Spatio-temporal activation of caspase revealed by indicator that is insensitive to environmental effects.
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.
Takemoto
Kiwamu
K
Laboratory for Cell Recovery Mechanisms, Advanced Technology Development Center, RIKEN Brain Science Institute, Wako, Saitama 351-0198, Japan.
Nagai
Takeharu
T
Miyawaki
Atsushi
A
Miura
Masayuki
M
D017209:Apoptosis / Q000187:drug effects / Q000502:physiology*; D053148:Caspase 3; D053453:Caspase 9; D020169:Caspases / Q000378:metabolism*; D002457:Cell Extracts / Q000494:pharmacology; D002467:Cell Nucleus / Q000187:drug effects / Q000201:enzymology*; D002712:Chlorides / Q000378:metabolism; D003600:Cytosol / Q000187:drug effects / Q000201:enzymology; D005057:Eukaryotic Cells / Q000187:drug effects / Q000201:enzymology*; D031541:Fluorescence Resonance Energy Transfer; D049452:Green Fluorescent Proteins; D006367:HeLa Cells; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007202:Indicators and Reagents / Q000737:chemistry; D008164:Luminescent Proteins; D018715:Microscopy, Video; D014409:Tumor Necrosis Factor-alpha / Q000494:pharmacology
D002457:Cell Extracts; D002712:Chlorides; D007202:Indicators and Reagents; D008164:Luminescent Proteins; D014409:Tumor Necrosis Factor-alpha; D049452:Green Fluorescent Proteins; C505407:CASP3 protein, human; C506433:CASP9 protein, human; D053148:Caspase 3; D053453:Caspase 9; D020169:Caspases