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Published on: June 7, 2019
Imaging calpain protease activity by multiphoton FRET in living mice
Daniel Stockholm1, Marc Bartoli, Guillaume Sillon
1Généthon, Centre National de la Recherche Scientifique UMR 8115, 1 bis rue de l'Internationale, 91000 Evry, France.
Abstract:
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.
Insights
Researchers developed a new method combining multiphoton microscopy and fluorescence resonance energy transfer (FRET) imaging to monitor calpain activity in living mouse muscle. This breakthrough allows quantitative analysis of calpain activation in vivo.
Area of Science:
- Molecular Imaging
- Biophysics
- Proteolysis
Background:
- Fluorescence resonance energy transfer (FRET) methods have successfully resolved molecular processes in cultured cells.
- However, FRET has not been previously combined with 3D intravital imaging for in vivo studies.
- Multiphoton microscopy (MPM) is a preferred technique for non-destructive investigation of living tissues.
Purpose of the Study:
- To evaluate the combination of MPM and FRET imaging for monitoring calpain proteolytic activity in living mouse muscle.
- To establish a method for quantitative, real-time assessment of calpain activation in vivo.
Main Methods:
- Utilized multiphoton microscopy (MPM) for intravital imaging of living mouse muscle.
- Employed fluorescence resonance energy transfer (FRET) imaging with a specific indicator to detect calpain activity.
- Monitored calpain activation kinetics following calcium influx at the cellular level.
Main Results:
- Successfully demonstrated the combined MPM-FRET approach for monitoring calpain activity in living mouse tissues.
- Showcased the efficient and quantitative tracking of calpain activation kinetics in vivo.
- Visualized calpain activity at the cellular level in response to calcium influx.
Conclusions:
- The developed MPM-FRET method enables visualization and quantitative monitoring of calpain activity in living tissues.
- This technique provides a powerful tool to investigate calpain's biological functions and roles in degenerative conditions.
- Offers a platform for evaluating the therapeutic efficacy of calpain inhibitors in vivo.
