Spatio-temporal activation of caspase revealed by indicator that is insensitive to environmental effects

J Cell Biol. 2003 Jan 20;160(2):235-43. doi: 10.1083/jcb.200207111. Epub 2003 Jan 13.


Indicator molecules for caspase-3 activation have been reported that use fluorescence resonance energy transfer (FRET) between an enhanced cyan fluorescent protein (the donor) and enhanced yellow fluorescent protein (EYFP; the acceptor). Because EYFP is highly sensitive to proton (H+) and chloride ion (Cl-) levels, which can change during apoptosis, this indicator's ability to trace the precise dynamics of caspase activation is limited, especially in vivo. Here, we generated an H+- and Cl--insensitive indicator for caspase activation, SCAT, in which EYFP was replaced with Venus, and monitored the spatio-temporal activation of caspases in living cells. Caspase-3 activation was initiated first in the cytosol and then in the nucleus, and rapidly reached maximum activation in 10 min or less. Furthermore, the nuclear activation of caspase-3 preceded the nuclear apoptotic morphological changes. In contrast, the completion of caspase-9 activation took much longer and its activation was attenuated in the nucleus. However, the time between the initiation of caspase-9 activation and the morphological changes was quite similar to that seen for caspase-3, indicating the activation of both caspases occurred essentially simultaneously during the initiation of apoptosis.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Apoptosis / drug effects
  • Apoptosis / physiology*
  • Caspase 3
  • Caspase 9
  • Caspases / metabolism*
  • Cell Extracts / pharmacology
  • Cell Nucleus / drug effects
  • Cell Nucleus / enzymology*
  • Chlorides / metabolism
  • Cytosol / drug effects
  • Cytosol / enzymology
  • Eukaryotic Cells / drug effects
  • Eukaryotic Cells / enzymology*
  • Fluorescence Resonance Energy Transfer
  • Green Fluorescent Proteins
  • HeLa Cells
  • Humans
  • Hydrogen-Ion Concentration
  • Indicators and Reagents / chemistry
  • Luminescent Proteins
  • Microscopy, Video
  • Tumor Necrosis Factor-alpha / pharmacology


  • Cell Extracts
  • Chlorides
  • Indicators and Reagents
  • Luminescent Proteins
  • Tumor Necrosis Factor-alpha
  • Green Fluorescent Proteins
  • CASP3 protein, human
  • CASP9 protein, human
  • Caspase 3
  • Caspase 9
  • Caspases