title: Live cell detection of caspase-3 activation by a Discosoma-red-fluorescent-protein-based fluorescence resonance energy transfer construct.
abstract: A probe consisting of Discosoma red fluorescent protein (DsRed) and enhanced yellow fluorescent protein (EYFP) linked by a 19-amino-acid chain containing the caspase-3 cleavage site Asp-Glu-Val-Asp was developed to monitor caspase-3 activation in living cells. The expression of the tandem construct in mammalian cells yielded a strong red fluorescence when excited with 450- to 490-nm light or with a 488-nm argon ion laser line as a result of fluorescence resonance energy transfer (FRET) from donor EYFP to acceptor DsRed. The advantage over previous constructs using cyan fluorescent protein is that our construct can be used when excitation wavelengths lower than 488nm are not available. To validate the construct, murine HT-22 hippocampal neuronal cells were triggered to undergo CD95-induced neuronal death. An increase in caspase-3 activity was demonstrated by a reduction of FRET in cells transfected with the construct. This was manifested by a dequenching of EYFP fluorescence leading to an increase in EYFP emission and a corresponding decrease in DsRed fluorescence, which correlated with an increase in pro-caspase-3 processing. We conclude that CD95-induced caspase-3 activation in HT-22 cells was readily detected at the single-cell level using the DsRed-EYFP-based FRET construct, making this a useful technology to monitor caspase-3 activity in living cells.
authors.affiliation: School of Biomedical and Molecular Sciences, University of Surrey, Guildford GU2 7XH, UK.
authors.forename: Lucy M
authors.initials: LM
authors.lastname: Elphick
authors.forename: Annika
authors.initials: A
authors.lastname: Meinander
authors.forename: Andrey
authors.initials: A
authors.lastname: Mikhailov
authors.forename: Morgiane
authors.initials: M
authors.lastname: Richard
authors.forename: Nick J
authors.initials: NJ
authors.lastname: Toms
authors.forename: John E
authors.initials: JE
authors.lastname: Eriksson
authors.forename: George E N
authors.initials: GE
authors.lastname: Kass
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D017209:Apoptosis / Q000502:physiology; D053148:Caspase 3 / Q000032:analysis*; D002460:Cell Line; D004789:Enzyme Activation / Q000502:physiology; D031541:Fluorescence Resonance Energy Transfer / Q000295:instrumentation / Q000379:methods; D008164:Luminescent Proteins; D051379:Mice; D008969:Molecular Sequence Data; D019014:fas Receptor / Q000378:metabolism
chemical_list: D008164:Luminescent Proteins; D019014:fas Receptor; C413663:fluorescent protein 583; D053148:Caspase 3