Intra-nucleic and genomic distributions of liquid droplets formed by liquid-liquid phase separations

• Project/Area Number: 18K19297
• Research Category: Grant-in-Aid for Challenging Research (Exploratory)
• Allocation Type: Multi-year Fund
• Review Section: Medium-sized Section 43:Biology at molecular to cellular levels, and related fields
• Research Institution: Kyoto University
• Principal Investigator: Shirakawa Masahiro 京都大学, 工学研究科, 教授 (00202119)
• Project Period (FY): 2018-06-29 – 2021-03-31
• Project Status: Completed (Fiscal Year 2020)
• Budget Amount: ¥6,240,000 (Direct Cost: ¥4,800,000、Indirect Cost: ¥1,440,000); Fiscal Year 2020: ¥2,210,000 (Direct Cost: ¥1,700,000、Indirect Cost: ¥510,000); Fiscal Year 2019: ¥2,210,000 (Direct Cost: ¥1,700,000、Indirect Cost: ¥510,000); Fiscal Year 2018: ¥1,820,000 (Direct Cost: ¥1,400,000、Indirect Cost: ¥420,000)
• Keywords: 量子センシング / 光検出磁気共鳴 / １分子観察 / ナノ粒子 / 光検出磁気共鳴法 / ナノダイヤモンド / 細胞内温度計測 / 細胞内粘度計測 / 高分解能イメージング / 液滴様構造体 / ODMR-FIONA / エピジェネティックス / 液液相分離 / 細胞分化

Outline of Final Research Achievements

The aim of this study was establish the method to measure several physicoochemical parameters inside living cells by using optical detection of magnetic resonance (ODMR) of Nitrogen Vacancy Centers (NVCs) in nanodiamonds (NDs) at nanometer space resolution. For the preparation of quantum molecular sensors, we irradiated NDs (φ5-20 nm) by electron beams in order to generate NVCs. We then coated the surfaces of FNDs by polymers in order to avoid self-aggregations. We further modified the surfaces by several functional groups so that we can measure diffusion rates, temperatures, pH and so on by ODMR spectra of FNDs at space resolution of 10 nm orders. We also measured temperatures inside living cells, and analyzed movements of actin fibers ic cytoskeletons.

Academic Significance and Societal Importance of the Research Achievements

FNDの光検出磁気共鳴法(ODMR)によって、生細胞内の高分子の動態、拡散性、温度、ｐHなどの様々な物理パラメータを高い空間分解能で量子センシングするという手法の開発を進めた。その達成には、窒素空孔センターが持つの光三重項電子のを用いた光超偏極技術と光学検出装置の作成といった電子・光工学技術に始まって、ソフトウェア技術、nmオーダーのFNDの調製や官能基の導入といった化学的技術、さらに物理学的理論、分子・細胞生物学の技術、といった広範な学問分野を要する。このような分野横断的な新規技術を開発したことは、学術的意義が高い。更に生物、医学的分野に適用が進めば、その社会的意義は計り知れない。

