RT Journal Article
SR Electronic
T1 Developing fluorescence sensor probe to capture activated muscle-specific calpain-3 (CAPN3) in living muscle cells
JF Biology Open
JO Biology Open
FD Company of Biologists
SP bio.048975
DO 10.1242/bio.048975
A1 Ojima, Koichi
A1 Hata, Shoji
A1 Shinkai-Ouchi, Fumiko
A1 Oe, Mika
A1 Muroya, Susumu
A1 Sorimachi, Hiroyuki
A1 Ono, Yasuko
YR 2020
UL http://bio.biologists.org/content/early/2020/08/13/bio.048975.abstract
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 which 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.