2024/03/21 更新

写真a

ウチハシ タカユキ
内橋 貴之
UCHIHASHI Takayuki
所属
大学院理学研究科 理学専攻 物理学第一 教授
大学院担当
大学院理学研究科
学部担当
理学部 物理学科
職名
教授
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プロフィール
1993年3月 広島大学理学部物理学科卒業
1993年4月 広島大学大学院理学研究科物理学専攻博士前期課程入学
1995年3月 広島大学大学院理学研究科物理学専攻博士前期課程修了
1995年4月 広島大学大学院理学研究科物理学専攻博士後期課程進学
1997年4月 大阪大学大学院工学研究科電子工学専攻博士後期課程編入学
1998年3月 大阪大学大学院工学研究科電子工学専攻博士後期課程修了
1998年4月-2000年3月 アトムテクノロジー研究体 博士研究員
2000年4月-2002年7月 姫路工業大学工学部電子工学科 助手
2002年8月-2004年7月 ダブリン大学トリニティカレッジ SFIナノサイエンス研究所 シニアサイエンティスト
2004年4月-2006年3月 金沢大学自然科学研究科 助手
2006年4月-2008年3月 金沢大学自然科学研究科 助教授
2008年4月-2015年3月 金沢大学理工研究域 准教授
2015年4月-2017年3月 金沢大学理工研究域 教授 金沢大学理工研究域バイオAFM先端研究センター センター長(併任)
2016年4月-2017年3月 京都大学エネルギー理工学研究所 客員教授
2017年4月- 名古屋大学大学院理学研究科 教授
2018年4月-2020年3月分子科学研究所・生命・錯体分子科学研究領域 客員教授
2018年8月- 生命創成探究センター・生命分子動態計測グループ(連携研究グループ) 客員教授   
外部リンク

学位 1

  1. 博士(工学) ( 1998年3月   大阪大学 ) 

研究キーワード 11

  1. 一分子イメージング

  2. タンパク質

  3. 生物物理学

  4. 走査型プローブ顕微鏡+

  5. 高速原子間力顕微鏡

  6. 原子間力顕微鏡

  7. 走査型プローブ顕微鏡

  8. 表面物理学

  9. 生物物理学

  10. 一分子計測

  11. 高速原子間力顕微鏡

研究分野 3

  1. その他 / その他  / ナノ材料・ナノバイオサイエンス

  2. ライフサイエンス / 生物物理学

  3. ライフサイエンス / 生物物理学

経歴 14

  1. 東海国立大学機構糖鎖生命コア研究拠点(iGCORE)兼任   分子生理・動態部門

    2021年 - 現在

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    備考:兼任

  2. 名古屋大学   理学研究科物質理学専攻(物理系)   教授

    2017年4月 - 現在

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    国名:日本国

  3. 名古屋大学   理学研究科物質理学専攻(物理系)   教授

    2017年4月 - 現在

  4. 名古屋大学   大学院理学研究科理学専攻(物理科学領域)   教授

    2017年4月 - 現在

  5. 金沢大学   理工研究域   教授

    2015年4月 - 2017年3月

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    国名:日本国

  6. 金沢大学   理工研究域 バイオAFM先端研究センター   副総長

    2015年4月 - 2017年3月

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    国名:日本国

  7. 金沢大学   理工研究域   教授

    2015年4月 - 2017年3月

  8. 金沢大学   理工研究域 バイオAFM先端研究センター   センター長

    2015年4月 - 2017年3月

  9. 金沢大学   理工研究域   准教授

    2008年4月 - 2015年3月

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    国名:日本国

  10. 金沢大学   自然科学研究科   助教授

    2006年4月 - 2008年3月

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    国名:日本国

  11. 金沢大学   自然科学研究科   助教授

    2004年4月 - 2006年3月

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    国名:日本国

  12. ダブリン大学トリニティカレッジ   SFIナノサイエンス研究所   シニアリサーチャー

    2002年8月 - 2004年3月

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    国名:日本国

  13. 姫路工業大学   工学部電子工学科   助教授

    2000年4月 - 2002年7月

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    国名:日本国

  14. アトムテクノロジー研究体   博士研究員

    1998年4月 - 2000年3月

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    国名:日本国

▼全件表示

学歴 5

  1. 大阪大学   工学研究科   電子工学専攻

    - 1998年

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    国名: 日本国

  2. 大阪大学   工学研究科   電子工学専攻

    - 1998年

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    国名: 日本国

  3. 広島大学   理学研究科   物理学専攻

    - 1995年

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    国名: 日本国

  4. 広島大学   理学研究科   物理学専攻

    - 1995年

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    国名: 日本国

  5. 広島大学   理学部   物理学科

    - 1993年

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    国名: 日本国

所属学協会 11

  1. 日本物理学会

  2. 応用物理学会

  3. 日本生物物理学会

  4. 日本表面真空学会

  5. 日本化学会

  6. 日本分子生物学会

  7. Japanese Applied Physics Society

  8. Japanese Biophysical Society

  9. Japanese Surface Science Society

  10. 日本化学会

  11. 日本物理学会

▼全件表示

委員歴 5

  1. 日本生物物理学会   理事  

    2015年 - 現在   

  2. 日本生物物理学会 学会誌「生物物理」編集委員会   委員  

    2014年 - 2015年   

  3. 日本生物物理学会    代議員  

    2014年 - 2015年   

  4. 日本生物物理学会 分野別専門委員会   委員  

    2011年 - 現在   

  5. 日本生物物理学会 分野別専門委員会   委員  

    2011年 - 現在   

受賞 5

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

    2019年8月   IEEE 3M Nano  

    Takayuki Uchihashi

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    受賞区分:国際学会・会議・シンポジウム等の賞  受賞国:中華人民共和国

  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年4月   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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    受賞国:日本国

  3. 平成21年度 ナノプローブテクノロジー賞

    2009年8月   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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    受賞国:日本国

  4. 第29回応用物理学会論文賞

    2007年9月   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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    受賞国:日本国

  5. 平成21年度 ナノプローブテクノロジー賞

    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

 

論文 189

  1. <i>In Situ</i> Real-Time Observation of Photoinduced Nanoscale Azo-Polymer Motions Using High-Speed Atomic Force Microscopy Combined with an Inverted Optical Microscope 査読有り

    Yang, KS; Chan, FY; Watanabe, H; Yoshioka, S; Inouye, Y; Uchihashi, T; Ishitobi, H; Verma, P; Umakoshi, T

    NANO LETTERS   24 巻 ( 9 ) 頁: 2805 - 2811   2024年2月

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

    Tanaka, Y; Uchihashi, T; Nakamura, A

    ARCHIVES OF BIOCHEMISTRY AND BIOPHYSICS   752 巻   頁: 109854   2024年2月

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    記述言語:英語   出版者・発行元:Archives of Biochemistry and Biophysics  

    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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  3. Quantitative Analysis of Therapeutic Antibody Interactions with Fcγ Receptors Using High-Speed Atomic Force Microscopy. 査読有り

    Yanaka S, Watanabe H, Yogo R, Kongsema M, Kondo S, Yagi H, Uchihashi T, Kato K

    Biological & pharmaceutical bulletin   47 巻 ( 1 ) 頁: 334 - 338   2024年1月

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    担当区分:責任著者   記述言語:英語   出版者・発行元:公益社団法人 日本薬学会  

    DOI: 10.1248/bpb.b23-00751

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    CiNii Research

  4. [Protein degradation in bacteria: focus on the ClpP protease]. 査読有り

    Ishikawa F, Homma M, Tanabe G, Uchihashi T

    Nihon saikingaku zasshi. Japanese journal of bacteriology   79 巻 ( 1 ) 頁: 1 - 13   2024年

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    担当区分:責任著者   記述言語:日本語   出版者・発行元:日本細菌学会  

    <p>細胞内のタンパク質は生まれ(合成され),働いて,死んで(分解されて)いく。タンパク質の一生の中で,生まれることも重要であるが,どのように死んでいくかもまた等しく重要な生物の営みである。細胞外に分泌されるプロテアーゼは,外のタンパク質を分解することによって栄養物として取り込むために利用される。細胞内の不要になった,あるいは害になるようなタンパク質を分解するプロテアーゼも存在する。真核生物では,プロテアソームと呼ばれる巨大酵素複合体がそのタンパク質の分解を主に行う。原核生物である細菌も類似のシステムをもつ。細菌の細胞の中で,そのタンパク質の死に関与するATP依存的なプロテアーゼClpの複雑な分解系の制御と作動機構について,特に,ClpXPを中心に,また,細菌に対する抗生物質のターゲットとしての側面について解説したい。</p>

    DOI: 10.3412/jsb.79.1

    PubMed

    CiNii Research

  5. The two-step cargo recognition mechanism of heterotrimeric kinesin 査読有り

    Jiang, XG; Ogawa, T; Yonezawa, K; Shimizu, N; Ichinose, S; Uchihashi, T; Nagaike, W; Moriya, T; Adachi, N; Kawasaki, M; Dohmae, N; Senda, T; Hirokawa, N

    EMBO REPORTS   24 巻 ( 11 ) 頁: e56864   2023年11月

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    記述言語:英語   掲載種別:研究論文(学術雑誌)   出版者・発行元:EMBO Reports  

    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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  6. Measuring mechanical properties with high-speed atomic force microscopy 招待有り 査読有り

    Ganser, C; Uchihashi, T

    MICROSCOPY   73 巻 ( 1 ) 頁: 14 - 21   2023年10月

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    担当区分:最終著者, 責任著者   記述言語:英語  

    DOI: 10.1093/jmicro/dfad051

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  7. Elastic Polymer Coated Nanoparticles with Fast Clearance for <sup>19</sup>F MR Imaging 査読有り

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

    Angewandte Chemie - International Edition   62 巻 ( 40 ) 頁: e202308565   2023年10月

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    記述言語:英語   出版者・発行元:Angewandte Chemie - International Edition  

    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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  8. Multiple Roles of a Conserved Glutamate Residue for Unique Biophysical Properties in a New Group of Microbial Rhodopsins Homologous to TAT Rhodopsin: Multiple roles of Glu54 in Twin-peaked Rhodopsins 査読有り

    Mannen K., Nagata T., Rozenberg A., Konno M., del Carmen Marín M., Bagherzadeh R., Béjà O., Uchihashi T., Inoue K.

    Journal of Molecular Biology     頁: 168331 - 168331   2023年10月

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    記述言語:英語   掲載種別:研究論文(学術雑誌)   出版者・発行元:Journal of Molecular Biology  

    TAT rhodopsin, a microbial rhodopsin found in the marine SAR11 bacterium HIMB114, uniquely possesses a Thr–Ala–Thr (TAT) motif in the third transmembrane helix. Because of a low pKa value of the retinal Schiff base (RSB), TAT rhodopsin exhibits both a visible light-absorbing state with the protonated RSB and a UV-absorbing state with the deprotonated RSB at a neutral pH. The UV-absorbing state, in contrast to the visible light-absorbing one, converts to a long-lived photointermediate upon light absorption, implying that TAT rhodopsin functions as a pH-dependent light sensor. Despite detailed biophysical characterization and mechanistic studies on the TAT rhodopsin, it has been unknown whether other proteins with similarly unusual features exist. Here, we identified several new rhodopsin genes homologous to the TAT rhodopsin of HIMB114 (TATHIMB) from metagenomic data. Based on the absorption spectra of expressed proteins from these genes with visible and UV peaks similar to that of TATHIMB, they were classified as Twin-peaked Rhodopsin (TwR) family. TwR genes form a gene cluster with a set of 13 ORFs conserved in subclade IIIa of SAR11 bacteria. A glutamic acid in the second transmembrane helix, Glu54, is conserved in all of the TwRs. We investigated E54Q mutants of two TwRs and revealed that Glu54 plays critical roles in regulating the RSB pKa, oligomer formation, and the efficient photoreaction of the UV-absorbing state. The discovery of novel TwRs enables us to study the universality and individuality of the characteristics revealed so far in the original TATHIMB and contributes to further studies on mechanisms of unique properties of TwRs.

    DOI: 10.1016/j.jmb.2023.168331

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  9. Structure of the human ATAD2 AAA+ histone chaperone reveals mechanism of regulation and inter-subunit communication 査読有り 国際誌

    Cho, CR; Ganser, C; Uchihashi, T; Kato, K; Song, JJ

    COMMUNICATIONS BIOLOGY   6 巻 ( 1 ) 頁: 993 - 993   2023年9月

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    記述言語:英語   掲載種別:研究論文(学術雑誌)   出版者・発行元:Communications Biology  

    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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  10. Visualizing the membrane disruption action of antimicrobial peptides by cryo-electron tomography 査読有り 国際誌

    Chen, EHL; Wang, CH; Liao, YT; Chan, FY; Kanaoka, Y; Uchihashi, T; Kato, K; Lai, LS; Chang, YW; Ho, MC; Chen, RPY

    NATURE COMMUNICATIONS   14 巻 ( 1 ) 頁: 5464 - 5464   2023年9月

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    記述言語:英語   掲載種別:研究論文(学術雑誌)   出版者・発行元:Nature Communications  

    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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  11. Molecular Design of FRET Probes Based on Domain Rearrangement of Protein Disulfide Isomerase for Monitoring Intracellular Redox Status 査読有り

    Yagi-Utsumi, M; Miura, H; Ganser, C; Watanabe, H; Hiranyakorn, M; Satoh, T; Uchihashi, T; Kato, K; Okazaki, KI; Aoki, K

    INTERNATIONAL JOURNAL OF MOLECULAR SCIENCES   24 巻 ( 16 ) 頁: 12865 - 12865   2023年8月

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    記述言語:英語   掲載種別:研究論文(学術雑誌)   出版者・発行元:International Journal of Molecular Sciences  

    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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  12. Single microgel degradation governed by heterogeneous nanostructures 査読有り

    Nishizawa, Y; Yokoi, H; Uchihashi, T; Suzuki, D

    SOFT MATTER   19 巻 ( 27 ) 頁: 5068 - 5075   2023年7月

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    担当区分:責任著者   記述言語:英語   掲載種別:研究論文(学術雑誌)   出版者・発行元:Soft Matter  

    Although the degradation of colloidal particles is one of the most attractive phenomena in the field of biological and environmental science, the degradation mechanism of single particles remains to be elucidated. In this study, in order to clarify the impact of the structure of a single particle on the oxidative degradation processes, thermoresponsive colloidal particles with chemical cleavage points were synthesized as a model, and their degradation behavior was evaluated using high-speed atomic force microscopy (HS-AFM) as well as conventional scattering techniques. The real-time observation of single-particle degradation revealed that the degradation behavior of microgels is governed by their inhomogeneous nanostructure, which originates from the polymerization method and their hydrophilicity. Our findings can be expected to advance the design of carriers for drug-delivery and the understanding of the formation processes of micro (nano)plastics.

    DOI: 10.1039/d3sm00216k

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  13. Multistep, site-selective noncovalent synthesis of two-dimensional block supramolecular polymers 査読有り

    Sasaki, N; Kikkawa, J; Ishii, Y; Uchihashi, T; Imamura, H; Takeuchi, M; Sugiyasu, K

    NATURE CHEMISTRY   15 巻 ( 7 ) 頁: 922 - +   2023年7月

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    記述言語:英語   掲載種別:研究論文(学術雑誌)   出版者・発行元:Nature Chemistry  

    Although the principles of noncovalent bonding are well understood and form the basis for the syntheses of many intricate supramolecular structures, supramolecular noncovalent synthesis cannot yet achieve the levels of precision and complexity that are attainable in organic and/or macromolecular covalent synthesis. Here we show the stepwise synthesis of block supramolecular polymers from metal–porphyrin derivatives (in which the metal centre is Zn, Cu or Ni) functionalized with fluorinated alkyl chains. These monomers first undergo a one-dimensional supramolecular polymerization and cyclization process to form a toroidal structure. Subsequently, successive secondary nucleation, elongation and cyclization steps result in two-dimensional assemblies with concentric toroidal morphologies. The site selectivity endowed by the fluorinated chains, reminiscent of regioselectivity in covalent synthesis, enables the precise control of the compositions and sequences of the supramolecular structures, as demonstrated by the synthesis of several triblock supramolecular terpolymers. [Figure not available: see fulltext.].

    DOI: 10.1038/s41557-023-01216-y

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    その他リンク: https://www.nature.com/articles/s41557-023-01216-y

  14. 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 査読有り

    Chien, YC; Wang, YS; Sridharan, D; Kuo, CW; Chien, CT; Uchihashi, T; Kato, K; Angata, T; Meng, TC; Hsu, STD; Khoo, KH

    JACS AU   3 巻 ( 7 ) 頁: 1864 - 1875   2023年6月

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    記述言語:英語   掲載種別:研究論文(学術雑誌)   出版者・発行元:JACS Au  

    The intracellular phosphatase domain of the receptor-type protein tyrosine phosphatase alpha (PTPRA) is known to regulate various signaling pathways related to cell adhesion through c-Src kinase activation. In contrast, the functional significance of its relatively short, intrinsically disordered, and heavily glycosylated ectodomain remains unclear. Through detailed mass spectrometry analyses of a combination of protease and glycosidase digests, we now provide the first experimental evidence for its site-specific glycosylation pattern. This includes the occurrence of O-glycan at the N-glycosylation sequon among the more than 30 O-glycosylation sites confidently identified beside the 7 N-glycosylation sites. The closely spaced N- and O-glycans appear to have mutually limited the extent of further galactosylation and sialylation. An immature smaller form of full-length PTPRA was found to be deficient in O-glycosylation, most likely due to failure to transit the Golgi. N-glycosylation, on the other hand, is dispensable for cell surface expression and contributes less than the extensive O-glycosylation to the overall solution structure of the ectodomain. The glycosylation information is combined with the overall structural features of the ectodomain derived from small-angle X-ray scattering and high-speed atomic force microscopy monitoring to establish a dynamic structural model of the densely glycosylated PTPRA ectodomain. The observed high structural flexibility, as manifested by continuous transitioning from fully to partially extended and fold-back conformations, suggests that the receptor-type phosphatase is anchored to the membrane and kept mostly at a monomeric state through an ectodomain shaped and fully shielded by glycosylation.

    DOI: 10.1021/jacsau.3c00124

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  15. Closed-loop recycling of microparticle-based polymers 査読有り

    Watanabe, T; Minato, H; Sasaki, Y; Hiroshige, S; Suzuki, H; Matsuki, N; Sano, K; Wakiya, T; Nishizawa, Y; Uchihashi, T; Kureha, T; Shibayama, M; Takata, T; Suzuki, D

    GREEN CHEMISTRY   25 巻 ( 9 ) 頁: 3418 - 3424   2023年5月

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    掲載種別:研究論文(学術雑誌)   出版者・発行元:Green Chemistry  

    Contemporary polymer science is shifting toward the development of recycling systems to curb global resource depletion and environmental contamination. However, most methods of polymer recycling require cleavage of chemical bonds, which diminishes the quality of the polymers during recycling. Here, we propose a recycling strategy for tough polymers based on microparticles, which allows materials recycling without loss of their properties (‘closed-loop’ recycling). The polymer microparticles can be used to generate tough polymer films by controlling the interparticle physical cross-linking, and subsequently recycled on demand by disassembling into individual microparticles without chemical reactions. Our “microparticle-based concept” for polymer recycling circumvents the infamous trade-off between mechanical stability and degradability of polymers and be expected to open new avenues for closed-loop recycling of polymer materials.

