Realization of nano-dynamics imaging of protein molecules in extremely soft membrane environments

• Project/Area Number: 17H06121
• Research Category: Grant-in-Aid for Scientific Research (S)
• Allocation Type: Single-year Grants
• Research Field: Nanobioscience
• Research Institution: Kanazawa University
• Principal Investigator: Ando Toshio 金沢大学, ナノ生命科学研究所, 特任教授 (50184320)
• Project Period (FY): 2017-05-31 – 2022-03-31
• Project Status: Completed (Fiscal Year 2021)
• Budget Amount: ¥164,320,000 (Direct Cost: ¥126,400,000、Indirect Cost: ¥37,920,000); Fiscal Year 2021: ¥24,960,000 (Direct Cost: ¥19,200,000、Indirect Cost: ¥5,760,000); Fiscal Year 2020: ¥26,780,000 (Direct Cost: ¥20,600,000、Indirect Cost: ¥6,180,000); Fiscal Year 2019: ¥30,550,000 (Direct Cost: ¥23,500,000、Indirect Cost: ¥7,050,000); Fiscal Year 2018: ¥33,410,000 (Direct Cost: ¥25,700,000、Indirect Cost: ¥7,710,000); Fiscal Year 2017: ¥48,620,000 (Direct Cost: ¥37,400,000、Indirect Cost: ¥11,220,000)
• Keywords: バイオイメージング / 高速AFM / 走査型イオン伝導顕微鏡 / 膜タンパク質 / 膜蛋白質 / ダイナミクス

Outline of Final Research Achievements

As typically exemplified by membrane proteins embedded in extremely soft membranes, there are many biological samples that are still difficult to observe with the current high-speed AFM. To make it possible to perform dynamic imaging of such specimens, we carried out various technical developments. Consequently, we accomplished the following achievements: (a)faster and higher resolution imaging capability of scanning ion conductance microscopy that allows non-contact imaging, as well as Nernst potential microscopy with both fast, high resolution and non-contact imaging capabilities, (b)enhanced low-disturbance imaging capability of high-speed AFM and thus faster imaging performance, and (c)high-speed/high-resolution/non-invasive imaging of very soft/fragile biological samples including liquid droplet-like protein complexes produced by liquid-liquid phase separation, intrinsically disordered proteins, a transporter membrane protein, cancer cells, and others.

Academic Significance and Societal Importance of the Research Achievements

代表者が開発した高速AFMは、機能動作中のタンパク質分子の構造・プロセスを直接観察できるため、その製品は世界に普及し、従来技術では得られない新しい発見を続々と生んでいる。だが、より脆弱で柔らかいバイオ試料系の観察は困難であるという問題を抱えていた。本研究は、高速AFMの高速性能・低侵襲性能の更なる向上と、SICM（及びSNPM）の高速化・高解像化を実現し、高解像動態観察可能な試料系を大幅に拡大させ得ることを実証した。この成功は、分子・細胞レベルの生命科学研究の今後の発展に大きく寄与するものである。

Assessment Rating

Verification Result (Rating)

