Development of molecular-resolution imaging technique for proteins on a live cell surface and its application to cancer research

• Project/Area Number: 20H00345
• Research Category: Grant-in-Aid for Scientific Research (A)
• Allocation Type: Single-year Grants
• Section: 一般
• Review Section: Medium-sized Section 29:Applied condensed matter physics and related fields
• Research Institution: Kanazawa University
• Principal Investigator: 福間 剛士 金沢大学, ナノ生命科学研究所, 教授 (90452094)
• Project Period (FY): 2020-04-01 – 2024-03-31
• Project Status: Completed (Fiscal Year 2023)
• Budget Amount: ¥45,500,000 (Direct Cost: ¥35,000,000、Indirect Cost: ¥10,500,000); Fiscal Year 2023: ¥8,710,000 (Direct Cost: ¥6,700,000、Indirect Cost: ¥2,010,000); Fiscal Year 2022: ¥8,710,000 (Direct Cost: ¥6,700,000、Indirect Cost: ¥2,010,000); Fiscal Year 2021: ¥8,710,000 (Direct Cost: ¥6,700,000、Indirect Cost: ¥2,010,000); Fiscal Year 2020: ¥19,370,000 (Direct Cost: ¥14,900,000、Indirect Cost: ¥4,470,000)
• Keywords: 原子間力顕微鏡（AFM） / マイクロポーラスメンブレン（MPM） / MET受容体 / HGF / 分子分解能観察 / 原子間力顕微鏡 / 細胞観察 / タンパク質 / がん

Outline of Research at the Start

生きた真核細胞の表面で直接タンパク質のナノ動態を分子分解能観察できるAFM技術を開発する。さらに、それを分子レベルのがん研究に応用し、開発した技術が生命科学における重要な課題の解決に役立つことを実証する。具体的には、薬剤耐性を獲得したヒト肺腺癌細胞（PC-9）の表面において、過剰発現したMETの集合状態を直接観察し、薬剤耐性獲得に至る分子機構を解明する。

Outline of Annual Research Achievements

本研究では、生きた細胞の表面における分子スケールの原子間力顕微鏡（AFM）観察を実現し、その技術を用いてがんの悪性化に関係する受容体であるMET分子の集合状態を直接観察して明らかにすることを目標としている。これまでに、数μmの穴がアレイ状に配置された厚さ数百nmのマイクロポーラスメンブレン（MPM）上に細胞を培養し、穴に露出した細胞の基底面をAFM観察することで、約10 nmの分解能で表面構造観察が可能であることを示してきた。また、この技術を用いて化学固定が細胞表面構造にもたらす影響も明らかにした（Commun. Biol. 2022）。一方、MET分子をノックアウト（KO）したCHO/KO細胞と、その細胞にMET分子をノックインして過剰発現させたCHO/KI細胞を準備し、それらを無血清培養することで安定に表面構造観察できることを見出した。
本年度は、AFMの走査範囲を制限することで、より再現性良く分子スケール観察できる技術を確立した。また、カンチレバーの振動振幅や位相の探針－試料間距離依存性（フォースカーブ）を異なる振動振幅や速度で取得し、高分解能観察に適した条件を検討した。一方、CHO/KOおよびCHO/KI細胞の表面構造を比較し、後者の細胞表面には微絨毛がより多く存在すること、および、それがMET阻害剤により減少することを明らかにした。これは、活性なMET分子の存在により微絨毛の形成が促進され、細胞の遊走性、浸潤性が増すことを示唆しており、がんの悪性化機構に関わる新たな知見が得られた。これらの結果から、ＭET分子が結合する成長因子であるHGF分子を溶液に加えた場合、微絨毛が増すことが予想されるため、今後はそれを示すデータを取得して、それらの結果をまとめて論文として公表する予定である。

