGT) acts as a gatekeeper in the ER quality control system by specifically catalysing monoglucosylation onto incompletely folded glycoproteins, thereby enabling them to interact with lectin-chaperone complexes. Here we characterise the dynamic structure of this enzyme. Our crystallographic data demonstrate that the sensor region is composed of four thioredoxin-like domains followed by a beta-rich domain, which are arranged into a C-shaped structure with a large central cavity, while the C-terminal catalytic domain undergoes a ligand-dependent conformational alteration. Furthermore, small-angle X-ray scattering, cryo-electron microscopy and high-speed atomic force microscopy have demonstrated that UGGT has a flexible modular structure in which the smaller catalytic domain is tethered to the larger folding-sensor region with variable spatial arrangements. These findings provide structural insights into the working mechanism whereby UGGT operates as a folding-sensor against a variety of glycoprotein substrates through its flexible modular structure possessing extended hydrophobic surfaces for the recognition of unfolded substrates.

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  140. Interdomain flip-flop motion visualized in flavocytochrome cellobiose dehydrogenase using high-speed atomic force microscopy during catalysis

    Hirofumi Harada, Akira Onoda, Takayuki Uchihashi, Hiroki Watanabe, Naoki Sunagawa, Masahiro Samejima, Kiyohiko Igarashi, Takashi Hayashi

    CHEMICAL SCIENCE   Vol. 8 ( 9 ) page: 6561 - 6565   2017.9

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    Cellobiose dehydrogenase (CDH) is a dual domain flavocytochrome, which consists of a dehydrogenase (DH) domain containing a flavin adenine dinucleotide and a cytochrome (CYT) domain containing b-type heme. To directly visualize the dynamic domain motion of class-I CDH from Phanerochaete chrysosporium (PcCDH) during catalysis using high-speed atomic force microscopy, the apo-form of PcCDH was anchored to a heme-immobilized flat gold surface that can specifically fix the orientation of the CYT domain. The two domains of CDH are found to be immobile in the absence of cellobiose, whereas the addition of cellobiose triggers an interdomain flip-flop motion involving domain-domain association and dissociation. Our results indicate that dynamic motion of a dual domain enzyme during catalysis induces efficient electron transfer to an external electron acceptor.

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  141. Scanning Probe Microscopy FOREWORD

    Takahashi, T; Fukui, K; Kageshima, M; Komeda, T; Nakajima, K; Nakayama, T; Sumitomo, K; Uchihashi, T

    JAPANESE JOURNAL OF APPLIED PHYSICS   Vol. 56 ( 8 )   2017.8

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    DOI: 10.7567/JJAP.56.08L001

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  142. High-Resolution Imaging of a Single Gliding Protofilament of Tubulins by HS-AFM

    Jakia Jannat Keya, Daisuke Inoue, Yuki Suzuki, Toshiya Kozai, Daiki Ishikuro, Noriyuki Kodera, Takayuki Uchihashi, Arif Md. Rashedul Kabir, Masayuki Endo, Kazuki Sada, Akira Kakugo

    SCIENTIFIC REPORTS   Vol. 7 ( 1 ) page: 6166   2017.7

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    In vitro gliding assay of microtubules (MTs) on kinesins has provided us with valuable biophysical and chemo-mechanical insights of this biomolecular motor system. Visualization of MTs in an in vitro gliding assay has been mainly dependent on optical microscopes, limited resolution of which often render them insufficient sources of desired information. In this work, using high speed atomic force microscopy (HS-AFM), which allows imaging with higher resolution, we monitored MTs and protofilaments (PFs) of tubulins while gliding on kinesins. Moreover, under the HS-AFM, we also observed splitting of gliding MTs into single PFs at their leading ends. The split single PFs interacted with kinesins and exhibited translational motion, but with a slower velocity than the MTs. Our investigation at the molecular level, using the HS-AFM, would provide new insights to the mechanics of MTs in dynamic systems and their interaction with motor proteins.

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  143. High-Speed Atomic Force Microscopy Reveals Loss of Nuclear Pore Resilience as a Dying Code in Colorectal Cancer Cells

    Mahmoud Shaaban Mohamed, Akiko Kobayashi, Azuma Taoka, Takahiro Watanabe-Nakayama, Yosuke Kikuchi, Masaharu Hazawa, Toshinari Minamoto, Yoshihiro Fukumori, Noriyuki Kodera, Takayuki Uchihashi, Toshio Ando, Richard W. Wong

    ACS NANO   Vol. 11 ( 6 ) page: 5567 - 5578   2017.6

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    Nuclear pore complexes (NPCs) are the sole turnstile implanted in the nuclear envelope (NE), acting as a central nanoregulator of transport between the cytosol and the nucleus. NPCs consist of similar to 30 proteins, termed nucleoporins. About one-third of nucleoporins harbor natively unstructured, intrinsically disordered phenylalanine-glycine strings (FG-Nups), which engage in transport selectivity. Because the barriers insert deeply in the NPC, they are nearly inaccessible. Several in vitro barrier models have been proposed; however, the dynamic FG-Nups protein molecules themselves are imperceptible in vivo. We show here that high-speed atomic force microscopy (HS-AFM) can be used to directly visualize nanotopographical changes of the nuclear pore inner channel in colorectal cancer (CRC) cells. Furthermore, using MLN8237/alisertib, an apoptotic and autophagic inducer currently being tested in relapsed cancer clinical trials, we unveiled the functional loss of nucleoporins, particularly the deformation of the FG-Nups barrier, in dying cancer cells. We propose that the loss of this nanoscopic resilience is an irreversible dying code in cells. These findings not only illuminate the potential application of HS-AFM as an intracellular nanoendoscopy but also might aid in the design of future nuclear targeted nanodrug delivery tailored to the individual patient.

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  144. High-speed AFM revealed single-molecule blocking dynamics of a scorpion toxin on the KcsA potassium channel Reviewed

    Ayumi Sumino, Takayuki Uchihashi, Takashi Sumikama, Shigetoshi Oiki

    JOURNAL OF PHYSIOLOGICAL SCIENCES   Vol. 67   page: S117   2017.3

  145. Oriented Reconstitution of the Full-Length KcsA Potassium Channel in a Lipid Bilayer for AFM Imaging Reviewed

    Ayumi Sumino, Takayuki Uchihashi, Shigetoshi Oiki

    JOURNAL OF PHYSICAL CHEMISTRY LETTERS   Vol. 8 ( 4 ) page: 785 - 793   2017.2

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    Here, we have developed a method of oriented reconstitution of the KcsA potassium channel amenable to high-resolution AFM imaging. The solubilized liposome full-length KcsA channels with histidine-tagged (His-tag) C-terminal ends were attached to a Ni2+-coated mica surface, and then detergent-destabilized liposomes were added to fill the interchannel space. AFM revealed that the membrane embedded KcsA channels were oriented with their extracellular faces upward, seen as a tetrameric square shape. This orientation was corroborated,,by the visible binding of a peptide scorpion toxin, agitoxin-2. To observe the cytoplasmic side of the channel, a His-tag was inserted into the extracellular loop, and the oppositely oriented channels provided wholly different images. In either orientation, the channels were individually dispersed at acidic pH, whereas they were self-assembled at neutral pH, indicating that the oriented channels are allowed to diffuse in the membrane. This method is readily applicable to membrane proteins' in general for AFM imaging.

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  146. Nuclear Pore Selective Barrier Dynamics as Revealed by High-Speed Atomic Force Microscopy in Colorectal Cancer Cells.

    Mohamed, MS; Kobayashi, A; Taoka, A; Watanabe-Nakayama, T; Kikuchi, Y; Hazawa, M; Minamoto, T; Fukumori, Y; Kodera, N; Uchihashi, T; Ando, T; Wong, R

    MOLECULAR BIOLOGY OF THE CELL   Vol. 28   2017

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  147. High-speed atomic force microscopy imaging of live mammalian cells.

    Shibata M, Watanabe H, Uchihashi T, Ando T, Yasuda R

    Biophysics and physicobiology   Vol. 14 ( 0 ) page: 127-135 - 135   2017

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    <p>Direct imaging of morphological dynamics of live mammalian cells with nanometer resolution under physiological conditions is highly expected, but yet challenging. High-speed atomic force microscopy (HS-AFM) is a unique technique for capturing biomolecules at work under near physiological conditions. However, application of HS-AFM for imaging of live mammalian cells was hard to be accomplished because of collision between a huge mammalian cell and a cantilever during AFM scanning. Here, we review our recent improvements of HS-AFM for imaging of activities of live mammalian cells without significant damage to the cell. The improvement of an extremely long (~3 μm) AFM tip attached to a cantilever enables us to reduce severe damage to soft mammalian cells. In addition, a combination of HS-AFM with simple fluorescence microscopy allows us to quickly locate the cell in the AFM scanning area. After these improvements, we demonstrate that developed HS-AFM for live mammalian cells is possible to image morphogenesis of filopodia, membrane ruffles, pits open-close formations, and endocytosis in COS-7, HeLa cells as well as hippocampal neurons.</p>

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  148. "Clusterase" model of dynamin-mediated membrane fission.

    Takeda T., Kozai T., Yang H., Kaho S., Yusuke K., Tadashi A., Hiroshi Y., Uchihashi T., Ando T., Takei K.

    MOLECULAR BIOLOGY OF THE CELL   Vol. 28   page: .   2017

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  149. "Clusterase" model of dynamin-mediated membrane fission.

    Takeda T, Kozai T, Yang H, Kaho S, Yusuke K, Tadashi A, Hiroshi Y, Uchihashi T, Ando T, Takei K

    MOLECULAR BIOLOGY OF THE CELL   Vol. 28   page: .   2017

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  150. Visualization of Biological/Artificial Macromolecules with High-Speed Atomic Force Microscope

    Uchihashi Takayuki

    Abstract of annual meeting of the Surface Science of Japan   Vol. 37 ( 0 ) page: 254   2017

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    DOI: 10.14886/sssj2008.37.0_254

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  151. In-situ observation of biological molecules with high-speed AFM

    Uchihashi Takayuki

    Abstract of annual meeting of the Surface Science of Japan   Vol. 37 ( 0 ) page: 19   2017

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    DOI: 10.14886/sssj2008.37.0_19

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  152. A natural light-driven inward proton pump. Reviewed

    Inoue K, Ito S, Kato Y, Nomura Y, Shibata M, Uchihashi T, Tsunoda SP, Kandori H

    Nature communications   Vol. 7   page: 13415   2016.11

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    DOI: 10.1038/ncomms13415

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  153. Functional extension of high-speed AFM for wider biological applications. Reviewed

    Uchihashi T, Watanabe H, Fukuda S, Shibata M, Ando T

    Ultramicroscopy   Vol. 160   page: 182-96   2016.1

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  154. Potential Prepore Trimer Formation by the Bacillus thuringiensis Mosquito-specific Toxin: MOLECULAR INSIGHTS INTO A CRITICAL PREREQUISITE OF MEMBRANE-BOUND MONOMERS. Reviewed

    Sriwimol W, Aroonkesorn A, Sakdee S, Kanchanawarin C, Uchihashi T, Ando T, Angsuthanasombat C

    The Journal of biological chemistry   Vol. 290 ( 34 ) page: 20793-803   2015.8

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    DOI: 10.1074/jbc.M114.627554

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  155. Method of mechanical holding of cantilever chip for tip-scan high-speed atomic force microscope. Reviewed

    Fukuda S, Uchihashi T, Ando T

    The Review of scientific instruments   Vol. 86 ( 6 ) page: 063703   2015.6

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  156. Long-tip high-speed atomic force microscopy for nanometer-scale imaging in live cells. Reviewed

    Shibata M, Uchihashi T, Ando T, Yasuda R

    Scientific reports   Vol. 5   page: 8724   2015.3

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    DOI: 10.1038/srep08724

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  157. Probing structural dynamics of an artificial protein cage using high-speed atomic force microscopy. Reviewed

    Imamura M, Uchihashi T, Ando T, Leifert A, Simon U, Malay AD, Heddle JG

    Nano letters   Vol. 15 ( 2 ) page: 1331-5   2015.2

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  158. Real-time dynamic adsorption processes of cytochrome c on an electrode observed through electrochemical high-speed atomic force microscopy. Reviewed

    Takeda K, Uchihashi T, Watanabe H, Ishida T, Igarashi K, Nakamura N, Ohno H

    PloS one   Vol. 10 ( 2 ) page: e0116685   2015

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  159. Two-way traffic of glycoside hydrolase family 18 processive chitinases on crystalline chitin. Reviewed

    Igarashi K, Uchihashi T, Uchiyama T, Sugimoto H, Wada M, Suzuki K, Sakuda S, Ando T, Watanabe T, Samejima M

    Nature communications   Vol. 5   page: 3975   2014.6

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    DOI: 10.1038/ncomms4975

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  160. Single-molecule imaging analysis of elementary reaction steps of Trichoderma reesei cellobiohydrolase I (Cel7A) hydrolyzing crystalline cellulose Iα and IIII.

