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Auto-Luminescent Genetically-Encoded Ratiometric Indicator for Real-Time Ca2+ Imaging at the Single Cell Level


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タイトル: Auto-Luminescent Genetically-Encoded Ratiometric Indicator for Real-Time Ca2+ Imaging at the Single Cell Level
著者: Saito, Kenta 著作を一覧する
Hatsugai, Noriyuki 著作を一覧する
Horikawa, Kazuki 著作を一覧する
Kobayashi, Kentaro 著作を一覧する
Matsu-ura, Toru 著作を一覧する
Mikoshiba, Katsuhiko 著作を一覧する
Nagai, Takeharu 著作を一覧する
発行日: 2010年 4月 1日
出版者: Public Library of Science
誌名: PLoS One
巻: 5
号: 4
開始ページ: e9935
出版社 DOI: 10.1371/journal.pone.0009935
抄録: Background: Efficient bioluminescence resonance energy transfer (BRET) from a bioluminescent protein to a fluorescent protein with high fluorescent quantum yield has been utilized to enhance luminescence intensity, allowing single-cell imaging in near real time without external light illumination. Methodology/Principal Findings: We applied BRET to develop an autoluminescent Ca2+ indicator, BRAC, which is composed of Ca^[2+]-binding protein, calmodulin, and its target peptide, M13, sandwiched between a yellow fluorescent protein variant, Venus, and an enhanced Renilla luciferase, RLuc8. Adjusting the relative dipole orientation of the luminescent protein's chromophores improved the dynamic range of BRET signal change in BRAC up to 60%, which is the largest dynamic range among BRET-based indicators reported so far. Using BRAC, we demonstrated successful visualization of Ca2+ dynamics at the single-cell level with temporal resolution at 1 Hz. Moreover, BRAC signals were acquired by ratiometric imaging capable of canceling out Ca^[2+]-independent signal drifts due to change in cell shape, focus shift, etc. Conclusions/Significance: The brightness and large dynamic range of BRAC should facilitate high-sensitive Ca2+ imaging not only in single live cells but also in small living subjects.
資料タイプ: article
出現コレクション:雑誌発表論文等 (Peer-reviewed Journal Articles, etc)

提供者: 永井 健治


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