Real-time in vivo imaging of extracellular ATP in the brain with a hybrid-type fluorescent sensor
- The University of Tokyo, Japan
- Nihon University School of Medicine, Japan
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.
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All data generated or analyzed during this study are included in the manuscript and supporting files.
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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.
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).
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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Real-time in vivo imaging of extracellular ATP in the brain with a hybrid-type fluorescent sensor
eLife 9:e57544.
https://doi.org/10.7554/eLife.57544