    DOI: 10.1039/d3gc00090g

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  16. Antiparallel dimer structure of CELSR cadherin in solution revealed by high-speed atomic force microscopy 査読有り

    Nishiguchi, S; Kasai, RS; Uchihashi, T

    PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA   120 巻 ( 18 ) 頁: e2302047120   2023年4月

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    担当区分:最終著者, 責任著者   記述言語:英語   出版者・発行元: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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  17. Creation of single molecular conjugates of metal-organic cages and DNA 査読有り 国際誌

    Nakajo T., Kusaka S., Hiraoka H., Nomura K., Matsubara N., Baba R., Yoshida Y., Nakamoto K., Honma M., Iguchi H., Uchihashi T., Abe H., Matsuda R.

    Chemical Communications   59 巻 ( 33 ) 頁: 4974 - 4977   2023年4月

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    記述言語:英語   掲載種別:研究論文(学術雑誌)   出版者・発行元:Chemical Communications  

    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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  18. 高速原子間力顕微鏡で明らかになった細胞間接着分子カドヘリンのダイマー形成過程 招待有り 査読有り

    西口茂孝, 古田忠臣, 内橋貴之

    生物物理   63 巻 ( 2 ) 頁: 104 - 106   2023年4月

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    担当区分:責任著者   記述言語:日本語   掲載種別:研究論文(学術雑誌)   出版者・発行元:一般社団法人 日本生物物理学会  

    <p>多細胞動物のからだの形作りは,隣り合う細胞と細胞をつなぐカドヘリン間の結合によって制御されているが,その結合機構については不明な点が多かった.高速原子間力顕微鏡を用いて,溶液中におけるカドヘリンの結合過程を一分子のスケールで直接可視化することで,カドヘリンの結合機構を解析した.</p>

    DOI: 10.2142/biophys.63.104

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  19. 3D Structure of Ring-shaped Microtubule Swarms Revealed by High-speed Atomic Force Microscopy

    Rashid M.R., Ganser C., Akter M., Nasrin S.R., Kabir A.M.R., Sada K., Uchihashi T., Kakugo A.

    Chemistry Letters   52 巻 ( 2 ) 頁: 100 - 104   2023年2月

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    記述言語:英語   掲載種別:研究論文(学術雑誌)   出版者・発行元:Chemistry Letters  

    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 nanometerlevel 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.

    DOI: 10.1246/cl.220491

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

    Soon J.W., Oohora K., Uchihashi T., Hayashi T.

    Chemistry Letters   52 巻 ( 2 ) 頁: 105 - 109   2023年2月

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    記述言語:英語   掲載種別:研究論文(学術雑誌)   出版者・発行元:Chemistry Letters  

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

    Yue Y., Yokota Y., Uchihashi T.

    iScience   26 巻 ( 1 ) 頁: 105864   2023年1月

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    記述言語:英語   出版者・発行元:iScience  

    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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  22. Decoding of the ubiquitin code for clearance of colliding ribosomes by the RQT complex 国際誌

    Matsuo, Y; Uchihashi, T; Inada, T

    NATURE COMMUNICATIONS   14 巻 ( 1 ) 頁: 79 - 79   2023年1月

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    記述言語:英語   掲載種別:研究論文(学術雑誌)   出版者・発行元:Nature Communications  

    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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  23. Introduction of Session 2, "Advanced methods for retinal proteins"

    Uchihashi, T; Kandori, H

    BIOPHYSICS AND PHYSICOBIOLOGY   20 巻 ( Supplemental ) 頁: n/a   2023年

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    記述言語:英語   出版者・発行元:Biophysics and physicobiology  

    DOI: 10.2142/biophysico.bppb-v20.s022

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  24. 支部だより

    古谷 祐詞, 内橋 貴之, 矢木 真穂

    生物物理   63 巻 ( 5 ) 頁: 285 - 286   2023年

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    記述言語:日本語   出版者・発行元:一般社団法人 日本生物物理学会  

    DOI: 10.2142/biophys.63.285

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

    Takahashi K., Nishikino T., Kajino H., Kojima S., Uchihashi T., Homma M.

    Biophysics and physicobiology   20 巻 ( 2 ) 頁: n/a   2023年

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    記述言語:英語   掲載種別:研究論文(学術雑誌)   出版者・発行元:Biophysics and physicobiology  

    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 two-transmembrane 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.

    DOI: 10.2142/biophysico.bppb-v20.0028

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  26. Clarification of Surface Deswelling of Thermoresponsive Microgels by Electrophoresis

    Nishizawa, Y; Inui, T; Namioka, R; Uchihashi, T; Watanabe, T; Suzuki, D

    LANGMUIR   38 巻 ( 51 ) 頁: 16084 - 16093   2022年12月

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    記述言語:英語   掲載種別:研究論文(学術雑誌)   出版者・発行元:Langmuir  

    Although many investigations of thermoresponsive microgels have been reported, their surface properties, which are crucial in colloid science, are still not fully understood. In this study, microgels with surface-localized charged groups were synthesized by precipitation polymerization, and their electrophoretic behaviors were analyzed using a modified version of Ohshima's equation to obtain two surface properties of the soft particles: the softness parameter and the surface charge density. This systematic evaluation allows us to discuss the thermoresponsiveness of the overall microgels and their surfaces separately. Furthermore, the validity of the surface properties obtained from electrophoresis was verified by comparing them with the results of seeded emulsion polymerization in the presence of the microgels and the force-indentation curves obtained via high-speed atomic force microscopy (HS-AFM).

    DOI: 10.1021/acs.langmuir.2c02742

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

    Uchiyama, T; Uchihashi, T; Ishida, T; Nakamura, A; Vermaas, JV; Crowley, MF; Samejima, M; Beckham, GT; Igarashi, K

    SCIENCE ADVANCES   8 巻 ( 51 ) 頁: eade5155   2022年12月

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    記述言語:英語   掲載種別:研究論文(学術雑誌)   出版者・発行元:Science Advances  

    Efficient depolymerization of crystalline cellulose requires cooperation between multiple cellulolytic enzymes. Through biochemical approaches, molecular dynamics (MD) simulation, and single-molecule observations using high-speed atomic force microscopy (HS-AFM), we quantify and track synergistic activity for cellobiohydrolases (CBHs) with a lytic polysaccharide monooxygenase (LPMO) from Phanerochaete chrysosporium. Increasing concentrations of LPMO (AA9D) increased the activity of a glycoside hydrolase family 6 CBH, Cel6A, whereas the activity of a family 7 CBH (Cel7D) was enhanced only at lower concentrations of AA9D. MD simulation suggests that the result of AA9D action to produce chain breaks in crystalline cellulose can oxidatively disturb the crystalline surface by disrupting hydrogen bonds. HS-AFM observations showed that AA9D increased the number of Cel7D molecules moving on the substrate surface and increased the processivity of Cel7D, thereby increasing the depolymerization performance, suggesting that AA9D not only generates chain ends but also amorphizes the crystalline surface, thereby increasing the activity of CBHs.

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

    Chan, FY; Kurosaki, R; Ganser, C; Takeda, T; Uchihashi, T

    REVIEW OF SCIENTIFIC INSTRUMENTS   93 巻 ( 11 ) 頁: 113703 - 113703   2022年11月

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    記述言語:英語   掲載種別:研究論文(学術雑誌)   出版者・発行元:Review of Scientific Instruments  

    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.

    DOI: 10.1063/5.0111017

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  29. 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   61 巻 ( SL )   2022年9月

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    出版者・発行元:Japanese Journal of Applied Physics  

    DOI: 10.35848/1347-4065/ac7cc6

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  30. 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   61 巻 ( 18 ) 頁: 1936 - 1944   2022年8月

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    記述言語:英語   出版者・発行元:Biochemistry  

    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.

    DOI: 10.1021/acs.biochem.2c00328

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  31. Multiple dimeric structures and strand-swap dimerization of E-cadherin in solution visualized by high-speed atomic force microscopy 国際誌

    Nishiguchi, S; Furuta, T; Uchihashi, T

    PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA   119 巻 ( 30 ) 頁: e2208067119   2022年7月

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    記述言語:英語   掲載種別:研究論文(学術雑誌)   出版者・発行元:Proceedings of the National Academy of Sciences of the United States of America  

    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.

    DOI: 10.1073/pnas.2208067119

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  32. Protein Needles Designed to Self-Assemble through Needle Tip Engineering 査読有り

    Kikuchi, K; Fukuyama, T; Uchihashi, T; Furuta, T; Maeda, YT; Ueno, T

    SMALL   18 巻 ( 10 ) 頁: 2106401 - 2106401   2022年3月

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    記述言語:英語   掲載種別:研究論文(学術雑誌)   出版者・発行元:Small  

    The dynamic process of formation of protein assemblies is essential to form highly ordered structures in biological systems. Advances in structural and synthetic biology have led to the construction of artificial protein assemblies. However, development of design strategies exploiting the anisotropic shape of building blocks of protein assemblies has not yet been achieved. Here, the 2D assembly pattern of protein needles (PNs) is controlled by regulating their tip-to-tip interactions. The PN is an anisotropic needle-shaped protein composed of β-helix, foldon, and His-tag. Three different types of tip-modified PNs are designed by deleting the His-tag and foldon to change the protein–protein interactions. Observing their assembly by high-speed atomic force microscopy (HS-AFM) reveals that PN, His-tag deleted PN, and His-tag and foldon deleted PN form triangular lattices, the monomeric state with nematic order, and fiber assemblies, respectively, on a mica surface. Their assembly dynamics are observed by HS-AFM and analyzed by the theoretical models. Monte Carlo (MC) simulations indicate that the mica-PN interactions and the flexible and multipoint His-tag interactions cooperatively guide the formation of the triangular lattice. This work is expected to provide a new strategy for constructing supramolecular protein architectures by controlling directional interactions of anisotropic shaped proteins.

    DOI: 10.1002/smll.202106401

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    その他リンク: https://onlinelibrary.wiley.com/doi/full-xml/10.1002/smll.202106401

  33. Quantitative Visualization of the Interaction between Complement Component C1 and Immunoglobulin G: The Effect of C<sub>H</sub>1 Domain Deletion 査読有り 国際誌

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

    INTERNATIONAL JOURNAL OF MOLECULAR SCIENCES   23 巻 ( 4 )   2022年2月

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    記述言語:英語   掲載種別:研究論文(学術雑誌)   出版者・発行元:International Journal of Molecular Sciences  

    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.

    DOI: 10.3390/ijms23042090

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

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

    DALTON TRANSACTIONS   51 巻 ( 4 ) 頁: 1318 - 1324   2022年1月

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    記述言語:英語   掲載種別:研究論文(学術雑誌)   出版者・発行元:Dalton Transactions  

    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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  35. The Lipid-Binding Defective Dynamin 2 Mutant in Charcot-Marie-Tooth Disease Impairs Proper Actin Bundling and Actin Organization in Glomerular Podocytes 国際誌

    Hamasaki E., Wakita N., Yasuoka H., Nagaoka H., Morita M., Takashima E., Uchihashi T., Takeda T., Abe T., Lee J.W., Iimura T., Saleem M.A., Ogo N., Asai A., Narita A., Takei K., Yamada H.

    Frontiers in Cell and Developmental Biology   10 巻   頁: 884509 - 884509   2022年

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    記述言語:英語   掲載種別:研究論文(学術雑誌)   出版者・発行元:Frontiers in Cell and Developmental Biology  

    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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  36. Quantitative Visualization of the Interaction between Complement Component C1 and Immunoglobulin G: The Effect of CH1 Domain Deletion 査読有り

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

    International Journal of Molecular Sciences   23 巻 ( 4 ) 頁: 2090   2021年12月

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    記述言語:英語   掲載種別:研究論文(学術雑誌)   出版者・発行元:MDPI AG  

    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.

  37. Molecular Origin of the Anomalous pH Effect in Blue Proteorhodopsin 査読有り

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

    JOURNAL OF PHYSICAL CHEMISTRY LETTERS   12 巻 ( 51 ) 頁: 12225 - 12229   2021年12月

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    記述言語:英語   掲載種別:研究論文(学術雑誌)   出版者・発行元:Journal of Physical Chemistry Letters  

    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.

    DOI: 10.1021/acs.jpclett.1c03355

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  38. Desiccation-induced fibrous condensation of CAHS protein from an anhydrobiotic tardigrade 査読有り 国際誌

    Yagi-Utsumi, M; Aoki, K; Watanabe, H; Song, CH; Nishimura, S; Satoh, T; Yanaka, S; Ganser, C; Tanaka, S; Schnapka, V; Goh, EW; Furutani, Y; Murata, K; Uchihashi, T; Arakawa, K; Kato, K

    SCIENTIFIC REPORTS   11 巻 ( 1 ) 頁: 21328 - 21328   2021年11月

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    記述言語:英語   掲載種別:研究論文(学術雑誌)   出版者・発行元:Scientific Reports  

    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.

    DOI: 10.1038/s41598-021-00724-6

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  39. Construction of ferritin hydrogels utilizing subunit-subunit interactions 査読有り

    Yamanaka, M; Mashima, T; Ogihara, M; Okamoto, M; Uchihashi, T; Hirota, S

    PLOS ONE   16 巻 ( 11 ) 頁: e0259052 - e0259052   2021年11月

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    記述言語:英語   掲載種別:研究論文(学術雑誌)   出版者・発行元:PLoS ONE  

    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 80C, 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 Fe3+ ions, indicating that the hydrogel adsorbed the Fe3+ ions. The yellow-brown color of the Fe3 +-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 Co2+ and Cu2+ ions and released them in the presence of EDTA, while it adsorbed less Ni2+ ions; more Fe3+ 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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  40. Deformation of microtubules regulates translocation dynamics of kinesin 査読有り 国際共著 国際誌

    Nasrin, SR; Ganser, C; Nishikawa, S; Kabir, AMR; Sada, K; Yamashita, T; Ikeguchi, M; Uchihashi, T; Hess, H; Kakugo, A

    SCIENCE ADVANCES   7 巻 ( 42 ) 頁: eabf2211   2021年10月

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    記述言語:英語   掲載種別:研究論文(学術雑誌)   出版者・発行元:Science Advances  

    Microtubules, the most rigid components of the cytoskeleton, can be key transduction elements between external forces and the cellular environment. Mechanical forces induce microtubule deformation, which is presumed to be critical for the mechanoregulation of cellular events. However, concrete evidence is lacking. In this work, with high-speed atomic force microscopy, we unravel how microtubule deformation regulates the translocation of the microtubule-associated motor protein kinesin-1, responsible for intracellular transport. Our results show that the microtubule deformation by bending impedes the translocation dynamics of kinesins along them. Molecular dynamics simulation shows that the hindered translocation of kinesins can be attributed to an enhanced affinity of kinesins to the microtubule structural units in microtubules deformed by bending. This study advances our understanding of the role of cytoskeletal components in mechanotransduction.

    DOI: 10.1126/sciadv.abf2211

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  41. 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   60 巻 ( SE )   2021年9月

  42. 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 招待有り

    内橋貴之

    生化学   93 巻   頁: 526 - 561   2021年8月

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    担当区分:筆頭著者, 最終著者, 責任著者   記述言語:日本語   掲載種別:研究論文(学術雑誌)  

  43. Tardigrade Secretory-Abundant Heat-Soluble Protein Has a Flexible β-Barrel Structure in Solution and Keeps This Structure in Dehydration 査読有り 国際誌

    Miyazawa, K; Itoh, SG; Watanabe, H; Uchihashi, T; Yanaka, S; Yagi-Utsumi, M; Kato, K; Arakawa, K; Okumura, H

    JOURNAL OF PHYSICAL CHEMISTRY B   125 巻 ( 32 ) 頁: 9145 - 9154   2021年8月

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    記述言語:英語   掲載種別:研究論文(学術雑誌)   出版者・発行元:Journal of Physical Chemistry B  

    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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  44. Reconstruction of Three-Dimensional Conformations of Bacterial ClpB from High-Speed Atomic-Force-Microscopy Images 査読有り 国際誌

    Dasgupta, B; Miyashita, O; Uchihashi, T; Tama, F

    FRONTIERS IN MOLECULAR BIOSCIENCES   8 巻   頁: 704274 - 704274   2021年8月

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    記述言語:英語   掲載種別:研究論文(学術雑誌)   出版者・発行元:Frontiers in Molecular Biosciences  

    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.

    DOI: 10.3389/fmolb.2021.704274

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  45. JRAB/MICAL-L2 undergoes liquid-liquid phase separation to form tubular recycling endosomes 査読有り 国際誌

    Sakane, A; Yano, T; Uchihashi, T; Horikawa, K; Hara, Y; Imoto, I; Kurisu, S; Yamada, H; Takei, K; Sasaki, T

    COMMUNICATIONS BIOLOGY   4 巻 ( 1 ) 頁: 551 - 551   2021年5月

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    記述言語:英語   掲載種別:研究論文(学術雑誌)   出版者・発行元:Communications Biology  

    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.

    DOI: 10.1038/s42003-021-02080-7

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  46. Non-close-packed arrangement of soft elastomer microspheres on solid substrates 査読有り

    Sasaki, Y; Hiroshige, S; Takizawa, M; Nishizawa, Y; Uchihashi, T; Minato, H; Suzuki, D

    RSC ADVANCES   11 巻 ( 24 ) 頁: 14562 - 14567   2021年5月

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    記述言語:英語   掲載種別:研究論文(学術雑誌)   出版者・発行元:RSC Advances  

    Unlike rigid microparticles, soft and deformable elastomer (rubber) microspheres were found to exhibit a non-close-packed arrangement on solid substrates after the evaporation of water from their dispersions. The microscopic observation revealed that individual microspheres are ordered in regular intervals at the air/water interface of a sessile droplet and remain fixed on the substrate without being affected by the capillary forces during evaporation due to their deformability. Moreover, using the Langmuir-Blodgett method, thin films of non-close-packed structures could be successfully generated over large areas. Our findings may potentially help to control the arranged structures of elastomer microspheres, which can be expected to improve the nano-science and technology for the precise control for e.g. surface patterning. This journal is

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  47. Nanostructure and thermoresponsiveness of poly(<i>N</i>-isopropyl methacrylamide)-based hydrogel microspheres prepared <i>via</i> aqueous free radical precipitation polymerization 査読有り

    Nishizawa, Y; Minato, H; Inui, T; Saito, I; Kureha, T; Shibayama, M; Uchihashi, T; Suzuki, D

    RSC ADVANCES   11 巻 ( 22 ) 頁: 13130 - 13137   2021年4月

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    記述言語:英語   掲載種別:研究論文(学術雑誌)   出版者・発行元:RSC Advances  

    Thermoresponsive hydrogel microspheres (microgels) are smart materials that quickly respond to external stimuli, and their thermoresponsiveness can be tuned by varying the constituent chemical species. Although uniformly sized microgels can be preparedviaaqueous free radical precipitation polymerization, the nanostructure of the obtained microgels is complex and remains unclear so far. In the present study, the nanostructure and thermoresponsiveness of poly(N-isopropyl methacrylamide) (pNIPMAm)-based microgels, which have a volume-transition temperature of ∼43 °C, were evaluated mainly using temperature-controllable high-speed atomic force microscopy. These observations, which are characterized by high spatio-temporal resolution, revealed that the pNIPMAm microgels have a peculiar heterogeneous structure, for example a core-shell and non-thermoresponsive nanostructure in the core region, that originates from the precipitation polymerization process. Furthermore, it was found that the adsorption concentration of the microgels on the substrate is one of the keys for controlling their thermoresponsiveness. These findings can be expected to advance the design of new materials such as thermoresponsive nanosheets and stimuli-responsive coatings.