Research Products

• [Journal Article] Transient Diffusive Interactions with a Protein Crowder Affect Aggregation Processes of Superoxide Dismutase 1 β-Barrel (2021)
  • Author(s): Iwakawa Naoto、Morimoto Daichi、Walinda Erik、Leeb Sarah、Shirakawa Masahiro、Danielsson Jens、Sugase Kenji
  • Journal Title: The Journal of Physical Chemistry B Volume: 125 Issue: 10 Pages: 2521-2532
  • DOI: 10.1021/acs.jpcb.0c11162
  • Peer Reviewed / Int'l Joint Research
• [Journal Article] Structural Dynamic Heterogeneity of Polyubiquitin Subunits Affects Phosphorylation Susceptibility (2021)
  • Author(s): Morimoto Daichi、Walinda Erik、Takashima Shingo、Nishizawa Mayu、Iwai Kazuhiro、Shirakawa Masahiro、Sugase Kenji
  • Journal Title: Biochemistry Volume: 60 Issue: 8 Pages: 573-583
  • DOI: 10.1021/acs.biochem.0c00619
  • Peer Reviewed
• [Journal Article] Chain-length dependent physical properties and degradation of ubiquitin chains (2020)
  • Author(s): 森本 大智、白川 昌宏
  • Journal Title: 生化学 Volume: 92 Issue: 1 Pages: 7-13
  • DOI: 10.14952/SEIKAGAKU.2020.920007
  • ISSN: 0037-1017
  • Year and Date: 2020-02-25
• [Journal Article] Quantitative monitoring of ubiquitination/deubiquitination reaction cycles by 18O-incorporation (2020)
  • Author(s): Tanaka Yuka、Morimoto Daichi、Walinda Erik、Sugase Kenji、Shirakawa Masahiro
  • Journal Title: Biochemical and Biophysical Research Communications Volume: 529 Issue: 2 Pages: 418-424
  • DOI: 10.1016/j.bbrc.2020.06.008
  • Peer Reviewed
• [Journal Article] Nanodiamonds for bioapplications?specific targeting strategies (2020)
  • Author(s): Terada Daiki、Genjo Takuya、Segawa Takuya F.、Igarashi Ryuji、Shirakawa Masahiro
  • Journal Title: Biochimica et Biophysica Acta (BBA) - General Subjects Volume: 1864 Issue: 2 Pages: 129354-129354
  • DOI: 10.1016/j.bbagen.2019.04.019
  • Peer Reviewed / Int'l Joint Research
• [Journal Article] Visualizing protein motion in Couette flow by all-atom molecular dynamics (2020)
  • Author(s): Walinda Erik、Morimoto Daichi、Shirakawa Masahiro、Scheler Ulrich、Sugase Kenji
  • Journal Title: Biochimica et Biophysica Acta (BBA) - General Subjects Volume: 1864 Issue: 2 Pages: 129383-129383
  • DOI: 10.1016/j.bbagen.2019.06.006
  • Peer Reviewed / Int'l Joint Research
• [Journal Article] High resolution protein 3D structure determination in living eukaryotic cells (2019)
  • Author(s): Tanaka, T., Ikeya, T., Kamoshida, H., Suemoto, Y., Mishima, M., Shirakawa, M., Guentert, P., Ito, Y.
  • Journal Title: Angewandte Chemie International Edition Volume: in press Issue: 22 Pages: 1-7
  • DOI: 10.1002/anie.201900840
  • Peer Reviewed / Int'l Joint Research
• [Journal Article] An innate interaction between IL-18 and the propeptide that inactivates its precursor form (2019)
  • Author(s): Naotaka Tsutsumi, Ayumi Yokota, Takeshi Kimura, Zenichiro Kato, Toshiyuki Fukao, Masahiro Shirakawa, Hidenori Ohnishi & Hidehito Tochio
  • Journal Title: Scientific Reportsvolume Volume: 9 Issue: 1 Pages: 6160-6160
  • DOI: 10.1038/s41598-019-42661-5
  • NAID: 120007006235
  • Peer Reviewed / Open Access
• [Journal Article] Monodisperse Five-Nanometer-Sized Detonation Nanodiamonds Enriched in Nitrogen-Vacancy Centers (2019)
  • Author(s): Terada Daiki、Segawa Takuya F.、Shames Alexander I.、Onoda Shinobu、Ohshima Takeshi、O?sawa Eiji、Igarashi Ryuji、Shirakawa Masahiro
  • Journal Title: ACS Nano Volume: 13 Issue: 6 Pages: 6461-6468
  • DOI: 10.1021/acsnano.8b09383
  • Peer Reviewed
• [Journal Article] Contribution of Coiled-Coil Assembly to Ca2+/Calmodulin-Dependent Inactivation of TRPC6 Channel and its Impacts on FSGS-Associated Phenotypes (2019)
  • Author(s): Polat Onur K.、Uno Masatoshi、Maruyama Terukazu、Tran Ha Nam、Imamura Kayo、Wong Chee Fah、Sakaguchi Reiko、Ariyoshi Mariko、Itsuki Kyohei、Ichikawa Jun、Morii Takashi、Shirakawa Masahiro、Inoue Ryuji、Asanuma Katsuhiko、Reiser Jochen、Tochio Hidehito、Mori Yasuo、Mori Masayuki X.
  • Journal Title: Journal of the American Society of Nephrology Volume: 30 Issue: 9 Pages: 1587-1603
  • DOI: 10.1681/asn.2018070756
  • Peer Reviewed / Open Access / Int'l Joint Research
• [Journal Article] pH Nanosensor Using Electronic Spins in Diamond (2019)
  • Author(s): Fujisaku Takahiro、Tanabe Ryotaro、Onoda Shinobu、Kubota Ryou、Segawa Takuya F.、So Frederick T.-K.、Ohshima Takeshi、Hamachi Itaru、Shirakawa Masahiro、Igarashi Ryuji
  • Journal Title: ACS Nano Volume: 13 Issue: 10 Pages: 11726-11732
  • DOI: 10.1021/acsnano.9b05342
  • Peer Reviewed / Open Access