Research Progress Status

令和5年度が最終年度であるため、記入しない。

Strategy for Future Research Activity

令和5年度が最終年度であるため、記入しない。

Research Products

• [Int'l Joint Research] University of Washington(米国)
• [Int'l Joint Research] Aalto University(フィンランド)
• [Int'l Joint Research] EPFL(スイス)
• [Int'l Joint Research] Aalto University(フィンランド)
• [Journal Article] Dynamics of Molecular Self‐Assembly of Short Peptides at Liquid-Solid Interfaces-Effect of Charged Amino Acid Point Mutations (2024)
  • Author(s): Yurtsever Ayhan、Hirata Kaito、Kojima Ryohei、Miyazawa Keisuke、Miyata Kazuki、Kesornsit Sanhanut、Zareie Hadi、Sun Linhao、Maeda Katsuhiro、Sarikaya Mehmet、Fukuma Takeshi
  • Journal Title: Small Volume: 20 Issue: 25 Pages: 2400653-2400653
  • DOI: 10.1002/smll.202400653
  • Peer Reviewed / Open Access / Int'l Joint Research
• [Journal Article] Visualizing the inside of three-dimensional self-organizing systems by three-dimensional atomic force microscopy (2023)
  • Author(s): Fukuma Takeshi
  • Journal Title: Japanese Journal of Applied Physics Volume: 63 Issue: 1 Pages: 010801-010801
  • DOI: 10.35848/1347-4065/acf721
  • Peer Reviewed
• [Journal Article] Protocol for live imaging of intracellular nanoscale structures using atomic force microscopy with nanoneedle probes (2023)
  • Author(s): Ichikawa Takehiko、Alam Mohammad Shahidul、Penedo Marcos、Matsumoto Kyosuke、Fujita Sou、Miyazawa Keisuke、Furusho Hirotoshi、Miyata Kazuki、Nakamura Chikashi、Fukuma Takeshi
  • Journal Title: STAR Protocols Volume: 4 Issue: 3 Pages: 102468-102468
  • DOI: 10.1016/j.xpro.2023.102468
  • Peer Reviewed / Open Access / Int'l Joint Research
• [Journal Article] Actin-binding domain of Rng2 sparsely bound on F-actin strongly inhibits actin movement on myosin II (2022)
  • Author(s): Hayakawa Yuuki、Takaine Masak、Ngo Kien Xuan、Imai Taiga、Yamada Masafumi D、Behjat Arash Badami、Umeda Kenichi、Hirose Keiko、Yurtsever Ayhan、Kodera Noriyuki、Tokuraku Kiyotaka、Numata Osamu、Fukuma Takeshi、Ando Toshio、Nakano Kentaro、Uyeda Taro QP
  • Journal Title: Life Science Alliance Volume: 6 Issue: 1 Pages: e202201469-e202201469
  • DOI: 10.26508/lsa.202201469
  • Peer Reviewed / Open Access / Int'l Joint Research
• [Journal Article] Molecular insights on the crystalline cellulose-water interfaces via three-dimensional atomic force microscopy (2022)
  • Author(s): Yurtsever Ayhan、Wang Pei-Xi、Priante Fabio、Morais Jaques Ygor、Miyazawa Keisuke、MacLachlan Mark J.、Foster Adam S.、Fukuma Takeshi
  • Journal Title: Science Advances Volume: 8 Issue: 41
  • DOI: 10.1126/sciadv.abq0160
  • Peer Reviewed / Open Access / Int'l Joint Research
• [Journal Article] Probing the Structural Details of Chitin Nanocrystal-Water Interfaces by Three‐Dimensional Atomic Force Microscopy (2022)
  • Author(s): Yurtsever Ayhan、Wang Pei‐Xi、Priante Fabio、Morais Jaques Ygor、Miyata Kazuki、MacLachlan Mark J.、Foster Adam S.、Fukuma Takeshi
  • Journal Title: Small Methods Volume: 6 Issue: 9 Pages: 2200320-2200320
  • DOI: 10.1002/smtd.202200320
  • Peer Reviewed / Open Access / Int'l Joint Research