    DOI: 10.1039/d1ra01650d

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

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

    SURFACE SCIENCE   705 巻   2021年3月

  49. Dynamic Assembly/Disassembly of <i>Staphylococcus aureus</i> FtsZ Visualized by High-Speed Atomic Force Microscopy 査読有り 国際誌

    Fujita, J; Sugiyama, S; Terakado, H; Miyazaki, M; Ozawa, M; Ueda, N; Kuroda, N; Tanaka, S; Yoshizawa, T; Uchihashi, T; Matsumura, H

    INTERNATIONAL JOURNAL OF MOLECULAR SCIENCES   22 巻 ( 4 ) 頁: 1 - 10   2021年2月

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    記述言語:英語   掲載種別:研究論文(学術雑誌)   出版者・発行元:International Journal of Molecular Sciences  

    FtsZ is a key protein in bacterial cell division and is assembled into filamentous architec-tures. 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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  50. Nanostructures, Thermoresponsiveness, and Assembly Mechanism of Hydrogel Microspheres during Aqueous Free-Radical Precipitation Polymerization 査読有り 国際誌

    Nishizawa, Y; Minato, H; Inui, T; Uchihashi, T; Suzuki, D

    LANGMUIR   37 巻 ( 1 ) 頁: 151 - 159   2021年1月

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    記述言語:英語   掲載種別:研究論文(学術雑誌)   出版者・発行元:Langmuir  

    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.

    DOI: 10.1021/acs.langmuir.0c02654

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  51. Single-molecule dynamics observed by high-speed atomic force microscopy: Recent applications

    Uchihashi T.

    Seikagaku   93 巻 ( 4 ) 頁: 526 - 531   2021年

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    記述言語:日本語   出版者・発行元:Seikagaku  

    DOI: 10.14952/SEIKAGAKU.2021.930526

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  52. Structural insights into the mechanism of rhodopsin phosphodiesterase 査読有り 国際誌

    Ikuta, T; Shihoya, W; Sugiura, M; Yoshida, K; Watari, M; Tokano, T; Yamashita, K; Katayama, K; Tsunoda, SP; Uchihashi, T; Kandori, H; Nureki, O

    NATURE COMMUNICATIONS   11 巻 ( 1 ) 頁: 5605 - 5605   2020年11月

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    記述言語:英語   掲載種別:研究論文(学術雑誌)   出版者・発行元:Nature Communications  

    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.

    DOI: 10.1038/s41467-020-19376-7

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  53. Novel <i>Babesia bovis</i> exported proteins that modify properties of infected red blood cells 査読有り 国際誌

    Hakimi, H; Templeton, TJ; Sakaguchi, M; Yamagishi, J; Miyazaki, S; Yahata, K; Uchihashi, T; Kawazu, S; Kaneko, O; Asada, M

    PLOS PATHOGENS   16 巻 ( 10 ) 頁: e1008917   2020年10月

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    記述言語:英語   掲載種別:研究論文(学術雑誌)   出版者・発行元:PLoS Pathogens  

    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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  54. Single-molecule level dynamic observation of disassembly of the apo-ferritin cage in solution 査読有り 国際誌

    Maity, B; Li, ZP; Niwase, K; Ganser, C; Furuta, T; Uchihashi, T; Lu, DN; Ueno, T

    PHYSICAL CHEMISTRY CHEMICAL PHYSICS   22 巻 ( 33 ) 頁: 18562 - 18572   2020年9月

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    記述言語:英語   掲載種別:研究論文(学術雑誌)   出版者・発行元:Physical Chemistry Chemical Physics  

    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. This journal is

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  55. Thermoresponsive structural changes of single poly(<i>N</i>-isopropyl acrylamide) hydrogel microspheres under densely packed conditions on a solid substrate 査読有り

    Minato, H; Nishizawa, Y; Uchihashi, T; Suzuki, D

    POLYMER JOURNAL   52 巻 ( 9 ) 頁: 1137 - 1141   2020年9月

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    記述言語:日本語   掲載種別:研究論文(学術雑誌)   出版者・発行元:Polymer Journal  

    The thermoresponsiveness of hydrogel microspheres (microgels) was visualized in situ at the nanoscale using temperature-controllable high-speed atomic force microscopy (TC-HS-AFM). The morphological changes of the microgels are strongly affected by their packing conditions and by their adsorption on a solid substrate.

    DOI: 10.1038/s41428-020-0372-3

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    その他リンク: http://www.nature.com/articles/s41428-020-0372-3

  56. Convergent evolution of processivity in bacterial and fungal cellulases 査読有り 国際誌

    Uchiyama, T; Uchihashi, T; Nakamura, A; Watanabe, H; Kaneko, S; Samejima, M; Igarashi, K

    PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA   117 巻 ( 33 ) 頁: 19896 - 19903   2020年8月

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    記述言語:英語   掲載種別:研究論文(学術雑誌)   出版者・発行元:Proceedings of the National Academy of Sciences of the United States of America  

    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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  57. Direct visualization of the conformational change of FUS/TLS upon binding to promoter-associated non-coding RNA 査読有り 国際誌

    Hamad, N; Watanabe, H; Uchihashi, T; Kurokawa, R; Nagata, T; Katahira, M

    CHEMICAL COMMUNICATIONS   56 巻 ( 64 ) 頁: 9134 - 9137   2020年8月

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    記述言語:英語   掲載種別:研究論文(学術雑誌)   出版者・発行元:Chemical Communications  

    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. This journal is

    DOI: 10.1039/d0cc03776a

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  58. 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   59 巻   2020年8月

  59. Assembly Mechanism of a Supramolecular MS-Ring Complex To Initiate Bacterial Flagellar Biogenesis in Vibrio Species 査読有り 国際誌

    Terashima, H; Hirano, K; Inoue, Y; Tokano, T; Kawamoto, A; Kato, T; Yamaguchi, E; Namba, K; Uchihashi, T; Kojima, S; Homma, M

    JOURNAL OF BACTERIOLOGY   202 巻 ( 16 )   2020年8月

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    記述言語:英語   掲載種別:研究論文(学術雑誌)   出版者・発行元:Journal of Bacteriology  

    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 that is a part of the rotor. The number and locations 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 or on the contributions 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 a requirement of other factors for MS-ring assembly. Consequently, we investigated if FlhF aided FliF in MS-ring assembly. We found that FlhF allowed green fluorescent protein (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 coexpressed 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 that of 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, which is 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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  60. 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   60 巻 ( Suppl.1-2 ) 頁: S154 - S154   2020年8月

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    記述言語:英語   出版者・発行元:(一社)日本生物物理学会  

  61. Structural Dynamics of a Protein Domain Relevant to the Water-Oxidizing Complex in Photosystem II as Visualized by High-Speed Atomic Force Microscopy 査読有り 国際誌

    Tokano, T; Kato, Y; Sugiyama, S; Uchihashi, T; Noguchi, T

    JOURNAL OF PHYSICAL CHEMISTRY B   124 巻 ( 28 ) 頁: 5847 - 5857   2020年7月

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    記述言語:英語   掲載種別:研究論文(学術雑誌)   出版者・発行元:Journal of Physical Chemistry B  

    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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  62. Dynamic behavior of an artificial protein needle contacting a membrane observed by high-speed atomic force microscopy 査読有り 国際誌

    Ueno, T; Niwase, K; Tsubokawa, D; Kikuchi, K; Takai, N; Furuta, T; Kawano, R; Uchihashi, T

    NANOSCALE   12 巻 ( 15 ) 頁: 8166 - 8173   2020年4月

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    記述言語:英語   掲載種別:研究論文(学術雑誌)   出版者・発行元:Nanoscale  

    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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  63. Dynamics of oligomer and amyloid fibril formation by yeast prion Sup35 observed by high-speed atomic force microscopy 査読有り 国際誌

    Konno, H; Watanabe-Nakayama, T; Uchihashi, T; Okuda, M; Zhu, LW; Kodera, N; Kikuchi, Y; Ando, T; Taguchi, H

    PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA   117 巻 ( 14 ) 頁: 7831 - 7836   2020年4月

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    記述言語:英語   掲載種別:研究論文(学術雑誌)   出版者・発行元:Proceedings of the National Academy of Sciences of the United States of America  

    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.

    DOI: 10.1073/pnas.1916452117

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  64. Schizorhodopsins: A family of rhodopsins from Asgard archaea that function as light-driven inward H<SUP>+</SUP> pumps 査読有り 国際誌

    Inoue, K; Tsunoda, SP; Singh, M; Tomida, S; Hososhima, S; Konno, M; Nakamura, R; Watanabe, H; Bulzu, PA; Banciu, HL; Andrei, AS; Uchihashi, T; Ghai, R; Béjà, O; Kandori, H

    SCIENCE ADVANCES   6 巻 ( 15 ) 頁: eaaz2441   2020年4月

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    記述言語:英語   掲載種別:研究論文(学術雑誌)   出版者・発行元:Science Advances  

    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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  65. Recent advances in bioimaging with high-speed atomic force microscopy 査読有り 国際誌

    Uchihashi T., Ganser C.

    Biophysical Reviews   12 巻 ( 2 ) 頁: 363 - 369   2020年4月

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    記述言語:英語   掲載種別:研究論文(学術雑誌)   出版者・発行元:Biophysical Reviews  

    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.

    DOI: 10.1007/s12551-020-00670-z

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  66. Single-molecule imaging analysis reveals the mechanism of a high-catalytic-activity mutant of chitinase A from <i>Serratia marcescens</i> 査読有り 国際誌

    Visootsat, A; Nakamura, A; Vignon, P; Watanabe, H; Uchihashi, T; Iino, R

    JOURNAL OF BIOLOGICAL CHEMISTRY   295 巻 ( 7 ) 頁: 1915 - 1925   2020年2月

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    記述言語:英語   掲載種別:研究論文(学術雑誌)   出版者・発行元:Journal of Biological Chemistry  

    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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  67. 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   118 巻 ( 3 ) 頁: 236A - 236A   2020年2月

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  68. Construction of a Hexameric Hemoprotein Sheet and Direct Observation of Dynamic Processes of Its Formation 査読有り

    Oohora, K; Hirayama, S; Uchihashi, T; Hayashi, T

    CHEMISTRY LETTERS   49 巻 ( 2 ) 頁: 186 - 190   2020年2月

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    記述言語:英語   掲載種別:研究論文(学術雑誌)   出版者・発行元:Chemistry Letters  

    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 hostguest 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.

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  69. High-speed near-field fluorescence microscopy combined with high-speed atomic force microscopy for biological studies 査読有り 国際誌

    Umakoshi, T; Fukuda, S; Iino, R; Uchihashi, T; Ando, T

    BIOCHIMICA ET BIOPHYSICA ACTA-GENERAL SUBJECTS   1864 巻 ( 2 ) 頁: 129325   2020年2月

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    記述言語:英語   掲載種別:研究論文(学術雑誌)   出版者・発行元:Biochimica et Biophysica Acta - General Subjects  

    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 ~3 s/frame for SNOM imaging, more than 100-times higher than the typical SNOM imaging rate. We also demonstrated ~39 nm resolution in HS-SNOM imaging of fluorescently labeled DNA in solution.

    DOI: 10.1016/j.bbagen.2019.03.011

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  70. Supramolecular tholos-like architecture constituted by archaeal proteins without functional annotation 査読有り 国際誌

    Yagi-Utsumi, M; Sikdar, A; Song, CH; Park, J; Inoue, R; Watanabe, H; Burton-Smith, RN; Kozai, T; Suzuki, T; Kodama, A; Ishii, K; Yagi, H; Satoh, T; Uchiyama, S; Uchihashi, T; Joo, K; Lee, J; Sugiyama, M; Murata, K; Kato, K

    SCIENTIFIC REPORTS   10 巻 ( 1 ) 頁: 1540 - 1540   2020年1月

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    記述言語:英語   掲載種別:研究論文(学術雑誌)   出版者・発行元:Scientific Reports  

    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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  71. Thermoresponsive Micellar Assembly Constructed from a Hexameric Hemoprotein Modified with Poly(<i>N</i>-isopropylacrylamide) toward an Artificial Light-Harvesting System 査読有り 国際誌

    Hirayama, S; Oohora, K; Uchihashi, T; Hayashi, T

    JOURNAL OF THE AMERICAN CHEMICAL SOCIETY   142 巻 ( 4 ) 頁: 1822 - 1831   2020年1月

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    記述言語:英語   掲載種別:研究論文(学術雑誌)   出版者・発行元:Journal of the American Chemical Society  

    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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  72. Dynamics of Inter-Molecular Interactions Between Single Aβ<sub>42</sub> Oligomeric and Aggregate Species by High-Speed Atomic Force Microscopy (vol 431, pg 2687, 2019) 査読有り 国際誌

    Feng, L; Watanabe, H; Molino, P; Wallace, GG; Phung, SL; Uchihashi, T; Higgins, MJ

    JOURNAL OF MOLECULAR BIOLOGY   432 巻 ( 2 ) 頁: 633 - 633   2020年1月

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    記述言語:英語   出版者・発行元:Journal of Molecular Biology  

    The authors would like to correct the values of Koff (s−1) in ‘Table 1. Summary of properties’ for column of Aβ15–20nm and Aβ36nm, replacing 2.17 ± 0.87 with 1.82, 1.64 ± 0.64 with 1.39, respectively. Enclosed you will find correct version of Table 1. Also, the Koff (s−1) values appear in the sentence “A mean lifetime <t> = 0.55 ± 0.22 s, with dissociation rate constant koff (1/<t>) = 2.17 ± 0.87 s−1, for the Aβ15–20 nm ↔ Aβ15–20 nm compared to the Aβ15–20 nm ↔ Aβ36 nm with a mean lifetime = 0.72 ± 0.28 s (koff = 1.64 ± 0.64 s−1), indicating the latter to some extent forms more tightly bound complexes due to its slower dissociation rate.” on page 2693 in the article. The koff (1/<t>) values of 2.17 ± 0.87 s−1 and 1.64 ± 0.64 s−1 are now corrected to 1.82 s−1 and 1.39 s−1, respectively. The authors would like to apologise for any inconvenience caused. [Table presented]

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  73. Rad50 zinc hook functions as a constitutive dimerization module interchangeable with SMC hinge 査読有り 国際誌

    Tatebe, H; Lim, CT; Konno, H; Shiozaki, K; Shinohara, A; Uchihashi, T; Furukohri, A

    NATURE COMMUNICATIONS   11 巻 ( 1 ) 頁: 370 - 370   2020年1月

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    記述言語:英語   掲載種別:研究論文(学術雑誌)   出版者・発行元:Nature Communications  

    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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  74. On-Membrane Dynamic Interplay between Anti-GM1 IgG Antibodies and Complement Component C1q 査読有り 国際誌

    Yanaka, S; Yogo, R; Watanabe, H; Taniguchi, Y; Satoh, T; Komura, N; Ando, H; Yagi, H; Yuki, N; Uchihashi, T; Kato, K

    INTERNATIONAL JOURNAL OF MOLECULAR SCIENCES   21 巻 ( 1 )   2020年1月

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    記述言語:英語   掲載種別:研究論文(学術雑誌)   出版者・発行元:International Journal of Molecular Sciences  

    Guillain–Barré 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 CH2 and CH3 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–Barré syndrome and, more generally, into antigen-dependent interplay between antibodies and complement components on membranes.

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  75. Structural basis of nucleosome assembly by the Abo1 AAA+ ATPase histone chaperone 査読有り 国際誌

    Cho, C; Jang, J; Kang, YJ; Watanabe, H; Uchihashi, T; Kim, SJ; Kato, K; Lee, JY; Song, JJ

    NATURE COMMUNICATIONS   10 巻 ( 1 ) 頁: 5764 - 5764   2019年12月

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    記述言語:英語   掲載種別:研究論文(学術雑誌)   出版者・発行元:Nature Communications  

    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.

    DOI: 10.1038/s41467-019-13743-9

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  76. Crystal structure of heliorhodopsin 査読有り 国際誌

    Shihoya, W; Inoue, K; Singh, M; Konno, M; Hososhima, S; Yamashita, K; Ikeda, K; Higuchi, A; Izume, T; Okazaki, S; Hashimoto, M; Mizutori, R; Tomida, S; Yamauchi, Y; Abe-Yoshizumi, R; Katayama, K; Tsunoda, SP; Shibata, M; Furutani, Y; Pushkarev, A; Béjá, O; Uchihashi, T; Kandori, H; Nureki, O

    NATURE   574 巻 ( 7776 ) 頁: 132 - +   2019年10月

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    記述言語:英語   掲載種別:研究論文(学術雑誌)   出版者・発行元:Nature  

    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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  77. 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 招待有り 査読有り

    高分子   68 巻 ( 10 ) 頁: 564 - 568   2019年10月

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    記述言語:日本語   掲載種別:研究論文(学術雑誌)  

  78. Protein uptake into individual hydrogel microspheres visualized by high-speed atomic force microscopy 査読有り 国際誌

    Matsui, S; Hosho, K; Minato, H; Uchihashi, T; Suzuki, D

    CHEMICAL COMMUNICATIONS   55 巻 ( 68 ) 頁: 10064 - 10067   2019年9月

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    記述言語:英語   出版者・発行元:Chemical Communications  

    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.

    DOI: 10.1039/c9cc05116c

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  79. 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   258 巻   2019年8月

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

    Honda, K; Sazuka, Y; Iizuka, K; Matsui, S; Uchihashi, T; Kureha, T; Shibayama, M; Watanabe, T; Suzuki, D

    ABSTRACTS OF PAPERS OF THE AMERICAN CHEMICAL SOCIETY   258 巻   2019年8月

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    記述言語:英語  

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  81. The Fab portion of immunoglobulin G contributes to its binding to Fcγ receptor III 査読有り 国際誌

    Yogo, R; Yamaguchi, Y; Watanabe, H; Yagi, H; Satoh, T; Nakanishi, M; Onitsuka, M; Omasa, T; Shimada, M; Maruno, T; Torisu, T; Watanabe, S; Higo, D; Uchihashi, T; Yanaka, S; Uchiyama, S; Kato, K

    SCIENTIFIC REPORTS   9 巻 ( 1 ) 頁: 11957 - 11957   2019年8月

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    記述言語:英語   掲載種別:研究論文(学術雑誌)   出版者・発行元:Scientific Reports  

    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, FcγRIIIa. 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 FcγRIIIa, 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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  82. 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   58 巻 ( SI )   2019年8月

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    記述言語:日本語   出版者・発行元:Japanese Journal of Applied Physics  

    DOI: 10.7567/1347-4065/ab2864

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  83. 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     頁: 281 - 285   2019年8月

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    記述言語:日本語   出版者・発行元:2019 IEEE International Conference on Manipulation, Manufacturing and Measurement on the Nanoscale, 3M-NANO 2019 - Proceedings  

    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.

    DOI: 10.1109/3M-NANO46308.2019.8947429

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  84. 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     頁: 253 - 256   2019年8月

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    記述言語:日本語   出版者・発行元:2019 IEEE International Conference on Manipulation, Manufacturing and Measurement on the Nanoscale, 3M-NANO 2019 - Proceedings  

    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.

    DOI: 10.1109/3M-NANO46308.2019.8947375

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  85. Dynamic structural states of ClpB involved in its disaggregation function (vol 9, 2147, 2018) 査読有り 国際誌

    Uchihashi, T; Watanabe, YH; Nakazaki, Y; Yamasaki, T; Watanabe, H; Maruno, T; Ishii, K; Uchiyama, S; Song, CH; Murata, K; Iino, R; Ando, T

    NATURE COMMUNICATIONS   10 巻 ( 1 ) 頁: 3079 - 3079   2019年7月

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    記述言語:英語   掲載種別:研究論文(学術雑誌)   出版者・発行元:Nature Communications  

    The original version of this Article did not acknowledge Ryota Iino as a corresponding author. This has now been corrected in both the PDF and HTML versions of the Article.

