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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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The specificity of SCAT3 for activated caspase-3 in TNF-α/CHX-treated HeLa cell lysates. (A) In vitro cleavage analysis of SCAT3. SCAT3 synthesized in vitro was incubated with 1 U of purified activated caspase-3, -6, -8, or -9 for 1 h. The reaction mixture was subjected to Western blotting using an anti-myc mAb. (B) Immunodepletion of caspase-3 from TNF-α/CHX-treated HeLa cell lysates. 20 μg of immunodepleted lysates were subjected to Western blotting using an anti–caspase-3 rabbit pAb (Invitrogen). (C) Cleavage assay of SCAT3 in caspase-3–depleted lysates. SCAT3 synthesized in vitro was incubated with 12 μg caspase-3–depleted lysates prepared from TNF-α/CHX-treated HeLa cells for the indicated periods, and then its cleavage was examined by Western blotting using an anti-myc antibody.
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fig3: The specificity of SCAT3 for activated caspase-3 in TNF-α/CHX-treated HeLa cell lysates. (A) In vitro cleavage analysis of SCAT3. SCAT3 synthesized in vitro was incubated with 1 U of purified activated caspase-3, -6, -8, or -9 for 1 h. The reaction mixture was subjected to Western blotting using an anti-myc mAb. (B) Immunodepletion of caspase-3 from TNF-α/CHX-treated HeLa cell lysates. 20 μg of immunodepleted lysates were subjected to Western blotting using an anti–caspase-3 rabbit pAb (Invitrogen). (C) Cleavage assay of SCAT3 in caspase-3–depleted lysates. SCAT3 synthesized in vitro was incubated with 12 μg caspase-3–depleted lysates prepared from TNF-α/CHX-treated HeLa cells for the indicated periods, and then its cleavage was examined by Western blotting using an anti-myc antibody.

Mentions: Next, we examined the specificity of SCAT3 for activated caspases in vitro. In vitro-synthesized SCAT3 protein was incubated with several activated caspases (Fig. 3 A). It was efficiently cleaved by caspase-3 and partially cleaved by caspase-8 and caspase-9 in vitro. In contrast, little SCAT3 was cleaved by caspase-6. To investigate the specificity of SCAT3 in the TNF treated cell lysates, we performed immunodepletion experiments to remove caspase-3 from TNF-α/CHX-treated HeLa cell lysates. Caspase-3 activation was observed at TNF-α/CHX-treated lysates (Fig. 3 B), then caspase-3 precursor and its activated form were depleted from apoptotic lysates by an anti-caspase-3 antibody. As a control, we incubated TNF-α/CHX-treated lysates with an anti-GFP antibody. We then examined SCAT3 cleavage in these extracts. SCAT3 was effectively cleaved in the control TNF-α/CHX-treated lysates (whether or not they had been treated with the GFP antibody). On the other hand, only a small portion of the SCAT3 was cleaved in the caspase-3–depleted lysates. These results indicated that most of the SCAT3 cleavage in TNF-α/CHX-treated HeLa cell lysates was done by caspase-3.


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)

The specificity of SCAT3 for activated caspase-3 in TNF-α/CHX-treated HeLa cell lysates. (A) In vitro cleavage analysis of SCAT3. SCAT3 synthesized in vitro was incubated with 1 U of purified activated caspase-3, -6, -8, or -9 for 1 h. The reaction mixture was subjected to Western blotting using an anti-myc mAb. (B) Immunodepletion of caspase-3 from TNF-α/CHX-treated HeLa cell lysates. 20 μg of immunodepleted lysates were subjected to Western blotting using an anti–caspase-3 rabbit pAb (Invitrogen). (C) Cleavage assay of SCAT3 in caspase-3–depleted lysates. SCAT3 synthesized in vitro was incubated with 12 μg caspase-3–depleted lysates prepared from TNF-α/CHX-treated HeLa cells for the indicated periods, and then its cleavage was examined by Western blotting using an anti-myc antibody.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2172647&req=5

fig3: The specificity of SCAT3 for activated caspase-3 in TNF-α/CHX-treated HeLa cell lysates. (A) In vitro cleavage analysis of SCAT3. SCAT3 synthesized in vitro was incubated with 1 U of purified activated caspase-3, -6, -8, or -9 for 1 h. The reaction mixture was subjected to Western blotting using an anti-myc mAb. (B) Immunodepletion of caspase-3 from TNF-α/CHX-treated HeLa cell lysates. 20 μg of immunodepleted lysates were subjected to Western blotting using an anti–caspase-3 rabbit pAb (Invitrogen). (C) Cleavage assay of SCAT3 in caspase-3–depleted lysates. SCAT3 synthesized in vitro was incubated with 12 μg caspase-3–depleted lysates prepared from TNF-α/CHX-treated HeLa cells for the indicated periods, and then its cleavage was examined by Western blotting using an anti-myc antibody.
Mentions: Next, we examined the specificity of SCAT3 for activated caspases in vitro. In vitro-synthesized SCAT3 protein was incubated with several activated caspases (Fig. 3 A). It was efficiently cleaved by caspase-3 and partially cleaved by caspase-8 and caspase-9 in vitro. In contrast, little SCAT3 was cleaved by caspase-6. To investigate the specificity of SCAT3 in the TNF treated cell lysates, we performed immunodepletion experiments to remove caspase-3 from TNF-α/CHX-treated HeLa cell lysates. Caspase-3 activation was observed at TNF-α/CHX-treated lysates (Fig. 3 B), then caspase-3 precursor and its activated form were depleted from apoptotic lysates by an anti-caspase-3 antibody. As a control, we incubated TNF-α/CHX-treated lysates with an anti-GFP antibody. We then examined SCAT3 cleavage in these extracts. SCAT3 was effectively cleaved in the control TNF-α/CHX-treated lysates (whether or not they had been treated with the GFP antibody). On the other hand, only a small portion of the SCAT3 was cleaved in the caspase-3–depleted lysates. These results indicated that most of the SCAT3 cleavage in TNF-α/CHX-treated HeLa cell lysates was done by caspase-3.

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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