      Vol. 289 ( 20 ) page: 14056-65   2014.5

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    DOI: 10.1074/jbc.M113.546085

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  161. [Direct observation of rotary catalysis of rotorless F1-ATPase with high-speed AFM].

    Uchihashi T, Iino R, Ando T, Noji H

    Seikagaku. The Journal of Japanese Biochemical Society   Vol. 86 ( 2 ) page: 127-36   2014.4

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  162. Trade-off between processivity and hydrolytic velocity of cellobiohydrolases at the surface of crystalline cellulose.

    Nakamura A, Watanabe H, Ishida T, Uchihashi T, Wada M, Ando T, Igarashi K, Samejima M

    Journal of the American Chemical Society   Vol. 136 ( 12 ) page: 4584-92   2014.3

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    Language:English   Publishing type:Research paper (scientific journal)  

    DOI: 10.1021/ja4119994

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  163. Filming biomolecular processes by high-speed atomic force microscopy.

    Ando T, Uchihashi T, Scheuring S

    Chemical reviews   Vol. 114 ( 6 ) page: 3120-88   2014.3

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    Language:English   Publishing type:Research paper (scientific journal)  

    DOI: 10.1021/cr4003837

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  164. Role of trimer-trimer interaction of bacteriorhodopsin studied by optical spectroscopy and high-speed atomic force microscopy.

    Yamashita H, Inoue K, Shibata M, Uchihashi T, Sasaki J, Kandori H, Ando T

    Journal of structural biology   Vol. 184 ( 1 ) page: 2-11   2013.10

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    Language:English   Publishing type:Research paper (scientific journal)  

    DOI: 10.1016/j.jsb.2013.02.011

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  165. Real-time visualization of assembling of a sphingomyelin-specific toxin on planar lipid membranes.

    Yilmaz N, Yamada T, Greimel P, Uchihashi T, Ando T, Kobayashi T

    Biophysical journal   Vol. 105 ( 6 ) page: 1397-405   2013.9

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    Language:English   Publishing type:Research paper (scientific journal)  

    DOI: 10.1016/j.bpj.2013.07.052

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  166. High-speed atomic force microscope combined with single-molecule fluorescence microscope.

    Fukuda S, Uchihashi T, Iino R, Okazaki Y, Yoshida M, Igarashi K, Ando T

    The Review of scientific instruments   Vol. 84 ( 7 ) page: 073706   2013.7

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    Language:English   Publishing type:Research paper (scientific journal)  

    DOI: 10.1063/1.4813280

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  167. Wide-area scanner for high-speed atomic force microscopy.

    Watanabe H, Uchihashi T, Kobashi T, Shibata M, Nishiyama J, Yasuda R, Ando T

    The Review of scientific instruments   Vol. 84 ( 5 ) page: 053702   2013.5

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    Language:English   Publishing type:Research paper (scientific journal)  

    DOI: 10.1063/1.4803449

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  168. High-speed AFM and applications to biomolecular systems.

    Ando T, Uchihashi T, Kodera N

    Annual review of biophysics   Vol. 42   page: 393-414   2013

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    Language:English   Publishing type:Research paper (scientific journal)  

    DOI: 10.1146/annurev-biophys-083012-130324

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  169. Single-molecule imaging on living bacterial cell surface by high-speed AFM.

    Yamashita H, Taoka A, Uchihashi T, Asano T, Ando T, Fukumori Y

    Journal of molecular biology   Vol. 422 ( 2 ) page: 300-9   2012.9

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    Language:English   Publishing type:Research paper (scientific journal)  

    DOI: 10.1016/j.jmb.2012.05.018

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  170. Guide to video recording of structure dynamics and dynamic processes of proteins by high-speed atomic force microscopy.

    Uchihashi T, Kodera N, Ando T

    Nature protocols   Vol. 7 ( 6 ) page: 1193-206   2012.5

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    Language:English   Publishing type:Research paper (scientific journal)  

    DOI: 10.1038/nprot.2012.047

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  171. Direct Visualization of Cellobiohydrolase on Crystalline Cellulose using High-Speed Atomic Force Microscopy Reviewed

    Uchihashi Takayuki, Igarashi Kiyohiko, Koivula Anu, Wada Masahisa, Penttil Merja, Samejima Masahiro, Ando Toshio

    BIOPHYSICAL JOURNAL   Vol. 102 ( 3 ) page: 585A-586A   2012.1

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  172. Visualization of cellobiohydrolase I from Trichoderma reesei moving on crystalline cellulose using high-speed atomic force microscopy.

    Methods in enzymology   Vol. 510   page: 169-82   2012

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    Language:English   Publishing type:Research paper (scientific journal)  

    DOI: 10.1016/B978-0-12-415931-0.00009-4

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  173. Traffic jams reduce hydrolytic efficiency of cellulase on cellulose surface.

    Science (New York, N.Y.)   Vol. 333 ( 6047 ) page: 1279-82   2011.9

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    Language:English   Publishing type:Research paper (scientific journal)  

    DOI: 10.1126/science.1208386

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  174. High-speed atomic force microscopy reveals rotary catalysis of rotorless F₁-ATPase.

      Vol. 333 ( 6043 ) page: 755-8   2011.8

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    Language:English   Publishing type:Research paper (scientific journal)  

    DOI: 10.1126/science.1205510

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  175. Structural changes in bacteriorhodopsin in response to alternate illumination observed by high-speed atomic force microscopy.

    Shibata M, Uchihashi T, Yamashita H, Kandori H, Ando T

    Angewandte Chemie (International ed. in English)   Vol. 50 ( 19 ) page: 4410-3   2011.5

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    Language:English   Publishing type:Research paper (scientific journal)  

    DOI: 10.1002/anie.201007544

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  176. High-speed atomic force microscopy and biomolecular processes.

    Uchihashi T, Ando T

    Methods in molecular biology (Clifton, N.J.)   Vol. 736   page: 285-300   2011

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    Language:English   Publishing type:Research paper (scientific journal)  

    DOI: 10.1007/978-1-61779-105-5_18

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  177. High-speed atomic force microscopy shows dynamic molecular processes in photoactivated bacteriorhodopsin Reviewed

    Shibata Mikihiro, Yamashita Hayato, Uchihashi Takayuki, Kandori Hideki, Ando Toshio

    NATURE NANOTECHNOLOGY   Vol. 5 ( 3 ) page: 208-212   2010.3

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    Language:English   Publishing type:Research paper (scientific journal)  

    DOI: 10.1038/NNANO.2010.7

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  178. Dynamics of bacteriorhodopsin 2D crystal observed by high-speed atomic force microscopy

    内橋 貴之

    JOURNAL OF STRUCTURAL BIOLOGY   Vol. 167 ( 153-158 ) page: .   2009

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  179. High resonance frequency force microscope scanner using inertia balance support Reviewed

    Fukuma Takeshi, Okazaki Yasutaka, Kodera Noriyuki, Uchihashi Takayuki, Ando Toshio

    APPLIED PHYSICS LETTERS   Vol. 92 ( 24 )   2008.6

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    Language:English   Publishing type:Research paper (scientific journal)  

    DOI: 10.1063/1.2951594

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  180. Visualization of intrinsically disordered regions of proteins by high-speed atomic force microscopy

    CHEMPHYSCHEM   Vol. 9 ( 13 ) page: 1859-1866   2008

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    Publishing type:Research paper (scientific journal)  

    DOI: 10.1002/cphc.200800210

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  181. High-speed AFM and nano-visualization of biomolecular processes

    内橋 貴之

    Pflugers Archiv -Eur. J. Physiol.   Vol. 456   page: .   2008

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  182. High resonance frequency force microscope scanner using inertia balance support

    内橋 貴之

    APPLIED PHYSICS LETTERS   Vol. 92 ( 243119 ) page: .   2008

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  183. High-speed atomic force microscopy for nano-visualization of dynamic biomolecular processes

    内橋 貴之

    PROGRESS IN SURFACE SCIENCE   Vol. 83 ( 337-437 ) page: .   2008

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  184. High-speed atomic force microscopy for observing dynamic biomolecular processes.

    Ando T, Uchihashi T, Kodera N, Yamamoto D, Taniguchi M, Miyagi A, Yamashita H

    Journal of molecular recognition : JMR   Vol. 20 ( 6 ) page: 448-58   2007.11

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    Language:English   Publishing type:Research paper (scientific journal)  

    DOI: 10.1002/jmr.843

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  185. Tip-sample distance control using photothermal actuation of a small cantilever for high-speed atomic force microscopy.

    Yamashita H, Kodera N, Miyagi A, Uchihashi T, Yamamoto D, Ando T

    The Review of scientific instruments   Vol. 78 ( 8 ) page: 083702   2007.8

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    Language:English   Publishing type:Research paper (scientific journal)  

    DOI: 10.1063/1.2766825

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  186. High-speed atomic force microscopy for observing dynamic biomolecular processes

    内橋 貴之

    JOURNAL OF MOLECULAR RECOGNITION   Vol. 20 ( 448-458 ) page: .   2007

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  187. Capacitance XAFS method: A new site-selective and microscopic X-ray absorption Spectroscopy Reviewed

    Ishii Masashi, Nakao Aiko, Uchihashi Takayuki

    PHYSICA SCRIPTA   Vol. T115   page: 97-101   2005

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Books 27

  1. Microtubule Preparation for Investigation with High-Speed Atomic Force Microscopy

    Ganser C., Uchihashi T.

    Methods in Molecular Biology  2022 

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    High-speed atomic force microscopy (AFM) is a versatile method that can visualize proteins and protein systems on the nanometer scale and at a temporal resolution of 100 ms. The application to microtubules can not only reveal structural information with single-tubulin resolution but can also extract mechanical information and allows to study single motor proteins walking on microtubules, among others. This chapter provides a step-by-step guide from microtubule polymerization to successful observation with high-speed AFM.

    DOI: 10.1007/978-1-0716-1983-4_22

    Scopus

    PubMed

  2. 実験医学別冊 「創薬研究のための相互作用解析パーフェクト: 低中分子・抗体創薬におけるスクリーニング戦略と実例、in silico解析、一歩進んだ分析技術まで」

    内橋貴之( Role: Contributor ,  第3章 ひとつ進んだ相互作用の理解をめざして: 5.高速原子間力顕微鏡によるタンパク質間動的相互作用の一分子計測)

    羊土社  2021.12  ( ISBN:978-4-7581-2256-6

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    Language:Japanese Book type:Scholarly book

  3. Single-molecule methods applied to circadian proteins with special emphasis on atomic force microscopy

    Mori T., Uchihashi T.