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  86. Dynamics of Inter-Molecular Interactions Between Single Aβ<sub>42</sub> Oligomeric and Aggregate Species by High-Speed Atomic Force Microscopy 査読有り 国際誌

    Feng, L; Watanabe, H; Molino, P; Wallace, GG; Phung, SL; Uchihashi, T; Higgins, MJ

    JOURNAL OF MOLECULAR BIOLOGY   431 巻 ( 15 ) 頁: 2687 - 2699   2019年7月

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    記述言語:英語   出版者・発行元:Journal of Molecular Biology  

    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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  87. 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 査読有り 国際誌

    Krayukhina, E; Yokoyama, M; Hayashihara, KK; Maruno, T; Noda, M; Watanabe, H; Uchihashi, T; Uchiyama, S

    JOURNAL OF PHARMACEUTICAL SCIENCES   108 巻 ( 7 ) 頁: 2278 - 2287   2019年7月

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    記述言語:英語   掲載種別:研究論文(学術雑誌)   出版者・発行元:Journal of Pharmaceutical Sciences  

    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.

    DOI: 10.1016/j.xphs.2019.02.002

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  88. 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   5 巻 ( 7 )   2019年7月

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    記述言語:日本語   出版者・発行元:Science Advances  

    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.

    DOI: 10.1126/sciadv.aax0495

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  89. High-speed AFM reveals accelerated binding of agitoxin-2 to a K+ channel by induced fit 査読有り

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

    Science Advances   5 巻 ( 7 ) 頁: 1 - 10   2019年7月

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    記述言語:英語   掲載種別:研究論文(学術雑誌)  

  90. Non-Thermoresponsive Decanano-sized Domains in Thermoresponsive Hydrogel Microspheres Revealed by Temperature-Controlled High-Speed Atomic Force Microscopy 査読有り 国際誌

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

    ANGEWANDTE CHEMIE-INTERNATIONAL EDITION   58 巻 ( 26 ) 頁: 8809 - 8813   2019年6月

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    記述言語:英語   出版者・発行元:Angewandte Chemie - International Edition  

    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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  91. Non‐Thermoresponsive Decanano‐sized Domains in Thermoresponsive Hydrogel Microspheres Revealed by Temperature‐Controlled High‐Speed Atomic Force Microscopy 査読有り

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

    Angewandte Chemie   131 巻 ( 26 ) 頁: 8901 - 8905   2019年6月

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    掲載種別:研究論文(学術雑誌)   出版者・発行元:Wiley  

    DOI: 10.1002/ange.201903483

  92. Hydrogel Microellipsoids that Form Robust String-Like Assemblies at the Air/Water Interface 査読有り 国際誌

    Honda, K; Sazuka, Y; Iizuka, K; Matsui, S; Uchihashi, T; Kureha, T; Shibayama, M; Watanabe, T; Suzuki, D

    ANGEWANDTE CHEMIE-INTERNATIONAL EDITION   58 巻 ( 22 ) 頁: 7294 - 7298   2019年5月

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    記述言語:英語   掲載種別:研究論文(学術雑誌)   出版者・発行元:Angewandte Chemie - International Edition  

    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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  93. Mutational and Combinatorial Control of Self-Assembling and Disassembling of Human Proteasome Subunits 査読有り 国際誌

    Sekiguchi, T; Satoh, T; Kurimoto, E; Song, CH; Kozai, T; Watanabe, H; Ishii, K; Yagi, H; Yanaka, S; Uchiyama, S; Uchihashi, T; Murata, K; Kato, K

    INTERNATIONAL JOURNAL OF MOLECULAR SCIENCES   20 巻 ( 9 )   2019年5月

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    記述言語:英語   掲載種別:研究論文(学術雑誌)   出版者・発行元:International Journal of Molecular Sciences  

    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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  94. Construction of a Quadrangular Tetramer and a Cage-Like Hexamer from Three-Helix Bundle-Linked Fusion Proteins 査読有り 国際誌

    Miyamoto, T; Hayashi, Y; Yoshida, K; Watanabe, H; Uchihashi, T; Yonezawa, K; Shimizu, N; Kamikubo, H; Hirota, S

    ACS SYNTHETIC BIOLOGY   8 巻 ( 5 ) 頁: 1112 - 1120   2019年5月

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    記述言語:英語   掲載種別:研究論文(学術雑誌)   出版者・発行元:ACS Synthetic Biology  

    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.

    DOI: 10.1021/acssynbio.9b00019

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  95. Inner lumen proteins stabilize doublet microtubules in cilia and flagella 査読有り 国際誌

    Owa, M; Uchihashi, T; Yanagisawa, H; Yamano, T; Iguchi, H; Fukuzawa, H; Wakabayashi, K; Ando, T; Kikkawa, M

    NATURE COMMUNICATIONS   10 巻 ( 1 ) 頁: 1143 - 1143   2019年3月

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    記述言語:英語   出版者・発行元:Nature Communications  

    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.

    DOI: 10.1038/s41467-019-09051-x

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

    Oohora, K; Kajihara, R; Fujimaki, N; Uchihashi, T; Hayashi, T

    CHEMICAL COMMUNICATIONS   55 巻 ( 11 ) 頁: 1544 - 1547   2019年2月

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    記述言語:英語   掲載種別:研究論文(学術雑誌)   出版者・発行元:Chemical Communications  

    Engineered cytochrome b562, a small hemoprotein, with an externally-attached heme moiety via a moderately long linker at a suitable position predominantly forms a thermodynamically stable ring-shaped trimer in dilute solution. In an equilibrium between supramolecular polymerization and depolymerization, the ring-shaped trimer is kinetically trapped even in a concentrated solution.

    DOI: 10.1039/c8cc09314h

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  97. Microtubule self- healing and defect creation investigated by in- line force measurements during high- speed atomic force microscopy imaging 査読有り

    Ganser, C; Uchihashi, T

    NANOSCALE   11 巻 ( 1 ) 頁: 125 - 135   2019年1月

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    記述言語:英語   出版者・発行元:Royal Society of Chemistry ({RSC})  

    Microtubules are biopolymers composed of tubulin and play diverse roles in a wide variety of biological processes such as cell division, migration and intracellular transport in eukaryotic cells. To perform their functions, microtubules are mechanically stressed and, thereby, susceptible to structural defects. Local variations in mechanical properties caused by these defects modulate their biological functions, including binding and transportation of microtubule-associated proteins. Therefore, assessing the local mechanical properties of microtubules and analyzing their dynamic response to mechanical stimuli provide insight into fundamental processes. It is, however, not trivial to control defect formation, gather mechanical information at the same time, and subsequently image the result at a high temporal resolution at the molecular level with minimal delay. In this work, we describe the so-called in-line force curve mode based on high-speed atomic force microscopy. This method is directly applied to create defects in microtubules at the level of tubulin dimers and monitor the following dynamic processes around the defects. Furthermore, force curves obtained during defect formation provide quantitative mechanical information to estimate the bonding energy between tubulin dimers.

    DOI: 10.1039/c8nr07392a

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  98. Single-Unit Imaging of Membrane Protein-Embedded Nanodiscs from Two Oriented Sides by High-Speed Atomic Force Microscopy 査読有り

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

    STRUCTURE   27 巻 ( 1 ) 頁: 152 - +   2019年1月

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    記述言語:英語   掲載種別:研究論文(学術雑誌)   出版者・発行元:Structure  

    Membrane proteins play important roles in various cellular functions. To analyze membrane proteins, nanodisc technology using membrane scaffold proteins allows single membrane protein units to be embedded into the lipid bilayer disc without detergents. Recent advancements in high-speed atomic force microscopy (HS-AFM) have enabled us to monitor the real-time dynamics of proteins in solution at the nanometer scale. In this study, we report HS-AFM imaging of membrane proteins reconstituted into nanodiscs using two membrane protein complexes, SecYEG complex and MgtE dimer. The observed images showed single particles of membrane protein-embedded nanodiscs in an end-up orientation whereby the membrane was fixed parallel to the supporting solid surface and in a side-on orientation whereby the membrane plane was vertically fixed to the solid surface, enabling the elucidation of domain fluctuations in membrane proteins. This technique provides a basic method for the high-resolution imaging of single membrane proteins by HS-AFM.

    DOI: 10.1016/j.str.2018.09.005

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  99. Metastable asymmetrical structure of a shaftless V<sub>1</sub> motor 査読有り

    Maruyama, S; Suzuki, K; Imamura, M; Sasaki, H; Matsunami, H; Mizutani, K; Saito, Y; Imai, FL; Ishizuka-Katsura, Y; Kimura-Someya, T; Shirouzu, M; Uchihashi, T; Ando, T; Yamato, I; Murata, T

    SCIENCE ADVANCES   5 巻 ( 1 ) 頁: eaau8149 - eaau8149   2019年1月

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    記述言語:英語   掲載種別:研究論文(学術雑誌)   出版者・発行元:Science Advances  

    V 1 -ATPase is an ATP-driven rotary motor that is composed of a ring-shaped A 3 B 3 complex and a central DF shaft. The nucleotide-free A 3 B 3 complex of Enterococcus hirae, composed of three identical A 1 B 1 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 3 B 3 . The nucleotide-free mutant A 3 B 3 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 3 B 3 forms a metastable (stressed) asymmetric structure composed of unstable A 1 B 1 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.

    DOI: 10.1126/sciadv.aau8149

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  100. 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 招待有り

    内橋貴之

    高分子   68 巻 ( 01 ) 頁: 564 - 568   2019年

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    担当区分:筆頭著者, 最終著者, 責任著者   記述言語:日本語   掲載種別:研究論文(学術雑誌)  

  101. 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)     頁: 253 - 256   2019年

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  102. 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)     頁: 281 - 285   2019年

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  103. 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)     頁: 281 - 285   2019年

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    記述言語:英語   掲載種別:研究論文(国際会議プロシーディングス)   出版者・発行元:IEEE  

    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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  104. Versatility of RNA-Binding Proteins in Living Cells 査読有り

    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   5 巻 ( 1 ) 頁: 7 - 7   2019年

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    記述言語:英語   掲載種別:研究論文(学術雑誌)   出版者・発行元:Science Publishing Group  

    DOI: 10.11648/j.bs.20190501.12

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

    Fukui, T; Uchihashi, T; Sasaki, N; Watanabe, H; Takeuchi, M; Sugiyasu, K

    ANGEWANDTE CHEMIE-INTERNATIONAL EDITION   57 巻 ( 47 ) 頁: 15465 - 15470   2018年11月

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    記述言語:英語   掲載種別:研究論文(学術雑誌)   出版者・発行元:Angewandte Chemie - International Edition  

    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.

    DOI: 10.1002/anie.201809165

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  106. Monitoring Thermoresponsive Morphological Changes in Individual Hydrogel Microspheres 査読有り 国際誌

    Matsui, S; Nishizawa, Y; Uchihashi, T; Suzuki, D

    ACS OMEGA   3 巻 ( 9 ) 頁: 10836 - 10842   2018年9月

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    記述言語:英語   掲載種別:研究論文(学術雑誌)   出版者・発行元:ACS Omega  

    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.

    DOI: 10.1021/acsomega.8b01770

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

    Oohora, K; Fujimaki, N; Kajihara, R; Watanabe, H; Uchihashi, T; Hayashi, T

    JOURNAL OF THE AMERICAN CHEMICAL SOCIETY   140 巻 ( 32 ) 頁: 10145 - 10148   2018年8月

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    記述言語:英語   掲載種別:研究論文(学術雑誌)   出版者・発行元:Journal of the American Chemical Society  

    A supramolecular assembly of units of cytochrome b562 with externally attached heme having intermolecular linkages formed via the heme-heme pocket interaction was investigated in an effort to construct a well-defined structure. The engineered site for surface attachment of heme at Cys80 in an N80C mutant of cytochrome b562 provides the primary basis for the formation of the periodic assembly structure, which is characterized herein by circular dichroism (CD) spectroscopy and high-speed atomic force microscopy (AFM). This assembly represents the first example of the observation of a split-type Cotton effect by heme-heme exciton coupling in an artificial hemoprotein assembly system. Molecular dynamics simulations validated by simulated CD spectra, AFM images, and mutation experiments reveal that the assembly has a periodic helical structure with 3 nm pitches, suggesting the formation of the assembled structure is driven not only by the heme-heme pocket interaction but also by additional secondary hydrogen bonding and/or electrostatic interactions at the protein interfaces of the assembly.

    DOI: 10.1021/jacs.8b06690

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

    Mori, T; Sugiyama, S; Byrne, M; Johnson, CH; Uchihashi, T; Ando, T

    NATURE COMMUNICATIONS   9 巻 ( 1 ) 頁: 3245   2018年8月

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    記述言語:英語   掲載種別:研究論文(学術雑誌)   出版者・発行元:Nature Communications  

    The circadian clock proteins KaiA, KaiB, and KaiC reconstitute a remarkable circa-24 h oscillation of KaiC phosphorylation that persists for many days in vitro. Here we use high-speed atomic force microscopy (HS-AFM) to visualize in real time and quantify the dynamic interactions of KaiA with KaiC on sub-second timescales. KaiA transiently interacts with KaiC, thereby stimulating KaiC autokinase activity. As KaiC becomes progressively more phosphorylated, KaiA’s affinity for KaiC weakens, revealing a feedback of KaiC phosphostatus back onto the KaiA-binding events. These non-equilibrium interactions integrate high-frequency binding and unbinding events, thereby refining the period of the longer term oscillations. Moreover, this differential affinity phenomenon broadens the range of Kai protein stoichiometries that allow rhythmicity, explaining how the oscillation is resilient in an in vivo milieu that includes noise. Therefore, robustness of rhythmicity on a 24-h scale is explainable by molecular events occurring on a scale of sub-seconds.

    DOI: 10.1038/s41467-018-05438-4

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  109. 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   57 巻 ( 8 )   2018年8月

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    記述言語:日本語   出版者・発行元:Japanese Journal of Applied Physics  

    DOI: 10.7567/JJAP.57.08N001

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

    Umakoshi, T; Udaka, H; Uchihashi, T; Ando, T; Suzuki, M; Fukuda, T

    COLLOIDS AND SURFACES B-BIOINTERFACES   167 巻   頁: 267 - 274   2018年7月

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    記述言語:英語   掲載種別:研究論文(学術雑誌)   出版者・発行元:Colloids and Surfaces B: Biointerfaces  

    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.

    DOI: 10.1016/j.colsurfb.2018.04.015

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  111. Translating MOF chemistry into supramolecular chemistry: soluble coordination nanofibers showing efficient photon upconversion

    Hosoyamada, M; Yanai, N; Okumura, K; Uchihashi, T; Kimizuka, N

    CHEMICAL COMMUNICATIONS   54 巻 ( 50 ) 頁: 6828 - 6831   2018年6月

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    記述言語:英語   掲載種別:研究論文(学術雑誌)   出版者・発行元:Chemical Communications  

    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.

    DOI: 10.1039/c8cc01594e

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

    Uchihashi, T; Watanabe, Y; Nakazaki, Y; Yamasaki, T; Watanabe, H; Maruno, T; Ishii, K; Uchiyama, S; Song, CH; Murata, K; Iino, R; Ando, T

    NATURE COMMUNICATIONS   9 巻 ( 1 ) 頁: 2147   2018年6月

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    記述言語:英語   掲載種別:研究論文(学術雑誌)   出版者・発行元:Nature Communications  

    The ATP-dependent bacterial protein disaggregation machine, ClpB 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 ClpB unfold and thread its protein substrate through the central pore. However, their function-related structural dynamics has remained elusive. Here we directly visualize ClpB 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 ClpB to disentangle protein aggregates.

    DOI: 10.1038/s41467-018-04587-w

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  113. Sweeping of Adsorbed Therapeutic Protein on Prefillable Syringes Promotes Micron Aggregate Generation 査読有り

    Maruno, T; Watanabe, H; Yoneda, S; Uchihashi, T; Adachi, S; Arai, K; Sawaguchi, T; Uchiyama, S

    JOURNAL OF PHARMACEUTICAL SCIENCES   107 巻 ( 6 ) 頁: 1521 - 1529   2018年6月

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    記述言語:英語   掲載種別:研究論文(学術雑誌)   出版者・発行元:Journal of Pharmaceutical Sciences  

    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/m 2 , 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.

    DOI: 10.1016/j.xphs.2018.01.021

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

    Shibata, M; Inoue, K; Ikeda, K; Konno, M; Singh, M; Kataoka, C; Abe-Yoshizumi, R; Kandori, H; Uchihashi, T

    SCIENTIFIC REPORTS   8 巻 ( 1 ) 頁: 8262   2018年5月

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    記述言語:英語   掲載種別:研究論文(学術雑誌)   出版者・発行元:Scientific Reports  

    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.

    DOI: 10.1038/s41598-018-26606-y

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  115. Structural properties determining low K<SUP>+</SUP> affinity of the selectivity filter in the TWIK1 K<SUP>+</SUP> channel

    Tsukamoto, H; Higashi, M; Motoki, H; Watanabe, H; Ganser, C; Nakajo, K; Kubo, Y; Uchihashi, T; Furutani, Y

    JOURNAL OF BIOLOGICAL CHEMISTRY   293 巻 ( 18 ) 頁: 6969 - 6984   2018年5月

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    記述言語:英語   掲載種別:研究論文(学術雑誌)   出版者・発行元:Journal of Biological Chemistry  

    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.

    DOI: 10.1074/jbc.RA118.001817

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  116. Construction of a Triangle-Shaped Trimer and a Tetrahedron Using an -Helix-Inserted Circular Permutant of Cytochrome <i>c</i><sub>555</sub>

    Oda, A; Nagao, S; Yamanaka, M; Ueda, I; Watanabe, H; Uchihashi, T; Shibata, N; Higuchi, Y; Hirota, S

    CHEMISTRY-AN ASIAN JOURNAL   13 巻 ( 8 ) 頁: 964 - 967   2018年4月

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    記述言語:英語   掲載種別:研究論文(学術雑誌)   出版者・発行元:Chemistry - An Asian Journal  

    Highly-ordered protein structures have gained interest for future uses for biomaterials. Herein, we constructed a building block protein (BBP) by the circular permutation of the hyperthermostable Aquifex aeolicus cytochrome (cyt) c555, and assembled BBP into a triangle-shaped trimer and a tetrahedron. The angle of the intermolecular interactions of BBP was controlled by cleaving the domain-swapping hinge loop of cyt c555 and connecting the original N- and C-terminal α-helices with an α-helical linker. We obtained BBP oligomers up to ≈40 mers, with a relatively large amount of trimers. According to the X-ray crystallographic analysis of the BBP trimer, the N-terminal region of one BBP molecule interacted intermolecularly with the C-terminal region of another BBP molecule, resulting in a triangle-shaped structure with an edge length of 68 Å. Additionally, four trimers assembled into a unique tetrahedron in the crystal. These results demonstrate that the circular permutation connecting the original N- and C-terminal α-helices with an α-helical linker may be useful for constructing organized protein structures.

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

    Terahara, N; Inoue, Y; Kodera, N; Morimoto, YV; Uchihashi, T; Imada, K; Ando, T; Namba, K; Minamino, T

    SCIENCE ADVANCES   4 巻 ( 4 ) 頁: eaao7054   2018年4月

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    記述言語:英語   掲載種別:研究論文(学術雑誌)   出版者・発行元:Science Advances  

    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.

