Development of single-molecule tracking using fluorescent silicon nanoparticles and its application to signaling mechanisms in clathrin-coated pits

• Project/Area Number: 21570167
• Research Category: Grant-in-Aid for Scientific Research (C)
• Allocation Type: Single-year Grants
• Section: 一般
• Research Field: Biophysics
• Research Institution: Kyoto University
• Principal Investigator: FUJIWARA Takahiro 京都大学, 物質-細胞統合システム拠点, 特定拠点講師 (80423060)
• Project Period (FY): 2009 – 2011
• Project Status: Completed (Fiscal Year 2011)
• Budget Amount: ¥4,810,000 (Direct Cost: ¥3,700,000、Indirect Cost: ¥1,110,000); Fiscal Year 2011: ¥910,000 (Direct Cost: ¥700,000、Indirect Cost: ¥210,000); Fiscal Year 2010: ¥910,000 (Direct Cost: ¥700,000、Indirect Cost: ¥210,000); Fiscal Year 2009: ¥2,990,000 (Direct Cost: ¥2,300,000、Indirect Cost: ¥690,000)
• Keywords: 1分子追跡 / 蛍光プローブ / 細胞膜 / 受容体 / クラスリン被覆ピット / シグナル伝達

Research Abstract

Single receptor molecules in the plasma membrane were specifically labeled with fluorescent silicon nanoparticles without blinking or photobleaching, making it possible for the first time to visualize individual internalization events through clathrin-coated pits. A method for conjugating ligands for enzyme-based protein tag to nanoparticle surface was developed, so that the versatility of the probe was enhanced. A new camera system was further developed, allowing us to detect each elementary process of receptor association and signaling complex formation at a 0. 1-ms resolution.