    Circadian Rhythms in Bacteria and Microbiomes  2021.6  ( ISBN:9783030721572

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    Single-molecule (SM) techniques have emerged as important tools to study biomolecules and chemical reactions in biophysics and biochemistry, providing detailed microscopic information about dynamic processes of molecules. In the field of circadian biology, the SM approach has rarely been taken to study the properties of clock components. In this chapter, we briefly overview the SM experimental tools available for studying circadian clocks and introduce our biophysical study on the in vitro KaiABC oscillator using high-speed atomic force microscopy (HS-AFM). Our HS-AFM observation, for the first time, visualized dynamic interactions of clock proteins working in real time at the single-molecule level. The KaiA dimer binds to the KaiC hexamer at the C-terminus of KaiC, and the affinity of the KaiA dimer binding to the KaiC hexamer depends on the phosphorylation state of the KaiC hexamer; KaiA has higher affinity for less phosphorylated KaiC hexamers. Mathematical modeling and simulations revealed that the phosphoform-dependent differential affinity (PDDA) supports rhythmicity over a broad range of Kai protein stoichiometries, serving as a buffer against noise. Additionally, we demonstrate real-time observations of the binding of fold-switched KaiB (fsKaiB) to the CI domain of KaiC hexamers and the sequestration of the KaiA dimer into the KaiB-KaiC complex.

    DOI: 10.1007/978-3-030-72158-9_9

    Scopus

  4. 図説 表面分析ハンドブック

    内橋貴之( Role: Contributor ,  23.8「高速原子間力顕微鏡」)

    朝倉書店  2021.6  ( ISBN:978-4-254-20170-3

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    Total pages:576   Language:Japanese Book type:Dictionary, encyclopedia

  5. 現代化学

    内橋貴之( Role: Sole author ,  はたらく分子マシン(9) 分子の姿と動きを直接視て操作する顕微鏡技術)

    東京化学同人  2021.5 

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    Responsible for pages:33-37   Language:Japanese Book type:General book, introductory book for general audience

  6. Circadian Rhythms in Bacteria and Microbiomes Reviewed International journal

    Tetsuya Mori and Takayuki Uchihashi( Role: Contributor ,  Single-Molecule Methods Applied to Circadian Proteins with Special Emphasis on Atomic Force Microscopy)

    Springer  2021 

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    Responsible for pages:147-178   Language:English Book type:Scholarly book

  7. 膜タンパク質工学ハンドブック

    内橋貴之( Role: Contributor ,  第1編 第2章15「高速原子間力顕微鏡によるタンパク質の構造ダイナミクス解析」)

    株式会社 エヌ・ティー・エス  2020.4  ( ISBN:978-4-86043-537-0

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    Language:Japanese Book type:Scholarly book

  8. 自己修復・自己組織化材料の開発と応用事例

    内橋貴之( Role: Contributor ,  第5章 第12節「高速原子間力顕微鏡による分子の自己組織化過程のリアルタイムでの観察」)

    技術情報協会  2020.3  ( ISBN:978-4861047817

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    Language:Japanese Book type:Scholarly book

  9. 実験医学増刊 Vol.36 No.20, 「生きてるものは全部観る! イメージングの選び方・使い方100」

    内橋貴之( Role: Contributor ,  第5章 走査型プローブ顕微鏡「原子間力顕微鏡 ⅰ.高速原子間力顕微鏡」)

    羊土社  2018.12  ( ISBN:978-4-7581-0375-6

  10. パリティ 2018年1月号

    内橋 貴之( Role: Contributor ,  高速原子間力顕微鏡によるタンパク質の動画撮影)

    丸善出版  2018.1 

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    Responsible for pages:71-73   Language:Japanese

  11. Compendium of Surface and Interface Analysis

    Takayuki Uchihashi( Role: Contributor)

    The Surface Science Society of Japan  2018 

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    Responsible for pages:263-267   Language:English Book type:Scholarly book

  12. Compendium of Surface and Interface Analysis (The Surface Science Society of Japan, Eds)

    Takayuki Uchihashi( Role: Contributor ,  High-Speed Atomic Force Microcopy)

    Springer  2018  ( ISBN:9789811061554

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    Responsible for pages:263-267   Language:English Book type:Dictionary, encyclopedia

  13. Optimum substrates for imaging biological molecules with high-speed atomic force microscopy

    Uchihashi T., Watanabe H., Kodera N.

    Methods in Molecular Biology  2018 

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    Recent progresses in high-speed atomic force microscopy (HS-AFM) have enabled us to directly visualize dynamic processes of various proteins in liquid conditions. One of the key factors leading to successful HS-AFM observations is the selection of an appropriate substrate depending on molecules to be observed. For the HS-AFM imaging, a target molecule must be absorbed on a substrate by controlling its orientation without impairing the dynamics or physiological function of the molecule. In this chapter, we describe protocols for preparation of substrates that have been used for HS-AFM and then introduce observation examples on dynamic processes of biological molecules.

    DOI: 10.1007/978-1-4939-8591-3_10

    Scopus

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  14. 光と生命の事典

    内橋貴之( Role: Contributor ,  第5章 「光による生命現象の計測」177節 高速原子間力顕微鏡)

    朝倉書店  2016.2  ( ISBN:978-4-254-17161-7

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  15. Noncontact Atomic Force Microscopy Vol.3

    Takayuyki Uchihashi, Noriyuki Kodera, and Toshio Ando( Role: Contributor ,  Chapter 22: High-speed Atomic Force Microscopy)

    Springer  2015 

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    Responsible for pages:481-518   Language:English Book type:Scholarly book

  16. Atomic Force Microscopy in Nanobiology

    Takayuki Uchihahsi, Noriyuki Kodera, Toshio Ando( Role: Contributor ,  " Chapter 8: Development of High-speed AFM and Its Biological Applications)

    Pan Stanford Publishing  2014  ( ISBN:978-981-4411-59-2

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    Total pages:437   Responsible for pages:143-176  

  17. 膜タンパク質構造研究

    内橋貴之, 安藤敏夫( Role: Contributor ,  23章: 原子間力顕微鏡による膜タンパク質のダイナミクス研究)

    化学同人  2013.10  ( ISBN:4759815619

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    Language:Japanese Book type:Scholarly book

  18. Nanoscale Liquid Interfaces: Wetting, Patterning, and Force Microscopy at the Molecular Scale

    Toshio Ando, Takayuki Uchihashi( Role: Contributor ,  Chapter 19: High-speed AFM and Imaging of Biomolecular Processes)

    Pan Stanford Publishing  2013  ( ISBN:9789814316453

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    Language:English Book type:Scholarly book

    DOI: 10.4032/9789814364485

  19. Single-molecule Studies of Proteins (Biophysics for the Life Sciences) Vol 2

    Takayuki Uchihashi, Noriyuki Kodera, Toshio. Ando( Role: Contributor ,  Chapter 5 : Nanovisualization of proteins in action using high-speed AFM)

    Springer  2013  ( ISBN:978-1-4614-4920-1

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    Responsible for pages:119-147   Language:English Book type:Scholarly book

    DOI: 10.1007/978-1-4614-4921-8

  20. Atomic force microscopy in liquid: Biological Applications

    ( Role: Contributor ,  Toshio Ando, Takayuki Uchihashi, Noriyuki Kodera)

    Wiley-VCH Verlag GmbH  2012  ( ISBN:9783527327584

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    Responsible for pages:189-209   Language:English Book type:Scholarly book

    DOI: 10.1002/9783527649808

  21. Life at the Nanoscale - Atomic force Microscopy of Live Cells

    Takayuki Uchihashi, Toshio Ando( Role: Contributor)

    Chapter 8 : High-speed atomic force microscopy for dynamic biological imaging  2011  ( ISBN:9789814267960

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    Responsible for pages:163-184   Language:English Book type:Scholarly book

    DOI: 10.4032/9789814267977

  22. Atomic force microscopy in Biomedical Research: Methods and Protocols

    Takayuki Uchihashi, Toshio Ando( Role: Contributor ,  Chapter 18 : High-speed Atomic Force Microscopy and Biomolecular Processes)

    Humana Press  2011 

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    Responsible for pages:285-300   Language:English Book type:Scholarly book

  23. 酵素利用技術大系

    内橋貴之, 安藤敏夫( Role: Contributor ,  第2編6節:AFMを用いた酵素反応解析)

    株式会社 エヌ・ティー・エス  2010.4  ( ISBN:4860432711

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  24. Single Molecule Dynamics in Life Science

    Toshio Ando, Takayuki Uchihashi, Noriyuki Kodera, Daisuke Yamamoto, Masaaki Taniguchi, Atsushi Miyagi, Hayato Yamashita( Role: Contributor ,  Chapter 12: High-speed atomic force microscopy for nano-visualization of biomolecular processes)

    Wiley-VCH Verlag GmbH  2008  ( ISBN:978-3-527-31288-7

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    Responsible for pages:277-296   Language:English Book type:Scholarly book

  25. Scanning Probe Microscopy: Characterization, Nanofabrication and Device Application of Functional Materials (NATO SCIENCE SERIES II: Mathematics, Physics and Chemistry)

    Seizo Morita, Takayuki Uchihashi, Kenji Okamoto, Masayuki Abe, and Yasuhiro Sugawara(Nanoscale Contact Charging on a Silicon Oxide)

    Springer Netherlands  2005  ( ISBN:978-1-4020-3019-2

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    Responsible for pages:289-308   Language:English Book type:Scholarly book

    DOI: 10.1007/1-4020-3019-3

  26. Fundamentals of Tribology and Bridging the Gap between Micro-and Micro/Nanoscales (NATO SCIENCE SERIES: Mathematics, Physics and Chemistry)

    Seizo Morita, Yasuhiro Sugawara, Kousuke Yokoyama, and Takayuki Uchihashi(Atomic Scale Origins of Force Interaction")

    Springer  2001  ( ISBN:978-94-010-0736-8

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    Responsible for pages:103-120   Language:English Book type:Scholarly book

    DOI: 10.1007/978-94-010-0736-8

  27. Forces in Scanning Probe Methods" (NATO Advanced Science Institutes Series E: Applied Science -Vol.286)

    Yasuhiro Sugawara, Seizo Morita, Yoshinobu Fukano, Takayuki Uchihashi, Takahiro Okusako, Ayumi Chayahara, Yoshiki Yamanishi, and Takahiko Oasa( Role: Contributor ,  Time Dependence and its Spatial Distribution of Densely Contact-Electrified Electrons on a Thin Silicon Oxide)

    Springer  1995  ( ISBN:978-94-010-4027-3

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    Responsible for pages:505-506   Language:English Book type:Scholarly book

    DOI: 10.1007/978-94-011-0049-6

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MISC 19

  1. Data for: Antiparallel dimer structure of CELSR cadherin in solution revealed by high-speed atomic force microscopy

    Shigetaka Nishiguchi, Rinshi S. Kasai, Takayuki Uchihashi

    Mendeley Data     2023.3

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    Language:English  

    DOI: 10.17632/vbpdtyxbtn.3

  2. 病原性大腸菌EPECが有するIII型分泌装置のATPase複合体の機能解析

    鈴木綾, 上野博史, 黒崎涼, 内橋貴之, 野地博行

    日本蛋白質科学会年会プログラム・要旨集   Vol. 21st   2021

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  3. Observation of substrate binding Sec translocon and structural change of SecA with HS-AFM

    長池航, 板家成良, 春山隆充, 塚崎智也, 内橋貴之, 内橋貴之

    日本生体エネルギー研究会討論会講演要旨集   Vol. 46th   2020

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  4. Structures of heliorhodopsin and schizorhodopsin elucidate the structural diversity of microbial rhodopsins

