Apoptosis plays an important role in many physiological and
pathological processes. The initiation and execution of the cell death
program requires activation of multiple caspases in a stringently
temporal order. Here we describe a method that allows real-time
observation of caspase activation in situ in live cells based on
fluorescent resonance energy transfer (FRET) measurement using the
prism and reflector imaging spectroscopy system (PARISS). When a fusion
protein consisting of CFP connected to YFP via an intervening caspase
substrate that has been targeted to a specific subcellular location is
excited with a light source whose wavelength matches the cyan
fluorescent protein (CFP) excitation peak, the energy absorbed by the
CFP fluorophore is not emitted as fluorescence. Instead, the excitation
energy is absorbed by the nearby yellow fluorescent protein (YFP)
fluorophore that is covalently linked to CFP through a short peptide
containing the caspase substrate. Cleavage of the linker peptide by
caspases results in loss of FRET due to the separation of CFP and YFP
fluorophores. Using a mitochondrially targeted CFP–caspase 3
substrate–YFP construct (mC3Y), we demonstrate for the first time
that there is caspase-3-like activity in the mitochondrial matrix of
some cells at very late stage of apoptosis.
Fluorogenic Substrates for In Situ Monitoring of Caspase-3 Activity in Live Cells
