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

Abstract

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.

On-Line Monitoring of Apoptosis in Insulin-Secreting Cells

“…For further development of the method, the ␤-cell line RINm5F was used. For shortening the time for on-line experiments, staurosporine was used as a well-known stimulator of apoptosis (24). Figure 1C shows that incubation of RINm5F cells with 6 mol/l staurosporine for only 3 h gave rise to a DNA laddering pattern typical for apoptosis.…”

Cell death: A dynamic response concept

“…This approach may be aided by: (i) increased utilization of live cell imaging techniques which report the dynamics of dye exclusion between the onset of a leaky membrane up to the loss of its integrity during necrosis or (ii) assessment of the time scale of caspase activation through fluorescence resonance energy transfer (FRET) microscopy. 45,46 To allow increased understanding of such dynamic events on a cell population level, this approach may be strengthened by data generated from living cells with fluorescence-activated cell sorting, particularly when tandem dyes are available. 47 …”

Plant caspase-like proteases in plant programmed cell death

“…26 During the process of staurosporine-induced apoptosis of COS-7 cells, FRET technique has been successfully employed to reveal the timescale of the activation of capase-3 at the single cell level by measuring the extent of FRET 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). 27 Likewise, FRET has been successfully used to detect caspase 1, caspase 7, caspase 8 and caspase 9 activation during apoptosis in animal cell induced by different stimulis. 28 Excitingly, a FRET probe to monitor caspase-3-like proteases activation in single living plant cells in real time has been constructed in Xing's laboratory.…”