    SHIHOYA Wataru, INOUE Keiichi, MANISH Singh, HIGUCHI Akimitsu, KONNO Masae, YOSHIZUMI Rei, UCHIHASHI Takayuki, KANDORI Hideki, NUREKI Osamu

    生物物理(Web)   Vol. 60 ( Supplement 1-2 )   2020

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  5. Structural and Physicochemical Study on Heliorhodopsin

    SHIHOYA Wataru, INOUE Keiichi, INOUE Keiichi, INOUE Keiichi, SINGH Manish, KONNO Masae, HOSOSHIMA Shoko, YAMASHITA Keitaro, YAMASHITA Keitaro, IKEDA Kento, HIGUCHI Akimitsu, IZUME Tamaki, OKAZAKI Sae, HASHIMOTO Masanori, MIZUTORI Ritsu, TOMIDA Sahoko, YAMAUCHI Yumeka, ABE-YOSHIZUMI Rei, KATAYAMA Kota, TSUNODA Satoshi P., TSUNODA Satoshi P., SHIBATA Mikihiro, FURUTANI Yuji, FURUTANI Yuji, FURUTANI Yuji, PUSHKAREV Alina, BEJA Oded, UCHIHASHI Takayuki, UCHIHASHI Takayuki, KANDORI Hideki, NUREKI Osamu

    日本化学会春季年会講演予稿集(CD-ROM)   Vol. 100th   2020

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  6. Assembly of rod-shaped hydrogel microspheres at the air/water interface

    Kenshiro Honda, Yuka Sazuka, Kojiro Iizuka, Shusuke Matsui, Takayuki Uchihashi, Takuma Kureha, Mitsuhiro Shibayama, Takumi Watanabe, Daisuke Suzuki

    ABSTRACTS OF PAPERS OF THE AMERICAN CHEMICAL SOCIETY   Vol. 258   2019.8

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    Language:English   Publishing type:Research paper, summary (international conference)   Publisher:AMER CHEMICAL SOC  

    Web of Science

  7. Structure and biophysical characterization of the heliorhodopsin

    SHIHOYA Wataru, INOUE Keiichi, INOUE Keiichi, INOUE Keiichi, MANISH Singh, KONNO Masae, HOSOSHIMA Shoko, YAMASHITA Keitaro, IKEDA Kento, HIGUCHI Akimitsu, OKAZAKI Sae, TAMAKI Izume, HASHIMOTO Masanori, MIZUTORI Ritsu, TOMIDA Sahoko, YAMAUCHI Yumeka, ABE-YOSHIZUMI Rei, KATAYAMA Kota, TSUNODA P. Satoshi, SHIBATA Mikihiro, FURUTANI Yuji, FURUTANI Yuji, FURUTANI Yuji, PUSHKAREV Alina, BEJA Oded, UCHIHASHI Takayuki, KANDORI Hideki, NUREKI Osamu

    生物物理(Web)   Vol. 59 ( Supplement 1-2 )   2019

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  8. The Transport Mechanism of a New Light-driven Inward Proton Pump, Schizorhodopsin (SzR)

    INOUE Keiichi, INOUE Keiichi, INOUE Keiichi, TSUNODA Satoshi P., TSUNODA Satoshi P., SINGH Manish, KONNO Masae, TOMIDA Sahoko, NAKAMURA Ryoko, WATANABE Hiroki, WATANABE Hiroki, UCHIHASHI Takayuki, GHAI Rohit, BEJA Oded, KANDORI Hideki, KANDORI Hideki

    日本化学会春季年会講演予稿集(CD-ROM)   Vol. 99th   2019

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  9. 新奇ロドプシンファミリーSchizorhodopsin(SzR)による光駆動内向きプロトン輸送

    Keiichi Inoue, Satoshi P. Tsunoda, SINGH Manish, Masae Konno, Sahoko Tomida, Ryoko nakamura, Daiki Watanabe, Takayuki Uchihashi, GHAI Rohit, BEJA Oded, Hideki Kandori

    生体分子科学討論会講演要旨集   Vol. 46th   page: 18‐19   2019

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    Language:Japanese  

    J-GLOBAL

  10. 膜を舞台にする抗体機能の高速原子間力顕微鏡解析

    與語理那, 與語理那, 與語理那, 谷中冴子, 谷中冴子, 谷中冴子, 渡辺大輝, 矢木宏和, 内橋貴之, 内橋貴之, 加藤晃一, 加藤晃一, 加藤晃一

    日本薬学会年会要旨集(CD-ROM)   Vol. 139th   2019

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  11. IgGとFc受容体の相互作用におけるFab領域の新規結合部位の同定

    與語理那, 與語理那, 山口祐希, 渡辺大輝, 矢木宏和, 佐藤匡史, 中西真人, 鬼塚正義, 大政健史, 嶋田麻里, 丸野孝浩, 鳥巣哲生, 渡邊史生, 肥後大輔, 内橋貴之, 内橋貴之, 谷中冴子, 谷中冴子, 内山進, 内山進, 加藤晃一, 加藤晃一

    日本生化学会大会(Web)   Vol. 92nd   2019

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  12. 新奇光駆動型内向きプロトンポンプ型ロドプシンSchizorhodopsin(SzR)とその輸送メカニズムの分光研究

    INOUE Keiichi, TSUNODA Satoshi, SINGH Manish, KONNO Masae, TOMIDA Sahoko, NAKAMURA Ryoko, WATANABE Daiki, UCHIHASHI Takayuki, GHAI Rohit, BEJA Oded, KANDORI Hideki

    日本生体エネルギー研究会討論会講演要旨集   Vol. 44th   page: 24‐25   2018.12

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    Language:Japanese  

    J-GLOBAL

  13. 高速AFM・ネイティブ質量分析・超遠心分析・電子顕微鏡の複合解析で明らかにする分子シャペロンClpBの多量体構造とダイナミクス

    内橋貴之, 渡辺洋平, 内山進, 内山進, 村田和義, 飯野亮太, 飯野亮太

    日本分子生物学会年会プログラム・要旨集(Web)   Vol. 41st   page: ROMBUNNO.2PW2‐10‐5 (WEB ONLY)   2018

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    Language:Japanese  

    J-GLOBAL

  14. プレフィルドシリンジに対する抗体医薬の吸着と凝集体生成の関係

    丸野孝浩, 丸野孝浩, 渡辺大輝, 米田早紀, 内橋貴之, 足達慧, 荒井邦仁, 澤口太一, 内山進, 内山進

    日本蛋白質科学会年会プログラム・要旨集   Vol. 18th   2018

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  15. コロイド微粒子はやわらかいほど速く基板上に吸着する

    鈴木大介, 内橋貴之

    『動的秩序と機能』ニュースレター   Vol. 48   page: 1   2017.8

  16. GTP加水分解に共役したダイナミン依存的膜切断機構の高速原子間力顕微鏡解析

    竹田哲也, 石黒大輝, 楊恵然, 小財稔矢, 背山佳穂, 熊谷祐介, 山田浩司, 内橋貴之, 安藤敏夫, 竹居孝二

    日本細胞生物学会大会(Web)   Vol. 69th   2017

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  17. 高速原子間力顕微鏡で可視化するタンパク質の動的秩序

    内橋貴之, 杉山翔吾, 小財稔矢, 與語理那, 谷中冴子, 佐藤匡史, 矢木和宏, 盛徹也, JOHNSON Carl, 安藤敏夫, 加藤晃一, 加藤晃一

    日本蛋白質科学会年会プログラム・要旨集   Vol. 17th   2017

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  18. 高速AFMを用いた膜上での抗原抗体複合体形成過程の観測

    與語理那, 與語理那, 谷中冴子, 谷中冴子, 矢木宏和, 内橋貴之, 加藤晃一, 加藤晃一

    日本薬学会年会要旨集(CD-ROM)   Vol. 137th   2017

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  19. ダイナミン-コルタクチンらせん状複合体の解析:機械的なアクチン線維束形成とアクチン脱重合保護作用

    阿部匡史, 山田浩司, 竹田哲也, 内橋貴之, 安藤敏夫, 竹居孝二

    日本生化学会大会(Web)   Vol. 90th   2017

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Presentations 46

  1. 高速原子間顕微鏡による膜輸送装置Secの動態観察

    長池航, 板家成良, 春山隆充, 塚崎智也, 内橋貴之

    日本生体エネルギー研究会第46回討論会  2020.12.10  日本生体エネルギー研究会

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    Event date: 2020.12

    Language:Japanese   Presentation type:Oral presentation (general)  

    Venue:金沢   Country:Japan  

  2. 高速原子間力顕微鏡を用いた光化学系IIの動態観察

    戸叶貴也, 加藤祐樹, 杉山翔吾, 野口巧, 内橋貴之

    日本生体エネルギー研究会第46回討論会  2020.12.10  日本生体エネルギー研究会

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    Event date: 2020.12

    Language:Japanese   Presentation type:Poster presentation  

    Venue:金沢   Country:Japan  

  3. 「見て触って理解するタンパク質の動きと機能 ~高速原子間力顕微鏡技術の基礎 から生物応用まで~ 」 Invited

    内橋貴之

    慶應義塾大学 大学院講義「先端研究  2020.12.2  慶應義塾大学

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    Event date: 2020.12

    Language:Japanese   Presentation type:Public lecture, seminar, tutorial, course, or other speech  

    Venue:オンライン   Country:Japan  

  4. Development of Tip-Scan Atomic Force Microscope for Stretching Elastic Substrates

    F.-Y. ChanR. Kurosaki, T. Uchihashi

    International Colloquium on Scanning Probe Microscopy (ICSPM28)  2020.12.10 

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    Event date: 2020.12

    Language:English   Presentation type:Poster presentation  

    Country:Japan  

  5. 高速AFMによる酸素発生光化学系II のドメイン構造揺らぎの可視化 Invited

    内橋貴之

    ブルカージャパン オンラインシンポジウム 〜単一生体分子の動的プロセス評価に向けた高速AFM技術〜  2020.12.15  ブルカージャパン

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    Event date: 2020.11

    Language:Japanese   Presentation type:Public lecture, seminar, tutorial, course, or other speech  

    Venue:オンライン   Country:Japan  

  6. 高速原子間力顕微鏡でタンパク質の一分子動態を可視化する Invited

    内橋貴之

    新学術領域合同シンポジウム-ソフトロボット学と発動分子科学の境界  2020.11.4 

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    Event date: 2020.11

    Language:Japanese   Presentation type:Oral presentation (invited, special)  

    Venue:オンライン   Country:Japan  

  7. 高速原子間力顕微鏡で可視化する生体・人工分子のダイナミクス Invited

    内橋貴之

    岡山大学大学院医歯薬学総合研究科(薬学系) 先端薬学特論 公開セミナー  2020.10.9  岡山大学大学院医歯薬学総合研究科

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    Event date: 2020.10

    Language:Japanese   Presentation type:Public lecture, seminar, tutorial, course, or other speech  

    Venue:オンライン   Country:Japan  

  8. Observation of Substrate Binding Sec Translocon and Structural Change of SecA with HS-AFM International conference

    Wataru Nagaike, Takamitsu Haruyama, Tomoya Tsukazaki and Takayuki Uchihashi

    2020.9.16 

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    Event date: 2020.9

    Language:English   Presentation type:Poster presentation  

  9. Chasing the transformation between monomer and dimer structure of cadherin anchored to supported lipid bilayer by high-speed AFM International conference

    Shigetaka Nishiguchi, Hiroki Oda and Takayuki Uchihashi

    2020.9.16 

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    Event date: 2020.9

    Language:English   Presentation type:Poster presentation  

    Country:Japan  

  10. Structures of heliorhodopsin and schizorhodopsin elucidate the structural diversity of microbial rhodopsins

    Wataru Shihoya, Keiichi Inoue, Singh Manish, Akimitsu Higuchi, Masae Konno, Rei Yoshizumi, Takayuki Uchihashi, Hideki Kandori and Osamu Nureki

