Rapid caspase-3 activation during apoptosis revealed using fluorescence-resonance energy transfer

Caspase‐3 is a crucial component of the apoptotic machinery in many cell types. Here, we report the timescale of caspase‐3 activation in single living cells undergoing apoptosis. This was achieved by measuring the extent of fluorescence resonance energy transfer within a recombinant substrate contai...

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Bibliographic Details
Published in:EMBO reports 2000-09, Vol.1 (3), p.266-270
Main Authors: Tyas, Lorraine, Brophy, Victoria A, Pope, Andrew, Rivett, A Jennifer, Tavaré, Jeremy M
Format: Article
Language:English
Subjects:
Animals
Apoptosis
Caspase 3
Caspases - metabolism
COS Cells
Energy transfer
Enzyme Activation
Fluorescence
Kinetics
Membrane Potentials
Microscopy, Fluorescence
Mitochondria - metabolism
Peptide Fragments - genetics
Peptide Fragments - metabolism
Poly(ADP-ribose) Polymerases - metabolism
Recombinant Fusion Proteins - genetics
Recombinant Fusion Proteins - metabolism
Resonance
Scientific Report
Scientific Reports
Spectrometry, Fluorescence
Staurosporine - pharmacology
Transfection
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Summary:Caspase‐3 is a crucial component of the apoptotic machinery in many cell types. Here, we report the timescale of caspase‐3 activation in single living cells undergoing apoptosis. This was achieved by measuring the extent of fluorescence resonance energy transfer within a recombinant substrate containing cyan fluorescent protein (CFP) linked by a short peptide possessing the caspase‐3 cleavage sequence, DEVD, to yellow fluorescent protein (YFP; i.e. CFP– DEVD –YFP). We demonstrate that, once initiated, the activation of caspase‐3 is a very rapid process, taking 5 min or less to reach completion. Furthermore, this process occurs almost simultaneously with a depolarization of the mitochondrial membrane potential. These events occur just prior to the characteristic morphological changes associated with apoptosis. Our results clearly demonstrate that, once initiated, the commitment of cells to apoptosis is a remarkably rapid event when visualized at the single cell level.
ISSN:1469-221X
1469-3178
DOI:10.1093/embo-reports/kvd050