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Research Products

• [Int'l Joint Research] Moleculaire Biofysica, Zernike Instituut/Groningen Biomol. Sci. Biotechnol. Inst.(オランダ)
• [Int'l Joint Research] The University of Michigan/University of Florida(米国)
• [Int'l Joint Research] Technische University of Munchen(ドイツ)
• [Int'l Joint Research] Aix-Marseille University and CNRS(フランス)
• [Int'l Joint Research] State University of Milano-Bicocca(イタリア)
• [Int'l Joint Research] McGill University(カナダ)
• [Int'l Joint Research] Dalian University of Technology/Chinese Academy of Sciences/Shanghai Institute of Applied Physics(中国)
• [Int'l Joint Research] UC Los Angels(米国)
• [Int'l Joint Research] Max Planck Institute/University of Freiburg(ドイツ)
• [Int'l Joint Research] Monash University(カナダ)
• [Int'l Joint Research] Central South University(中国)
• [Int'l Joint Research] University of Michigan(米国)
• [Int'l Joint Research] Vanderbilt University/Lawrence Livermore National Laboratory(米国)
• [Int'l Joint Research] Lawrence Livermore National Laboratory/University of Michigan(米国)
• [Int'l Joint Research] KU Leuven-University of Leuven(Belgium)
• [Int'l Joint Research] Gdansk University of Technology(Poland)
• [Int'l Joint Research] Shanghai Institute of Applied Physics(China)
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• [Journal Article] A high-speed piezoelectric Z-scanner with a resonance frequency above 1.1 MHz for high-speed atomic force microscopy. (2022)
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• [Journal Article] High-speed atomic force microscopy reveals a three-state elevator mechanism in the citrate transporter CitS (2022)
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  • Journal Title: Proceedings of the National Academy of Sciences Volume: 119 Issue: 6 Pages: 1-9
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• [Journal Article] Two-State Exchange Dynamics in Membrane-Embedded Oligosaccharyltransferase Observed in Real-Time by High-Speed AFM (2020)
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• [Journal Article] High-Speed Atomic Force Microscopy Reveals Factors Affecting the Processivity of Chitinases during Interfacial Enzymatic Hydrolysis of Crystalline Chitin (2020)
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• [Journal Article] High-Speed AFM Reveals Molecular Dynamics of Human Influenza A Hemagglutinin and Its Interaction with Exosomes (2020)
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• [Journal Article] Spatiotemporally tracking of nano-biofilaments inside the nuclear pore complex core (2020)
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• [Journal Article] Self- and Cross-Seeding on α-Synuclein Fibril Growth Kinetics and Structure Observed by High-Speed Atomic Force Microscopy (2020)
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• [Journal Article] Capturing transient antibody conformations with DNA origami epitopes (2020)
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• [Journal Article] Diversity of physical properties of bacterial extracellular membrane vesicles revealed through atomic force microscopy phase imaging (2020)
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  • Journal Title: Nanoscale Volume: 12 Issue: 14 Pages: 7950-7959
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• [Journal Article] Dynamics of oligomer and amyloid fibril formation by yeast prion Sup35 observed by high-speed atomic force microscopy (2020)
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• [Journal Article] Phase separation organizes the site of autophagosome formation (2020)
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• [Journal Article] High-speed near-field fluorescence microscopy combined with high-speed atomic force microscopy for biological studies (2019)
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• [Journal Article] Development of high-speed ion conductance microscopy (2019)
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• [Journal Article] High-speed atomic force microscopy (2019)
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• [Journal Article] Structural insights into the substrate specificity switch mechanism of the type III protein export apparatus (2019)
