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.