Detection of single cell epigenetic status using single molecule imaging

• Project/Area Number: 24570191
• Research Category: Grant-in-Aid for Scientific Research (C)
• Allocation Type: Multi-year Fund
• Section: 一般
• Research Field: Biophysics
• Research Institution: The Institute of Physical and Chemical Research
• Principal Investigator: FUJITA Hideaki 独立行政法人理化学研究所, 生命システム研究センター, 客員研究員 (50318804)
• Project Period (FY): 2012-04-01 – 2015-03-31
• Project Status: Completed (Fiscal Year 2014)
• Budget Amount: ¥5,460,000 (Direct Cost: ¥4,200,000、Indirect Cost: ¥1,260,000); Fiscal Year 2014: ¥1,820,000 (Direct Cost: ¥1,400,000、Indirect Cost: ¥420,000); Fiscal Year 2013: ¥1,820,000 (Direct Cost: ¥1,400,000、Indirect Cost: ¥420,000); Fiscal Year 2012: ¥1,820,000 (Direct Cost: ¥1,400,000、Indirect Cost: ¥420,000)
• Keywords: 一分子イメージング / ＥＳ細胞 / 分化 / Histac / ヒストン / アセチル化 / 蛍光観察 / エピジェネティクス / 一分子観察 / 蛍光イメージング / 筋肉

Outline of Final Research Achievements

Aim of this study is to construct a method to report the epigenetical change using single molecule imaging. I have achieved this by measuring the diffusion property of fluorescent protein within the nucleus, and by using reporter for histone acetylation, Histac.
I found that fluorescent protein dimer change its diffusion dynamics by the differentiation status of the cell. Fluorescent protein dimer show higher diffusion in fully reprogramed iPS than in partially reprogrammed iPS. Modified Histac capable of single molecule imaging was constructed by replacing Venus with photo-activatable GFP. ES cell expressing this modified Histac increased FRET efficiency by treatment with trichostatin A, which shows that this modified Histac is capable of reporting the histone acetylation status.