    DOI: 10.1126/sciadv.aao7054

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  118. 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) 査読有り

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

    Chemistry - An Asian Journal   13 巻 ( 10 ) 頁: 1229   2018年4月

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    出版者・発行元:Wiley  

    DOI: 10.1002/asia.201800587

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

    Matsui, S; Uchihashi, T; Suzuki, D

    ABSTRACTS OF PAPERS OF THE AMERICAN CHEMICAL SOCIETY   255 巻   頁: .   2018年3月

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    記述言語:英語   掲載種別:研究論文(学術雑誌)  

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

    Matsui, S; Uchihashi, T; Suzuki, D

    ABSTRACTS OF PAPERS OF THE AMERICAN CHEMICAL SOCIETY   255 巻   頁: .   2018年3月

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    記述言語:英語   掲載種別:研究論文(学術雑誌)  

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  121. 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   255 巻   頁: .   2018年3月

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    記述言語:英語   掲載種別:研究論文(学術雑誌)  

  122. 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   255 巻   頁: .   2018年3月

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    記述言語:英語   掲載種別:研究論文(学術雑誌)  

  123. Negatively Charged Lipids Are Essential for Functional and Structural Switch of Human 2-Cys Peroxiredoxin II 査読有り

    Haruyama, T; Uchihashi, T; Yamada, Y; Kodera, N; Ando, T; Konno, H

    JOURNAL OF MOLECULAR BIOLOGY   430 巻 ( 5 ) 頁: 602 - 610   2018年3月

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    記述言語:英語   掲載種別:研究論文(学術雑誌)  

    DOI: 10.1016/j.jmb.2017.12.020

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  124. Rate constants, processivity, and productive binding ratio of chitinase A revealed by single-molecule analysis 国際誌

    Nakamura, A; Tasaki, T; Okuni, Y; Song, CH; Murata, K; Kozai, T; Hara, M; Sugimoto, H; Suzuki, K; Watanabe, T; Uchihashi, T; Noji, H; Iino, R

    PHYSICAL CHEMISTRY CHEMICAL PHYSICS   20 巻 ( 5 ) 頁: 3010 - 3018   2018年2月

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    記述言語:英語   掲載種別:研究論文(学術雑誌)   出版者・発行元:Physical Chemistry Chemical Physics  

    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, kfastoff (3.2 s-1, 78%) and kslowoff (0.38 s-1, 22%), corresponding to bindings to different crystal surfaces. From the kon, kfastoff, kslowoff 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.

    DOI: 10.1039/c7cp04606e

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  125. Rate constants, processivity, and productive binding ratio of chitinase A revealed by single-molecule analysis (vol 20, pg 3010, 2018) 国際誌

    Nakamura, A; Tasaki, T; Okuni, Y; Song, C; Murata, K; Kozai, T; Hara, M; Sugimoto, H; Suzuki, K; Watanabe, T; Uchihashi, T; Noji, H; Iino, R

    PHYSICAL CHEMISTRY CHEMICAL PHYSICS   20 巻 ( 5 ) 頁: 3844 - 3844   2018年2月

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    記述言語:英語   掲載種別:研究論文(学術雑誌)  

    DOI: 10.1039/c8cp90024h

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

    Sugiyama, S; Mori, T; Byrne, M; Uchihashi, T; Johnson, CH; Ando, T

    BIOPHYSICAL JOURNAL   114 巻 ( 3 ) 頁: 68A - 68A   2018年2月

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    記述言語:英語   掲載種別:研究論文(学術雑誌)  

    DOI: 10.1016/j.bpj.2017.11.421

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  127. 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   114 巻 ( 3 ) 頁: 68A-68A   2018年2月

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    記述言語:英語   掲載種別:研究論文(学術雑誌)  

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

    Uchihashi, T; Scheuring, S

    BIOCHIMICA ET BIOPHYSICA ACTA-GENERAL SUBJECTS   1862 巻 ( 2 ) 頁: 229 - 240   2018年2月

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    記述言語:英語   掲載種別:研究論文(学術雑誌)   出版者・発行元:Biochimica et Biophysica Acta - General Subjects  

    Background Many biological processes in a living cell are consequences of sequential and hierarchical dynamic events of biological macromolecules such as molecular interactions and conformational changes. Hence, knowledge of structures, assembly and dynamics of proteins is the foundation for understanding how biological molecules work. Among several techniques to analyze dynamics of proteins, high-speed atomic force microscopy (HS-AFM) is unique to provide direct information about both structure and dynamics of single proteins at work. Scope of review The scope of this review is overviewing recent progresses of HS-AFM for studying dynamic processes of biomolecular systems. In the technical descriptions, key developments enabling fast and non-invasive imaging of biological samples are briefly mentioned. Then recent successful applications of HS-AFM are overviewed to showcase the power of HS-AFM in biological research. Major conclusions We discuss examples where HS-AFM movies captured important dynamic biological processes, including conformational dynamics of membrane proteins, processive movements of enzymes, assembly and disassembly processes of protein supramolecular structures, and dynamics in a two-dimensional protein crystal. These examples demonstrate the usability of HS-AFM to reveal biomolecular processes at high spatiotemporal (nanometer and subsecond) resolution. General significance Real-time movies of unlabeled proteins at work captured by HS-AFM allowed us to directly gain insights into mechanisms of molecular actions. Together with further functional extensions, HS-AFM will enable researchers to investigate more complex biological systems involving multiple proteins and will become an indispensable technique for life science. This article is part of a Special Issue entitled “Biophysical Exploration of Dynamical Ordering of Biomolecular Systems” Guest Editor: Dr., Professor Koichi Kato.

    DOI: 10.1016/j.bbagen.2017.07.010

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  129. Dynamic clustering of dynamin-amphiphysin helices regulates membrane constriction and fission coupled with GTP hydrolysis 査読有り

    Takeda, T; Kozai, T; Yang, H; Ishikuro, D; Seyama, K; Kumagai, Y; Abe, T; Yamada, H; Uchihashi, T; Ando, T; Takei, K

    ELIFE   7 巻   2018年1月

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    記述言語:英語   掲載種別:研究論文(学術雑誌)   出版者・発行元:eLife  

    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.

    DOI: 10.7554/eLife.30246

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  130. Conversion of functionally undefined homopentameric protein PbaA into a proteasome activator by mutational modification of its C-terminal segment conformation 査読有り 国際誌

    Yagi-Utsumi, M; Sikdar, A; Kozai, T; Inoue, R; Sugiyama, M; Uchihashi, T; Yagi, H; Satoh, T; Kato, K

    PROTEIN ENGINEERING DESIGN & SELECTION   31 巻 ( 1 ) 頁: 29 - 36   2018年1月

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    記述言語:英語   掲載種別:研究論文(学術雑誌)   出版者・発行元:Protein Engineering, Design and Selection  

    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 crystallographically 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.

    DOI: 10.1093/protein/gzx066

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

    Uchihashi Takayuki

    JOURNAL OF COMPUTER CHEMISTRY-JAPAN   17 巻 ( 1 ) 頁: 20-30 - 30   2018年

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    記述言語:英語   掲載種別:研究論文(学術雑誌)   出版者・発行元:日本コンピュータ化学会  

    <p>最近の高速原子間力顕微鏡(Atomic Force Microscopy: AFM)の進展によって, 生理溶液中でのタンパク質の構造とその時間発展,分子の離散集合などダイナミクスをリアルタイムで可視化できるようになってきた. これまで様々な計測手法による実験事実の積み重ねから推定されてきた事実を動画として提示することで, タンパク質の動的振る舞いを介した機能発現の分子機構を直接的かつ直感的に把握できるようになった. 動画は静止画に比べて極めて多くの情報を与えるが, 一方で, 単に動画鑑賞に終わることなく, そこから有益な情報を客観的な指標を基に抽出すること, すなわち動画解析が次のステップには重要である. 本稿では, これまで高速AFMで観察されてきた代表的なタンパク質の動的現象をi)一分子の構造変化, ii)分子の直進運動と速度解析, iii)二分子間の結合と解離, の3つに大別し, それぞれについて筆者らの実験結果をデータの解釈と解析の効率化のために開発した画像解析法とともに解説する.</p>

    DOI: 10.2477/jccj.2018-0001

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

    Uchihashi, T; Watanabe, H; Kodera, N

    NANOSCALE IMAGING: METHODS AND PROTOCOLS   1814 巻   頁: 159 - 179   2018年

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    記述言語:英語   出版者・発行元:Methods in Molecular Biology  

    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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  133. High-speed scanning near-field optical microscopy

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

    Optics InfoBase Conference Papers   Part F125-JSAP 2018 巻   2018年

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    記述言語:日本語   出版者・発行元:Optics InfoBase Conference Papers  

    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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  134. Optimum Substrates for Imaging Biological Molecules with High-Speed Atomic Force Microscopy 査読有り

    Takayuki Uchihashi, Hiroki Watanabe, Noriyuki Kodera

    Methods in Molecular Biology   1814 巻   頁: 159 - 179   2018年

  135. 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   2017年度 巻   頁: [4AW17 - 2]   2017年12月

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    記述言語:英語   出版者・発行元:生命科学系学会合同年次大会運営事務局  

  136. Two-step process for disassembly mechanism of proteasome α7 homo-tetradecamer by α6 revealed by high-speed atomic force microscopy 国際誌

    Kozai, T; Sekiguchi, T; Satoh, T; Yagi, H; Kato, K; Uchihashi, T

    SCIENTIFIC REPORTS   7 巻 ( 1 ) 頁: 15373 - 15373   2017年11月

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    記述言語:英語   掲載種別:研究論文(学術雑誌)   出版者・発行元:Scientific Reports  

    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.

    DOI: 10.1038/s41598-017-15708-8

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

    Shibata, M; Nishimasu, H; Kodera, N; Hirano, S; Ando, T; Uchihashi, T; Nureki, O

    NATURE COMMUNICATIONS   8 巻 ( 1 ) 頁: 1430   2017年11月

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    記述言語:英語   掲載種別:研究論文(学術雑誌)   出版者・発行元:Nature Communications  

    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.

    DOI: 10.1038/s41467-017-01466-8

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  138. Na<SUP>+</SUP>-induced structural transition of MotPS for stator assembly of the <i>Bacillus</i> flagellar motor

    Terahara, N; Kodera, N; Uchihashi, T; Ando, T; Namba, K; Minamino, T

    SCIENCE ADVANCES   3 巻 ( 11 ) 頁: eaao4119   2017年11月

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    記述言語:英語   掲載種別:研究論文(学術雑誌)   出版者・発行元:Science Advances  

    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.

    DOI: 10.1126/sciadv.aao4119

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  139. 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   29 巻 ( 6 ) 頁: 542 - 545   2017年11月

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    記述言語:日本語  

    DOI: 10.1016/j.ajoms.2017.06.007

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

    Matsui, S; Kureha, T; Hiroshige, S; Shibata, M; Uchihashi, T; Suzuki, D

    ANGEWANDTE CHEMIE-INTERNATIONAL EDITION   56 巻 ( 40 ) 頁: 12146 - 12149   2017年9月

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    記述言語:英語   掲載種別:研究論文(学術雑誌)   出版者・発行元:Angewandte Chemie - International Edition  

    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.

    DOI: 10.1002/anie.201705808

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

    Satoh, T; Song, C; Zhu, T; Toshimori, T; Murata, K; Hayashi, Y; Kamikubo, H; Uchihashi, T; Kato, K

    SCIENTIFIC REPORTS   7 巻 ( 1 ) 頁: 12142   2017年9月

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    記述言語:英語   掲載種別:研究論文(学術雑誌)   出版者・発行元:Scientific Reports  

    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 β-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.

    DOI: 10.1038/s41598-017-12283-w

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

    Harada, H; Onoda, A; Uchihashi, T; Watanabe, H; Sunagawa, N; Samejima, M; Igarashi, K; Hayashi, T

    CHEMICAL SCIENCE   8 巻 ( 9 ) 頁: 6561 - 6565   2017年9月

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    記述言語:英語   掲載種別:研究論文(学術雑誌)   出版者・発行元:Chemical Science  

    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.

    DOI: 10.1039/c7sc01672g

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  143. 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   56 巻 ( 8 )   2017年8月

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    記述言語:日本語   出版者・発行元:Japanese Journal of Applied Physics  

    DOI: 10.7567/JJAP.56.08L001

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  144. 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   7 巻 ( 1 ) 頁: 6166   2017年7月

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    記述言語:英語   掲載種別:研究論文(学術雑誌)  

    DOI: 10.1038/s41598-017-06249-1

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  145. High-Speed Atomic Force Microscopy Reveals Loss of Nuclear Pore Resilience as a Dying Code 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, RW

    ACS NANO   11 巻 ( 6 ) 頁: 5567 - 5578   2017年6月

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    記述言語:英語   掲載種別:研究論文(学術雑誌)  

    DOI: 10.1021/acsnano.7b00906

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  146. High-speed AFM revealed single-molecule blocking dynamics of a scorpion toxin on the KcsA potassium channel 査読有り

    Ayumi Sumino, Takayuki Uchihashi, Takashi Sumikama, Shigetoshi Oiki

    JOURNAL OF PHYSIOLOGICAL SCIENCES   67 巻   頁: S117   2017年3月

  147. Oriented Reconstitution of the Full-Length KcsA Potassium Channel in a Lipid Bilayer for AFM Imaging 査読有り

    Sumino, A; Uchihashi, T; Oiki, S

    JOURNAL OF PHYSICAL CHEMISTRY LETTERS   8 巻 ( 4 ) 頁: 785 - 793   2017年2月

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    記述言語:英語   掲載種別:研究論文(学術雑誌)  

    DOI: 10.1021/acs.jpclett.6b03058

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  148. 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   28 巻   2017年

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    記述言語:日本語  

    Web of Science

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

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

    Biophysics and physicobiology   14 巻 ( 0 ) 頁: 127-135 - 135   2017年

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    記述言語:英語   掲載種別:研究論文(学術雑誌)   出版者・発行元:一般社団法人 日本生物物理学会  

    <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>

    DOI: 10.2142/biophysico.14.0_127

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  150. "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   28 巻   頁: .   2017年

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    記述言語:英語   掲載種別:研究論文(学術雑誌)  

    Web of Science

  151. "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   28 巻   頁: .   2017年

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    記述言語:英語   掲載種別:研究論文(学術雑誌)  

  152. 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

    内橋 貴之

    表面科学学術講演会要旨集   37 巻 ( 0 ) 頁: 254   2017年

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    記述言語:日本語   出版者・発行元:公益社団法人 日本表面真空学会  

    高速原子間力顕微鏡(HS-AFM)は1frame/s以上のフレームレートで溶液環境にある分子の様々なダイナミクスをリアルタイムで可視化でき、これまで多くのタンパク質の機能動態観察に成功している。本講演では、HS-AFMの要素技術について概説するとともに、最近の生体分子および人工超分子に応用した例について紹介する。

    DOI: 10.14886/sssj2008.37.0_254

    CiNii Research

  153. 高速AFMを用いた生体分子のその場観察

    内橋 貴之

    表面科学学術講演会要旨集   37 巻 ( 0 ) 頁: 19   2017年

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    記述言語:日本語   出版者・発行元:公益社団法人 日本表面真空学会  

    高速原子間力顕微鏡により生体分子の機能動態のリアルタイムイメージングと一分子力学操作について紹介する。

    DOI: 10.14886/sssj2008.37.0_19

    CiNii Research

  154. A natural light-driven inward proton pump. 査読有り

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

    Nature communications   7 巻   頁: 13415   2016年11月

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    記述言語:英語   掲載種別:研究論文(学術雑誌)  

    DOI: 10.1038/ncomms13415

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  155. Functional extension of high-speed AFM for wider biological applications. 査読有り

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

    Ultramicroscopy   160 巻   頁: 182-96   2016年1月

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    記述言語:英語   掲載種別:研究論文(学術雑誌)  

    DOI: 10.1016/j.ultramic.2015.10.017

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

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

    The Journal of biological chemistry   290 巻 ( 34 ) 頁: 20793-803   2015年8月

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    記述言語:英語   掲載種別:研究論文(学術雑誌)  

    DOI: 10.1074/jbc.M114.627554

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  157. Method of mechanical holding of cantilever chip for tip-scan high-speed atomic force microscope. 査読有り

    Fukuda S, Uchihashi T, Ando T

    The Review of scientific instruments   86 巻 ( 6 ) 頁: 063703   2015年6月

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    記述言語:英語   掲載種別:研究論文(学術雑誌)  

    DOI: 10.1063/1.4922381

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  158. Long-tip high-speed atomic force microscopy for nanometer-scale imaging in live cells. 査読有り

    Shibata M, Uchihashi T, Ando T, Yasuda R

    Scientific reports   5 巻   頁: 8724   2015年3月

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    記述言語:英語   掲載種別:研究論文(学術雑誌)  

    DOI: 10.1038/srep08724

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  159. Probing structural dynamics of an artificial protein cage using high-speed atomic force microscopy. 査読有り

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

    Nano letters   15 巻 ( 2 ) 頁: 1331-5   2015年2月

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    記述言語:英語   掲載種別:研究論文(学術雑誌)  

    DOI: 10.1021/nl5045617

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  160. Real-time dynamic adsorption processes of cytochrome c on an electrode observed through electrochemical high-speed atomic force microscopy. 査読有り

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

    PloS one   10 巻 ( 2 ) 頁: e0116685   2015年

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    記述言語:英語   掲載種別:研究論文(学術雑誌)  

    DOI: 10.1371/journal.pone.0116685

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  161. Two-way traffic of glycoside hydrolase family 18 processive chitinases on crystalline chitin. 査読有り

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

    Nature communications   5 巻   頁: 3975   2014年6月

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    記述言語:英語   掲載種別:研究論文(学術雑誌)  

    DOI: 10.1038/ncomms4975

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

      289 巻 ( 20 ) 頁: 14056-65   2014年5月

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    記述言語:英語   掲載種別:研究論文(学術雑誌)  

    DOI: 10.1074/jbc.M113.546085

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  163. [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   86 巻 ( 2 ) 頁: 127-36   2014年4月

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    記述言語:日本語   掲載種別:研究論文(学術雑誌)  

    PubMed

  164. 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   136 巻 ( 12 ) 頁: 4584-92   2014年3月

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    記述言語:英語   掲載種別:研究論文(学術雑誌)  

    DOI: 10.1021/ja4119994

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

    Ando T, Uchihashi T, Scheuring S

    Chemical reviews   114 巻 ( 6 ) 頁: 3120-88   2014年3月

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    記述言語:英語   掲載種別:研究論文(学術雑誌)  

    DOI: 10.1021/cr4003837

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  166. 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   184 巻 ( 1 ) 頁: 2-11   2013年10月

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    記述言語:英語   掲載種別:研究論文(学術雑誌)  

    DOI: 10.1016/j.jsb.2013.02.011

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  167. 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   105 巻 ( 6 ) 頁: 1397-405   2013年9月

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    記述言語:英語   掲載種別:研究論文(学術雑誌)  

    DOI: 10.1016/j.bpj.2013.07.052

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  168. 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   84 巻 ( 7 ) 頁: 073706   2013年7月

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    記述言語:英語   掲載種別:研究論文(学術雑誌)  

    DOI: 10.1063/1.4813280

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  169. 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   84 巻 ( 5 ) 頁: 053702   2013年5月

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    記述言語:英語   掲載種別:研究論文(学術雑誌)  

    DOI: 10.1063/1.4803449

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

    Ando T, Uchihashi T, Kodera N

    Annual review of biophysics   42 巻   頁: 393-414   2013年

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    記述言語:英語   掲載種別:研究論文(学術雑誌)  

    DOI: 10.1146/annurev-biophys-083012-130324

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  171. 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   422 巻 ( 2 ) 頁: 300-9   2012年9月

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    記述言語:英語   掲載種別:研究論文(学術雑誌)  

    DOI: 10.1016/j.jmb.2012.05.018

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  172. 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   7 巻 ( 6 ) 頁: 1193-206   2012年5月

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    記述言語:英語   掲載種別:研究論文(学術雑誌)  

