Genetically encoded far-red fluorescent sensors for caspase-3 activity

Area of Science:

• Biochemistry
• Cell Biology
• Molecular Imaging

Background:

• Caspase-3 is a crucial enzyme in apoptosis, but existing fluorescent sensors have limitations for advanced imaging techniques.
• Current sensors operate at shorter wavelengths, hindering multiparameter microscopy and whole-body imaging applications.

Purpose of the Study:

• To develop genetically encoded fluorescent sensors for caspase-3 activity with red-shifted spectra for improved imaging.
• To assess the performance of these novel sensors in mammalian cell apoptosis models.

Main Methods:

• Development of Förster resonance energy transfer (FRET) based sensors using far-red and infrared fluorescent proteins linked by a caspase-3 cleavage site (DEVD).
• Testing of mKate2-DEVD-iRFP and eqFP650-DEVD-iRFP sensors in staurosporine-induced apoptosis in HeLa and CT26 cells.
• Utilizing fluorescence lifetime imaging (FLIM) to detect caspase-3 activation and performing simultaneous imaging with EGFP-Bax translocation.

Main Results:

• Both developed sensors demonstrated robust responses to caspase-3 activation, indicated by increased donor fluorescence intensity.
• The mKate2-DEVD-iRFP sensor exhibited suitability for multiparameter imaging and showed potential for in vivo applications.
• Fluorescence lifetime imaging successfully detected caspase-3 activation using the mKate2-DEVD-iRFP sensor.

Conclusions:

• The novel red-shifted caspase-3 sensors, particularly mKate2-DEVD-iRFP, overcome limitations of existing tools.
• These sensors are well-suited for advanced imaging techniques, including multiparameter and in vivo imaging of apoptosis.
• The developed sensors represent a significant advancement for studying apoptosis in complex biological systems.