Live cell detection of caspase-3 activation by a Discosoma-red-fluorescent-protein-based fluorescence resonance energy transfer construct

LM Elphick, A Meinander, A Mikhailov, M Richard, NJ Toms, JE Eriksson, GEN Kass*

*Corresponding author for this work

    Research output: Contribution to journalArticle

    15 Citations (Scopus)

    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. (c) 2005 Elsevier Inc. All rights reserved.

    Original languageEnglish
    Pages (from-to)148-155
    Number of pages8
    JournalAnalytical Biochemistry
    Volume349
    Issue number1
    DOIs
    Publication statusPublished - 1 Feb 2006

    Keywords

    • green fluorescent protein
    • FRET
    • apoptosis
    • CD95
    • ischemia
    • cytochrome c
    • caspase
    • ACETAMINOPHEN-INDUCED APOPTOSIS
    • FOCAL CEREBRAL-ISCHEMIA
    • CYTOCHROME-C RELEASE
    • DEATH RECEPTOR
    • GRANZYME-B
    • RAT-BRAIN
    • FAS
    • EXPRESSION
    • BAX
    • FAMILY

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