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• [Journal Article] Insight into structural remodeling of the FlhA ring responsible for bacterial flagellar type III protein export (2018)
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  • Author(s): Shibata Mikihiro、Nishimasu Hiroshi、Kodera Noriyuki、Hirano Seiichi、Ando Toshio、Uchihashi Takayuki、Nureki Osamu
  • Journal Title: Nature Communications Volume: 8 Issue: 1 Pages: 1430-1430
  • DOI: 10.1038/s41467-017-01466-8
  • NAID: 120006374160
  • Peer Reviewed / Open Access
• [Journal Article] Na+-induced structural transition of MotPS for stator assembly of the Bacillus flagellar motor (2017)
  • Author(s): Terahara Naoya、Kodera Noriyuki、Uchihashi Takayuki、Ando Toshio、Namba Keiichi、Minamino Tohru
  • Journal Title: Science Advances Volume: 3 Issue: 11 Pages: 4119-4119
  • DOI: 10.1126/sciadv.aao4119
  • Peer Reviewed / Open Access
• [Journal Article] High-speed atomic force microscopy imaging of live mammalian cells (2017)
  • Author(s): Mikihiro Shibata, Hiroki Watanabe, Takayuki Uchihashi, Toshio Ando, and Ryohei Yasuda
  • Journal Title: Biophysics and Physicobiology Volume: 14 Issue: 0 Pages: 127-135
  • DOI: 10.2142/biophysico.14.0_127
  • NAID: 130005997921
  • ISSN: 2189-4779
  • Peer Reviewed / Int'l Joint Research
• [Journal Article] High-speed XYZ nanopositioner for scanning ion conductance microscopy (2017)
  • Author(s): S. Watanabe and T. Ando
  • Journal Title: Appl. Phys. Lett. Volume: 111 Issue: 11 Pages: 113106-113106
  • DOI: 10.1063/1.4993296
  • NAID: 120006370925
  • Peer Reviewed
• [Journal Article] Directly watching biomolecules in action by high-speed atomic force microscopy (2017)
  • Author(s): T. Ando
  • Journal Title: Biophys. Rev. Volume: 9 Issue: 4 Pages: 421-429
  • DOI: 10.1007/s12551-017-0281-7
  • NAID: 120006374161
  • Peer Reviewed
• [Journal Article] High-Speed Atomic Force Microscopy Reveals Loss of Nuclear Pore Resilience as a Dying Code in Colorectal Cancer Cells. (2017)
  • Author(s): 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
  • Journal Title: ACS Nano. Volume: 11(6) Issue: 6 Pages: 5567-5578
  • DOI: 10.1021/acsnano.7b00906
  • Peer Reviewed / Open Access
• [Journal Article] Imaging modes of atomic force microscopy for application in molecular and cell biology (2017)
  • Author(s): Yves F. Dufrene, Toshio Ando, Ricardo Garcia, David Alsteens, David Martinez-Martin, Andreas Engel, Christoph Gerber and Daniel J. Muller
  • Journal Title: Nature Nanotechnology Volume: 12 Issue: 4 Pages: 295-307
  • DOI: 10.1038/nnano.2017.45
  • Peer Reviewed / Int'l Joint Research / Acknowledgement Compliant
• [Journal Article] High-speed atomic force microscopy for observing protein molecules in dynamic action (2017)
  • Author(s): T. Ando
  • Journal Title: Proceedings of SPIE Volume: 10328 Pages: 103281R-103281R
  • DOI: 10.1117/12.2268795
  • NAID: 120006306248
  • Peer Reviewed
• [Presentation] 高速AFMが拓く新生命科学 (2022)
  • Author(s): 安藤敏夫
  • Organizer: 第25回アディポサンエンスシンポジウム
  • Invited
• [Presentation] 高速AFM技術バイオ応用研究の進展 (2021)
  • Author(s): 安藤敏夫
  • Organizer: 第2回高速AFMオンラインシンポジウム
  • Invited
• [Presentation] Structural and dynamics analysis of intrinsically disordered proteins by high-speed AFM (2021)
  • Author(s): Toshio Ando
  • Organizer: Symposium "Time to Share: New Insights into High-speed AFM"
  • Int'l Joint Research / Invited
• [Presentation] Keynote speech "High-speed AFM for life sciences" (2020)
  • Author(s): Toshio Ando
  • Organizer: 8th Multifrequency AFM Conference
  • Int'l Joint Research / Invited
• [Presentation] 高速AFMとそのバイオ応用研究の概観 (2020)
  • Author(s): 安藤 敏夫
  • Organizer: ブルッカージャパンナノ計測事業部主催シンポジウム
  • Invited
• [Presentation] High-speed AFM revealing dynamic biomolecular processes (2020)
  • Author(s): T. Ando
  • Organizer: Biophysical Society Annual Meeting: Nanoscale Approaches to Biology Subgroup meeting (San Diego, USA)
  • Int'l Joint Research / Invited
• [Presentation] Early preautophagosomal structure is formed by liquid-liquid phase separation through weak intermolecular interactions between Atg 13 and Atg17 (2020)
  • Author(s): T. Ando
