Spatio-temporal activation of caspase revealed by indicator that is insensitive to environmental effects |
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Authors: | Takemoto Kiwamu Nagai Takeharu Miyawaki Atsushi Miura Masayuki |
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Affiliation: | Laboratory for Cell Recovery Mechanisms, Advanced Technology Development Center, RIKEN Brain Science Institute, Wako, Saitama 351-0198, Japan. |
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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+) 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. |
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Keywords: | FRET caspase-3 caspase-9 nuclear activation of caspase-3 apoptosis |
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