Research Products

• [Journal Article] Comprehensive chemical secretory measurement of single cells trapped in a micro-droplet array with mass spectrometry (2015)
  • Author(s): Hideaki Fujita, Tsuyoshi Esaki, Tsutomu Masujima, Akitsu Hotta, Soo Hyeon Kim, Hiroyuki Noji, Tomonobu M. Watanabe
  • Journal Title: RSC Adv. Volume: 5 Issue: 22 Pages: 16968-16971
  • DOI: 10.1039/c4ra12021c
  • Peer Reviewed / Open Access
• [Journal Article] Fluid driving system for a micropump by differentiating iPS cells into cardiomyocytes on a tent-like structure. (2015)
  • Author(s): Tanaka Y., Fujita H.
  • Journal Title: Sens. Actuators B Chem. Volume: 210 Pages: 267-272
  • DOI: 10.1016/j.snb.2014.12.069
  • Peer Reviewed
• [Journal Article] Gene dynamics of core transcription factors for pluripotency in embryonic stem cells (2014)
  • Author(s): Brit G. David, Kazuko Okamoto, Taishi Kakizuka, Taro Ichimura, Tomonobu M. Watanabe, Hideaki Fujita
  • Journal Title: J. Biosci. Bioeng Volume: 119 Issue: 4 Pages: 406-409
  • DOI: 10.1016/j.jbiosc.2014.09.011
  • NAID: 110009942819
  • Peer Reviewed / Open Access
• [Journal Article] Four-dimensional spatial nanometry of single particles in living cells using polarized quantum rods (2013)
  • Author(s): Watanabe TM, Fujii F, Jin T, Umemoto E, Miyasaka M, Fujita H, Yanagida T.
  • Journal Title: Biophys J Volume: 105 Issue: 3 Pages: 555-564
  • DOI: 10.1016/j.bpj.2013.07.001
  • Peer Reviewed
• [Journal Article] Glycine insertion makes yellow fluorescent protein sensitive to hydrostatic pressure. (2013)
  • Author(s): Watanabe TM, Imada K, Yoshizawa K, Nishiyama M, Kato C, Abe F, Morikawa TJ, Kinoshita M, Fujita H, Yanagida T.
  • Journal Title: PLoS One Volume: 8 Issue: 8 Pages: e73212-e73212
  • DOI: 10.1371/journal.pone.0073212
  • NAID: 120005328377
  • Peer Reviewed
• [Journal Article] Heart extracellular matrix supports cardiomyocyte differentiation of mouse embryonic stem cells. (2013)
  • Author(s): Higuchi S, Lin Q, Wang J, Lim TK, Joshi SB, Anand GS, Chung MC, Sheetz MP, Fujita H.
  • Journal Title: J Biosci Bioeng Volume: 115(3) Issue: 3 Pages: 320-325
  • DOI: 10.1016/j.jbiosc.2012.10.004
  • NAID: 110009596108
  • Peer Reviewed
• [Journal Article] Chromatin plasticity as a differentiation index during muscle differentiation of C2C12 myoblasts. (2012)
  • Author(s): Tomonobu M. Watanabe et al.
  • Journal Title: Biochemical and Biophysical Research Communications Volume: 418 Issue: 4 Pages: 742-747
  • DOI: 10.1016/j.bbrc.2012.01.091
  • Peer Reviewed
• [Journal Article] p130Cas-dependent actin remodeling regulates myogenic differentiation (2012)
  • Author(s): Kawauchi K, Tan WW, Araki K, Bakar F, Kim M, Fujita H, Hirata H, Sawada Y
  • Journal Title: Biochemical Journal Volume: 445(3) Issue: 3 Pages: 323-332
  • DOI: 10.1042/bj20112169
  • Peer Reviewed
• [Journal Article] Engineering strain-sensitive yellow fluorescent protein. (2012)
  • Author(s): Taro Ichimura et al.
  • Journal Title: Chemical Communications Volume: 48 Issue: 63 Pages: 7871-7873
  • DOI: 10.1039/c2cc32541a
  • Peer Reviewed
• [Journal Article] Distinct modulated pupil function system for real-time imaging of living cells. (2012)
  • Author(s): Tomonobu M Watanabe et al.
  • Journal Title: PLoS ONE Volume: 7 Issue: 9 Pages: e44028-e44028
  • DOI: 10.1371/journal.pone.0044028
  • Peer Reviewed
• [Presentation] SH3 domain of C-Src regulates its dynamic behaviour in the cell membrane (2015)
  • Author(s): Machiyama H., Yamaguchi T., Watanabe TM., Fujita H.
  • Organizer: 59th Biophysical Society Annual Meeting
  • Place of Presentation: Baltimore, USA
  • Year and Date: 2015-02-07 – 2015-02-11
• [Presentation] Evaluating intracellular crowded with a glycine-inserted mutant fluorescent protein (2014)
  • Author(s): Takamitsu Morikawa, Hiroaki Machiyama, Kazuko Okamoto, Keiko Yoshizawa, Hideaki Fujita, Taro Ichimura, Katsumi Imada, Takaharu Nagai, Tohio Yanagida, Tomonobu Watanabe
  • Organizer: Biophysical Society Meeting, USA
  • Place of Presentation: サンフランシスコ・アメリカ合衆国
• [Presentation] Modeling for T cell-mediated immune regulation (2013)
  • Author(s): Tomoyuki Yamaguchi, Hiroaki Machiyama, Shimon Sakaguchi, Hideaki Fujita
  • Organizer: 日本免疫学会総会
  • Place of Presentation: 幕張メッセ
• [Presentation] Intracellular measurement of protein-crowding condition by a gene-encoded indicator (2013)
  • Author(s): Takamitsu Morikawa, Keiko Yoshizawa, Hideaki Fujita, Katsumi Imada, Takeharu Nagai, Toshio Yanagida, Tomonobu Watanabe
  • Organizer: 日本生物物理学会年会
  • Place of Presentation: 京都国際会館
• [Presentation] Actin filament remodeling in cell-sheet by mechanical stretch (2013)
  • Author(s): Madoka Suzuki, Keiko Kawauchi, Ee Chu Chai, Shota Yamauchi, Shin'ichi Ishiwata, Hideaki Fujita
  • Organizer: 日本生物物理学会年会
  • Place of Presentation: 京都国際会館
• [Presentation] Visualization of the molecular-crowding effects in living cell on cellular functions using a FRET-based biosensor (2013)
  • Author(s): Hiroaki Machiyama, Takamitsu Morikawa, Tomoyuki Yamaguchi, Toshio Yanagida, Tomonobu Watanabe, Hideaki Fujita
  • Organizer: 日本生物物理学会年会
  • Place of Presentation: 京都国際会館
• [Presentation] Describing the ES cell differentiation pathway using Raman microscopy (2013)
  • Author(s): Taro Ichimura, Liang-da Chiu, Katsumasa Fujita, Satoshi Kawata, Tomonobu M. Watanabe, and Hideaki Fujita
  • Organizer: EMBO meeting
  • Place of Presentation: アムステルダム・オランダ
• [Presentation] 電気刺激による高機能人工筋組織の作製 (2012)
  • Author(s): 山本泰徳、井藤彰、河邉佳典、藤田英明、長森英二、上平正道
  • Organizer: 日本生物工学会大会
  • Place of Presentation: 神戸国際会議場
• [Presentation] Change in the nucleus plasticity during C2C12 differentiation measured by fluorescent microscope (2012)
  • Author(s): Watanabe T, Ichimura T, Fujita H
  • Organizer: The 4th EMBO meeting
  • Place of Presentation: Nice, France
• [Presentation] 力を感じて色が変わる蛍光タンパク質の開発 (2012)
  • Author(s): 市村垂生、藤田英明、慶澤景子、渡邉朋信
  • Organizer: 平成24年度日本分光学会年次講演会
  • Place of Presentation: 東京工業大学
• [Presentation] ラマン分光顕微鏡を用いて細胞の分化状態を識別する (2012)
  • Author(s): 吉田宗生、市村垂生、邱亮達、藤田克昌、渡邉朋信、藤田英明
  • Organizer: 第50回日本生物物理学会年会
  • Place of Presentation: 名古屋大学 東山キャンパス
• [Presentation] Soft substrates are effective in establishment of iPS cells without preparation of feeder cells (2012)
  • Author(s): 樋口清香、渡邉朋信、市村垂生、藤田英明
  • Organizer: The 45th Annual Meeting of JSDB and The 64th Annual Meeting of JSCB
  • Place of Presentation: 神戸国際会議場
• [Patent(Industrial Property Rights)] 体細胞のリプログラミングを亢進させる方法、及び細胞作製キット (2012)
  • Inventor(s): 藤田英明、樋口清香、渡邉朋信
  • Industrial Property Rights Holder: 藤田英明、樋口清香、渡邉朋信
  • Industrial Property Rights Type: 特許
  • Industrial Property Number: 2012-263735
  • Filing Date: 2012-11-30