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- Title
Spatio-temporal activation of caspase revealed by indicator that is sensitive to environmental effects.
- Authors
Takemoto, Kiwamu; Nagai, Takeharu; Miyawaki, Atsushi; Miura, Masayuki
- Abstract
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[sup +]) and chloride ion (Cl[sup -])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[sup +]- and CI[sup -]-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 cytosoi 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.
- Subjects
ENZYME activation; PROTEINS; APOPTOSIS
- Publication
Journal of Cell Biology, 2003, Vol 160, Issue 2, p235
- ISSN
0021-9525
- Publication type
Article
- DOI
10.1083/jcb.200207111