1. Biochemistry and Chemical Biology
  2. Neuroscience

Real-time in vivo imaging of extracellular ATP in the brain with a hybrid-type fluorescent sensor

  1. Nami Kitajima
  2. Kenji Takikawa
  3. Hiroshi Sekiya
  4. Kaname Satoh
  5. Daisuke Asanuma
  6. Hirokazu Sakamoto
  7. Shodai Takahashi
  8. Kenjiro Hanaoka
  9. Yasuteru Urano
  10. Shigeyuki Namiki
  11. Masamitsu Iino
  12. Kenzo Hirose  Is a corresponding author
  1. The University of Tokyo, Japan
  2. Nihon University School of Medicine, Japan
https://doi.org/10.7554/eLife.57544
Share this article
Cite this article
  1. Nami Kitajima
  2. Kenji Takikawa
  3. Hiroshi Sekiya
  4. Kaname Satoh
  5. Daisuke Asanuma
  6. Hirokazu Sakamoto
  7. Shodai Takahashi
  8. Kenjiro Hanaoka
  9. Yasuteru Urano
  10. Shigeyuki Namiki
  11. Masamitsu Iino
  12. Kenzo Hirose
(2020)
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
  2. Download BibTeX
  3. Download .RIS

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

  1. Nami Kitajima

    Department of Pharmacology, The University of Tokyo, Bunkyo-ku, Japan
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0001-9838-832X

  2. Kenji Takikawa

    Department of Pharmacology, The University of Tokyo, Bunkyo-ku, Japan
    Competing interests
    The authors declare that no competing interests exist.

  3. Hiroshi Sekiya

    Department of Physiology, The University of Tokyo, Bunkyo-ku, Japan
    Competing interests
    The authors declare that no competing interests exist.

  4. Kaname Satoh

    Department of Pharmacology, The University of Tokyo, Bunkyo-ku, Japan
    Competing interests
    The authors declare that no competing interests exist.

  5. Daisuke Asanuma

    Department of Pharmacology, The University of Tokyo, Bunkyo-ku, Japan
    Competing interests
    The authors declare that no competing interests exist.

  6. Hirokazu Sakamoto

    Department of Pharmacology, The University of Tokyo, Bunkyo-ku, Japan
    Competing interests
    The authors declare that no competing interests exist.

  7. Shodai Takahashi

    Graduate School of Pharmaceutical Sciences, The University of Tokyo, Bunkyo-ku, Japan
    Competing interests
    The authors declare that no competing interests exist.

  8. Kenjiro Hanaoka

    Graduate School of Pharmaceutical Sciences, The University of Tokyo, Bunkyo-ku, Japan
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0003-0797-4038

  9. Yasuteru Urano

    Graduate School of Pharmaceutical Sciences, The University of Tokyo, Bunkyo-ku, Japan
    Competing interests
    The authors declare that no competing interests exist.

  10. Shigeyuki Namiki

    Department of Pharmacology, The University of Tokyo, Bunkyo-ku, Japan
    Competing interests
    The authors declare that no competing interests exist.

  11. Masamitsu Iino

    Cellular and Molecular Pharmacology, Nihon University School of Medicine, Tokyo, Japan
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0001-6426-4206

  12. Kenzo Hirose

    Department of Pharmacology, The University of Tokyo, Bunkyo-ku, Japan
    For correspondence
    kenzoh@m.u-tokyo.ac.jp
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0002-8944-6513

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.

Metrics

  • 10,574
    views
  • 1,195
    downloads
  • 43
    citations

Views, downloads and citations are aggregated across all versions of this paper published by eLife.

Download links

A two-part list of links to download the article, or parts of the article, in various formats.