  • Organizer: XXII Linz Winter Workshop (Bio-AFM Workshop (Johannes Kepler University Linz, Linz Austria)
  • Int'l Joint Research / Invited
• [Presentation] Keynote speech "High-speed AFM in Bio-Med: Its current state and future prospects" (2019)
  • Author(s): T. Ando
  • Organizer: AFM BioMed Conference 2019 (Münster, Germany)
• [Presentation] High-speed AFM (2019)
  • Author(s): T. Ando
  • Organizer: Keystone Symposia Conference: Imaging across scales leveraging the revolution in resolution. (Snowbird, Utah, USA)
  • Int'l Joint Research / Invited
• [Presentation] Direct visualization of protein molecules during their functional activity (2019)
  • Author(s): 安藤敏夫
  • Organizer: 蛋白質科学会・シンポジウム「蛋白質科学の新常識：ナノスケールから細胞まで」
  • Invited
• [Presentation] High-speed AFM: directly visualizing protein molecules during their functional activity (2019)
  • Author(s): T. Ando
  • Organizer: 15th JSPS German and Japanese Colloquium: Nano-LifeScience (Harnack Hous of the Max Planck Institute, Berlin)
  • Int'l Joint Research / Invited
• [Presentation] Protein machinery enabling life (2019)
  • Author(s): T. Ando
  • Organizer: Max von Laue Colloquium of the Physical Society of Berlin (Physikalisch-Technische Bundesanstalt Hörsaal im Hermann-von-Helmholtz-Bau, Berlin)
  • Int'l Joint Research / Invited
• [Presentation] High-speed atomic force microscopy for observing biological molecules in dynamic action (2019)
  • Author(s): T. Ando
  • Organizer: Seminar at the Fritz Harber Institute of the Max Planck Society (Berlin)
  • Int'l Joint Research / Invited
• [Presentation] Dynamic processes in pore-forming Streptolysin O on lipid membrane (2019)
  • Author(s): T. Ando
  • Organizer: XXI Linz Winter Workshop (Bio-AFM Workshop (Johaness Kepler University Linz, Linz Austria)
  • Int'l Joint Research / Invited
• [Presentation] High-speed AFM and SICM: Watching biological samples in dynamic action (2019)
  • Author(s): T. Ando
  • Organizer: Bio-AFM Workshop on "AFM and Related Techniques for Biological Research" (Empa, St. Gallen, Switzerland)
  • Int'l Joint Research / Invited
• [Presentation] High-speed AFM: Visializing protein molecules during their functional activity (2019)
  • Author(s): T. Ando
  • Organizer: 19th International Microscopy Congress (Sydney, Australia)
  • Int'l Joint Research / Invited
• [Presentation] Dynamic GroEL-GroES interaction and its dependence on substrate protein (2018)
  • Author(s): T. Ando
  • Organizer: XX Linz Winter Workshop
  • Int'l Joint Research / Invited
• [Presentation] High-speed atomic force microscopy: Visualizing protein molecules in action (2017)
  • Author(s): T. Ando
  • Organizer: Physics & Life Science Colloquium (Lawrence Livermore National Laboratory)
  • Int'l Joint Research / Invited
• [Presentation] Structural analysis of IDPs by their visualization with high-speed AFM (2017)
  • Author(s): T. Ando
  • Organizer: CECAM Workshop on "Disordered protein segments: revisiting the structure-function paradigm"
  • Int'l Joint Research / Invited
• [Presentation] Special Lecture "Nano-visualization of protein molecules in action by high-speed AFM" (2017)
  • Author(s): T. Ando
  • Organizer: 15th International Conference on Na, K-ATPase and Related Transport ATPases
  • Int'l Joint Research / Invited
• [Presentation] Directly watching biomolecules in action by high-speed atomic force microscopy (2017)
  • Author(s): T. Ando
  • Organizer: 19th IUPAB Congress & 11th EBSA Congress
  • Int'l Joint Research / Invited
• [Presentation] Direct visualization of biological nanomachines in action by high-speed AFM (2017)
  • Author(s): T. Ando
  • Organizer: 9th International Conference on Engineering of Chemical Complexity
  • Int'l Joint Research / Invited
• [Presentation] Dynamic GroEL-GroES interaction revealed by high-speed atomic force microscopy (2017)
  • Author(s): T. Ando
  • Organizer: Royal Society Discussion Meeting on Allostery and Molecular Machines
  • Int'l Joint Research / Invited
• [Presentation] High-speed atomic force microscopy and its future prospects (2017)
  • Author(s): T. Ando
  • Organizer: 19th International Scanning Probe Microscopy Conference
  • Int'l Joint Research / Invited