• [Journal Article] Computed Three-Dimensional Atomic Force Microscopy Images of Biopolymers Using the Jarzynski Equality (2022)
  • Author(s): Sumikama Takashi、Federici Canova Filippo、Gao David Z.、Penedo Marcos、Miyazawa Keisuke、Foster Adam S.、Fukuma Takeshi
  • Journal Title: The Journal of Physical Chemistry Letters Volume: 13 Issue: 23 Pages: 5365-5371
  • DOI: 10.1021/acs.jpclett.2c01093
  • Peer Reviewed / Open Access / Int'l Joint Research
• [Journal Article] Unraveling the host-selective toxic interaction of cassiicolin with lipid membranes and its cytotoxicity (2022)
  • Author(s): Ngo Kien Xuan、Nguyen Phuong Doan N.、Furusho Hirotoshi、Miyata Makoto、Shimonaka Tomomi、Chau Nguyen Ngoc Bao、Vinh Nguyen Phuong、Nghia Nguyen Anh、Mohammed Tareg Omer、Ichikawa Takehiko、Kodera Noriyuki、Konno Hiroki、Fukuma Takeshi、Quoc Nguyen Bao
  • Journal Title: Phytopathology Volume: - Issue: 7 Pages: 000-000
  • DOI: 10.1094/phyto-09-21-0397-r
  • Peer Reviewed / Open Access / Int'l Joint Research
• [Journal Article] Chemical fixation creates nanoscale clusters on the cell surface by aggregating membrane proteins (2022)
  • Author(s): Ichikawa Takehiko、Wang Dong、Miyazawa Keisuke、Miyata Kazuki、Oshima Masanobu、Fukuma Takeshi
  • Journal Title: Communications Biology Volume: 5 Issue: 1 Pages: 487-487
  • DOI: 10.1038/s42003-022-03437-2
  • Peer Reviewed / Open Access
• [Journal Article] Structural and mechanical characteristics of exosomes from osteosarcoma cells explored by 3D-atomic force microscopy (2021)
  • Author(s): Yurtsever Ayhan、Yoshida Takeshi、Badami Behjat Arash、Araki Yoshihiro、Hanayama Rikinari、Fukuma Takeshi
  • Journal Title: Nanoscale Volume: 13 Issue: 13 Pages: 6661-6677
  • DOI: 10.1039/d0nr09178b
  • Peer Reviewed / Int'l Joint Research
• [Journal Article] Cell penetration efficiency analysis of different atomic force microscopy nanoneedles into living cells (2021)
  • Author(s): Penedo Marcos、Shirokawa Tetsuya、Alam Mohammad Shahidul、Miyazawa Keisuke、Ichikawa Takehiko、Okano Naoko、Furusho Hirotoshi、Nakamura Chikashi、Fukuma Takeshi
  • Journal Title: Scientific Reports Volume: 11 Issue: 1 Pages: 7756-7756
  • DOI: 10.1038/s41598-021-87319-3
  • Peer Reviewed / Open Access / Int'l Joint Research
• [Journal Article] Visualizing intracellular nanostructures of living cells by nanoendoscopy-AFM (2021)
  • Author(s): Penedo Marcos、Miyazawa Keisuke、Okano Naoko、Furusho Hirotoshi、Ichikawa Takehiko、Alam Mohammad Shahidul、Miyata Kazuki、Nakamura Chikashi、Fukuma Takeshi
  • Journal Title: Science Advances Volume: 7 Issue: 52
  • DOI: 10.1126/sciadv.abj4990
  • Peer Reviewed / Open Access / Int'l Joint Research
• [Journal Article] Photothermal excitation efficiency enhancement of cantilevers by electron beam deposition of amorphous carbon thin films (2020)
  • Author(s): Penedo Marcos、Yurtsever Ayhan、Miyazawa Keisuke、Furusho Hirotoshi、Ishii Kiyo-Aki、Fukuma Takeshi
  • Journal Title: Scientific Reports Volume: 10 Issue: 1 Pages: 17436-17436
  • DOI: 10.1038/s41598-020-74433-x
  • Peer Reviewed / Open Access
• [Journal Article] Tip dependence of three-dimensional scanning force microscopy images of calcite?water interfaces investigated by simulation and experiments (2020)
