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

Takemoto K, Nagai T, Miyawaki A, Miura M - J. Cell Biol. (2003)

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: Laboratory for Cell Recovery Mechanisms, Advanced Technology Development Center, RIKEN Brain Science Institute, Wako, Saitama 351-0198, Japan.

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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Specificity of SCAT9 for activated caspase-9 in apoptotic HeLa cell extract. (A) In vitro cleavage analysis of SCAT9. SCAT9 synthesized in vitro was cleaved by purified activated caspase-3, -6, -8, or -9 for 1 h. The reaction mixture was then subjected to Western blotting using an anti-myc mAb. (B) Immunodepletion of caspase-9 from dATP/cytochrome c activated apoptotic HeLa extract. Immunodepleted extracts (18 μg) were subjected to Western blotting using an anti-caspase-9 mouse mAb. Both the precursor and activated forms of caspase-9 could be depleted from the extracts. (C) Cleavage assay of SCAT9 in caspase-9–depleted apoptotic extracts. SCAT9 synthesized in vitro was incubated with 18 μg caspase-9–depleted apoptotic extracts for the indicated periods. The reacted lysates were then examined by Western blotting using an anti-myc antibody.
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fig6: Specificity of SCAT9 for activated caspase-9 in apoptotic HeLa cell extract. (A) In vitro cleavage analysis of SCAT9. SCAT9 synthesized in vitro was cleaved by purified activated caspase-3, -6, -8, or -9 for 1 h. The reaction mixture was then subjected to Western blotting using an anti-myc mAb. (B) Immunodepletion of caspase-9 from dATP/cytochrome c activated apoptotic HeLa extract. Immunodepleted extracts (18 μg) were subjected to Western blotting using an anti-caspase-9 mouse mAb. Both the precursor and activated forms of caspase-9 could be depleted from the extracts. (C) Cleavage assay of SCAT9 in caspase-9–depleted apoptotic extracts. SCAT9 synthesized in vitro was incubated with 18 μg caspase-9–depleted apoptotic extracts for the indicated periods. The reacted lysates were then examined by Western blotting using an anti-myc antibody.

Mentions: The caspase-9–mediated mitochondrial pathway is critical for the physiological apoptosis that occurs in normal development (Cecconi et al., 1998; Hakem et al., 1998; Kuida et al., 1998; Yoshida et al., 1998). To examine the caspase-9 activation by real-time imaging analysis, we constructed SCAT9, which contains a caspase-9 cleavage-site sequence, LEHD, in its linker region. To test whether SCAT9 could be cleaved specifically by caspase-9, the SCAT9 protein was generated in vitro and incubated with recombinant activated caspase-3, caspase-6, caspase-8, and caspase-9. SCAT9 was cleaved by caspase-8 and caspase-9 with the same efficiency but not by caspase-3 or caspase-6 (Fig. 6 A). We then first examined SCAT9 specificity in TNF-α/CHX-treated HeLa cell lysate. Because of the weak enzymatic activities of caspase-9 in apoptotic extracts from TNF-α/CHX-treated HeLa cells, only the small amount of SCAT9 cleavage was observed in this lysate. In stead, we examined its specificity in apoptotic HeLa extract induced by cytochrome c and dATP. Most of pro- and activated caspase-9 was immunodepleted from apoptotic lysate (Fig. 6 B), and SCAT9 cleavage was examined using caspase-9 depleted lysate. SCAT9 was cleaved in the apoptotic lysates but its cleavage was significantly but not completely blocked by the immunodepletion of caspase-9 (Fig. 6 C). Because we could detect a small amount of caspase-9 in the caspase-9–depleted lysates, it is possible that residual caspase-9 cleaved the SCAT9 in the caspase-9–depleted apoptotic lysate. However, we cannot exclude the possibility that other caspases were involved in this cleavage.


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

Takemoto K, Nagai T, Miyawaki A, Miura M - J. Cell Biol. (2003)

Specificity of SCAT9 for activated caspase-9 in apoptotic HeLa cell extract. (A) In vitro cleavage analysis of SCAT9. SCAT9 synthesized in vitro was cleaved by purified activated caspase-3, -6, -8, or -9 for 1 h. The reaction mixture was then subjected to Western blotting using an anti-myc mAb. (B) Immunodepletion of caspase-9 from dATP/cytochrome c activated apoptotic HeLa extract. Immunodepleted extracts (18 μg) were subjected to Western blotting using an anti-caspase-9 mouse mAb. Both the precursor and activated forms of caspase-9 could be depleted from the extracts. (C) Cleavage assay of SCAT9 in caspase-9–depleted apoptotic extracts. SCAT9 synthesized in vitro was incubated with 18 μg caspase-9–depleted apoptotic extracts for the indicated periods. The reacted lysates were then examined by Western blotting using an anti-myc antibody.
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fig6: Specificity of SCAT9 for activated caspase-9 in apoptotic HeLa cell extract. (A) In vitro cleavage analysis of SCAT9. SCAT9 synthesized in vitro was cleaved by purified activated caspase-3, -6, -8, or -9 for 1 h. The reaction mixture was then subjected to Western blotting using an anti-myc mAb. (B) Immunodepletion of caspase-9 from dATP/cytochrome c activated apoptotic HeLa extract. Immunodepleted extracts (18 μg) were subjected to Western blotting using an anti-caspase-9 mouse mAb. Both the precursor and activated forms of caspase-9 could be depleted from the extracts. (C) Cleavage assay of SCAT9 in caspase-9–depleted apoptotic extracts. SCAT9 synthesized in vitro was incubated with 18 μg caspase-9–depleted apoptotic extracts for the indicated periods. The reacted lysates were then examined by Western blotting using an anti-myc antibody.
Mentions: The caspase-9–mediated mitochondrial pathway is critical for the physiological apoptosis that occurs in normal development (Cecconi et al., 1998; Hakem et al., 1998; Kuida et al., 1998; Yoshida et al., 1998). To examine the caspase-9 activation by real-time imaging analysis, we constructed SCAT9, which contains a caspase-9 cleavage-site sequence, LEHD, in its linker region. To test whether SCAT9 could be cleaved specifically by caspase-9, the SCAT9 protein was generated in vitro and incubated with recombinant activated caspase-3, caspase-6, caspase-8, and caspase-9. SCAT9 was cleaved by caspase-8 and caspase-9 with the same efficiency but not by caspase-3 or caspase-6 (Fig. 6 A). We then first examined SCAT9 specificity in TNF-α/CHX-treated HeLa cell lysate. Because of the weak enzymatic activities of caspase-9 in apoptotic extracts from TNF-α/CHX-treated HeLa cells, only the small amount of SCAT9 cleavage was observed in this lysate. In stead, we examined its specificity in apoptotic HeLa extract induced by cytochrome c and dATP. Most of pro- and activated caspase-9 was immunodepleted from apoptotic lysate (Fig. 6 B), and SCAT9 cleavage was examined using caspase-9 depleted lysate. SCAT9 was cleaved in the apoptotic lysates but its cleavage was significantly but not completely blocked by the immunodepletion of caspase-9 (Fig. 6 C). Because we could detect a small amount of caspase-9 in the caspase-9–depleted lysates, it is possible that residual caspase-9 cleaved the SCAT9 in the caspase-9–depleted apoptotic lysate. However, we cannot exclude the possibility that other caspases were involved in this cleavage.

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: Laboratory for Cell Recovery Mechanisms, Advanced Technology Development Center, RIKEN Brain Science Institute, Wako, Saitama 351-0198, Japan.

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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