PT  - JOURNAL ARTICLE
AU  - Ojima, Koichi
AU  - Hata, Shoji
AU  - Shinkai-Ouchi, Fumiko
AU  - Oe, Mika
AU  - Muroya, Susumu
AU  - Sorimachi, Hiroyuki
AU  - Ono, Yasuko
TI  - Developing fluorescence sensor probe to capture activated muscle-specific calpain-3 (CAPN3) in living muscle cells
AID  - 10.1242/bio.048975
DP  - 2020 Sep 15
TA  - Biology Open
PG  - bio048975
VI  - 9
IP  - 9
4099  - http://bio.biologists.org/content/9/9/bio048975.short
4100  - http://bio.biologists.org/content/9/9/bio048975.full
SO  - Biology Open2020 Sep 15; 9
AB  - 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.