    2020.9.16 

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    Event date: 2020.9

    Language:English   Presentation type:Poster presentation  

    Country:Japan  

  11. Characterization of the enzymatic property and structural dynamics of the T3SS ATPase from Enteropathogenic Escherichia coli International conference

    Aya Suzuki, Hiroshi Ueno, Ryo Kurosaki, Takayuki Uchihashi and Hiroyuki Noji

    2020.9.16 

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    Event date: 2020.9

    Language:English   Presentation type:Poster presentation  

    Country:Japan  

  12. HS-AFM observation of Amyloid β elongation and inhibition by antibodies International conference

    Shogo Miyajima, Maho Yagi-Utsumi, Takayuki Uchihashi and Koichi Kato

    2020.9.16 

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    Event date: 2020.9

    Language:English   Presentation type:Poster presentation  

    Country:Japan  

  13. HS-AFM as a versatile tool to study dynamical and mechanical properties of proteins International conference

    Christian Ganser, Kimitoshi Takeda, Ryota Iino, Koichi Kato and Takayuki Uchihashi

    2020.9.18 

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    Event date: 2020.9

    Language:English   Presentation type:Oral presentation (invited, special)  

    Country:Japan  

  14. Structural insights into the mechanism of rhodopsin phosphodiesterase Invited International conference

    Wataru Shihoya, Tatsuya Ikuta, Masahiro Sugiura, Kazuho Yoshida, Masahito Watari, Takaya Tokano, Kota Katayama, Satoshi Tsunoda, Takayuki Uchihashi, Hideki kandori and Osamu Nureki

    2020.9.17 

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    Event date: 2020.9

    Language:English   Presentation type:Oral presentation (invited, special)  

    Country:Japan  

  15. High-speed atomic force microscopy – a versatile tool to study protein dynamics and more

    Christian Ganser, Kimitoshi Takeda, Ryota Iino, Koichi Kato and Takayuki Uchihashi

    2020.2.7 

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    Event date: 2020.2

    Language:English   Presentation type:Oral presentation (general)  

    Country:Japan  

  16. 高速AFMを用いた生体分子のその場観察 Invited

    内橋 貴之

    2017年真空・表面科学合同講演会, 合同シンポジウム「バイオ表面・界面,細胞,生体組織のオペランド計測」  2017.8.17 

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    Language:Japanese   Presentation type:Oral presentation (invited, special)  

    Venue:横浜市立大学 金沢八景キャンパス, 神奈川  

  17. 高速AFMの開発とタンパク質の動態計測 Invited

    内橋 貴之

    第58回生物物理若手の会  2018.8.29 

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    Language:Japanese   Presentation type:Oral presentation (invited, special)  

    Venue:ぎふ長良川 ホテルパーク, 岐阜  

  18. Imaging and Manipulation of Biological Molecules with High-Speed Atomic Force Microscopy Invited International conference

    UCHIHASHI Takayuki

    ICN-T 2018  2018.7.22 

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    Language:English   Presentation type:Oral presentation (invited, special)  

    Venue:BVV Trade Fairs, Brno, Czech Republic  

  19. Image Processing and Quantitative Analysis of High-Speed-AFM Data for Studying Single-Molecule Dynamics International conference

    UCHIHASHI Takayuki

    第56回日本生物物理学会年会, Sympojium on "Multiple Approaches for Analyses of Protein Complexes -Methods and Applications"  2018.9.16 

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    Language:English   Presentation type:Oral presentation (invited, special)  

  20. High-speed atomic force microscopy: A tool for visualizing dynamic behavior from proteins to cells Invited International conference

    UCHIHASHI Takayuki

    The 28th 2017 International Symposium on Micro-NanoMechanical and Human Science  2017.12.6 

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    Language:English   Presentation type:Oral presentation (keynote)  

    Venue:Nagoya University, Aichi  

  21. High-speed Atomic Force Microscopy: A tool for direct visualization of single-molecule dynamics Invited International conference

    UCHIHASHI Takayuki

    Minisymposium on “Advanced Atomic Force Microscopy”  2018.7.18 

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    Language:English   Presentation type:Oral presentation (invited, special)  

    Venue:Montanuniversität Leoben, Austria  

  22. High-Speed Atomic Force Microscopy for Visualization of Dynamic Processes in Biological and Artificial Supramolecules Invited International conference

    UCHIHASHI Takayuki

    International Scanning Probe Microscopy 2018  2018.5.8 

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    Language:English   Presentation type:Oral presentation (invited, special)  

    Venue:the Arizona State University Tempe Campus, USA  

  23. High-Speed Atomic Force Microscopy for Visualization and Manipulation of Biological and Artificial Molecules Invited International conference

    Takayuki Uchihash

    SPMonSPM 2018  2018.8.20 

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    Language:English   Presentation type:Oral presentation (invited, special)  

    Venue:Catholic University of Leuven, Leuven, Belgium  

  24. High-speed atomic force microscopy for direct visualization of biological macromolecules at work Invited International conference

    Takayuki Uchihashi

    The 79th Okazaki Conference "Synthetic, Biological, and Hybrid Molecular Engines"  2018.8.31 

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    Language:English   Presentation type:Oral presentation (invited, special)  

    Venue:Okazaki Conference Center, Okazaki  

  25. High-Speed AFM Observation of Domain Flexibility Related to Enzymatic Function of CRISPR-Cas9 International conference

    Takayuki Uchihashi, Mikihiro Shibata, Hiroshi Nishimasu, Noriyuki Kodera, Seiichi Hirano, Toshio Ando, Osamu Nureki

    2017.9.20 

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    Language:English   Presentation type:Symposium, workshop panel (nominated)  

  26. High-Resolution Imaging of a Single Gliding Protofilament of Tubulins by HS-AFM.

    Keya JJ, Inoue D, Suzuki Y, Kozai T, Ishikuro D, Kodera N, Uchihashi T, Kabir AMR, Endo M, Sada K, Kakugo A

    Scientific reports  2017.7.21 

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    Language:English   Presentation type:Oral presentation (general)  

  27. High speed atomic force microscopy for a tool to visualize dynamic events on biological systems from single molecules to living cells Invited International conference

    UCHIHASHI Takayuki

    Workshop on “Nanofluidics in Biological Systems”  2017.9.13 

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    Language:English   Presentation type:Oral presentation (invited, special)  

    Venue:Durham University, UK  

  28. Dynamic Structural States of Molecular Disaggregation Machine ClpB Revealed by High-Speed Atomic Force Microscopy Invited International conference

    UCHIHASHI Takayuki

    Frontier Bioorganization Forum 2018  2018.7.8 

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    Language:English   Presentation type:Oral presentation (invited, special)  

    Venue:Yamate Campus, ExCELLS, Okazaki  

  29. Direct visualization of single molecule dynamics by high-speed atomic force microscopy Invited International conference

    UCHIHASHI Takayuki

    Telluride Science Research Center Workshop on Protein Dynamics  2017.7.30 

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    Language:English   Presentation type:Oral presentation (invited, special)  

    Venue:Telluride Intermediate School, Telluride, USA  

  30. Direct visualization of dynamic molecular interactions using HS-AFM Invited International conference

    UCHIHASHI Takayuki

    Frontier Bioorganization Forum 2017: Dynamical ordering and integrated functions of biomolecular systems  2017.4.24 

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    Language:English   Presentation type:Oral presentation (invited, special)  

    Venue:Academia Sinica, Taipei, Taiwan  

  31. Direct observation of single molecule dynamics at work with high-speed atomic force microscopy Invited International conference

    UCHIHASHI Takayuki

    Frontier in Single Molecule Biophysics 2017  2017.10.15 

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    Language:English   Presentation type:Oral presentation (invited, special)  

    Venue:Neve Ilan Hotel, Israel  

  32. Direct observation of self-assembly process of biological and artificial fibrils using high-speed atomic force microscopy Invited International conference

    UCHIHASHI Takayuki

    Interhierarchical understanding of materials and life through molecular observation  2018.3.24 

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    Language:English   Presentation type:Oral presentation (invited, special)  

    Venue:Institute of Molecular Science, Okazaki  

  33. 高速原子間力顕微鏡で可視化する Kai タンパク質複合体のダイナミクス

    内橋 貴之

    CyanoClock 1.0,  2018.6.29 

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    Language:Japanese   Presentation type:Oral presentation (invited, special)  

    Venue:名古屋大学  

  34. 高速原子間力顕微鏡で可視化する Kai タンパク質間相互作用のダイナミクス Invited

    内橋 貴之

    第24回日本時間生物学会学術大会 シンポジウム「24時間の創出原理」  2017.10.29 

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    Language:Japanese   Presentation type:Oral presentation (invited, special)  

    Venue:京都大学吉田キャンパス, 京都  

  35. 高速原子間力顕微鏡で可視化するタンパク質の動的秩序 Invited

    内橋 貴之

    第17回 日本蛋白質科学会年会, ワークショップ「蛋白質動的秩序のマルチプローブを用いた統合的解析」  2017.6.22 

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    Language:Japanese   Presentation type:Oral presentation (invited, special)  

    Venue:仙台国際センター, 宮城  

  36. 高速原子間力顕微鏡で可視化する生体・人工高分子の動態 Invited

    内橋 貴之

    2017年真空・表面科学合同講演会, 表面:プローブ顕微鏡研究部会「走査プローブ顕微鏡によるナノ表面科学の最前線」  2017.8.19 

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    Language:Japanese   Presentation type:Oral presentation (invited, special)  

    Venue:横浜市立大学 金沢八景キャンパス, 神奈川  

  37. 高速原子間力顕微鏡で可視化する生体分子のナノ動態

    内橋 貴之

    NSIセミナー・アドバンス生命理学特論  2018.4.12 

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    Language:Japanese   Presentation type:Public lecture, seminar, tutorial, course, or other speech  

    Venue:名古屋大学  

  38. 高速原子間力顕微鏡で可視化する生体膜反応ダイナミクス Invited

    内橋 貴之

    017年度 生命科学系合同年次大会 ワークショップ「最先端の表面科学手法による生体膜反応の実動作下計測」  2017.12.8 

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    Language:Japanese   Presentation type:Symposium, workshop panel (nominated)  

    Venue:神戸ポートピアホテル, 兵庫  

  39. 高速原子間力顕微鏡による生体分子のダイナミクス計測 Invited

    内橋 貴之

    新世代研究所 バイオ単分子研究会「タンパク質の作動原理の理解へ向けて - 機能する姿を活写する -」  2017.9.11 

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    Language:Japanese   Presentation type:Public lecture, seminar, tutorial, course, or other speech  

    Venue:呉羽ハイツ, 富山  

  40. 高速原子間力顕微鏡を用いた溶液環境下での分子のダイナミクス計測 Invited

    内橋 貴之

    平成30年度実践セミナー 『光・ナノ計測実践セミナーⅢ』  2018.6.19 

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    Language:Japanese   Presentation type:Public lecture, seminar, tutorial, course, or other speech  

    Venue:産業技術総合研究所, つくば  

  41. Oligomeric state and conformational dynamics of eubacterial ion-pumping rhodopsin studied by high-speed AFM International conference

    UCHIHASHI Takayuki

    KAKENHI International Symposium on “Studying the Function of Soft Molecular Systems”  2017.6.26 

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    Language:English   Presentation type:Symposium, workshop panel (nominated)  

    Venue:Royton Sapporo, Sapporo  

  42. 高速AFMで明らかにするKaiタンパク質間の動的相互作用 Invited

    内橋 貴之

    第69回 日本細胞生物学会大会, シンポジウム「分子の集合・離脱がつかさどる動的な細胞機能」  2017.6.13 

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    Language:Japanese   Presentation type:Oral presentation (invited, special)  