    DOI: 10.1038/nprot.2012.047

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  173. Direct Visualization of Cellobiohydrolase on Crystalline Cellulose using High-Speed Atomic Force Microscopy 査読有り

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

    BIOPHYSICAL JOURNAL   102 巻 ( 3 ) 頁: 585A-586A   2012年1月

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    記述言語:英語   掲載種別:研究論文(学術雑誌)  

    Web of Science

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

    Methods in enzymology   510 巻   頁: 169-82   2012年

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    記述言語:英語   掲載種別:研究論文(学術雑誌)  

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

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

    Science (New York, N.Y.)   333 巻 ( 6047 ) 頁: 1279-82   2011年9月

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    記述言語:英語   掲載種別:研究論文(学術雑誌)  

    DOI: 10.1126/science.1208386

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

      333 巻 ( 6043 ) 頁: 755-8   2011年8月

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    記述言語:英語   掲載種別:研究論文(学術雑誌)  

    DOI: 10.1126/science.1205510

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  177. 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)   50 巻 ( 19 ) 頁: 4410-3   2011年5月

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    記述言語:英語   掲載種別:研究論文(学術雑誌)  

    DOI: 10.1002/anie.201007544

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

    Uchihashi T, Ando T

    Methods in molecular biology (Clifton, N.J.)   736 巻   頁: 285-300   2011年

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    記述言語:英語   掲載種別:研究論文(学術雑誌)  

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

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  179. High-speed atomic force microscopy shows dynamic molecular processes in photoactivated bacteriorhodopsin 査読有り

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

    NATURE NANOTECHNOLOGY   5 巻 ( 3 ) 頁: 208-212   2010年3月

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    記述言語:英語   掲載種別:研究論文(学術雑誌)  

    DOI: 10.1038/NNANO.2010.7

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

    内橋 貴之

    JOURNAL OF STRUCTURAL BIOLOGY   167 巻 ( 153-158 ) 頁: .   2009年

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    掲載種別:研究論文(学術雑誌)  

  181. High resonance frequency force microscope scanner using inertia balance support 査読有り

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

    APPLIED PHYSICS LETTERS   92 巻 ( 24 )   2008年6月

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    記述言語:英語   掲載種別:研究論文(学術雑誌)  

    DOI: 10.1063/1.2951594

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

    MIYAGI Atsushi, TSUNAKA Yasuo, TSUNAKA Yasuo, UCHIHASHI Takayuki, UCHIHASHI Takayuki, MAYANAGI Kouta, MAYANAGI Kouta, MAYANAGI Kouta, HIROSE Susumu, MORIKAWA Kosuke, MORIKAWA Kosuke, ANDO Toshio, ANDO Toshio

    CHEMPHYSCHEM   9 巻 ( 13 ) 頁: 1859-1866   2008年

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    掲載種別:研究論文(学術雑誌)  

    DOI: 10.1002/cphc.200800210

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

    内橋 貴之

    Pflugers Archiv -Eur. J. Physiol.   456 巻   頁: .   2008年

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    掲載種別:研究論文(学術雑誌)  

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

    内橋 貴之

    APPLIED PHYSICS LETTERS   92 巻 ( 243119 ) 頁: .   2008年

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    掲載種別:研究論文(学術雑誌)  

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

    内橋 貴之

    PROGRESS IN SURFACE SCIENCE   83 巻 ( 337-437 ) 頁: .   2008年

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    掲載種別:研究論文(学術雑誌)  

  186. 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   20 巻 ( 6 ) 頁: 448-58   2007年11月

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    記述言語:英語   掲載種別:研究論文(学術雑誌)  

    DOI: 10.1002/jmr.843

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  187. 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   78 巻 ( 8 ) 頁: 083702   2007年8月

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    記述言語:英語   掲載種別:研究論文(学術雑誌)  

    DOI: 10.1063/1.2766825

    PubMed

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

    内橋 貴之

    JOURNAL OF MOLECULAR RECOGNITION   20 巻 ( 448-458 ) 頁: .   2007年

     詳細を見る

    掲載種別:研究論文(学術雑誌)  

  189. Capacitance XAFS method: A new site-selective and microscopic X-ray absorption Spectroscopy 査読有り

    Ishii Masashi, Nakao Aiko, Uchihashi Takayuki

    PHYSICA SCRIPTA   T115 巻   頁: 97-101   2005年

     詳細を見る

    記述言語:英語   掲載種別:研究論文(学術雑誌)  

    Web of Science

▼全件表示

書籍等出版物 27

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

    Ganser C., Uchihashi T.

    Methods in Molecular Biology  2022年 

     詳細を見る

    記述言語:英語

    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( 担当: 分担執筆 ,  範囲: 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年12月  ( ISBN:978-4-7581-2256-6

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    記述言語:日本語 著書種別:学術書

  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年6月  ( 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( 担当: 分担執筆 ,  範囲: 23.8「高速原子間力顕微鏡」)

    朝倉書店  2021年6月  ( ISBN:978-4-254-20170-3

     詳細を見る

    総ページ数:576   記述言語:日本語 著書種別:事典・辞書

  5. 現代化学

    内橋貴之( 担当: 単著 ,  範囲: 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年5月 

     詳細を見る

    担当ページ:33-37   記述言語:日本語 著書種別:一般書・啓蒙書

  6. Circadian Rhythms in Bacteria and Microbiomes 査読有り 国際共著

    Tetsuya Mori and Takayuki Uchihashi( 担当: 分担執筆 ,  範囲: Single-Molecule Methods Applied to Circadian Proteins with Special Emphasis on Atomic Force Microscopy)

    Springer  2021年 

     詳細を見る

    担当ページ:147-178   記述言語:英語 著書種別:学術書

  7. 膜タンパク質工学ハンドブック

    内橋貴之( 担当: 分担執筆 ,  範囲: 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年4月  ( ISBN:978-4-86043-537-0

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    記述言語:日本語 著書種別:学術書

  8. 自己修復・自己組織化材料の開発と応用事例

    内橋貴之( 担当: 分担執筆 ,  範囲: 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年3月  ( ISBN:978-4861047817

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    記述言語:日本語 著書種別:学術書

  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( 担当: 分担執筆 ,  範囲: 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年12月  ( ISBN:978-4-7581-0375-6

  10. パリティ 2018年1月号

    内橋 貴之( 担当: 分担執筆 ,  範囲: 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年1月 

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    担当ページ:71-73   記述言語:日本語

  11. Compendium of Surface and Interface Analysis

    Takayuki Uchihashi( 担当: 分担執筆)

    The Surface Science Society of Japan  2018年 

     詳細を見る

    担当ページ:263-267   記述言語:英語 著書種別:学術書

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

    ( 担当: 分担執筆 ,  範囲: High-Speed Atomic Force Microcopy)

    Springer  2018年  ( ISBN:9789811061554

     詳細を見る

    担当ページ:263-267   記述言語:英語 著書種別:事典・辞書

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

    Uchihashi T., Watanabe H., Kodera N.

    Methods in Molecular Biology  2018年 

     詳細を見る

    記述言語:日本語

    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

    PubMed

  14. 光と生命の事典

    内橋貴之( 担当: 分担執筆 ,  範囲: 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年2月  ( ISBN:978-4-254-17161-7

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    記述言語:日本語 著書種別:学術書

  15. Noncontact Atomic Force Microscopy Vol.3

    Takayuyki Uchihashi, Noriyuki Kodera, and Toshio Ando( 担当: 分担執筆 ,  範囲: Chapter 22: High-speed Atomic Force Microscopy)

    Springer  2015年 

     詳細を見る

    担当ページ:481-518   記述言語:英語 著書種別:学術書

  16. Atomic Force Microscopy in Nanobiology

    Takayuki Uchihahsi, Noriyuki Kodera, Toshio Ando( 担当: 分担執筆 ,  範囲: " Chapter 8: Development of High-speed AFM and Its Biological Applications)

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

     詳細を見る

    総ページ数:437   担当ページ:143-176  

  17. 膜タンパク質構造研究

    内橋貴之, 安藤敏夫( 担当: 分担執筆 ,  範囲: 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年10月  ( ISBN:4759815619

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    記述言語:日本語 著書種別:学術書

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

    Toshio Ando, Takayuki Uchihashi( 担当: 分担執筆 ,  範囲: Chapter 19: High-speed AFM and Imaging of Biomolecular Processes)

    Pan Stanford Publishing  2013年  ( ISBN:9789814316453

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    記述言語:英語 著書種別:学術書

    DOI: 10.4032/9789814364485

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

    Takayuki Uchihashi, Noriyuki Kodera, Toshio. Ando( 担当: 分担執筆 ,  範囲: Chapter 5 : Nanovisualization of proteins in action using high-speed AFM)

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

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    担当ページ:119-147   記述言語:英語 著書種別:学術書

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

  20. Atomic force microscopy in liquid: Biological Applications

    ( 担当: 分担執筆 ,  範囲: Toshio Ando, Takayuki Uchihashi, Noriyuki Kodera)

    Wiley-VCH Verlag GmbH  2012年  ( ISBN:9783527327584

     詳細を見る

    担当ページ:189-209   記述言語:英語 著書種別:学術書

    DOI: 10.1002/9783527649808

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

    Takayuki Uchihashi, Toshio Ando( 担当: 分担執筆)

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

     詳細を見る

    担当ページ:163-184   記述言語:英語 著書種別:学術書

    DOI: 10.4032/9789814267977

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

    Takayuki Uchihashi, Toshio Ando( 担当: 分担執筆 ,  範囲: Chapter 18 : High-speed Atomic Force Microscopy and Biomolecular Processes)

    Humana Press  2011年 

     詳細を見る

    担当ページ:285-300   記述言語:英語 著書種別:学術書

  23. 酵素利用技術大系

    内橋貴之, 安藤敏夫( 担当: 分担執筆 ,  範囲: 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年4月  ( ISBN:4860432711

     詳細を見る

    記述言語:日本語 著書種別:学術書

  24. Single Molecule Dynamics in Life Science

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

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

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    担当ページ:277-296   記述言語:英語 著書種別:学術書

  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( 範囲: Nanoscale Contact Charging on a Silicon Oxide)

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

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    担当ページ:289-308   記述言語:英語 著書種別:学術書

    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( 範囲: Atomic Scale Origins of Force Interaction")

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

     詳細を見る

    担当ページ:103-120   記述言語:英語 著書種別:学術書

    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( 担当: 分担執筆 ,  範囲: Time Dependence and its Spatial Distribution of Densely Contact-Electrified Electrons on a Thin Silicon Oxide)

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

     詳細を見る

    担当ページ:505-506   記述言語:英語 著書種別:学術書

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

▼全件表示

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年3月

     詳細を見る

    記述言語:英語  

    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21st 巻   2021年

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  3. ヘリオロドプシンの構造生物学的および物理化学的研究

    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)100th 巻   2020年

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  4. 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

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

    生物物理(Web)60 巻 ( Supplement 1-2 )   2020年

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  5. 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, 内橋貴之  

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

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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 SOCIETY258 巻   2019年8月

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    記述言語:英語   掲載種別:研究発表ペーパー・要旨(国際会議)   出版者・発行元:AMER CHEMICAL SOC  

    Web of Science

  7. 新奇光駆動型内向きプロトンポンプ型ロドプシンSchizorhodopsin(SzR)の輸送メカニズム

    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)99th 巻   2019年

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  8. 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

    井上圭一, 角田聡, SINGH Manish, 今野雅恵, 富田紗穂子, 中村良子, 渡辺大輝, 内橋貴之, GHAI Rohit, BEJA Oded, 神取秀樹  

    生体分子科学討論会講演要旨集46th 巻   頁: 18‐19   2019年

     詳細を見る

    記述言語:日本語  

    J-GLOBAL

  9. ヘリオロドプシンの構造と生物物理学的解析

    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)59 巻 ( Supplement 1-2 )   2019年

     詳細を見る

  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)139th 巻   2019年

     詳細を見る

  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)92nd 巻   2019年

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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

    井上圭一, 角田聡, SINGH Manish, 今野雅恵, 富田紗穂子, 中村良子, 渡辺大輝, 内橋貴之, GHAI Rohit, BEJA Oded, 神取秀樹  

    日本生体エネルギー研究会討論会講演要旨集44th 巻   頁: 24‐25   2018年12月

     詳細を見る

    記述言語:日本語  

    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)41st 巻   頁: ROMBUNNO.2PW2‐10‐5 (WEB ONLY)   2018年

     詳細を見る

    記述言語:日本語  

    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18th 巻   2018年

     詳細を見る

  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48 巻   頁: 1   2017年8月

  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)69th 巻   2017年

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  17. 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)90th 巻   2017年

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  18. 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17th 巻   2017年

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  19. 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)137th 巻   2017年

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▼全件表示

講演・口頭発表等 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年12月10日  日本生体エネルギー研究会

     詳細を見る

    開催年月日: 2020年12月

    記述言語:日本語   会議種別:口頭発表(一般)  

    開催地:金沢   国名:日本国  

  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年12月10日  日本生体エネルギー研究会

     詳細を見る

    開催年月日: 2020年12月

    記述言語:日本語   会議種別:ポスター発表  

    開催地:金沢   国名:日本国  

  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~ 」 招待有り

    内橋貴之

    慶應義塾大学 大学院講義「先端研究  2020年12月2日  慶應義塾大学

     詳細を見る

    開催年月日: 2020年12月

    記述言語:日本語   会議種別:公開講演,セミナー,チュートリアル,講習,講義等  

    開催地:オンライン   国名:日本国  

  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年12月10日 

     詳細を見る

    開催年月日: 2020年12月

    記述言語:英語   会議種別:ポスター発表  

    国名:日本国  

  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 招待有り

    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年12月15日  ブルカージャパン

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    開催年月日: 2020年11月

    記述言語:日本語   会議種別:公開講演,セミナー,チュートリアル,講習,講義等  

    開催地:オンライン   国名:日本国  

  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 招待有り

    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年11月4日 

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    開催年月日: 2020年11月

    記述言語:日本語   会議種別:口頭発表(招待・特別)  

    開催地:オンライン   国名:日本国  

  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 招待有り

    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年10月9日  岡山大学大学院医歯薬学総合研究科

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    開催年月日: 2020年10月

    記述言語:日本語   会議種別:公開講演,セミナー,チュートリアル,講習,講義等  

    開催地:オンライン   国名:日本国  

  8. Observation of Substrate Binding Sec Translocon and Structural Change of SecA with HS-AFM 国際会議

    Wataru Nagaike, Takamitsu Haruyama, Tomoya Tsukazaki and Takayuki Uchihashi

    2020年9月16日 

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    開催年月日: 2020年9月

    記述言語:英語   会議種別:ポスター発表  

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

    Shigetaka Nishiguchi, Hiroki Oda and Takayuki Uchihashi

    2020年9月16日 

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    開催年月日: 2020年9月

    記述言語:英語   会議種別:ポスター発表  

    国名:日本国  

  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年9月16日 

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    開催年月日: 2020年9月

    記述言語:英語   会議種別:ポスター発表  

    国名:日本国  

  11. Characterization of the enzymatic property and structural dynamics of the T3SS ATPase from Enteropathogenic Escherichia coli 国際会議

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

    2020年9月16日 

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    開催年月日: 2020年9月

    記述言語:英語   会議種別:ポスター発表  

    国名:日本国  

  12. HS-AFM observation of Amyloid β elongation and inhibition by antibodies 国際会議

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

    2020年9月16日 

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    開催年月日: 2020年9月

    記述言語:英語   会議種別:ポスター発表  

    国名:日本国  

  13. HS-AFM as a versatile tool to study dynamical and mechanical properties of proteins 国際会議

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

    2020年9月18日 

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    開催年月日: 2020年9月

    記述言語:英語   会議種別:口頭発表(招待・特別)  

    国名:日本国  

  14. Structural insights into the mechanism of rhodopsin phosphodiesterase 招待有り 国際会議

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

    2020年9月17日 

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    開催年月日: 2020年9月

    記述言語:英語   会議種別:口頭発表(招待・特別)  

    国名:日本国  

  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年2月7日 

     詳細を見る

    開催年月日: 2020年2月

    記述言語:英語   会議種別:口頭発表(一般)  

    国名:日本国  

  16. 高速AFMを用いた生体分子のその場観察 招待有り

    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年8月17日 

     詳細を見る

    記述言語:日本語   会議種別:口頭発表(招待・特別)  

    開催地: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 招待有り

    内橋 貴之

    第58回生物物理若手の会  2018年8月29日 

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    記述言語:日本語   会議種別:口頭発表(招待・特別)  

    開催地:ぎふ長良川 ホテルパーク, 岐阜  

  18. 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 招待有り

    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年6月19日 

     詳細を見る

    記述言語:日本語   会議種別:公開講演,セミナー,チュートリアル,講習,講義等  

    開催地:産業技術総合研究所, つくば  

  19. 高速AFMで明らかにするKaiタンパク質間の動的相互作用 招待有り

    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年6月13日 

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    記述言語:日本語   会議種別:口頭発表(招待・特別)  

    開催地:仙台国際センター, 宮城  

  20. 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 ~ 招待有り

    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年3月9日 

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    記述言語:日本語   会議種別:公開講演,セミナー,チュートリアル,講習,講義等  

    開催地:自然科学研究機構、東京  

  21. 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 招待有り

    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年9月11日 

     詳細を見る

    記述言語:日本語   会議種別:シンポジウム・ワークショップ パネル(指名)  

    開催地:同志社大学, 京都  

  22. Visualization of Single-Molecule Dynamics Using High-Speed Atomic Force Microscopy 招待有り 国際会議

    内橋 貴之

    The 2nd Korea-Japan Joint Symposium on Single-Molecule Biophysics 2017  2017年11月8日 

     詳細を見る

    記述言語:英語   会議種別:口頭発表(招待・特別)  

    開催地:Seoul National University, Korea  

  23. Structural Flexibility and Chaperone Activity of TClpB revealed by High-Speed AFM 招待有り 国際会議

    XIX. Annual Linz Winter Workshop  2017年2月3日 

     詳細を見る

    記述言語:英語   会議種別:口頭発表(招待・特別)  

    開催地:Sommerhotel Julius-Raab-Heim, Linz, Austria  

  24. Oligomeric state and conformational dynamics of eubacterial ion-pumping rhodopsin studied by high-speed AFM 国際会議

    内橋 貴之

    KAKENHI International Symposium on “Studying the Function of Soft Molecular Systems”  2017年6月26日 

     詳細を見る

    記述言語:英語   会議種別:シンポジウム・ワークショップ パネル(指名)  

    開催地:Royton Sapporo, Sapporo  

  25. Imaging and Manipulation of Biological Molecules with High-Speed Atomic Force Microscopy 招待有り 国際会議

    内橋 貴之

    ICN-T 2018  2018年7月22日 

     詳細を見る

    記述言語:英語   会議種別:口頭発表(招待・特別)  

    開催地:BVV Trade Fairs, Brno, Czech Republic  

  26. Image Processing and Quantitative Analysis of High-Speed-AFM Data for Studying Single-Molecule Dynamics 国際会議

    内橋 貴之

    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年9月16日 

     詳細を見る

    記述言語:英語   会議種別:口頭発表(招待・特別)  

    開催地:岡山大学津島キャンパス, 岡山  

  27. High-speed atomic force microscopy: A tool for visualizing dynamic behavior from proteins to cells 招待有り 国際会議

    内橋 貴之

    The 28th 2017 International Symposium on Micro-NanoMechanical and Human Science  2017年12月6日 

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    記述言語:英語   会議種別:口頭発表(基調)  

    開催地:Nagoya University, Aichi  

  28. High-speed Atomic Force Microscopy: A tool for direct visualization of single-molecule dynamics 招待有り 国際会議

    内橋 貴之

    Minisymposium on “Advanced Atomic Force Microscopy”  2018年7月18日 

     詳細を見る

    記述言語:英語   会議種別:口頭発表(招待・特別)  

