Improved unroofing method in AFM and cryo-electron microscopy to observe the interaction between organelles and cytoskeleton.

• Project/Area Number: 26650049
• Research Category: Grant-in-Aid for Challenging Exploratory Research
• Allocation Type: Multi-year Fund
• Research Field: Biophysics
• Research Institution: Nagoya University
• Principal Investigator: Usukura Jiro 名古屋大学, 理学(系)研究科(研究院), 研究員 (30143415)
• Project Period (FY): 2014-04-01 – 2016-03-31
• Project Status: Completed (Fiscal Year 2015)
• Budget Amount: ¥3,900,000 (Direct Cost: ¥3,000,000、Indirect Cost: ¥900,000); Fiscal Year 2015: ¥1,560,000 (Direct Cost: ¥1,200,000、Indirect Cost: ¥360,000); Fiscal Year 2014: ¥2,340,000 (Direct Cost: ¥1,800,000、Indirect Cost: ¥540,000)
• Keywords: 細胞膜剥離法 / 細胞骨格 / 滑面小胞体 / クライオ電顕 / 原子間力顕微鏡 / アクチン / 膜 / アクチン線維 / クラスリン / 電子顕微鏡 / バイオイメージング

Outline of Final Research Achievements

Two unroofing methods via tearing off cell membrane with adhesive mesh and sonication developed and used for AFM and cryo-electron microscopy instead of vitreous ice section. An improved unroofing method enabled the cantilever of AFM to reach directly into a cell to visualize the intracellular cytoskeletal actin filaments, microtubules, clathrin coats, and caveolae in PBS at molecular resolution. The actin filaments clearly exhibited a short periodicity of approximately 5-6 nm. In cryo electron microscopy, unroofing enabled us to view membrane cytoskeleton panoramically with extremely high contrast in native state. Many actin filaments and microtubules were observed clearly on the cytoplasmic surface of the plasma membrane at extremely high contrast, because soluble components of cytoplasm flowed out on breaking off the cells. Furthermore, Cryo-EM combined with unroofing revealed a network of smooth ER beneath the cell membrane first time in native ordinary cells.