    Venue:仙台国際センター, 宮城  

  43. 生命の構成部品を直接みて理解する ~ 顕微鏡技術で可視化するタンパク質のダイナミクス現象 ~ Invited

    内橋 貴之

    自然科学研究機構 機構長プレス懇談会 「生きているとは何か?」~みる・よむ・つくる研究領域~  2018.3.9 

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    Language:Japanese   Presentation type:Public lecture, seminar, tutorial, course, or other speech  

    Venue:自然科学研究機構、東京  

  44. 原子間力顕微鏡でリアルタイム可視化する生体/人工超分子の重合ダイナミクス Invited

    内橋 貴之

    日本物理学会 2018年秋季大会 領域9, 5合同シンポジウム「時間分解プローブを駆使した表面・界面科学及び結晶成長の進展と展望 」  2018.9.11 

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    Language:Japanese   Presentation type:Symposium, workshop panel (nominated)  

    Venue:同志社大学, 京都  

  45. Visualization of Single-Molecule Dynamics Using High-Speed Atomic Force Microscopy Invited International conference

    UCHIHASHI Takayuki

    The 2nd Korea-Japan Joint Symposium on Single-Molecule Biophysics 2017  2017.11.8 

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    Language:English   Presentation type:Oral presentation (invited, special)  

    Venue:Seoul National University, Korea  

  46. Structural Flexibility and Chaperone Activity of TClpB revealed by High-Speed AFM Invited International conference

    Takayuki Uchihashi, Yo-hei Watanabe, Ryota Iino, Toshio Ando

    XIX. Annual Linz Winter Workshop  2017.2.3 

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    Language:English   Presentation type:Oral presentation (invited, special)  

    Venue:Sommerhotel Julius-Raab-Heim, Linz, Austria  

▼display all

Works 4

  1. 高速AFMによる機能性材料の動的解析

    2007

  2. Dynamic Analysis of Functional Materials using HIgh-speed AFM

    2007

  3. 高速AFM法による膜タンパク質のダイナミクス計測

    2004

  4. Dynamics of membrane proteins by high-speed AFM

    2004

KAKENHI (Grants-in-Aid for Scientific Research) 30

  1. 単一タンパク質の構造と局所物性のダイナミクスを可視化できる顕微鏡技術の開発

    Grant number:22K18943  2022.6 - 2024.3

    日本学術振興会  科学研究費補助金  挑戦的研究(萌芽)

  2. High-Speed-AFM Analysis of Structural and Mechanical Properties of Single Protein under Mechanical Stimulation

    Grant number:21H01772  2021.4 - 2024.3

    Japan Society for the Promotion of Science  Grants-in-Aid for Scientific Research  Grant-in-Aid for Scientific Research (B)

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    Authorship:Principal investigator 

    Grant amount:\17420000 ( Direct Cost: \13400000 、 Indirect Cost:\4020000 )

  3. 単一ゲル微粒子の強靭化に基づくミクロ空間移動科学の構築

    Grant number:21H01999  2021.4 - 2024.3

    日本学術振興会  科学研究費助成事業  基盤研究(B)

    鈴木 大介, 内橋 貴之, 中薗 和子, 呉羽 拓真, 藤本 和士

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    私達の体の中にある血管はミクロ空間であり、血液が高速で流動している。そのような場において、自在な移動を可能とする新たな高分子微粒子(ハイドロゲル微粒子)を開発する。従来、トレードオフの関係にあった柔らかさと耐久性を兼ね備えた、新規ゲル微粒子を開発し、ゲル微粒子のナノ構造と力学特性の関係を解明する。そのための手法として、実験的手法と計算機シミュレーションを併用する。以上を通じ、血管のようなミクロ流動場を、自在に移動できるゲル微粒子の実現を目指す。
    初年度に引き続き、水溶性を示す擬ロタキサンを活用することで、新規ゲル微粒子の合成検討を実施した。この際、擬ロタキサンが重合中に構造を崩壊してしまうという課題に直面したため、重合法の全面的な見直しを行った。その結果、擬ロタキサン構造をできるだけ維持したうえで、ゲル微粒子に導入する主張を見出し、その結果、目的とするロタキサン架橋構造を有する新規ゲル微粒子の合成に成功した。
    更に、構造が明確なロタキサン架橋剤をゲル微粒子に導入することにも成功した。この際、ミニエマルション重合法を適用した。すなわち、微小水滴内にモノマーや架橋剤を溶解させ、ラジカル重合法を実施することでゲル微粒子を得た。一般的に、ミニエマルション重合法により得られる微粒子のサイズ分布が広いのに対し、かなり粒子径分布が狭いゲル微粒子を得ることに成功した。これらの強靭性を確かめるために、ゲル微粒子をフィルム化して力学試験を実施した。特に、一軸伸長試験を行ったところ、一般的な化学架橋を施したゲル微粒子からなるフィルムと比較し、2倍以上も破断ひずみが増加した。すなわち、強靭なゲル微粒子フィルムを作成することができた。
    その他にも、別の観点から、本ゲル微粒子の力学特性評価を実施した。特に、原子間力顕微鏡法を活用することで、単一ゲル微粒子の強靭性の評価を実施した。従来の化学架橋を施したゲル微粒子と比較し、本開発ゲル微粒子は、壊れにくい事が分かった。
    構造が明確なロタキサン架橋剤を導入したゲル微粒子を合成することに成功したため。また、得られたゲル微粒子が、構造由来の特異的な力学機能の発現をすることを見出したため。こうした微粒子をさらに構造設計することにより、より高度な力学機能の発現に挑戦できる。
    構造が明確なロタキサン架橋を有するゲル微粒子の多様化に挑戦する。このことにより、ゲル微粒子の力学特性の全容解明を目指していく。最終的には、高速流動場において柔らかさの有効性を発揮するうえで十分な強靭性を有するゲル微粒子を開発する。重合法によって制御したゲル微粒子内の微細構造を、高速AFMや放射光散乱測定により定量評価する。そして、ナノ構造と強靭性の相関関係の解明に挑む。さらに、血管を模倣したマイクロ流路の実験を通じ、ゲル微粒子の流動制御に重要な因子を解明する。

  4. Elucidation of working mechanisms of a molecular-engine complex responsible for protein membrane permeation by imaging of the conformational dynamics

    Grant number:21H00393  2021.4 - 2023.3

    Japan Society for the Promotion of Science  Grants-in-Aid for Scientific Research  Grant-in-Aid for Scientific Research on Innovative Areas (Research in a proposed research area)

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    Authorship:Principal investigator 

    Grant amount:\5980000 ( Direct Cost: \4600000 、 Indirect Cost:\1380000 )

  5. Study for the mechanism of functional expression and dysfunction of membrane proteins through direct observations of single molecular fluctuation

    Grant number:20K07279  2020.4 - 2023.3

    Japan Society for the Promotion of Science  Grants-in-Aid for Scientific Research  Grant-in-Aid for Scientific Research (C)

    Sohma Yoshiro

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    CFTR is an anion channel plays a central role in the transepithelial ions and water transport, which mutations cause a congenital disease Cystic Fibrosis (CF). In this study, we investigated impact of molecular fluctuation of CFTR molecule for its function and expression in in health and disease using high speed atomic force microscopy (HS-AFM) and molecular dynamics simulation (MD).
    HS-AFM showed a large fluctuation of two NBDs which drive the channel gating, suggesting that it might be a stochastic rather than predicable process.
    F508del mutation located at the NBD1-ICL4 interface is the most frequent in Caucasians. It is known the F508del-CFTR is degraded and poorly expressed because of its structural instability. The MD study indicated that the Japanese CF mutations in NBD1 and ICL4 as well as the F508del mutation affect the molecular fluctuations of CFTR protein. Thus the molecular fluctuation underlies the function and expression of CFTR in health and disease.

  6. High-Speed AFM Visualization of Structural Dynamics of Porous Coordination Polymers Induced External Stimuli

    Grant number:20H04669  2020.4 - 2022.3

    Japan Society for the Promotion of Science  Grants-in-Aid for Scientific Research  Grant-in-Aid for Scientific Research on Innovative Areas (Research in a proposed research area)

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    Authorship:Principal investigator 

    Grant amount:\5330000 ( Direct Cost: \4100000 、 Indirect Cost:\1230000 )

  7. 再構成アプローチで解明するダイナミンの膜切断機構とその破綻に起因する疾患発症機序

    Grant number:19KK0180  2019.10 - 2024.3

    日本学術振興会  科学研究費助成事業  国際共同研究加速基金(国際共同研究強化(B))

    竹田 哲也, 内橋 貴之, 竹居 孝二, 西上 幸範, 谷 知己

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    Authorship:Coinvestigator(s) 

    本研究では、日本国内5名(代表者:竹田、分担者:内橋、谷、竹居、西上)と、海外共同研究者(Harvey McMahon、MRC分子生物学研究所)で構成する学際的かつ国際的な共同研究チームを組織し、ダイナミンによる膜切断機構の作動原理とBARドメイン蛋白質による制御機構、さらにダイナミンの機能破綻によって起こる難治性疾患の発症機序を、in vitro再構成系を用いた「ボトムアップ型」の構成生物学的なアプローチで解明する。
    先天性ミオパチーは,筋力や筋緊張の低下を伴う筋疾患である.先天性ミオパチーの一つである中心核ミオパチー(Centronuclear Myopathy; CNM)は,T管やTriadの形成異常により,骨格筋の興奮-収縮連関が正常に起こらない.先行研究で,膜リモデリング分子であるBIN1 (Amphiphysin 2)とDynamin 2をコードするBIN1,DNM2の各遺伝子の一塩基変異(SNV)が,CNM発症に関与することが知られていた.しかし,膜リモデリング異常によりCNMが発症するメカニズムは不明であった.代表者は現在までに,(1) BIN1とDynamin 2の膜リモデリング機能を定量的に解析することができるin vitroおよびin celluloのT管様構造の再構成系を確立した.またこれらの再構成系を用いて,(2) CNM変異型のBIN1およびDynamin 2が膜リモデリング機能異常を示すこと,(3) Dynamin 2の膜切断機能に必要なGTPアーゼ活性が,CNM変異型Dynamin 2では恒常的に亢進し,T管様構造が過度に切断されることを明らかにした.以上の成果については,原著論文2報(Fujise et al., Journal of Biological Chemistry 2021; Fujise et al., Human Mutation 2021),関連する総説1報(Fujise et al., IJMS 2022)と著書1報(竹田、医学のあゆみ 2022)に発表した.さらに,研究代表者がオーガナイズした第45回日本分子生物学会年会のワークショップ「生体膜の構造機能を制御する分子の秩序と集合機構」に国際共同研究者のMcMahon博士(MRC分子生物学研究所)を招聘し,最新の知見についての講演をしていただいた.また研究代表者は,2022/9/12-9/16まで,MRC分子生物学研究所に滞在し,9/14にセミナーを行ったほか,McMahon博士と今後の研究方針についてのディスカッションを行った.
    Covid-19の影響で延期していたイギリスへの渡航,海外共同研究者の日本への招聘など,予定していたイベントをおこなった.また国内において実施可能な研究計画についても,成果を挙げることができている.
    代表者(竹田)は研究統括および膜リモデリング分子の細胞生物学的解析を行う.また,海外共同研究者(McMahon博士)や分担者(竹居)が開発した膜リモデリングのin vitro再構成系を用い,電子顕微鏡による構造解析(竹居),高速AFMによる分子動態解析(内橋),蛍光偏光顕微鏡解析(谷),数理モデリング(西上)を行う。今年度も,代表者(竹田)はMcMahon研究室に滞在し,疾患型ダイナミンのin vitro膜リモデリング解析を行う。万が一,イギリスへの渡航が困難な状況になった場合でも,国内のリソースを利用しながら構造解析や分子間相互作用解析などを進めるとともに,MRC分子生物学研究所の研究者とのネットワーキングをオンラインで積極的に行い,学術的・人的な基盤づくりを行う.

