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Research Article
Developing fluorescence sensor probe to capture activated muscle-specific calpain-3 (CAPN3) in living muscle cells
Koichi Ojima, Shoji Hata, Fumiko Shinkai-Ouchi, Mika Oe, Susumu Muroya, Hiroyuki Sorimachi, Yasuko Ono
Biology Open 2020 9: bio048975 doi: 10.1242/bio.048975 Published 4 September 2020
Koichi Ojima
1Muscle Biology Research Unit, Division of Animal Products Research, Institute of Livestock and Grassland Science, NARO, 305-0901 Tsukuba, Japan
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  • ORCID record for Koichi Ojima
  • For correspondence: koojima@affrc.go.jp
Shoji Hata
2Calpain Project, Tokyo Metropolitan Institute of Medical Science, 156-8506 Tokyo, Japan
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Fumiko Shinkai-Ouchi
2Calpain Project, Tokyo Metropolitan Institute of Medical Science, 156-8506 Tokyo, Japan
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Mika Oe
1Muscle Biology Research Unit, Division of Animal Products Research, Institute of Livestock and Grassland Science, NARO, 305-0901 Tsukuba, Japan
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Susumu Muroya
1Muscle Biology Research Unit, Division of Animal Products Research, Institute of Livestock and Grassland Science, NARO, 305-0901 Tsukuba, Japan
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Hiroyuki Sorimachi
2Calpain Project, Tokyo Metropolitan Institute of Medical Science, 156-8506 Tokyo, Japan
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Yasuko Ono
2Calpain Project, Tokyo Metropolitan Institute of Medical Science, 156-8506 Tokyo, Japan
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ABSTRACT

Calpain-3 (CAPN3) is a muscle-specific type of calpain whose protease activity is triggered by Ca2+. Here, we developed CAPN3 sensor probes (SPs) to detect activated-CAPN3 using a fluorescence/Förster resonance energy transfer (FRET) technique. In our SPs, partial amino acid sequence of calpastatin, endogenous CAPN inhibitor but CAPN3 substrate, is inserted between two different fluorescence proteins that cause FRET. Biochemical and spectral studies revealed that CAPN3 cleaved SPs and changed emission wavelengths of SPs. Importantly, SPs were scarcely cleaved by CAPN1 and CAPN2. Furthermore, our SP successfully captured the activation of endogenous CAPN3 in living myotubes treated with ouabain. Our SPs would become a promising tool to detect the dynamics of CAPN3 protease activity in living cells.

Footnotes

  • Competing interests

    The authors declare no competing or financial interests.

  • Author contributions

    Conceptualization: K.O., H.S., Y.O.; Methodology: K.O., H.S., Y.O.; Validation: K.O.; Formal analysis: K.O.; Investigation: K.O., S.H., F.S.-O., M.O., S.M.; Resources: K.O., S.H., F.S.-O., H.S., Y.O.; Data curation: K.O., S.H., F.S.-O., M.O., S.M., Y.O.; Writing - original draft: K.O., Y.O.; Writing - review & editing: K.O., Y.O.; Project administration: K.O.; Funding acquisition: K.O.

  • Funding

    This work was supported in part by the Japan Society for the Promotion of Science KAKENHI [no.23500477 and 19H04014] and Naito Foundation to K.O.

  • Received October 24, 2019.
  • Accepted July 28, 2020.
  • © 2020. Published by The Company of Biologists Ltd
http://creativecommons.org/licenses/by/4.0

This is an Open Access article distributed under the terms of the Creative Commons Attribution License (https://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution and reproduction in any medium provided that the original work is properly attributed.

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Keywords

  • Calpain
  • Calpain-3
  • Skeletal muscle
  • Calpainopathy
  • Limb-girdle muscular dystrophy type 2A
  • Proteolysis

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Research Article
Developing fluorescence sensor probe to capture activated muscle-specific calpain-3 (CAPN3) in living muscle cells
Koichi Ojima, Shoji Hata, Fumiko Shinkai-Ouchi, Mika Oe, Susumu Muroya, Hiroyuki Sorimachi, Yasuko Ono
Biology Open 2020 9: bio048975 doi: 10.1242/bio.048975 Published 4 September 2020
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Research Article
Developing fluorescence sensor probe to capture activated muscle-specific calpain-3 (CAPN3) in living muscle cells
Koichi Ojima, Shoji Hata, Fumiko Shinkai-Ouchi, Mika Oe, Susumu Muroya, Hiroyuki Sorimachi, Yasuko Ono
Biology Open 2020 9: bio048975 doi: 10.1242/bio.048975 Published 4 September 2020

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