Research Products

• [Journal Article] Archipelago architecture of the focal adhesion : Membrane molecules freely enter and exit from the focal adhesion zone (2012)
  • Author(s): A. C. E. Shibata, T. K. Fujiwara, L. Chen, K. G. N. Suzuki, Y. Ishikawa, Y. L. Nemoto, Y. Miwa, R. Chadda, K. Naruse, and A. Kusumi
  • Journal Title: Cytoskeleton Volume: (in press)
  • Peer Reviewed
• [Journal Article] Confining domains lead to reaction bursts : reaction kinetics in the plasma membrane (2012)
  • Author(s): Z.Kalay, T.Fujiwara, A.Kusumi
  • Journal Title: PLoS One Volume: 7 Issue: 3 Pages: e32948-e32948
  • DOI: 10.1371/journal.pone.0032948
  • NAID: 120004161078
  • Peer Reviewed
• [Journal Article] Archipelago architecture of the focal adhesion : Membrane molecules freely enter and exit from the focal adhesion zone (2012)
  • Author(s): A.C.E.Shibata, T.K.Fujiwara, L.Chen, K.G.N.Suzuki, Y.Ishikawa, Y.L.Nemoto, Y.Miwa, R.Chadda, K.Naruse, A.Kusuni
  • Journal Title: Cytoskeleton Volume: (印刷中)
  • Peer Reviewed
• [Journal Article] Hierarchical meso-scale somain organization of the plasma membrane (2011)
  • Author(s): A.Kusumi, K.G.N.Suzuki, R.S.Kasai, K.Ritchie, T.K.Fujiwara
  • Journal Title: Trends Biochem.Sci. Volume: 36 Issue: 11 Pages: 604-615
  • DOI: 10.1016/j.tibs.2011.08.001
  • Peer Reviewed
• [Journal Article] Full characterization of GPCR monomer-dimer dynamic equilibrium by single molecule imaging (2011)
  • Author(s): R. S. Kasai, K. G. N. Suzuki, E. R. Prossnitz, I. Koyama-Honda, C. Nakada, T. K. Fujiwara, and A. Kusumi
  • Journal Title: Journal of Cell Biology Volume: Vol.192 Issue: 3 Pages: 463-480
  • DOI: 10.1083/jcb.201009128
  • Peer Reviewed
• [Journal Article] Hierarchical organization of the plasma membrane : Investigations by single-molecule tracking vs. fluorescence correlation spectroscopy (2010)
  • Author(s): A. Kusumi, Y. M. Shirai, I. Koyama-Honda, K. G. Suzuki, and T. K. Fujiwara
  • Journal Title: FEBS Letters Volume: 584 Issue: 9 Pages: 1814-1823
  • DOI: 10.1016/j.febslet.2010.02.047
  • Peer Reviewed
• [Journal Article] Hierarchical organization of the plasma membrane : Investigations by single-molecule tracking vs.fluorescence correlation spectroscopy (2010)
  • Author(s): A.Kusumi, Y.M.Shirai, I.Koyama-Honda, K.G.Suzuki, T.K.Fujiwara
  • Journal Title: FEBS Letters Volume: 584 Pages: 1814-1823
  • Peer Reviewed
• [Journal Article] (2009)
  • Author(s): 高橋陽一郎(楠見明弘、藤原敬宏)
  • Journal Title: 「ランダムな運動に法則を見出す」math stories変化をとらえる(東京図書) Pages: 238-247
• [Presentation] 膜骨格の仕切りによる細胞膜の組織化:高速1分子追跡による研究 (2011)
  • Author(s): 藤原敬宏
  • Organizer: 第4回JSBi応用システムバイオロジー研究会
  • Place of Presentation: 神戸理研計算科学研究機構
  • Year and Date: 2011-11-07
• [Presentation] 膜骨格の仕切りによる細胞膜の組織化:高速1分子追跡による研究 (2011)
  • Author(s): 藤原敬宏
  • Organizer: 第4回JSBi応用システムバイオロジー研究会「細胞環境の測定とモデリング」ワークショップ
  • Place of Presentation: 兵庫県神戸市神戸理研計算科学研究機構(招待講演)
  • Year and Date: 2011-11-07
• [Presentation] Enhanced confinement of activated EGF receptor in the plasma membrane compartments revealed by ultra high-speed single-molecule tracking (2011)
  • Author(s): T. K. Fujiwara, Z. Kalay, S. Shibutani, K. P. Ritchie, S. Takeuchi, K. G. N. Suzuki, and A. Kusumi
  • Organizer: 第49回日本生物物理学会年会
  • Place of Presentation: 兵庫県立大学・姫路書写キャンパス
  • Year and Date: 2011-09-17
• [Presentation] Enhanced confinement of activated EGF receptor in the plasma membrane compartments revealed by ultra high-speed single-molecule tracking (2011)
  • Author(s): T.K.Fujiwara, Z.Kalay, S.Shibutani, K.P.Ritchie, S.Takeuchi, K.G.N.Suzuki, A.Kusumi
  • Organizer: 第49回日本生物物理学会年会
  • Place of Presentation: 兵庫県姫路市兵庫県立大学姫路書写キャンパス
  • Year and Date: 2011-09-17
• [Presentation] Super-speed single fluorescent-molecule imaging directly revealed in tercompartmental hop diffusion of membrane molecules (2010)
  • Author(s): T. K. Fujiwara, S. Takeuchi, Y. Nagai, K. Hanaka, K. Iwasawa, K. G. N. Suzuki, and A. Kusumi
  • Organizer: APRUリサーチシンポジウム"Interface Between Molecular Biology and Nano-Biology"
  • Place of Presentation: 京都大学
  • Year and Date: 2010-11-25
• [Presentation] Super-speed single fluorescent-molecule imaging directly revealed intercompartmental hop diffusion of membrane molecules (2010)
  • Author(s): T.K.Fujiwara, S.Takeuchi, Y.Nagai, K.Hanaka, K.Iwasawa, K.G.N.Suzuki, A.Kusumi
  • Organizer: APRUリサーチシンポジウム"Interface Between Molecular Biology and Nano-Biology"
  • Place of Presentation: 京都大学(京都)
  • Year and Date: 2010-11-25
• [Presentation] Direct observation of hop diffusion of membrane molecules by developing ultra high-speed single fluorescent-molecule imaging (2010)
  • Author(s): T. K. Fujiwara, S. Takeuchi, Y. Nagai, K. Hanaka, K. Iwasawa, K. G. N. Suzuki, and A. Kusumi
  • Organizer: 第48回日本生物物理学会年会
  • Place of Presentation: 東北大学
  • Year and Date: 2010-09-20
• [Presentation] Direct observation of hop diffusion of membrane molecules by developing ultra high-speed single fluorescent-molecule imaging (2010)
  • Author(s): T.K.Fujiwara, S.Takeuchi, Y.Nagai, K.Hanaka, K.Iwasawa, K.G.N.Suzuki, A.Kusumi
  • Organizer: 第48回日本生物物理学会年会
  • Place of Presentation: 東北大学(仙台)
  • Year and Date: 2010-09-20
• [Presentation] Hop diffusion of membrane lipids and proteins in the plasma membrane as directly observed by high-speed single fluorescent-molecule tracking (2010)
  • Author(s): T. Fujiwara
  • Organizer: 13th International Membrane Research Forum
  • Place of Presentation: ホテルフジタ京都
  • Year and Date: 2010-01-29
• [Presentation] Hop diffusion of membrane lipids and proteins in the plasma membrane as directly observed by high-speed single fluorescent-molecule tracking (2010)
  • Author(s): T.Fujiwara
  • Organizer: 13^<th> International Membrane Research Forum
  • Place of Presentation: ホテルフジタ京都
  • Year and Date: 2010-01-29
• [Presentation] Compartmentalization of the plasma membrane by actin-based membrane skeleton as revealed by high-speed single fluorescent-molecule tracking (2009)
  • Author(s): T. Fujiwara, S. Takeuchi, Y. Nagai, K. Hanaka, K. Iwasawa, K. Suzuki, and A. Kusumi
  • Organizer: 第82回日本生化学会大会
  • Place of Presentation: 神戸ポートアイランド
  • Year and Date: 2009-10-21
• [Presentation] 超高速1蛍光分子追跡法による、コンパートメント化された細胞膜内でのリン脂質のホップ拡散の検出 (2009)
  • Author(s): 藤原敬宏
  • Organizer: 第61回日本細胞生物学会大会ランチョンセミナー
  • Place of Presentation: 名古屋国際会議場
  • Year and Date: 2009-06-02
• [Book] トランスポートソームの組織化における細胞骨格系の役割(トランスポートソームの世界-膜輸送研究の源流から未来へ-) (2011)
  • Author(s): 木下専, 藤原敬宏
  • Publisher: 京都廣川書店
• [Book] トランスポートソームの組織化における細胞骨格系の役割「トランスポートソームの世界-膜輸送研究の源流から未来へ-」 (2011)
  • Author(s): 木下専、藤原敬宏
  • Publisher: 京都廣川書店
• [Book] ランダムな運動に法則を見出す(Math Stories変化をとらえる) (2009)
  • Author(s): 楠見明弘, 藤原敬宏
  • Publisher: 東京図書