Downloads (link to download the article as PDF)

Open citations (links to open the citations from this article in various online reference manager services)

Cite this article (links to download the citations from this article in formats compatible with various reference manager tools)

  1. Nami Kitajima
  2. Kenji Takikawa
  3. Hiroshi Sekiya
  4. Kaname Satoh
  5. Daisuke Asanuma
  6. Hirokazu Sakamoto
  7. Shodai Takahashi
  8. Kenjiro Hanaoka
  9. Yasuteru Urano
  10. Shigeyuki Namiki
  11. Masamitsu Iino
  12. Kenzo Hirose
(2020)
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

Share this article

https://doi.org/10.7554/eLife.57544

Categories and tags

Research organism

Further reading

    1. Biochemistry and Chemical Biology

    Disordered proteins interact with the chemical environment to tune their protective function during drying

    Shraddha KC, Kenny H Nguyen ... Thomas C Boothby
    Research Article

    The conformational ensemble and function of intrinsically disordered proteins (IDPs) are sensitive to their solution environment. The inherent malleability of disordered proteins, combined with the exposure of their residues, accounts for this sensitivity. One context in which IDPs play important roles that are concomitant with massive changes to the intracellular environment is during desiccation (extreme drying). The ability of organisms to survive desiccation has long been linked to the accumulation of high levels of cosolutes such as trehalose or sucrose as well as the enrichment of IDPs, such as late embryogenesis abundant (LEA) proteins or cytoplasmic abundant heat-soluble (CAHS) proteins. Despite knowing that IDPs play important roles and are co-enriched alongside endogenous, species-specific cosolutes during desiccation, little is known mechanistically about how IDP-cosolute interactions influence desiccation tolerance. Here, we test the notion that the protective function of desiccation-related IDPs is enhanced through conformational changes induced by endogenous cosolutes. We find that desiccation-related IDPs derived from four different organisms spanning two LEA protein families and the CAHS protein family synergize best with endogenous cosolutes during drying to promote desiccation protection. Yet the structural parameters of protective IDPs do not correlate with synergy for either CAHS or LEA proteins. We further demonstrate that for CAHS, but not LEA proteins, synergy is related to self-assembly and the formation of a gel. Our results suggest that functional synergy between IDPs and endogenous cosolutes is a convergent desiccation protection strategy seen among different IDP families and organisms, yet the mechanisms underlying this synergy differ between IDP families.

    1. Biochemistry and Chemical Biology
    2. Structural Biology and Molecular Biophysics

    Isobaric crosslinking mass spectrometry technology for studying conformational and structural changes in proteins and complexes

    Jie Luo, Jeff Ranish
    Tools and Resources

    Dynamic conformational and structural changes in proteins and protein complexes play a central and ubiquitous role in the regulation of protein function, yet it is very challenging to study these changes, especially for large protein complexes, under physiological conditions. Here, we introduce a novel isobaric crosslinker, Qlinker, for studying conformational and structural changes in proteins and protein complexes using quantitative crosslinking mass spectrometry. Qlinkers are small and simple, amine-reactive molecules with an optimal extended distance of ~10 Å, which use MS2 reporter ions for relative quantification of Qlinker-modified peptides derived from different samples. We synthesized the 2-plex Q2linker and showed that the Q2linker can provide quantitative crosslinking data that pinpoints key conformational and structural changes in biosensors, binary and ternary complexes composed of the general transcription factors TBP, TFIIA, and TFIIB, and RNA polymerase II complexes.

    1. Biochemistry and Chemical Biology
    2. Stem Cells and Regenerative Medicine

    Proteomic and functional comparison between human induced and embryonic stem cells

    Alejandro J Brenes, Eva Griesser ... Angus I Lamond
    Research Article

    Human induced pluripotent stem cells (hiPSCs) have great potential to be used as alternatives to embryonic stem cells (hESCs) in regenerative medicine and disease modelling. In this study, we characterise the proteomes of multiple hiPSC and hESC lines derived from independent donors and find that while they express a near-identical set of proteins, they show consistent quantitative differences in the abundance of a subset of proteins. hiPSCs have increased total protein content, while maintaining a comparable cell cycle profile to hESCs, with increased abundance of cytoplasmic and mitochondrial proteins required to sustain high growth rates, including nutrient transporters and metabolic proteins. Prominent changes detected in proteins involved in mitochondrial metabolism correlated with enhanced mitochondrial potential, shown using high-resolution respirometry. hiPSCs also produced higher levels of secreted proteins, including growth factors and proteins involved in the inhibition of the immune system. The data indicate that reprogramming of fibroblasts to hiPSCs produces important differences in cytoplasmic and mitochondrial proteins compared to hESCs, with consequences affecting growth and metabolism. This study improves our understanding of the molecular differences between hiPSCs and hESCs, with implications for potential risks and benefits for their use in future disease modelling and therapeutic applications.