  • Author(s): Miyazawa Keisuke、Tracey John、Reischl Bernhard、Spijker Peter、Foster Adam S.、Rohl Andrew L.、Fukuma Takeshi
  • Journal Title: Nanoscale Volume: 12 Issue: 24 Pages: 12856-12868
  • DOI: 10.1039/d0nr02043e
  • Peer Reviewed / Open Access / Int'l Joint Research
• [Journal Article] Wideband Magnetic Excitation System for Atomic Force Microscopy Cantilevers with Megahertz-Order Resonance Frequency (2020)
  • Author(s): Hirata Kaito、Igarashi Takumi、Suzuki Keita、Miyazawa Keisuke、Fukuma Takeshi
  • Journal Title: Scientific Reports Volume: 10 Issue: 1 Pages: 9133-9133
  • DOI: 10.1038/s41598-020-65980-4
  • Peer Reviewed / Open Access
• [Journal Article] Improvements in fundamental performance of in-liquid frequency modulation atomic force microscopy (2020)
  • Author(s): Fukuma Takeshi
  • Journal Title: Microscopy Volume: 69 Issue: 6 Pages: 340-349
  • DOI: 10.1093/jmicro/dfaa045
  • Peer Reviewed
• [Journal Article] Subnanometer-scale imaging of nanobio-interfaces by frequency modulation atomic force microscopy (2020)
  • Author(s): Fukuma Takeshi
  • Journal Title: Biochemical Society Transactions Volume: 48 Issue: 4 Pages: 1675-1682
  • DOI: 10.1042/bst20200155
  • Peer Reviewed
• [Presentation] 2D/3D-AFMによるバイオ材料界面と細胞内現象の観察 (2024)
  • Author(s): 福間剛士
  • Organizer: 日本顕微鏡学会SPM研究会2023
  • Invited
• [Presentation] 革新的原子間力顕微鏡技術による未踏ナノ領域の開拓と世界トップレベル研究拠点の形成 (2024)
  • Author(s): 福間剛士
  • Organizer: 第46回 JEOL R&Dエグゼクティブ交流会
  • Invited
• [Presentation] Subnanoscale Imaging of Nano-Bio Interfaces by 2D- and 3D-AFM Imaging Techniques (2023)
  • Author(s): Takeshi Fukuma
  • Organizer: 2023 MRS Fall Meeting & Exhibit
  • Int'l Joint Research / Invited
• [Presentation] ナノ内視鏡AFMによる生細胞内部のナノ動態・力学計測 (2023)
  • Author(s): 福間剛士
  • Organizer: ニコンソリューションズ／ブルカージャパン共催ウェビナー
  • Invited
• [Presentation] Atomic Force Microscopy Technique for Visualizing the Inside of Living Cells (2023)
  • Author(s): Takeshi Fukuma
  • Organizer: The 82nd Annual Meeting of the Japanese Cancer Association
  • Int'l Joint Research / Invited
• [Presentation] Molecular Ordering and Electrochemical Reactions at Solid-Liquid Interfaces Investigated by Atomic Force Microscopy (2023)
  • Author(s): Takeshi Fukuma
  • Organizer: The 20th International Microscopy Congress (IMC20)
  • Int'l Joint Research / Invited
• [Presentation] 液中原子間力顕微鏡技術の開発と生体分子・細胞計測への応用 (2023)
  • Author(s): 福間剛士
  • Organizer: 第6回分子サイバネティクス・第50回分子ロボティクス定例研究会
  • Invited
• [Presentation] Visualizing Inside of 3D Self-Organizing Systems by 3D-AFM (2023)
  • Author(s): Takeshi Fukuma
  • Organizer: 9th Multifrequency AFM Conference
  • Int'l Joint Research / Invited
• [Presentation] Visualizing Inside of 3D Self-Organizing Systems by 3D-AFM (2023)
  • Author(s): Takeshi Fukuma
  • Organizer: The 9th International Symposium on Organic and Inorganic Electronic Materials and Related Nanotechnologies (EM-NANO 2023)
  • Int'l Joint Research / Invited
• [Presentation] 原子間力顕微鏡を用いたウリ類炭疽病菌の付着器の膨圧計測 (2023)
  • Author(s): 松森海晴、宮澤佳甫、熊倉直祐、白須賢、福間剛士
  • Organizer: 第70回 応用物理学会春季学術講演会