  8. Cooperative regulation of cytoskeleton and membrane dynamics by novel mechanism of dynamin

    Grant number:19H03225  2019.4 - 2022.3

    Japan Society for the Promotion of Science  Grants-in-Aid for Scientific Research  Grant-in-Aid for Scientific Research (B)

    Takei Kohji

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    We found that Charcot-Marie-Tooth disease-associated mutations of dynamin 2 cause aberrant stress fibers, showing that dynamin is required for the formation and stabilization of stress fibers. And we reconstituted in vitro the actin bundle formation by dynamin. We also found that dynamin 1 bundles microtubules, and showed that this bundling is necessary for the primary processes formation and stabilization of cell morphology of renal glomerular podocytes. The microtubule-binding site of dynamin 1 was identified. Furthermore, regarding the regulation of membrane dynamics, we demonstrated that dynamin 2 and BIN1, a BAR protein, cooperatively function in T-tubule formation and stabilization of skeletal muscle cells.

  9. 発動分子の化学-力学エネルギー変換機構の解明に資する高速AFM技術の開発

    Grant number:19H05389  2019.4 - 2021.3

    日本学術振興会  科学研究費助成事業  新学術領域研究(研究領域提案型)

    内橋 貴之

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    Authorship:Principal investigator 

    Grant amount:\5980000 ( Direct Cost: \4600000 、 Indirect Cost:\1380000 )

    本領域で創成された発動分子やその集積体の構造動態や動的相互作用、秩序形成のダイナミクス観察に関して共同研究を推進する。同時に、計測技術・シミュレーション・理論班との協働により発動分子の作動原理を実験・理論両面から明らかにする。一方で、回転分子モーターV-ATPaseに焦点を絞り、高速AFM/一分子蛍光顕微鏡を用いてATPの結合と解離の検出と同時に分子の構造変化を測定する方法を確立し、化学-力学エネルギー変換の分子機構を明らかにする。また、高速AFM/一分子蛍光顕微鏡装置に実装する多機能基板の開発も進め、外部刺激による発動分子の動態応答に関する研究を推進する。
    i) フェリチンケージ分解過程の直接観察(A01-3上野Gとの領域内共同研究):鉄を貯蔵タンパク質フェリチンは自己集合によってかご状構造体を形成することが知られており, ナノ材料を調製するためのテンプレートとして広く使用されてきた。このかご状構造体は溶液のpHに依存して分解及び再構成されることが知られているが, それらの開始や中間状態に関与するダイナミクスは解明されていなかった。高速AFMで, 溶液中の単一のフェリチンケージのpH依存的な分解過程を観察した結果, かご状構造体が断片に分解する前に穴が形成されることを明らかにした。MDシミュレーションの結果, 穴はフェリチンタンパク質3個で構成される3回対称チャネルの開口形成でトリガーされることを明らかにした。(Basudev et al, PCCP 2020)
    ii) 微小管の変形によるキネシンの運動速度変化( B01 角五Gとの領域内共研究):マイカ基板にアイランド上に平面脂質膜を形成し、さらに脂質膜を構成する正電荷脂質の量を制御することで、微小管を屈曲して基板に固定することができた。この微小管上で微小管関連運動タンパク質であるキネシンの滑走運動を高速AFMで観察した結果、微小管の変形が細胞内輸送に関与する滑走速度を制御していることが明らかになった。屈曲の曲率が異なる微小管でのキネシンの移動速度を解析したところ、曲率が大きくなるほどキネシンの運動速度が低下した。分子動力学シミュレーションでキネシンの運動速度低下の要因を探ったところ、変形した微小管ではキネシンの親和性が高まっているためであることがわかった。この結果は、キネシンの移動を制御するためのメカノセンサーとしての微小管の役割を明らかにし、微小管の機械的変形がキネシンの移動を制御する役割を果たしていることを示唆している。
    令和2年度が最終年度であるため、記入しない。
    令和2年度が最終年度であるため、記入しない。

  10. High-speed AFM study on functional modulation of kinesin caused by structural defects of mictoruble

    Grant number:18H01837  2018.4 - 2021.3

    Grants-in-Aid for Scientific Research  Grant-in-Aid for Scientific Research (B)

    Uchihashi Takayuki

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    Authorship:Principal investigator 

    Grant amount:\17550000 ( Direct Cost: \13500000 、 Indirect Cost:\4050000 )

    In this study, we have developed an in-line force-curve mode, which is an advanced molecular manipulation function of high-speed atomic force microscopy (AFM), allowing us to form single tubulin dimer defects in microtubules with high controllability and to obtain force-distance curves in realtime. This technique enabled us to observe the self-healing process of structural defects created on a microtubule and the translational motility of kinesin molecules around the defects, and to quantify the binding energy between tubulins. Furthermore, we have extended this technique to the fast force mapping mode. Also, by observing the kinesin sliding motion in bent microtubules, we found that the kinesin translational velocity is significantly reduced in the bent region.

  11. 高速原子間力顕微鏡で明らかにする微小管の構造欠陥によるキネシンの機能変調

    2018.4 - 2021.3

    日本学術振興会  科学研究費補助金 基盤研究(B) 

    内橋 貴之

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    Authorship:Principal investigator  Grant type:Competitive

    Grant amount:\17550000 ( Direct Cost: \13500000 、 Indirect Cost:\4050000 )

  12. 高速AFMを基盤としたソフトクリスタルの構造物性ダイナミクス評価技術の確立

    2018.4 - 2020.3

    日本学術振興会  科学研究費補助金 新学術領域 

    内橋 貴之

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    Authorship:Principal investigator  Grant type:Competitive

    Grant amount:\5200000 ( Direct Cost: \4000000 、 Indirect Cost:\1200000 )

  13. Development of analytical techniques for structural property dynamics based on high-speed AFM

    Grant number:18H04512  2018.4 - 2020.3

    Grant-in-Aid for Scientific Research on Innovative Areas (Research in a proposed research area)

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    Grant amount:\5200000 ( Direct Cost: \4000000 、 Indirect Cost:\1200000 )

  14. Development of high-speed AFM/single-molecule FRET to visualize synthesis and degradation of carbohydrate chain

    Grant number:17K19519  2017.6 - 2019.3

    Grant-in-Aid for Challenging Research (Exploratory)

    Uchihashi Takayuki

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    Grant amount:\6240000 ( Direct Cost: \4800000 、 Indirect Cost:\1440000 )

    We have incorporated an image-splitting optical system into a combined high-speed AFM/ total-reflection fluorescent microscopy system which enables us to perform high-speed AFM imaging and single-molecule FRET simultaneously. We prepared the fluorescent labeling sample for single-molecule FRET measurements for bacterial chondroitin polymerase K4CP and glycoside hydrolase cellulase, TrCel6A, and succeeded in measuring changes of the FRET efficiently due to the extension of the chondroitin chain and the structural change of the cellulase. Although we tried to observe these molecule with the combined high-speed AFM and single-molecule FRET system, in the face of various problems, we have not reached the success within the period. However we are now establishing the optimum measurement conditions for the simultaneous observation and thus assume that we can gain novel insights about molecular mechanisms with detailed single-molecule analysis using the combined system.

  15. 糖鎖の合成と分解過程を可視化する高速AFM/一分子FRET技術の確立

    2017.6 - 2019.3

    日本学術振興会  科学研究費補助金 挑戦的研究(萌芽) 

    内橋 貴之

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    Authorship:Principal investigator  Grant type:Competitive

    Grant amount:\6240000 ( Direct Cost: \4800000 、 Indirect Cost:\1440000 )

  16. Elucidation of mechanical property and self-repair mechanism of microtubule with high-speed AFM

    Grant number:17F17701  2017.4 - 2019.3

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  17. 高速原子間力顕微鏡による微小管の自己修復機構の解明と機械特性評価

    2017.4 - 2019.3

    日本学術振興会  特別研究員奨励費 

    内橋 貴之

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    Authorship:Principal investigator  Grant type:Competitive

    Grant amount:\2300000 ( Direct Cost: \2300000 )

  18. 高速AFM計測によるKaiタンパク質のロバストな概日周期発生機構の解明

    2016.4 - 2018.3

    日本学術振興会  科学研究費補助金 新学術領域 

    内橋 貴之

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    Authorship:Principal investigator  Grant type:Competitive

    Grant amount:\6370000 ( Direct Cost: \4900000 、 Indirect Cost:\1470000 )

  19. 高速AFMで明らかにする真正細菌型イオンポンプロドプシンの多量体構造と機能動態

    2016.4 - 2018.3

    日本学術振興会  科学研究費補助金 新学術領域 

    内橋 貴之

      More details

    Authorship:Principal investigator  Grant type:Competitive

    Grant amount:\6890000 ( Direct Cost: \5300000 、 Indirect Cost:\1590000 )

  20. 高速AFMで明らかにする真正細菌型イオンポンプロドプシンの多量体構造と機能動態

    Grant number:16H00830  2016.4 - 2018.3

    新学術領域研究(研究領域提案型)

    内橋 貴之

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    Authorship:Principal investigator 

    Grant amount:\6890000 ( Direct Cost: \5300000 、 Indirect Cost:\1590000 )

    1.イオンポンプロドプシンの多量体構造の決定: 様々な微生物型ロドプシンを高速AFM法と円二色性(CD)分光法により網羅的に解析し、脂質膜に再構成された状態での多量体構造を決定した。高速AFM観察により、GR、KR2、FR、KrActR、QsActR等の真性細菌で見出された微生物型ロドプシンはリング状の5量体を形成しており、GPRは5量体と6量体が共存していることがわかった。また、センサリーロドプシンであるSRII、ハロロドプシンNpHRなどの古細菌型ロドプシンは三量体であることがわかった。一方、内向きH+ポンプであるPoXeRやセンサリーロドプシンASRは真性細菌で発見された微生物型ロドプシンであるが、アミノ酸残基の相同性からは古細菌型に分類され、実際多量体構造は三量体であった。また、CD分光で観察される三量体と五量体に特徴的なスペクトルは多量体構造に依存したレチナールの配向で説明できることもわかった。これらの結果から、微生物型プロドプシンの多量体構造は進化系統樹と深く関わっている一方、イオンポンプのイオン種や向きと相関がないことがわかった。
    2.KR2の光誘起構造変化の観察の観察: 外向きNa+ポンプであるKR2の光誘起による構造変化の観察に向けて、KR2五量体の観察面の決定およい高解像観察に適した試料の調製を行った。野生型に比べて光サイクルが100以上遅いN112A変異体を高解像観察しながら、周期的な光照射を照射したが明瞭な構造変化は観察できなかった。このことから、BRとは異なり、KR2の光サイクル中には大規模な構造変化は起きていない可能性が示唆された。
    29年度が最終年度であるため、記入しない。
    29年度が最終年度であるため、記入しない。

  21. 高速AFM計測によるKaiタンパク質のロバストな概日周期発生機構の解明

    Grant number:16H00758  2016.4 - 2018.3

    新学術領域研究(研究領域提案型)

    内橋 貴之

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    Authorship:Principal investigator 

    Grant amount:\6370000 ( Direct Cost: \4900000 、 Indirect Cost:\1470000 )

