Imaging calpain protease activity by multiphoton FRET in living mice.
Constant efforts are ongoing for the development of new imaging methods that allow the investigation of molecular processes in vivo. Protein-protein interactions, enzymatic activities and intracellular Ca2+ fluxes, have been resolved in cultured cells using a variety of fluorescence resonance energy transfer (FRET) detection methods. However, FRET has not been used so far in conjunction with 3D intravital imaging. We evaluated here a combination of multiphoton microscopy (MPM), method of choice for non-destructive living tissue investigation, and FRET imaging to monitor calpain proteolytic activity in living mice muscle. We show that kinetics of ubiquitous calpains activation can be efficiently and quantitatively monitored in living mouse tissues at cellular level with a FRET-based indicator upon calcium influx. The ability to visualize calpain activity in living tissue offers a unique opportunity to challenge remaining questions on the biological functions of calpains and to evaluate the therapeutic potential of calpain inhibitors in many degenerative conditions.
Stockholm
Daniel
D
Généthon, Centre National de la Recherche Scientifique UMR 8115, 1 bis rue de l'Internationale, 91000 Evry, France.
Bartoli
Marc
M
Sillon
Guillaume
G
Bourg
Nathalie
N
Davoust
Jean
J
Richard
Isabelle
I
D000818:Animals; D002118:Calcium / Q000494:pharmacology; D002138:Calibration; D002154:Calpain / Q000378:metabolism*; D031541:Fluorescence Resonance Energy Transfer / Q000295:instrumentation* / Q000379:methods*; D051379:Mice; D009132:Muscles / Q000187:drug effects / Q000201:enzymology; D017785:Photons
D002154:Calpain; D002118:Calcium