Visualization of subcellular thermoregulation in live cells using fluorescent coiled-coil proteins

• Project/Area Number: 24655151
• Research Category: Grant-in-Aid for Challenging Exploratory Research
• Allocation Type: Single-year Grants
• Research Field: Chemistry related to living body
• Research Institution: Kyoto University
• Principal Investigator: KIOYNAKA Shigeki 京都大学, 地球環境学堂, 准教授 (90422980)
• Project Period (FY): 2012-04-01 – 2014-03-31
• Project Status: Completed (Fiscal Year 2013)
• Budget Amount: ¥4,160,000 (Direct Cost: ¥3,200,000、Indirect Cost: ¥960,000); Fiscal Year 2013: ¥2,080,000 (Direct Cost: ¥1,600,000、Indirect Cost: ¥480,000); Fiscal Year 2012: ¥2,080,000 (Direct Cost: ¥1,600,000、Indirect Cost: ¥480,000)
• Keywords: サーモセンサー / 蛍光タンパク質 / coiled-coil / tsGFP / TWIX

Research Abstract

In mammals and birds, thermoregulation to conserve body temperature is vital to life. Multiple mechanisms of thermogeneration localized in different subcellular organelles have been proposed. However, previous studies have failed to observe thermogenesis directly in intact organelles. Here, we have visualized intracellular thermogenesis by using a fluorescent thermosensor, tsGFP. Specific targeting of tsGFPs enables visualization of thermogenesis in discrete organelles, notably mitochondria in brown adipocytes and the endoplasmic reticulum in myotubes. Strikingly, in HeLa cells, targeted tsGFP reveals heterogeneity in mitochondrial thermogenesis that is well correlated with the electrochemical gradient. Thus, tsGFPs directly demonstrate subcellular thermotransduction, and will serve as powerful tools to assess thermogenesis in vivo.

Research Products

• [Journal Article] Transnitrosylation directs TRPA1 selectivity in N-nitrosamine activators. (2014)
  • Author(s): Kozai D, Kabasawa Y, Ebert M, Kiyonaka S ; Finnan, Otani Y, Numata T, Takahashi N, Mori Y, Ohwada T.
  • Journal Title: Molecular Pharmacology Volume: 85 Issue: 1 Pages: 175-185
  • DOI: 10.1124/mol.113.088864
  • Peer Reviewed
• [Journal Article] Genetically encoded fluorescent thermosensors visualize subcellular thermoregulation in living cells (2013)
  • Author(s): Shigeki Kiyonaka, Taketoshi Kajimoto, Reiko Sakaguchi, Daisuke Shinmi, Mariko Omatsu-Kanbe, Hiroshi Matsuura, Hiromi Imamura, Takenao Yoshizaki, Itaru Hamachi, Takashi Morii, Yasuo Mori
  • Journal Title: Nature Methods Volume: 10 Issue: 12 Pages: 1232-1238
  • DOI: 10.1038/nmeth.2690
  • Peer Reviewed
• [Journal Article] Nuclear life of the voltage-gated Cacnb4 subunit and its role in gene transcription regulation. (2013)
  • Author(s): Ronjat M, Kiyonaka S, Barbado M, De Waard M, Mori Y
  • Journal Title: Channels Volume: 7 Issue: 2 Pages: 119-125
  • DOI: 10.4161/chan.23895
  • Peer Reviewed
• [Journal Article] Molecular determinants for cardiovascular TRPC6 channel regulation by Ca2+/calmodulin-dependent kinase II (2013)
  • Author(s): Juan Shi, Naomi Geshi, Shinichi Takahashi, Shigeki Kiyonaka, Jun Ichikawa, Yaopeng Hu, Yasuo Mori, Yushi Ito, Ryuji Inoue
  • Journal Title: Journal of Physiology (London) Volume: 591(11) Issue: 11 Pages: 2851-2866
  • DOI: 10.1113/jphysiol.2013.251249
  • Peer Reviewed
• [Journal Article] Cacnb4 directly couples electrical activity to gene expression, a process defective in juvenile epilepsy. (2012)
  • Author(s): Tadmouri A
  • Journal Title: The EMBO journal Volume: 31 Issue: 18 Pages: 3730-3744
  • DOI: 10.1038/emboj.2012.226
  • Peer Reviewed
• [Journal Article] Physical and Functional Interaction of the Active Zone Protein CAST/ERC2 and the β-subunit of the Voltage-dependent Ca2+Channel. (2012)
  • Author(s): Kiyonaka S
  • Journal Title: J. Biochem Volume: 152 Issue: 2 Pages: 149-159
  • DOI: 10.1093/jb/mvs054
  • NAID: 10031166124
  • Peer Reviewed
• [Journal Article] Physical and functional interaction of the active zone protein CAST/ERC2 and the β-subunit of the voltage-dependent Ca(2+) channel. (2012)
  • Author(s): Kiyonaka S
  • Journal Title: Journal of Biochemistry Volume: 152 Pages: 149-159
  • NAID: 10031166124
  • Peer Reviewed
• [Presentation] Genetically encoded fluorescent thermosensors visualize subcellular thermoregulation in living cells (2014)
  • Author(s): Shigeki Kiyonaka, Taketoshi Kajimoto, Reiko Sakaguchi, Daisuke Shinmi, Mariko Omatsu-Kanbe, Hiroshi Matsuura, Hiromi Imamura, Takenao Yoshizaki, Itaru Hamachi, Takashi Morii and Yasuo Mori
  • Organizer: the 58th Annual Meeting of the Biophysical Society
• [Presentation] 蛍光性サーモセンサーの開発と生体の恒常性を担う熱産生機構の可視化 (2013)
  • Author(s): 清中茂樹、梶本武利、坂口怜子、森井孝、森泰生
  • Organizer: 生理学研究所研究会「新規シグナル伝達分子とその生理学的可能性」
  • Year and Date: 2013-09-26
• [Presentation] 神経細胞における内在性グルタミン酸受容体のケミカルラベル (2013)
  • Author(s): 若山翔、清中茂樹、浜地格
  • Organizer: 第7回バイオ関連化学シンポジウム
• [Presentation] 神経細胞における内在性グルタミン酸受容体の蛍光ラベル化法の開発 (2013)
  • Author(s): 若山翔、清中茂樹、浜地格
  • Organizer: 第86回日本生化学会大会
• [Presentation] 神経伝達物質受容体のケミカルラベル (2013)
  • Author(s): 若山翔、清中茂樹、浜地格
  • Organizer: 日本化学会第93春季年会
• [Presentation] 蛍光性サーモセンサーの開発と生体の恒常性を担う熱産生機構の可視化 (2013)
  • Author(s): 清中 茂樹、梶本 武利、坂口 怜子、森井 孝、森 泰生
  • Organizer: 生理学研究所研究会「新規シグナル伝達分子とその生理学的可能性」
  • Place of Presentation: 生理学研究所
• [Presentation] 神経伝達物質受容体のケミカルラベル (2013)
  • Author(s): 若山 翔
  • Organizer: 日本化学会第93春季年会
  • Place of Presentation: 立命館大学（滋賀県）
• [Remarks]
  • URL: http://www.sbchem.kyoto-u.ac.jp/hamachi-lab/