    開催地:Montanuniversität Leoben, Austria  

  29. High-Speed Atomic Force Microscopy for Visualization of Dynamic Processes in Biological and Artificial Supramolecules 招待有り 国際会議

    内橋 貴之

    International Scanning Probe Microscopy 2018  2018年5月8日 

     詳細を見る

    記述言語:英語   会議種別:口頭発表(招待・特別)  

    開催地:the Arizona State University Tempe Campus, USA  

  30. High-Speed Atomic Force Microscopy for Visualization and Manipulation of Biological and Artificial Molecules 招待有り 国際会議

    内橋 貴之

    2018年8月20日 

     詳細を見る

    記述言語:英語   会議種別:口頭発表(招待・特別)  

    開催地:Catholic University of Leuven, Leuven, Belgium  

  31. High-speed atomic force microscopy for direct visualization of biological macromolecules at work 招待有り 国際会議

    The 79th Okazaki Conference "Synthetic, Biological, and Hybrid Molecular Engines"  2018年8月31日 

     詳細を見る

    記述言語:英語   会議種別:口頭発表(招待・特別)  

    開催地:Okazaki Conference Center, Okazaki  

  32. High-Speed AFM Observation of Domain Flexibility Related to Enzymatic Function of CRISPR-Cas9 国際会議

    55回日本生物物理学会年会 シンポジウム: "Softness and functions of biological molecules under various environments"  2017年9月20日 

     詳細を見る

    記述言語:英語   会議種別:シンポジウム・ワークショップ パネル(指名)  

    開催地:熊本大学 黒髪キャンパス, 熊本  

  33. 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年7月21日 

     詳細を見る

    記述言語:英語   会議種別:口頭発表(一般)  

  34. High speed atomic force microscopy for a tool to visualize dynamic events on biological systems from single molecules to living cells 招待有り 国際会議

    内橋 貴之

    Workshop on “Nanofluidics in Biological Systems”  2017年9月13日 

     詳細を見る

    記述言語:英語   会議種別:口頭発表(招待・特別)  

    開催地:Durham University, UK  

  35. Dynamic Structural States of Molecular Disaggregation Machine ClpB Revealed by High-Speed Atomic Force Microscopy 招待有り 国際会議

    内橋 貴之

    Frontier Bioorganization Forum 2018  2018年7月8日 

     詳細を見る

    記述言語:英語   会議種別:口頭発表(招待・特別)  

    開催地:Yamate Campus, ExCELLS, Okazaki  

  36. Direct visualization of single molecule dynamics by high-speed atomic force microscopy 招待有り 国際会議

    内橋 貴之

    Telluride Science Research Center Workshop on Protein Dynamics  2017年7月30日 

     詳細を見る

    記述言語:英語   会議種別:口頭発表(招待・特別)  

    開催地:Telluride Intermediate School, Telluride, USA  

  37. Direct visualization of dynamic molecular interactions using HS-AFM 招待有り 国際会議

    内橋 貴之

    Frontier Bioorganization Forum 2017: Dynamical ordering and integrated functions of biomolecular systems  2017年4月24日 

     詳細を見る

    記述言語:英語   会議種別:口頭発表(招待・特別)  

    開催地:Academia Sinica, Taipei, Taiwan  

  38. Direct observation of single molecule dynamics at work with high-speed atomic force microscopy 招待有り 国際会議

    内橋 貴之

    Frontier in Single Molecule Biophysics 2017  2017年10月15日 

     詳細を見る

    記述言語:英語   会議種別:口頭発表(招待・特別)  

    開催地:Neve Ilan Hotel, Israel  

  39. Direct observation of self-assembly process of biological and artificial fibrils using high-speed atomic force microscopy 招待有り 国際会議

    内橋 貴之

    Interhierarchical understanding of materials and life through molecular observation  2018年3月24日 

     詳細を見る

    記述言語:英語   会議種別:口頭発表(招待・特別)  

    開催地:Institute of Molecular Science, Okazaki  

  40. 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年6月29日 

     詳細を見る

    記述言語:日本語   会議種別:口頭発表(招待・特別)  

    開催地:名古屋大学  

  41. 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 招待有り

    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年10月29日 

     詳細を見る

    記述言語:日本語   会議種別:口頭発表(招待・特別)  

    開催地:京都大学吉田キャンパス, 京都  

  42. 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 招待有り

    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年6月22日 

     詳細を見る

    記述言語:日本語   会議種別:口頭発表(招待・特別)  

    開催地:仙台国際センター, 宮城  

  43. 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 招待有り

    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年8月19日 

     詳細を見る

    記述言語:日本語   会議種別:口頭発表(招待・特別)  

    開催地:横浜市立大学 金沢八景キャンパス, 神奈川  

  44. 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年4月12日 

     詳細を見る

    記述言語:日本語   会議種別:公開講演,セミナー,チュートリアル,講習,講義等  

    開催地:名古屋大学  

  45. 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 招待有り

    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年12月8日 

     詳細を見る

    記述言語:日本語   会議種別:シンポジウム・ワークショップ パネル(指名)  

    開催地:神戸ポートピアホテル, 兵庫  

  46. 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 招待有り

    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年9月11日 

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    記述言語:日本語   会議種別:公開講演,セミナー,チュートリアル,講習,講義等  

    開催地:呉羽ハイツ, 富山  

▼全件表示

Works(作品等) 4

  1. 高速AFMによる機能性材料の動的解析

    2007年

  2. Dynamic Analysis of Functional Materials using HIgh-speed AFM

    2007年

  3. 高速AFM法による膜タンパク質のダイナミクス計測

    2004年

  4. Dynamics of membrane proteins by high-speed AFM

    2004年

科研費 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

    研究課題/研究課題番号:22K18943  2022年6月 - 2024年3月

    日本学術振興会  科学研究費補助金  挑戦的研究(萌芽)

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    担当区分:研究代表者 

  2. 機械刺激を加えた単一タンパク質の構造および力学特性応答の高速AFM解析

    研究課題/研究課題番号:21H01772  2021年4月 - 2024年3月

    日本学術振興会  科学研究費助成事業  基盤研究(B)

    内橋 貴之

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    担当区分:研究代表者 

    配分額:17420000円 ( 直接経費:13400000円 、 間接経費:4020000円 )

    メカノバイオロジーの進展により、様々なタンパク質が機械的刺激を感知して機能を変化させることが明らかになってきた。しかしながら、機械的負荷に対するメカノセンサータンパク質の構造/力学応答と機能変調を単分子スケールで計測する手法はなかった。本研究では、高速AFM技術を基盤として、タンパク質に引張/圧縮による力学負荷を印加しながら、構造および力学特性の動態を解析できる技術を確立する。細胞骨格タンパク質フィラメントへの応力印加による構造・力学応答とフィラメント構造の安定性や結合タンパク質との親和性変化、細胞膜変形タンパク質の膜結合や集合体形成能と膜張力との関係を明らかにする。
    前年度に開発した一軸伸張機構付き探針走査型高速AFMを用いて、脂質膜の曲率を感知して膜に結合する分子であるBIN1の曲率依存的な膜への結合を観察した。PDMS基板に応力を加えると、基板表面に応力印加方向とは垂直方向に沿って表面リップル構造を曲率を制御して作ることができた。次にBIN1の脂質膜への結合ダイナミクスを観察したところ、平坦な脂質膜へは明らかな結合は確認できなかった。親和性の高いCNM関連変異体K436Xであっても観察できず、BIN1と平板脂質との相互作用はAFM探針からの外乱で容易に解離するほど弱いか、少なくとも結合時間は最速0.1秒/フレームのイメージング速度を用いた高速FMの時間分解能よりはるかに短いことを示している。一方、曲率を持つ脂質膜表面では0.5秒/フレームのイメージング速度でも、BIN1の脂質膜への結合が明確に観察された。ほとんどのBIN1分子は、脂質で覆われたリップル構造の頂上付近で安定に結合し、側面や底面ではあまり結合しておらず、正の曲率領域への優先的な結合が示唆された。異なる曲率での単分子の脂質膜上での滞留時間のヒストグラムを作り、単指数減衰関数でフィッティングしたところ、脂質膜の曲率が5.59μm-1での時定数は1.83±0.09秒、414.27μm-1での時定数は3.77±0.06秒となった。これは、脂質曲率の違いにより親和性に2倍近い差が生じることを示している。これまでの研究では、BIN1と脂質の相互作用は、脂質の曲率の違いによるBIN1の結合による蛍光強度の変化としてしか解析できなかったが、今回開発した一軸伸張機構付き探針走査型高速AFMにより、脂質の曲率に依存した結合・解離のダイナミクスを1分子レベルで直接測定することができるようになった。
    一軸伸張機構付き探針走査型高速AFMの開発がほぼ終了し、微小管の屈曲、アクチニンのアクチンへの結合やBIN1の脂質膜への結合など、当初予定したサンプルに対する実験を行えることを確認でき、これらの成果をReview Scientific Instrumentsに論文として発表することができたため。
    当初2022年度に予定していた、アクチン線維と結合タンパク質のアクチン繊維の屈曲による親和性変化についての実験が、脂質膜とBIN1の測定で予定よりも時間がかかったためにあまり進めることができなかった。最終年度はこの実験を集中して行う。弾性シート基板表面にビオチン化脂質を含んだ平面脂質二重膜を展開し、一部ビオチン化したアクチン線維をストレプトアビジン分子を介して脂質膜に固定する。アクチンストレスファイバーを模すために、α-アクチニンによるアクチン線維の架橋とバンドル化を行い基板に固定する。溶液中にはコフィリンあるいはミオシンIIのアクチン結合部位であるS1部を入れておく。弾性基板の引張でアクチンネットワークに力学負荷をかけながら、アクチン線維の構造変化(らせんピッチとG-アクチンの間隔)とコフィリンおよびS1の結合・解離を観察し、アクチン線維の局所構造の張力依存的変化と結合タンパク質の親和性変化の相関を明らかにする。

  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

    研究課題/研究課題番号:21H01999  2021年4月 - 2024年3月

    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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    担当区分:研究分担者 

    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. 動態イメージングによるタンパク質膜透過を担う発動分子複合体の作動メカニズムの解明

    研究課題/研究課題番号:21H00393  2021年4月 - 2023年3月

    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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    担当区分:研究代表者 

    配分額:5980000円 ( 直接経費:4600000円 、 間接経費:1380000円 )

    タンパク質の膜透過を担う膜タンパク質複合体は、細胞質にある基質タンパク質を掴んでチャネルに押し込み、さらに膜の逆側から引き抜くという、大きな構造変化を伴った複雑な動きにより機能を発揮する高次な発動分子複合体であると言える。本研究では、高速原子間力顕微鏡(AFM)および蛍光顕微鏡との複合機を駆使して、バクテリアのタンパク質膜透過装置Sec複合体が基質タンパク質を輸送する様子をリアルタイムに可視化し、さらに静止立体構造とのドッキングシミュレイションによる構造モデリングで、膜タンパク質透過の分子メカニズムを明らかにする。
    SecYAEGの動態観察: 前年度に膜輸送時にはSecAの一部が構造変化している様子を観察することに成功した。今年度はSecYAEGのモデル構造に対して様々な方位で擬似AFM像を形成させることで実AFM像の観察方位を決定する解析法を開発し、既知のPPXDの構造変化(Open/Closed)と高速AFMで観察された構造変化の対応関係を明らかにした。さらに、ATPやADP・Pi、ADP結合状態を模擬する各種ヌクレオチドアナログ存在下でのPPXDの構造を解析し、PPXDの構造状態とATP加水分解サイクルの対応を明らかにするとともに、構造変化の頻度からATP加水分解活性を定量することに成功した。
    <BR>
    細胞間接着分子カドヘリンのダイマー形成過程の観察
    カドヘリンは隣り合う細胞表面に提示されたカドヘリンと互いに結合することで細胞と細胞をつなぐものの、一分子のスケールでのカドヘリンの結合機構には未だ明らかになっていない部分も多い。そこで、高速AFMを用いて溶液中におけるカドヘリンの結合構造および結合過程を一分子のスケールで直接可視化した。その結果、カドヘリンは複数の異なる形状のダイマーを形成していることがわかった。これらのダイマーの形状とダイナミクスを解析すると、結合界面を頻繁に変えながらダイマー形状が過渡的に変化するS形状ダイマー、ダイマー形状が変化しない安定したW形状ダイマー、同じく安定したcross形状ダイマーの3種類の構造に分類することが出来た。変異体の形状解析から、W形状ダイマーとcross形状ダイマーは、それぞれ従来から知られていたストランドスワップダイマーとXダイマーに対応することがわかった。一方、ダイマー形状がダイナミックに変化するS形状ダイマーは,ストランドスワップダイマーとXダイマーとは異なる新規のダイマー構造であることが明らかになった。
    令和4年度が最終年度であるため、記入しない。
    令和4年度が最終年度であるため、記入しない。

  5. 1分子ゆらぎの直接観察による膜蛋白の機能発現と障害発生のメカニズムの解明

    研究課題/研究課題番号:20K07279  2020年4月 - 2023年3月

    日本学術振興会  科学研究費助成事業  基盤研究(C)

    相馬 義郎, 内橋 貴之, 古田 忠臣, 中川 大, 岩本 真幸, 大崎 寿久

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    担当区分:研究分担者 

    医学・生理学的に重要な生命プロセスの多くは、チャネルやトランスポータなどの膜蛋白および、それらの集合体である膜蛋白複合体の機能によって支えられおり、これらの動作機構の解明は、医学生理学の大きな進歩に繋がると期待されるが、その詳細なメカニズムはほとんど解っていない。
    本研究においては、細胞膜環境プラットフォームを備えた高速原子間力顕微鏡を用いて、生理的環境における膜蛋白の1分子から複合体までのメゾスコピック領域での「ゆらぎ」に注目した分子動態の直接観察とシミュレーションを用いた定量的解析を行ない、膜蛋白1分子および膜蛋白複合体の動作機構の解明をめざす。
    本研究では、白人種に多い遺伝疾患嚢胞繊維症の原因遺伝子産物であるCFTRチャネルの機能発現および病因性遺伝子変異による発現障害の発生における分子「ゆらぎ」の関与について、高速原子間力顕微鏡(高速AFM)および分子動力学シミュレーション(MD)を用いて調べた。その結果、ゲーティング機能に重要な2つのNBDの大きな「ゆらぎ」の高速AFMによる直接観察に成功した。白人最多の⊿F508変異はNBD1-ICL4接合面に存在するが、NBD1およびICL4に存在する日本人病因性変異も同様に、CFTR分子の「ゆらぎ」に影響を与えて発現障害をおこしている可能性が、MDによって明らかになった。
    蛋白およびそれらの高次機能複合体の動作機構の解明は、医学生理学の大きな進歩に繋がると期待されるが、その分子レベルでの詳細な動作メカニズムについては、ほとんど解っていない。
    研究代表者は、高速原子間力顕微鏡(高速AFM)を用いた蛋白1分子動態直接観察と分子動力学シミュレーションを組み合わせて、研究対象であるCFTRチャネルの機能発現における分子内ドメインのゆらぎの関与および、病因性遺伝子変異によって引き起こされる発現障害における分子ゆらぎの関与についての重要な知見を得た。
    これは現在までの組織・細胞レベルでの生理学や巨視的な生化学では得られない、新しい研究成果である。

  6. 高速AFMで可視化する外的刺激により誘起される多孔性結晶表面の構造ダイナミクス

    研究課題/研究課題番号:20H04669  2020年4月 - 2022年3月

    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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    担当区分:研究代表者 

    配分額:5330000円 ( 直接経費:4100000円 、 間接経費:1230000円 )

    吸着分離材、不均一系触媒などの多くの応用が期待されている多孔性結晶は、ゲスト分子の吸着や圧力、電場などの外的刺激に応答して結晶構造が柔軟に変化する。本研究では、外的刺激に応答する多孔性結晶表面のナノスケールダイナミクス現象を高速原子間力顕微鏡(AFM)で直接可視化し、I: MMFやMOFなど多孔性結晶へのゲスト分子の吸着と格子歪の結晶表面伝搬の可視化、II: MMF表面での金ナノ粒子の成長過程の追跡、III: 局所力学刺激による格子歪誘起と選択的ゲスト分子の吸着制御の3項目について研究を行い、多孔性結晶の機能設計や性能向上のために基盤的知見を得る。
    *多孔性結晶の高解像可視化技術の確立.
    当初目的としていたMOF結晶はDMF中での観察が結晶の安定化のために必須であるが、DMF中ではMOF結晶表面に数nmスケールの汚染物質が付着していることが判明し、汚染物質を除去できないことからMOF結晶のAFM観察は断念した。そこで、水中でも結晶が安定なピラーアレーン結晶表面での測定を試み、いくつかの結晶で結晶格子像を得ることができ、さらにステップ端面での分子の脱離と再結合などの興味深いダイナミクスを観察できた(A01 生越Gとの共同研究)。
    *高速AFMのソフトクリスタルへの応用
    領域内外の研究グループとの共同研究により高速AFM技術を様々なソフトクリスタル材料に応用した。当初目的としていたMMF結晶表面での金微粒子の成長過程のリアルタイム観察については成功し、論文としてまとめた。領域内共同研究として、ポルフィリン誘導体による超分子螺旋集合体の形成と融解過程の観察 (A02 杉安Gとの共同研究)や有機分子により触媒される金微粒子の成長過程の観察(A01 楽Gとの共同研究)に成功した。また、低分子ゲル化剤として機能するUrea-C13のゲル化初期過程の高速AFM観察を行ったところ、Urea-C13はグラファイト基板で単分子鎖膜を速やかに形成したあとに、単分子鎖が数本集合した中間ファイバーを経て、さらに太いバンドルファイバーを形成することがわかった。太いバンドルファイバーがゲルの直接的な構成要素であり、平均速度0.05 nm/sで非常に遅い速度で伸長と停止を繰り返しながら成長することがわかった。また、この伸長と停止を伴う成長様式をファイバー端面への分子の結合とバンドルによる安定化によって説明できることが数理モデルにより明らかにした(A03 山中Gとの共同研究)。
    令和3年度が最終年度であるため、記入しない。
    令和3年度が最終年度であるため、記入しない。

  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

    研究課題/研究課題番号:19KK0180  2019年10月 - 2024年3月

    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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    担当区分:研究分担者 

    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. ダイナミンの新規分子マシナリーによる細胞骨格と膜のダイナミクス制御の連携機構

    研究課題/研究課題番号:19H03225  2019年4月 - 2022年3月

    日本学術振興会  科学研究費助成事業  基盤研究(B)

    竹居 孝二, 山田 浩司, 竹田 哲也, 内橋 貴之

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    担当区分:研究分担者 

    ダイナミンはアクチン、微小管、生体膜を制御する。ダイナミンとそのターゲットは、重合脱重合、変形などダイナミック変化するため分子の作動実態は未解明である。我々は最近、ダイナミンの膜切断過程をin vitroで再現し、その働きを直接観察した(Takeda et al. eLife 2018)。in vitro再構成系と細胞レベルの解析により、アクチン、微小管、生体膜に対するダイナミンの作動実態と、ダイナミンのマルチ機能が細胞内で連携統合する仕組みを解析する。ダイナミンファミリー分子に共通する本質的分子マシナリーの解明、マルチ機能の統合の仕組みの解明、ダイナミン変異疾患の病態解明を目指す。
    ダイナミン2のCharcot-Marie-Tooth病変異によりストレスファイバーが異常になることを見出し、ダイナミンがストレスファイバーの形成、安定化に必要であることを示すとともに、ダイナミンによるアクチン線維束形成をin vitroで再現した。また、ダイナミン1が微小管を束化することを見出し、この微小管束化が腎糸球体ポドサイトの一次突起の形成と安定化や、細胞形態の維持に必要であることを示すとともに、ダイナミン1の微小管結合部位を同定した。さらに、膜ダイナミクス制御に関して、ダイナミン2とBAR タンパクBIN1が協働して骨格筋細胞のT管の形成、安定化に機能することを明らかにした。
    ダイナミンの変異が、てんかん性脳症や末梢神経変性疾患Charcot-Marie-Tooth病、先天性筋疾患の一つである中心核ミオパチーの原因となることがわかり、それらの疾患に関連するダイナミン変異も多数報告されている。ダイナミン分子の生理的機能、作動機構を解明するとともに、変異に伴う機能破綻のメカニズムを明らかにすることは、これらの難治性疾患の分子病態の解明や新規の診断、治療の開発に不可欠である。

