Real-time in vivo imaging of extracellular ATP in the brain with a hybrid-type fluorescent sensor
Abstract
Adenosine 5' triphosphate (ATP) is a ubiquitous extracellular signaling messenger. Here, we describe a method for in-vivo imaging of extracellular ATP with high spatiotemporal resolution. We prepared a comprehensive set of cysteine-substitution mutants of ATP-binding protein, Bacillus FoF1-ATP synthase e subunit, labeled with small-molecule fluorophores at the introduced cysteine residue. Screening revealed that the Cy3-labeled glutamine-105 mutant (Q105C-Cy3; designated ATPOS) shows a large fluorescence change in the presence of ATP, with submicromolar affinity, pH-independence, and high selectivity for ATP over ATP metabolites and other nucleotides. To enable in-vivo validation, we introduced BoNT/C-Hc for binding to neuronal plasma membrane and Alexa Fluor 488 for ratiometric measurement. The resulting ATPOS complex binds to neurons in cerebral cortex of living mice, and clearly visualized a concentrically propagating wave of extracellular ATP release in response to electrical stimulation. ATPOS should be useful to probe the extracellular ATP dynamics of diverse biological processes in vivo.
Data availability
All data generated or analyzed during this study are included in the manuscript and supporting files.
Article and author information
Author details
Funding
Ministry of Education, Culture, Sports, Science, and Technology (17H04029)
- Kenzo Hirose
Ministry of Education, Culture, Sports, Science, and Technology (17K08584)
- Shigeyuki Namiki
Japan Science and Technology Agency (JPMJPR17P1)
- Daisuke Asanuma
Takeda Science Foundation
- Nami Kitajima
Ministry of Education, Culture, Sports, Science, and Technology (19K22247)
- Kenzo Hirose
Ministry of Education, Culture, Sports, Science, and Technology (25221304)
- Masamitsu Iino
Ministry of Education, Culture, Sports, Science, and Technology (18K14915)
- Hiroshi Sekiya
Ministry of Education, Culture, Sports, Science, and Technology (17H04764)
- Daisuke Asanuma
Ministry of Education, Culture, Sports, Science, and Technology (18H04726)
- Daisuke Asanuma
Ministry of Education, Culture, Sports, Science, and Technology (19K16251)
- Hirokazu Sakamoto
Ministry of Education, Culture, Sports, Science, and Technology (18H04609)
- Kenjiro Hanaoka
Ministry of Education, Culture, Sports, Science, and Technology (19H05414)
- Kenjiro Hanaoka
The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.
Reviewing Editor
- Yamuna Krishnan, University of Chicago, United States
Ethics
Animal experimentation: All procedures used in animal experiments were in accordance with the guidelines established by the Animal Welfare Committee of the University of Tokyo (Medicine-P10-010, Medicine-P15-017 and Medicine-P19-092).
Version history
- Received: April 3, 2020
- Accepted: July 9, 2020
- Accepted Manuscript published: July 10, 2020 (version 1)
- Version of Record published: August 3, 2020 (version 2)
Copyright
© 2020, Kitajima et al.
This article is distributed under the terms of the Creative Commons Attribution License permitting unrestricted use and redistribution provided that the original author and source are credited.
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