Research Products

• [Journal Article] ROR1 sustains caveolae and survival signaling as a scaffold of cavin-1 and caveolin-1 (2016)
  • Author(s): Yamaguchi T, Lu C, Yanagisawa K, Can Lu, Usukura J, Cheng J, Hotta N, Shimada Y, Ida R, Isomura H, Suzuki M, Fujimoto T, Takahashi T
  • Journal Title: Nat Commun Volume: 7 Issue: 1 Pages: 10060-10060
  • DOI: 10.1038/ncomms10060
  • Peer Reviewed / Open Access
• [Journal Article] Imaging of the Subsurface Structures of “Unroofed” Plasmodium falciparum-infected Erythrocytes (2015)
  • Author(s): Eri H. Hayakawa, Fuyuki Tokumasub, Jiro Usukura, Hiroyuki Matsuoka, Takafumi Tsuboi, and Thomas E. Wellems
  • Journal Title: Experimental Parasitology Volume: 153 Pages: 174-179
  • DOI: 10.1016/j.exppara.2015.03.021
  • Peer Reviewed / Acknowledgement Compliant
• [Journal Article] Protein-4.1G-Mediated Membrane Trafficking Is Essential for Correct Rod Synaptic Location in the Retina and for Normal Visual Function. (2015)
  • Author(s): Sanuki R, Watanabe S, Sugita Y, Irie S, Kozuka T, Shimada M, Ueno S, Usukura J, Furukawa T.
  • Journal Title: Cell Reports Volume: 10 Issue: 5 Pages: 796-808
  • DOI: 10.1016/j.celrep.2015.01.005
  • Peer Reviewed / Open Access / Acknowledgement Compliant
• [Journal Article] Cooperative binding of the outer arm docking complex underlies the regular arrangement of outer arm dynein in the axoneme. (2014)
  • Author(s): Mikito Owa, Akane Furuta, Jiro Usukura, Fumio Arisaka, Stephen M. King, George B. Witman, Ritsu Kamiya, Ken-ichi Wakabayashi
  • Journal Title: Proc. Natl. Acad. Sci. USA Volume: 111(26) Issue: 26 Pages: 9455-9460
  • DOI: 10.1073/pnas.1403101111
  • Peer Reviewed / Open Access / Acknowledgement Compliant
• [Presentation] Unroofing methods in AFM imaging for intra-cellar cytoskeletons at molecular resolution (2015)
  • Author(s): 10.Usukura E, Narita A, Yagi A, Ito S, Usukura J
  • Organizer: Annual Meeting of the American Society for Cell Biology
  • Place of Presentation: Diego Convention Center, Sandiego, CA ,USA
  • Year and Date: 2015-12-12
  • Int'l Joint Research
• [Presentation] Development of low voltage cryo-electron microscope for simultaneous imaging of bright field, dark field STEM and SEM using unroofed frozen cells. (2015)
  • Author(s): Usukura J, Matsumoto T, Usukura E, Oharai Y, Sunaoshi T, Tamochi R, Narita A
  • Organizer: Annual Meeting of the American Society for Cell Biology
  • Place of Presentation: San Diego Convention Center, Sandiego, CA ,USA
  • Year and Date: 2015-12-12
  • Int'l Joint Research
• [Presentation] Development of low voltage cryo-electron microscope for simultaneous imaging of bright field, dark field STEM and SEM using unroofed frozen cells (2015)
  • Author(s): Usukura J
  • Organizer: Microscopy Conference 2015
  • Place of Presentation: Georg-August-University Gottingen, Germany
  • Year and Date: 2015-09-06
  • Int'l Joint Research / Invited
• [Presentation] High resolution AFM direct imaging of the intra-cellular cytoskeleton by using unroofed cells (2015)
  • Author(s): Usukura J
  • Organizer: Multinational Congress on Microscopy
  • Place of Presentation: Hotel Eger& Park, Eger, Hungary
  • Year and Date: 2015-08-23
  • Int'l Joint Research
• [Presentation] Use of the unroofing technique AFMdirect imaging of the intra-cellular structure at high resolution (2015)
  • Author(s): Usukura J, Usukura E, Narita A, Yagi A, Ito S
  • Organizer: Microscopy & Microanalysis
  • Place of Presentation: Oregon Convention Center Portland, Oregon, USA
  • Year and Date: 2015-08-02
  • Int'l Joint Research
• [Presentation] Spatial organization ofnuclear membrane cytoskeleton (2014)
  • Author(s): Jiro Usukura, Shiho Minakata
  • Organizer: Annal meeting of American Society of Cell Biology
  • Place of Presentation: Philadelphia, PA, USA
  • Year and Date: 2014-12-06 – 2014-12-10
• [Presentation] Simultaneous imaging of cry-bright field, dark field STEM and SEM using unroofed living cells. (2014)
  • Author(s): Jiro Usukura, Shiho Minakata
  • Organizer: International Microscopy Congress 2014
  • Place of Presentation: Prague, Czech Republic
  • Year and Date: 2014-09-07 – 2014-09-11
  • Invited
• [Presentation] Simultaneous imaging of cry-bright field, dark field STEM and SEM using unroofed living cells with special reference to membrane cytoskeletons. (2014)
  • Author(s): Jiro Usukura, Shiho Minakata
  • Organizer: Microscopy & Microanalysis 2014
  • Place of Presentation: Hartford, CT, USA
  • Year and Date: 2014-08-03 – 2014-08-07
• [Presentation] クライオSTEMによる細胞膜骨格の暗視野像、明視野像、虹電子像の同時計測 (2014)
  • Author(s): 臼倉治郎、南方志帆
  • Organizer: 日本電子顕微鏡学会
  • Place of Presentation: 幕張メッセ
  • Year and Date: 2014-05-11 – 2014-05-13
• [Remarks] 理学研究科構造生物学研究センター 臼倉機器開発プロジェクト
  • URL: http://str.bio.nagoya-u.ac.jp