  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

    研究課題/研究課題番号:19H05389  2019年4月 - 2021年3月

    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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    担当区分:研究代表者 

    配分額:5980000円 ( 直接経費:4600000円 、 間接経費:1380000円 )

    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. 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

    研究課題/研究課題番号:18H01837  2018年4月 - 2021年3月

    科学研究費助成事業  基盤研究(B)

    内橋 貴之

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    担当区分:研究代表者 

    配分額:17550000円 ( 直接経費:13500000円 、 間接経費:4050000円 )

    本研究では、高速原子間力顕微鏡(AFM)による分子操作機能を高度化したインラインフォースカーブモードを開発し、微小管に高い制御性で単一チューブリンダイマー欠陥を形成できるとともに、力―距離曲線を取得できるようになった。この手法により、微小管の自己修復過程や欠陥周囲のキネシン分子の運動挙動を観察できるようになり、また、チューブリン間の結合エネルギーの定量化に成功した。さらに、この手法を発展させた高速フォースマッピング法の観察にも成功した。屈曲した微小管のキネシン滑走運動を観察した結果、屈曲した領域ではキネシンの移動速度が低下することを見出した。
    本研究で開発された手法は、微小管の機械特性によるMAPsの機能変調を解析できる唯一手法であり、今後微小管に限らず様々なタンパク質の一分子構造操作や機能解析が可能になることから、生物学の重要な計測ツールとなり学術的意義は高い。

  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年4月 - 2021年3月

    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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    担当区分:研究代表者  資金種別:競争的資金

    配分額:17550000円 ( 直接経費:13500000円 、 間接経費:4050000円 )

  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年4月 - 2020年3月

    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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    担当区分:研究代表者  資金種別:競争的資金

    配分額:5200000円 ( 直接経費:4000000円 、 間接経費:1200000円 )

  13. 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

    研究課題/研究課題番号:18H04512  2018年4月 - 2020年3月

    新学術領域研究(研究領域提案型)

    内橋 貴之

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    担当区分:研究代表者 

    配分額:5200000円 ( 直接経費:4000000円 、 間接経費:1200000円 )

    本研究課題では、高速AFMを用いて外部刺激に応答するソフトクリスタルの表面・物性構造のダイナミクスを高時空間分解能で可視化する基盤技術を確立し、ソフトクリスタルの機能創発の分子機構を明らかにすることを目的としている。以下に今年度の成果を項目ごとに記述する。
    1. 高速力学マッピング法の開発:高速AFMでイメージング中に任意の位置での定量機械特性を取得可能な”in-line-force-curve”法を開発した。この手法を用いてチューブリンが重合したタンパク質ポリマーである微小管の単一チューブリンダイマーの引き抜きによる局所構造操作に成功し、チューブリンダイマー間の結合エネルギーの定量化を行った。さらに、”in-line-force-curve”を拡張し、機械特性(弾性率・凝着力)を1フレーム3秒程度でマッピングできるシステムを開発した。
    2. 高分子ゲル微粒子の昇温収縮の過程:温度制御型高速AFMと位相計測を組み合わせて、熱応答性のpNiPAmハイドロゲル微粒子の温度依存的収縮過程を観察したところ、微粒子内部に非熱応答性のナノサイズのドメインが存在することを見出した。また、この非熱応答性ドメインは沈殿重合法、あるいは体積相転移温度以下での逆相ミニエマルション重合法で重合した微粒子に存在し、体積相転移温度よりも高い温度で重合された微粒子ではナノドメイン微粒子内に均一に分布することを明らかにした。
    3. 六量体ヘムタンパク質のシート構造形成過程の観察:六量体ヘムタンパク質(HTTP)のモノマーにCys残基を導入してトリペプチドFGGタグを付与し、さらに、ククルビットウリル8(CB8)をFGGタグのホストとすることで、HTTPのシート状自己組織化構造を作製することに成功した。また、シート形成過程を高速AFMでモニターし、シート構造形成の詳細を明らかにした。
    令和元年度が最終年度であるため、記入しない。
    令和元年度が最終年度であるため、記入しない。

  14. 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

    研究課題/研究課題番号:17K19519  2017年6月 - 2019年3月

    挑戦的研究(萌芽)

    内橋 貴之

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    担当区分:研究代表者 

    配分額:6240000円 ( 直接経費:4800000円 、 間接経費:1440000円 )

    高速AFM/全反射照明蛍光顕微鏡複合機にイメージスプリッティング光学系を組み込み、高速AFMと一分子FRETの同時計測が可能なシステムを開発した。糖鎖合成酵素K4CPと多糖分解酵素セルラーゼTrCel6Aの一分子FRET計測のための蛍光標識試料を調製し、糖鎖の伸張やセルラーゼの構造変化に伴うFRET効率の変化を計測することができ、同時観察用試料の調製法を確立した。これらの試料に対し、高速AFMと一分子FRETの同時観察を試みたが、様々な問題に直面し、期間内に成功には至らなかったが、現在、同時計測を可能にするための測定条件の確立を急いでおり、近い将来、同時計測が可能になると期待される。
    個々のタンパク質の構造とそのダイナミクス計測は、タンパク質の機能発現のメカニズムの直接的理解を可能にすることから、生命現象を理解する上で必須である。高速原子間力顕微鏡と一分子FRET計測の融合は、両手法の欠点を補いタンパク質一分子のダイナミクスの詳細が定量的に解析できる。この手法でこれまで困難であった、タンパク質のダイナミクス現象を計測できるようになり、生命科学分野の発展に大きなインパクトを与える手法になると考えられる。本研究成果は、複合計測の実現可能性と萌芽を示すものであり、学術的意義は大きいと考える。

  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年6月 - 2019年3月

    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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    担当区分:研究代表者  資金種別:競争的資金

    配分額:6240000円 ( 直接経費:4800000円 、 間接経費:1440000円 )

  16. 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

    研究課題/研究課題番号:17F17701  2017年4月 - 2019年3月

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    担当区分:その他 

    I. インラインフォースカーブモードの開発:高速AFMでタンパク質の動態を可視化しながら、任意の位置で試料の力学操作と機械特性計測が可能なインラインフォースカーブモードを開発した。具体的には、イメージング中にPCに表示された画像で決めた位置で画像取得を停止し、プローブ-試料間距離を変化させてフォースカーブを取得し、引き続きイメージングを行い局所外力印加後の分子の動態を観察できるシステムを構築した。これにより微小管への局所外力の印加とそれによる構造変化やナノ力学特性の定量的解析が可能になった。
    II. 微小管への欠陥生成と機械特性評価:インラインフォースカーブモードを使って、微小管からチューブリンダイマーを引き抜き、同時にフォースカーブを計測することに成功した。さらに、欠陥のサイズを変えながらフォースカーブを取得し、チューブリン間の結合力を定量化した。これにより、これまで理論的にしか研究されていなかった微小管の機械特性を解析することができた。
    III. 微小管の自己修復過程の可視化:微小管にチューブリンダイマーの欠陥を形成後、その欠陥が再びチューブリンの再結合による修復される、自己修復と呼ばれる現象を捉えることに成功した。この測定により、微小管の自己修復過程は微小管の中空内で拡散しているチューブリンの結合で生じることを明らかにした。
    IV. 欠陥周囲のキネシンの運動解析:微小管の構造欠陥周辺でのキネシンの運動観察を行った。微小管に沿って進んできたキネシンが欠陥直前で微小管から解離したり、プロトフィラメントを乗り換えて、欠陥を迂回して進んでいく様子が見られた。さらに再現性の高い実験を可能にするため、多数の微小管を平行に並べてストライプ状に配列させる基板条件の検討を行った。その結果、適切なバッファ条件と基板に脂質二重膜を用いることで微小管をストライプ状に整列させることに成功した。
    平成30年度が最終年度であるため、記入しない。
    平成30年度が最終年度であるため、記入しない。

  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年4月 - 2019年3月

    日本学術振興会  特別研究員奨励費 

    内橋 貴之

      詳細を見る

    担当区分:研究代表者  資金種別:競争的資金

    配分額:2300000円 ( 直接経費:2300000円 )

  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年4月 - 2018年3月

    日本学術振興会  科学研究費補助金 新学術領域 

    内橋 貴之

      詳細を見る

    担当区分:研究代表者  資金種別:競争的資金

    配分額:6370000円 ( 直接経費:4900000円 、 間接経費:1470000円 )

  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年4月 - 2018年3月

    日本学術振興会  科学研究費補助金 新学術領域 

    内橋 貴之

      詳細を見る

    担当区分:研究代表者  資金種別:競争的資金

    配分額:6890000円 ( 直接経費:5300000円 、 間接経費:1590000円 )

  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

    研究課題/研究課題番号:16H00830  2016年4月 - 2018年3月

    新学術領域研究(研究領域提案型)

    内橋 貴之

      詳細を見る

    担当区分:研究代表者 

    配分額:6890000円 ( 直接経費:5300000円 、 間接経費:1590000円 )

    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

    研究課題/研究課題番号:16H00758  2016年4月 - 2018年3月

    新学術領域研究(研究領域提案型)

    内橋 貴之

      詳細を見る

    担当区分:研究代表者 

    配分額:6370000円 ( 直接経費:4900000円 、 間接経費:1470000円 )

    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. 放線菌由来セルラーゼの結晶性基質の効率的分解に関わる機構解明

    研究課題/研究課題番号:15K07383  2015年4月 - 2018年3月

    日本学術振興会  科学研究費助成事業  基盤研究(C)

    内山 拓, 内橋 貴之, 石田 拓也

      詳細を見る

    担当区分:連携研究者 

    「バイオリファイナリー」の実現に向けて、その鍵を握る酵素糖化の効率を高めることを目標とし,セルラーゼの効率的な結晶性セルロース分解に関わる酵素反応メカニズムを解明することを試みた。具体的には放線菌由来のセルラーゼSaCel6Bの生化学実験、高速原子間力顕微鏡観察、タンパク質結晶構造解析を通じて、そのようなメカニズムの解明を試みた。実験や観察の結果、セルラーゼSaCel6Bの効率的な結晶性セルロース分解に関わるアミノ酸残基2つを見出し、これらの重要性を明らかにした。このようなアミノ酸残基の情報を元に、糖化効率の高いセルラーゼの発見や、糖化効率の高い新規セルラーゼの合成を試みることが可能となった。

  23. 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

    研究課題/研究課題番号:15H03540  2015年4月 - 2018年3月

    内橋 貴之

      詳細を見る

    担当区分:研究代表者 

    配分額:16770000円 ( 直接経費:12900000円 、 間接経費:3870000円 )

    室温から約45℃の温度制御可能な高速AFMシステムを開発した。実際に、41度で固相から液相に相転移する脂質二重膜(DPPC)を用いて高温観察の評価を行い、相転移温度以上で膜の流動性が増加し脂質二重膜の形状が変化する様子を確認できた。温度制御システムを使って、べん毛の輸送タンパク質の一部であるFliIの観察を行ったところ、モノマーから六量体形成していく過程やFliI六量体の構造が変化する様子も観察できた。シアノバクテリアの概日周期タンパク質であるKaiCとKaiAの結合解離過程の温度依存性の測定にも成功した。

  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年4月 - 2018年3月

    日本学術振興会  科学研究費補助金 基盤研究(B) 

    内橋 貴之

      詳細を見る

    担当区分:研究代表者  資金種別:競争的資金

    配分額:16770000円 ( 直接経費:12900000円 、 間接経費:3870000円 )

  25. 動的平衡状態にある膜蛋白複合体を生理的条件下で直接観察するための技術基盤の開発

    研究課題/研究課題番号:15K15035  2015年4月 - 2017年3月

    日本学術振興会  科学研究費助成事業  挑戦的萌芽研究

    相馬 義郎, 内橋 貴之, 西坂 崇之, 櫻井 実, 佐藤 主税, 余 盈君, 黄 自強, 加藤 孝信, 藤村 章子

      詳細を見る

    医学生理学の大きな進歩に繋がると期待される、脂質2重膜中で動的平衡状態にある膜蛋白複合体の1分子動態を、高速原子間力顕微鏡(高速AFM)を用いて生理的条件下で直接観察するための技術基盤の開発を行なった。
    その結果、生理的細胞膜環境AFMプラットフォームは、ナノディスクを基盤に開発を進めるのが有望であることが分かった。 また、高速AFMで得られた1分子動態および分子間相互作用の動画データの解析理論の開発過程において、1分子レベルと巨視レベル間におけるメゾスコピックな領域において、分子間相互作用に関する未知の重要な分子動態プロセスが起こっている可能性が示唆された。

  26. 高速AFMの高度化技術の開発とタンパク質の動作機序解析

    研究課題/研究課題番号:26119003  2014年7月 - 2019年3月

    新学術領域研究(研究領域提案型)

    安藤 敏夫

      詳細を見る

    担当区分:連携研究者 

    本研究の目的は大きく2つに分けられる。(1)高速AFMのバイオ応用研究を自ら推進するだけでなく、班員と領域外の研究者にも装置を開放して、彼らと協力してタンパク質分子が動作する姿を活写する動的構造生命科学を幅広く新規開拓すること、(2)装置の高度化を更に進めて、従来の高速AFMでは観察不可能な分子レベルで起こる現象を観察可能にすることにある。前者については、非常に多様なタンパク質系の高速AFM観察に成功し、機能プロセスのメカニズムの理解に繋がった。後者については、高速AFMと光ピンセットとの複合機、及び、高速AFM装置に基づく超解像蛍光顕微鏡の開発に成功した。これらの応用展開は今後の課題である。
    タンパク質分子は生命の機能素子であり、その機能する仕組の解明は生命現象及び疾病の理解に必須である。代表者が開発した高速AFMは機能中のタンパク質分子を直接観察することを可能にし、機能メカニズムの理解を促進する。だが、多くの対象を観察するには、多くの研究者との共同研究が必要であった。また、現在の高速AFMでさえ観察できない現象があり、装置の高度化は必須であった。本研究の実施は、多くの多様なタンパク質系の理解を可能にしただけでなく、この顕微鏡を利用できる人材の育成にも繋がった。高速AFMの高度化は新規観察を可能にした。その本格的応用は今後の課題だが、更なる生命現象の理解に貢献するものと期待される。

  27. 高速AFMを用いた1分子動態観察によるABCトランスポータの動作機構の解明

    研究課題/研究課題番号:25293049  2013年4月 - 2017年3月

    日本学術振興会  科学研究費助成事業  基盤研究(B)

    相馬 義郎, 内橋 貴之, 西坂 崇之, 櫻井 実, 佐藤 主税, 山下 隼人, 余 盈君, 黄 自強

      詳細を見る

    医学・生理的に重要な役割を果たしているABCトランスポータ・スーパーファミリーに共通したATP 依存性駆動ドメイン(NBD エンジン)の作動メカニズムとその異常についての研究を、CFTRチャネルをモデルとして、高速原子間力顕微鏡(高速AFM)を含む複数の1分子測定法を組み合わせて行なった。
    その結果、CFTR分子の細胞内側に存在する、活性調節(R)ドメインを含めたNBDエンジン部分の1分子動態観察に成功した。さらに分子間相互作用の高速AFM観察技術の確立をめざした抗原-抗体反応の直接観察および病因性CFTR変異体の分子生理学・薬理学的研究で成果を挙げることができた。

  28. 植物細胞壁合成酵素および分解酵素を用いた細胞外情報処理空間の動的可視化

    研究課題/研究課題番号:24114008  2012年6月 - 2017年3月

    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)

    五十嵐 圭日子, 内橋 貴之

      詳細を見る

    植物の細胞壁は多様な高分子物質が互いに作用しあった複雑な構造を有しており、詳細は明らかになっていない。細胞壁を構成するそれぞれの成分は細胞の酵素によって合成された後、自己組織化によって複合化することで細胞外に複雑な細胞壁構造を構築する。一方、細胞壁を栄養源として生育するきのこ等の生物は自身が生産する分解酵素により細胞壁の複雑な構造を分解することで、栄養を獲得している。そこで本研究ではこれらの生物が有する酵素と細胞壁を構成する成分の組織化との関係性を顕微鏡技術や生化学技術によって明らかにし、複雑な植物細胞壁の理解、利用に向けた知見を得た。

  29. 高速バイオAFMが拓く新構造生物学

    研究課題/研究課題番号:24227005  2012年5月 - 2017年3月

    日本学術振興会  科学研究費助成事業  基盤研究(S)

    安藤 敏夫, 内橋 貴之, 福森 義宏, 福間 剛士, 古寺 哲幸, 紺野 宏記, ウオング リチャード, 村上 聡, 小椋 光, 豊島 陽子, 神取 秀樹

      詳細を見る

    三つの課題に取り組んだ。課題1では、既に確立した高速AFMを利用して多様な蛋白質系で起こる動的プロセスを観察し、機能メカニズムに迫るとともに、従来技術では困難な天然変性蛋白質の構造解析が高速AFMで可能であることを実証した。課題2では、振動を起こさずに広域を高速走査する技術やイメージング中に試料を操作可能なインターラクティブ高速AFMを開発し、その有効性を実証した。また、カンチレバー走査方式の高速AFMと蛍光顕微鏡との複合機を開発し、蛍光像と高速AFM像の同時取得を実現した。課題3では、非接触観察可能な走査型イオン伝導顕微鏡の高速化に向け要素技術を開発し、約100倍の高速化に成功した。

  30. Development of high-speed atromic force microscope for biological system

    2004年

    Cooperative Research 

      詳細を見る

    資金種別:競争的資金

▼全件表示

産業財産権 3

  1. 走査型プローブ顕微鏡

    内橋 貴之, 柴田 幹大, 古寺 哲幸

     詳細を見る

    出願人:国立大学法人金沢大学

    出願番号:特願2016-234584  出願日:2016年12月

    公開番号:特開2018-091695  公開日:2018年6月

    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

     詳細を見る

    出願人:国立大学法人金沢大学

    出願番号:特願2016-233494  出願日:2016年11月

    公開番号:特開2018-091666  公開日:2018年6月

    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

     詳細を見る

    出願人:国立大学法人金沢大学

    出願番号:特願2016-232100  出願日:2016年11月

    公開番号:特開2018-085975  公開日:2018年6月

    J-GLOBAL

 

担当経験のある科目 (本学以外) 7

  1. 電磁気学演習II

    金沢大学)

  2. 電磁気学演習I

    金沢大学)

  3. 生物物理学I

    名古屋大学)

  4. 物理学演習II

    名古屋大学)

  5. 物理学I

    金沢大学)

  6. 力学演習II

    金沢大学)

  7. 力学演習I

    金沢大学)

▼全件表示

 

学術貢献活動 2

  1. AFM BioMed Conference 2022 国際学術貢献

    役割:企画立案・運営等

    2022年8月 - 2022年9月

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