• [Presentation] Visualizing Inside of 3D Self-Organizing Systems by 3D Atomic Force Microscopy (2022)
  • Author(s): Takeshi Fukuma
  • Organizer: 2022 JAIST International Symposium of Nanomaterials and Devices Research Area
  • Int'l Joint Research / Invited
• [Presentation] Intracellular Structures and Mechanics of Living Cells Investigated by Nanoendoscopy-AFM (2022)
  • Author(s): Takeshi Fukuma
  • Organizer: 6th NanoLSI International Symposium
  • Int'l Joint Research / Invited
• [Presentation] Introduction to Research Activities at Nano Life Science Institute (WPI-NanoLSI) (2022)
  • Author(s): Takeshi Fukuma
  • Organizer: The Computational Biophysics of Atomic Force Microscopy
  • Int'l Joint Research / Invited
• [Presentation] Development of Cross-Scale In-cell AFM Technologies for Elucidating Intra-Cellular Structures and Dynamics (2022)
  • Author(s): 福間剛士
  • Organizer: 学術変革領域A第２回領域会議
  • Invited
• [Presentation] Nanoendoscopy AFM for visualizing nanodynamics and nanomechanics inside living cells (2022)
  • Author(s): Takeshi Fukuma
  • Organizer: Engineering Mechanics of Cell & Tissue Morphogenesis
  • Int'l Joint Research / Invited
• [Presentation] 世界トップレベル研究拠点プログラム －金沢大学ナノ生命科学研究所－ (2022)
  • Author(s): 福間剛士
  • Organizer: NanoLSI Special Seminar at Takaramachi Campus
  • Invited
• [Presentation] Development of a Method for Visualizing Nanometer-Scale Three-Dimensional Structures of Chromosomes by Three-Dimensional Atomic Force Microscopy (2022)
  • Author(s): R. Kojima, K. Miyazawa, K. Teramae, T. Sumikama, M. Meguro, K. Imadate, N. Okano, S. Horike, K. Hirahara T. Fukuma
  • Organizer: ISSP Workshop「機能的走査プローブ顕微鏡の新展開」
  • Int'l Joint Research
• [Presentation] Direct observation of focal adhesion by nanoendoscopy-AFM in live cells (2022)
  • Author(s): M. S. Alam, T. Shirokawa, T. Ichikawa, K. Miyazawa, K. Miyata, T. Fukuma
  • Organizer: AVS 68th INTERNATIONAL SYMPOSIUM & EXHIBITION
  • Int'l Joint Research
• [Presentation] Direct observation of focal adhesions by nanoendoscopy-AFM in live cells (2022)
  • Author(s): M. S. Alam, M. Penedo, T. Shirokawa, T. Ichikawa, M. M. Hosain, K. Miyazawa, K. Miyata, T. Fukuma
  • Organizer: The 22nd international vacuum congress IVC-22
  • Int'l Joint Research
• [Presentation] Development of nanoendoscopy-AFM for visualizing intracellular nanostructures of living cells (2022)
  • Author(s): K. Miyazawa, M. Penedo, N. Okano, H. Furusho, T. Ichikawa, M. S. Alam, K. Miyata, C. Nakamura, T Fukuma
  • Organizer: The 22nd International Vacuum Congress (IVC-22)
  • Int'l Joint Research
• [Presentation] Development of Humidity-controlled Atomic Force Microscopy for measurement of Humidity Dependence of Elasticity Distribution of a Single Human Corneocyte (2022)
  • Author(s): T. Kawabata, K. Takeuchi, M. Toyoda, F. Komatsu, M. Harada, M. Suzuki, N. Ohara, K. Miyata, K. Miyazawa and T. Fukuma
  • Organizer: AFM BioMed Conference 2022
  • Int'l Joint Research
• [Presentation] Visualizing focal adhesion by nanoendoscopy-AFM in live cells (2022)
  • Author(s): M. S. Alam, T. Shirokawa, T. Ichikawa, K. Miyazawa, C. M. Franz and T. Fukuma