• [Presentation] Keynote speech "High-speed AFM for filming protein molecules in action" (2017)
  • Author(s): T. Ando
  • Organizer: 8th International Conference on Advanced Materials and Nanotechnology
  • Int'l Joint Research / Invited
• [Presentation] 特別講演「タンパク質分子の機能動態を直接撮影する高速AFM」 (2017)
  • Author(s): 安藤敏夫
  • Organizer: 日本薬学会「生体膜と薬物の相互作用」シンポジウム
  • Invited
• [Book] High-speed Atomic Force Microscopy in Biology (2022)
  • Author(s): T. Ando
  • Total Pages: 319
  • Publisher: Springer Berlin Heidelberg
  • ISBN: 9783662647837
• [Book] "High-speed atomic force microscopy to study myosin motility" in Myosins: A superfamility of Molecular Motors (2020)
  • Author(s): Noriyuki Kodera、Toshio Ando
  • Publisher: Springer
  • ISBN: 9781402065163
• [Book] 高速AFMの技術革新と注目の成果 in 実験医学増刊 Vol.38 No.5 イメージング時代の構造生命科学 (2020)
  • Author(s): 安藤敏夫
  • Total Pages: 8
  • Publisher: 羊土社
  • ISBN: 9784758103855
• [Book] Direct imaging of walking myosin V by high-speed atomic force microscopy", a chapter in Molecular Motor (Methods in Molecular Biology) (2019)
  • Author(s): N. Kodera and T. Ando
  • Total Pages: 20
  • Publisher: Springer
  • ISBN: 9781493985548
• [Remarks] 金沢大学 生物物理学グループへようこそ
  • URL: http://biophys.w3.kanazawa-u.ac.jp/index_J.htm
• [Remarks] Welcome to Kanazawa Biophysics Goup
  • URL: http://biophys.w3.kanazawa-u.ac.jp/index.htm
• [Remarks] 高速原子間力顕微鏡の世界初の「教科書」完成！
  • URL: https://nanolsi.kanazawa-u.ac.jp/category/announcements/news/
• [Remarks] 世界初！マイコプラズマの滑走運動における分子モーター の動きを - - -
  • URL: https://nanolsi.kanazawa-u.ac.jp/post-16986/
• [Remarks] 高速AFMのさらなる高速化・低侵襲化に成功
  • URL: https://nanolsi.kanazawa-u.ac.jp/achievements/achievements-16204/
• [Remarks] Faster and less-invasive atomic force microscopy
  • URL: https://nanolsi.kanazawa-u.ac.jp/en/achievements/achievements-16204/
• [Remarks] High-speed atomic force microscopy takes on - -
  • URL: https://nanolsi.kanazawa-u.ac.jp/en/achievements/high-speed-atomic-force-microscopy-takes-on-intrinsically-disordered-proteins/
• [Remarks] 天然変性タンパク質の動的かつ多様な構造の定量的解析に成功
  • URL: https://nanolsi.kanazawa-u.ac.jp/achievements/high-speed-atomic-force-microscopy-takes-on-intrinsically-disordered-proteins/
• [Remarks] EurekAlert: High-speed atomic force - -
  • URL: https://www.eurekalert.org/pub_releases/2020-12/ku-haf122720.php
• [Remarks] 液－液相分離がオートファジーを制御する仕組みを発見 ～オートファジー研究は次のフェーズへ～
  • URL: https://nanolsi.kanazawa-u.ac.jp/post-9787/
• [Remarks] ミトコンドリアへのタンパク質搬入口TOM複合体の精密構造と働く仕組みを解明
  • URL: https://nanolsi.kanazawa-u.ac.jp/achievements/achievements-8472/
• [Remarks] 世界初！細菌べん毛タンパク質輸送の交通整理のしくみを解明
  • URL: https://nanolsi.kanazawa-u.ac.jp/achievements/achievements-2184/
• [Remarks] 金沢大学の研究：ピペットを完全に満たす
  • URL: https://www.prnewswire.com/news-releases/kanazawa-university-research-complete-filling-of-batches-of-nanopipettes-300979509.html
• [Remarks] 解像度を下げずにSICMのスピードを上げる
  • URL: http://biophys.w3.kanazawa-u.ac.jp/paper/Scilight-RSI-2019.pdf
• [Remarks] 安藤敏夫 研究者紹介 金沢大学ナノ生命科学研究所
  • URL: https://nanolsi.kanazawa-u.ac.jp/research/researchers/toshio-ando/
• [Remarks] Prof. Toshio Ando, Nano Life Science Institute
  • URL: https://nanolsi.kanazawa-u.ac.jp/en/research/researchers/toshio-ando/
• [Remarks] 研究成果の紹介 金沢大学ナノ生命科学研究所
  • URL: https://nanolsi.kanazawa-u.ac.jp/achievements/
• [Remarks] Ｋｅｙ Ｆｉｎｄｉｎｇｓ Ｎａｎｏ Ｌｉｆｅ Ｓｃｉｅｎｃｅ Ｉｎｓｔｉｔｕｔｅ
  • URL: https://nanolsi.kanazawa-u.ac.jp/en/achievements/
• [Remarks] Welcome to Kanazawa Biophysics Lab
  • URL: http://biophys.w3.kanazawa-u.ac.jp/
• [Remarks] Nano LSI WPI Kanazawa University
  • URL: https://nanolsi.kanazawa-u.ac.jp/en/
• [Remarks] 金沢大学 生物物理学研究室にようこそ
  • URL: http://biophys.w3.kanazawa-u.ac.jp/index_J.htm
• [Remarks] 金沢大学ナノ生命科学研究所
  • URL: https://nanolsi.kanazawa-u.ac.jp/
• [Patent(Industrial Property Rights)] 液体充填方法、ＳＩＣＭ用プローブの製造方法、ＳＩＣＭ用プローブ及び ＳＩＣＭ (2021)
  • Inventor(s): Sun Linhao, 執行航希, 渡辺信嗣, 安藤敏夫
  • Industrial Property Rights Holder: 金沢大学
  • Industrial Property Rights Type: 特許
  • Filing Date: 2021
  • Acquisition Date: 2021
• [Patent(Industrial Property Rights)] 原子間力顕微鏡、及び、その制御方法、ならびに、プログラミングに関する (2020)
  • Inventor(s): 安藤敏夫、福田真悟
  • Industrial Property Rights Holder: 金沢大学
  • Industrial Property Rights Type: 特許
  • Industrial Property Number: 2020-199938
  • Filing Date: 2020