    Kaiタンパク質の動的相互作用: KaiCのリン酸化状態に依存してKaiAとの相互作用が概日周期的に変動することを見出した(Phase Dependent Differential Affinity: PDDAと名付けた )。KaiCのリン酸化概日周期について、実験で得られたパラメーターを用いて数理シミュレイションを行い、PDDAが概日周期にどのような影響を及ぼすのかを調べた。PDDAが無い場合には、KaiAとKaiCの濃度比が変動すると概日周期が消失するのに対して、PDDAがある場合には概日周期が維持される濃度比が3倍程度大きくなった。このことから、PDDAは細胞内でのタンパク質濃度の揺らぎに対するKaiシステムの頑強性に寄与していることが明らかになった。また、温度制御下でKaiA-KaiCの相互作用を調べたところ、25-29℃の温度範囲では動的親和性に大きな変化は見られえず、30℃以上では、KaiAとKaiCの親和性が大きく変化することがわかった。
    プロテアソームα7ホモ14量体のα6による2ステップ解体過程:領域内共同研究としてプロテアソーム構成タンパク質α7ホモ14量体がα6により解体される過程を観察した。α7-14量体をアミノシランで化学修飾したマイカに強固に吸着させると14量体が自発的に7量体に分離する様子が見られた。さらに,α7-7量体リングの中心孔にα6サブユニットが結合・解離を繰り返し、時間経過とともにα6が中心孔に強固に結合することが分かった。また、積層した7量体リング間に隙間が経時的に生じ、そこにα6が結合する様子が観察された。これらのことから、α7-14量体のα6サブユニットによる解体は、リング積層間隙へのα6の結合と解離、7量体リング中心孔へのα6の強固な結合によるダブルリングの再生阻止の2段階の過程を経ていることを明らかにした。
    29年度が最終年度であるため、記入しない。
    29年度が最終年度であるため、記入しない。

  22. Analysis of the mechanism of efficient degradation of crystalline substrate by cellulases from Actinomycetes

    Grant number:15K07383  2015.4 - 2018.3

    Japan Society for the Promotion of Science  Grants-in-Aid for Scientific Research  Grant-in-Aid for Scientific Research (C)

    Uchiyama Taku, UCHIHASHI TAKAYUKI, ISHIDA TAKUYA

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    Authorship:Collaborating Investigator(s) (not designated on Grant-in-Aid) 

    Toward to the realization of “Biorefinery”, we aimed to increase the efficiency of enzymatic saccharification, and attempted to elucidate the enzymatic reaction mechanism involved in cellulases efficient crystalline cellulose degradation. Specifically, we attempted to elucidate such a mechanism through the biochemical experiment, HS-AFM observation, and protein crystal structure analysis of cellulase SaCel6B derived from Actinomycetes. As a result of experiments and observations, we found two amino acid residues involved in efficient crystalline cellulose degradation of cellulase SaCel6B and clarified the importance of these residues. Based on information of such amino acid residues, it could be possible to try to discover cellulases with highly saccharification efficiency or synthesize novel cellulases with high efficiency of saccharification.

  23. Development of variable-temperature high-speed AFM and its application to temperature-dependent ATPases

    Grant number:15H03540  2015.4 - 2018.3

    UCHIHASHI TAKAYUKI

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    Authorship:Principal investigator 

    Grant amount:\16770000 ( Direct Cost: \12900000 、 Indirect Cost:\3870000 )

    We have developed variable-temperature high-speed atomic force microscopy (VT-HS-AFM) with which the experimental temperature can be controlled ranging from a room temperature to around 40 ℃. The performance of VT-HS-AFM was confirmed by observation of fluidity of DPPC lipid bilayer with the phase-transition temperature of 41℃ from gel phase to liquid crystal phase. We applied the VT-HS-AFM system to observation of a flagellar protein, FliI, which is an ATPase with the activity optimum temperature over 40℃. HS-AFM images captured oligomerization processes of the FliI monomers to the hexamer and also conformational changes of the oligomers. Also we observed temperature dependent interaction between KaiC and KaiA which are proteins responsible to the circadian rhythm of Cyanobacteria.

  24. 温度可変高速AFMの開発と温度依存的ATPaseの構造機能相関の解明

    2015.4 - 2018.3

    日本学術振興会  科学研究費補助金 基盤研究(B) 

    内橋 貴之

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    Authorship:Principal investigator  Grant type:Competitive

    Grant amount:\16770000 ( Direct Cost: \12900000 、 Indirect Cost:\3870000 )

  25. Development of technical basis for direct observations of membrane protein complex in dynamic equilibrium under a physiological condition

    Grant number:15K15035  2015.4 - 2017.3

    Japan Society for the Promotion of Science  Grants-in-Aid for Scientific Research  Grant-in-Aid for Challenging Exploratory Research

    SOHMA Yoshiro, UCHIHASHI Takayuki, NISHIZAKA Takayuki, SAKURAI Minoru, SATO Chikara, YU Ying-Chun, HWANG Tzyh-Chang, KATO Takanobu, FUJIMURA Shoko

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    Membrane proteins functions with forming a complex in the plasma membrane. The mechanism of the membrane protein complexes is one of the most important in the physiology and medicine. We attempted to develop the technical basis for the direct observation of membrane protein complexes working in non-equilibrium dynamics under a physiological condition using the high-speed atomic force microscopy (HS-AFM).
    We found that the ‘Nanodisc’ should be the most likely candidate for the central parts of the ‘physiological AFM platform’ allowing us the successful direct observation for ‘in vivo’-like dynamics of membrane protein complexes. We also attempted to establish a fundamental theory for interpreting the HS-AFM movie data of the intermolecular interaction. Several issues appeared in the theoretical study suggested the existence of an unknown important process for the intermolecular interaction in a mesoscopic level between the single-molecular and the macroscopic levels

  26. Development of advanced high-speed AFM and analysis of proteins' action mechanism

    Grant number:26119003  2014.7 - 2019.3

    Grant-in-Aid for Scientific Research on Innovative Areas (Research in a proposed research area)

    Ando Toshio

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    Authorship:Collaborating Investigator(s) (not designated on Grant-in-Aid) 

    The aim of this study is largely classified into two: (1) Extensively exploring a new research field of “dynamic structural biology” that seeks to film protein molecules in dynamic action with high-speed AFM, not only by conducting the study by myself but also by collaborating with researchers of the project group as well as external researchers, and (2) enabling observation of new molecular phenomena that have been impossible with the current high-speed AFM, by advancing the capability of high-speed AFM. For the aim (1), our instruments were opened to these researchers. Through many collaborations, we succeeded in observing a variety of proteins, providing mechanistic insights into their molecular processes. For the aim (2), we succeeded in developing high-speed AFM combined with optical tweezers and super-resolution fluorescence microscopy based on high-speed AFM. The actual application studies of these new instruments are subjects to be done in the future.

  27. Study for mechanism of ABC transporters based on single molecular direct observations using high speed Atomic Force Microscopy.

    Grant number:25293049  2013.4 - 2017.3

    Japan Society for the Promotion of Science  Grants-in-Aid for Scientific Research  Grant-in-Aid for Scientific Research (B)

    SOHMA Yoshiro, UCHIHASHI Takayuki, NISHIZAKA Takayuki, SAKURAI Minoru, SATO Chikara, YAMASHITA Hayato, YU Ying-Chun, HWANG Tzyh-Chang

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    ABC transporter superfamily is one of the biggest protein families which members play essential physiological roles in human. The members in the ABC transporter superfamily share highly-conserved Nucleotide Binding Domain (NBD) underlying their ATP-driven mechanisms. We investigated molecular mechanism of the ‘NBD engine’ in an ABC member, Cystic Fibrosis Transmembrane Regulator (CFTR) using single-molecular measuring methods including high-speed atomic force microscopy (HS-AFM).
    We succeeded the first direct observation of dynamics of the intracellular complex consisting of two NBDs and regulatory (R) domain in CFTR using HS-AFM. We also obtained results in the direct observations of binding/unbinding process between AQP4 and anti-AQP4 antibody as a model of the molecule-molecule interactions. In addition, we performed several molecular physiological/pharmacological studies for disease-associated CFTR mutants.

  28. Dynamic visualization of extracellular information processing space using synthetic and degrading enzymes of plant cell wall components

    Grant number:24114008  2012.6 - 2017.3

    Japan Society for the Promotion of Science  Grants-in-Aid for Scientific Research  Grant-in-Aid for Scientific Research on Innovative Areas (Research in a proposed research area)

    IGARASHI Kiyohiko, UCHIHASHI Takayuki

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    The plant cell wall has a complicated structure in which various polymeric materials interact with each other, and the details are not clarified. Each component constituting the cell wall is synthesized by the enzyme of the cell and then complexed by self-assembly to construct a complicated cell wall structure outside the cell. On the other hand, organisms such as mushrooms that grow with the cell wall as a nutrient source acquire nutrition by decomposing the complicated structure of the cell wall by the degrading enzyme. Therefore, in this study, the relationship between the enzymes of these organisms and the organization of components constituting the cell wall was clarified by microscope technology and biochemical technology, and knowledge was obtained for understanding and utilizing complex plant cell wall.

  29. Opening up New Structural Biology by High-speed AFM

    Grant number:24227005  2012.5 - 2017.3

    Japan Society for the Promotion of Science  Grants-in-Aid for Scientific Research  Grant-in-Aid for Scientific Research (S)

    Ando Toshio, UCHIHASHI Takayuki, FUKUMORI Yoshihiro, FUKUMA Takeshi, KODERA Noriyuki, KONNO Hiroki, WONG Richard, MURAKAMI Satoshi, OGURA Teru, TOYOSHIMA Yoko, KANDORI Hideki

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    We worked on three subjects. In subject 1, we elucidated the functional mechanism of various protein systems by observing their dynamic processes using high-speed AFM (HS-AFM) that we had established before. Besides, we demonstrated that HS-AFM can analyze the structure of intrinsically disordered proteins that are difficult to analyze with conventional methods. In subject 2, we developed fast/wide-area scanning techniques without generation of unwanted vibrations and an interactive HS-AFM technique that can manipulate the sample during imaging. The usefulness of these new techniques were demonstrated. In addition, we developed cantilever-scan type of HS-AFM combined with fluorescence microscopy, and thereby, made it possible to capture HS-AFM and fluorescence images simultaneously. In subject 3, we developed high-speed scanning ion conductance microscopy that can image the sample without contact with the sample. The imaging speed reached a level 100-times faster than before.

  30. Development of high-speed atromic force microscope for biological system

    2004

    Cooperative Research 

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    Grant type:Competitive

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Industrial property rights 3

  1. 走査型プローブ顕微鏡

    内橋 貴之, 柴田 幹大, 古寺 哲幸

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    Applicant:国立大学法人金沢大学

    Application no:特願2016-234584  Date applied:2016.12

    Announcement no:特開2018-091695  Date announced:2018.6

    J-GLOBAL

  2. 昇温ホルダおよびプローブ顕微鏡

    内橋 貴之, 足立 彗, 古寺 哲幸

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    Applicant:国立大学法人金沢大学

    Application no:特願2016-233494  Date applied:2016.11

    Announcement no:特開2018-091666  Date announced:2018.6

    J-GLOBAL

  3. チャンバーアレイの製造方法

    古寺 哲幸, 豐田 貴大, 内橋 貴之

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    Applicant:国立大学法人金沢大学

    Application no:特願2016-232100  Date applied:2016.11

    Announcement no:特開2018-085975  Date announced:2018.6

    J-GLOBAL

 

Teaching Experience (Off-campus) 7

  1. 物理学演習II

    Nagoya University)

  2. 物理学I

    Kanazawa University)

  3. 力学演習II

    Kanazawa University)

  4. 力学演習I

    Kanazawa University)

  5. 生物物理学I

    Nagoya University)

  6. 電磁気学演習II

    Kanazawa University)

  7. 電磁気学演習I

    Kanazawa University)

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Academic Activities 2

  1. AFM BioMed Conference 2022 International contribution

    Role(s):Planning, management, etc.

    2022.8 - 2022.9

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    Type:Academic society, research group, etc. 

  2. JST さきがけ[高次構造体]細胞の動的高次構造体 | アドバイザー

    Role(s):Review, evaluation