  • Organizer: AFM BioMed Conference 2022
  • Int'l Joint Research
• [Presentation] Visualizing 3D internal structure of cell nucleus by 3D-AFM (2022)
  • Author(s): R. Kojima, K. Miyazawa, A. M. Shahidul, K. Sivashanmugan, T. Sumikama, R. Omura and T. Fukuma
  • Organizer: AFM BioMed Conference 2022
  • Int'l Joint Research
• [Presentation] Chemical fixation creates nanoscale clusters on the cell surface by aggregating membrane proteins (2022)
  • Author(s): T. Ichikawa, D. Wang, K. Miyazawa, K. Miyata, M. Oshima, T. Fukuma
  • Organizer: AFM BioMed Conference 2022
  • Int'l Joint Research
• [Presentation] Low-voltage Cryo Scanning Electron Microscopy Imaging of Rubber Leaves with Corynespora Cassiicola (2022)
  • Author(s): H. Furusho, KX. Ngo, NB. Quoc, T. Fukuma,
  • Organizer: The 78th Annual Meeting of The Japanese Society of Microscopy
  • Int'l Joint Research
• [Presentation] A Self-making TEM Holder Designed for Evaluating Cantilevers of Scanning Probe Microscopy (2022)
  • Author(s): H. Furusho, MS. Alam, R. Kojima, T. Shirokawa, MM. Hosain, K. Teramae, Y. Asano, K. Hirata, K. Miyazawa, T. Fukuma
  • Organizer: The 78th Annual Meeting of The Japanese Society of Microscopy
  • Int'l Joint Research
• [Presentation] 化学固定は膜タンパク質を凝集することにより細胞表面にナノスケールのクラスターを形成する (2022)
  • Author(s): 市川 壮彦, Dong Wang, 宮澤 佳甫, 宮田 一輝, 大島 正伸, 福間 剛士
  • Organizer: 定量生物学の会第十回年会
• [Presentation] 原子間力顕微鏡を用いた生きた細胞のナノスケール表面構造体及び内部構造体観察方法の開発 (2022)
  • Author(s): 市川 壮彦, Marcos Penedo, 宮澤 佳甫, 古庄 公寿, Alam Mohammad Shahidul, 宮田 一輝,中村 史, 福間 剛士
  • Organizer: 第60回日本生物物理学会年会
• [Presentation] 細胞核内部の3 次元構造を可視化する3D-AFM の開発 (2022)
  • Author(s): 児島 亮平，宮澤 佳甫，Alam Mohammad Shahidul，Kundan Sivashanmugan，炭竈 享司，太村 理沙，福間 剛士
  • Organizer: 第83回応用物理学会秋季学術講演会
• [Presentation] Visualizing Unexplored Nanoworld by Innovative In-liquid AFM Technologies (2022)
  • Author(s): Takeshi Fukuma
  • Organizer: The 99th CEMS Colloquium
  • Invited
• [Presentation] 革新的液中原子間力顕微鏡技術による未踏ナノ領域の可視化 (2021)
  • Author(s): 福間剛士
  • Organizer: ナノ科学シンポジウム2021
  • Invited
• [Presentation] 金沢大学ナノ生命科学研究所における活動の紹介 (2021)
  • Author(s): 福間剛士
  • Organizer: ナノプローブテクノロジー第167委員会第98回研究会
  • Invited
• [Presentation] 革新的ナノプローブ技術によるナノ生命科学研究の新展開 (2021)
  • Author(s): 福間剛士
  • Organizer: 第73回日本細胞生物学会大会
  • Invited
• [Presentation] 化学固定は膜タンパク質を凝集することにより細胞表面にナノスケールのクラスターを形成する (2021)
  • Author(s): 市川 壮彦, Dong Wang, 宮澤 佳甫, 宮田 一輝, 大島 正伸, 福間 剛士
  • Organizer: 第５９回日本生物物理学会
• [Presentation] 液中AFMによる肺がん細胞の薬剤耐性獲得に伴うナノスケール表面構造変化の解明 (2021)
  • Author(s): 北村 太樹, 福田 康二, 宮澤 佳甫, 日笠山 拓, 原田 昌征, 矢野 聖二, 福間 剛士
  • Organizer: 第59回日本生物物理学会年会
• [Presentation] 液中AFMによる界面現象や生命現象のオペランド計測と「次世代」の方向性 (2020)
  • Author(s): 福間剛士
  • Organizer: JST-CRSD科学技術未来戦略ワークショップ
  • Invited
• [Remarks] 福間研究室ホームページ
  • URL: https://fukuma.w3.kanazawa-u.ac.jp/
• [Remarks] ナノ生命科学研究所ホームページ
  • URL: https://nanolsi.kanazawa-u.ac.jp/
• [Remarks] 金沢大学福間研究室ホームページ
  • URL: http://fukuma.w3.kanazawa-u.ac.jp/
• [Remarks] 金沢大学ナノ生命科学研究所
  • URL: https://nanolsi.kanazawa-u.ac.jp/
• [Remarks] 金沢大学ナノ生命科学研究所ホームページ
  • URL: https://nanolsi.kanazawa-u.ac.jp/