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

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Title: Auto-Luminescent Genetically-Encoded Ratiometric Indicator for Real-Time Ca2+ Imaging at the Single Cell Level
Authors: Saito, Kenta Browse this author
Hatsugai, Noriyuki Browse this author
Horikawa, Kazuki Browse this author
Kobayashi, Kentaro Browse this author
Matsu-ura, Toru Browse this author
Mikoshiba, Katsuhiko Browse this author
Nagai, Takeharu Browse this author →KAKEN DB
Issue Date: 1-Apr-2010
Publisher: Public Library of Science
Journal Title: PLoS One
Volume: 5
Issue: 4
Start Page: e9935
Publisher DOI: 10.1371/journal.pone.0009935
Abstract: 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.
Type: article
Appears in Collections:電子科学研究所 (Research Institute for Electronic Science) > 雑誌発表論文等 (Peer-reviewed Journal Articles, etc)

Submitter: 永井 健治

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