Prediction and control of cellular response by measuring the conformation of RAF

• Project/Area Number: 24770161
• Research Category: Grant-in-Aid for Young Scientists (B)
• Allocation Type: Multi-year Fund
• Research Field: Biophysics
• Research Institution: The Institute of Physical and Chemical Research
• Principal Investigator: HIBINO Kayo 独立行政法人理化学研究所, 生命システム研究センター, 研究員 (40435673)
• Project Period (FY): 2012-04-01 – 2015-03-31
• Project Status: Completed (Fiscal Year 2014)
• Budget Amount: ¥4,680,000 (Direct Cost: ¥3,600,000、Indirect Cost: ¥1,080,000); Fiscal Year 2013: ¥2,340,000 (Direct Cost: ¥1,800,000、Indirect Cost: ¥540,000); Fiscal Year 2012: ¥2,340,000 (Direct Cost: ¥1,800,000、Indirect Cost: ¥540,000)
• Keywords: 細胞内情報処理機構 / RAF / 細胞状態 / 細胞操作 / 細胞内1分子計測 / 1細胞計測 / FRET / 細胞動態計測 / ライブセルイメージング / 蛍光スペクトル計測 / 蛍光プローブ / 細胞内1分子解析 / 1分子FRET

Outline of Final Research Achievements

Uncontrollability of cell behaviors is a major obstacle to the therapeutic application of regenerative biology. To regulate cell behaviors, it is important to understand how cellular system works and to find the key factors to monitor and manipulate the cell-states.
Here, I focus on the signaling molecule RAS and RAF as a candidate factor to monitor and predict cell-states. RAS-RAF signaling involves proliferation, differentiation.
By using RAF-FRET probes, I found that the cellular response correlated with the ensemble RAF-conformation in individual cells before growth factor (GF)-stimulation and that the RAF-conformation depends on the phosphorylations. The results suggest that the phosphorylation state, therefore the ensemble RAF-conformation differs in individual cells and the difference induces varied responsibility to GF. The phosphorylation states of RAF could work as cellular memory which reflects the past extracellular environment and intracellular reactions.

Research Products

• [Journal Article] Visualizing specific protein glycoforms by transmembrane fluorescence resonance energy transfer (2012)
  • Author(s): Haga, Y., Ishii, K., Hibino, K., Sako, Y., Ito, Y., Taniguchi, N., Suzuki, T
  • Journal Title: Nature Communications Volume: 3 Issue: 1 Pages: 907-907
  • DOI: 10.1038/ncomms1906
  • Peer Reviewed
• [Journal Article] Live cell single-molecule detection in systems biology (2011)
  • Author(s): Sako, Y, Hiroshima, M., Pack, C.-G., Okamoto, K., Hibino, K., Yamamoto, A.
  • Journal Title: WIREs Systems Biology and Medicine Volume: 4 Issue: 2 Pages: 183-192
  • DOI: 10.1002/wsbm.161
  • Peer Reviewed
• [Presentation] Single-cell imaging analysis of conformation and function of disease-associated RAF mutants (2015)
  • Author(s): Hibino, K., Ueda, M., and Sako, Y.
  • Organizer: 第67回日本細胞生物学会大会
  • Place of Presentation: タワーホール船堀（東京・江戸川区）
  • Year and Date: 2015-06-30 – 2015-07-02
• [Presentation] Conformation and function of disease-associated RAF mutants (2014)
  • Author(s): Hibino, K., Ueda, M., and Sako, Y.
  • Organizer: 第52回日本生物物理学会年会
  • Place of Presentation: 札幌コンベンションセンター（北海道・札幌市）
  • Year and Date: 2014-09-25 – 2014-09-27
• [Presentation] Polymorphism of a signaling protein RAF regulates cellular responses. (2013)
  • Author(s): Hibino, K., Okamoto, K., Ueda, M., and Sako, Y.
  • Organizer: 第51回日本生物物理学会年会
  • Place of Presentation: 京都
• [Presentation] Single-molecule analysis of signaling protein RAF-conformation in living cells. (2013)
  • Author(s): Hibino, K., Okamoto, K., Ueda, M., and Sako, Y.
  • Organizer: 日本顕微鏡学会 第69回学術講演会
  • Place of Presentation: 大阪
• [Presentation] Signaling Molecule RAF: its conformation and cellular response (2012)
  • Author(s): 日比野佳代
  • Organizer: 定量生物学の会第五回年会
  • Place of Presentation: 東京大学 駒場IIキャンパス 生産技術研究所(東京)
• [Presentation] The conformation of signaling molecule RAF and the cellular response (2012)
  • Author(s): Hibino, K., Ueda, M., and Sako, M.
  • Organizer: RIKEN Quantitative Biology Center Inaugural Symposium "Towards Whole-Cell Modeling"
  • Place of Presentation: Kobe International Conference Center (Kobe)
• [Presentation] 細胞内スペクトル計測を用いた情報伝達タンパク質RAFの構造とEGF応答の相関解析 (2012)
  • Author(s): 日比野佳代
  • Organizer: 理論と実験研究会
  • Place of Presentation: 広島大学理学部(西条)
• [Presentation] Correlation between the molecular conformation and EGF response of RAF by FRET imaging in individual cells (2012)
  • Author(s): 日比野佳代、上田昌宏、佐甲靖志
  • Organizer: 第50回日本生物物理学会年会
  • Place of Presentation: 名古屋大学(名古屋)
• [Presentation] Correlation analysis of the molecular conformation and signal-responsiveness of RAF by FRET imaging in individual cells (2012)
  • Author(s): Hibino, K., Ueda, M., and Sako, M.
  • Organizer: Joint Meeting of The 45th Annual Meeting of the Japanese Society of Developmental Biologists & The 64th Annual Meeting of the Japan Society for Cell Biology
  • Place of Presentation: Kobe International Conference Center (Kobe)
• [Book] 1分子FRET計測，「1分子生物学」 (2014)
  • Author(s): 日比野佳代
  • Total Pages: 9
  • Publisher: 化学同人
• [Book] 細胞内情報処理反応の1分子計測：タンパク質ダイナミクスと分子認識 「１分子ナノバイオ計測」 (2014)
  • Author(s): 佐甲靖志、廣島通夫、日比野佳代
  • Total Pages: 10
  • Publisher: 化学同人
• [Book] Single-Molecule Imaging Measurements of Protein-Protein Interactions in Living Cells. (2013)
  • Author(s): Hibino, K., Hiroshima, M., Nakamura Y., and Sako Y.
  • Total Pages: 22
  • Publisher: InTech
• [Remarks] QBIC Quantitative Biology Center, RIKEN
  • URL: http://www.qbic.riken.jp/csd/ja/publication/index.html
• [Remarks] 理化学研究所 生命システム研究センター 細胞シグナル動態研究グループ
  • URL: http://www.qbic.riken.jp/csd/ja/index.html