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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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Single-cell imaging analysis of SCAT3-expressing living HeLa cells. (A) Western blot analysis of SCAT3-expressing HeLa cells exposed to TNF-α/CHX. HeLa cells in a 6-well plate were transfected with 1 μg pcDNA-SCAT3. Cells were exposed to 50 ng/ml TNF-α and 10 μg/ml CHX 18 h after transfection. The cells were then lysed with sample buffer at the indicated times. The lysates were examined by Western blotting using an anti-myc antibody. (B) Ratio images of the SCAT3- expressing cells. HeLa cells were transfected with 0.5 μg pcDNA-SCAT3. Imaging analysis was started 18 h after transfection. (C) Venus/ECFP emission ratio changes of individual cells examined in B. Arrowheads indicate the time cells first showed the early apoptotic cell death morphology, including membrane blebbing and cell shrinkage.
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fig4: Single-cell imaging analysis of SCAT3-expressing living HeLa cells. (A) Western blot analysis of SCAT3-expressing HeLa cells exposed to TNF-α/CHX. HeLa cells in a 6-well plate were transfected with 1 μg pcDNA-SCAT3. Cells were exposed to 50 ng/ml TNF-α and 10 μg/ml CHX 18 h after transfection. The cells were then lysed with sample buffer at the indicated times. The lysates were examined by Western blotting using an anti-myc antibody. (B) Ratio images of the SCAT3- expressing cells. HeLa cells were transfected with 0.5 μg pcDNA-SCAT3. Imaging analysis was started 18 h after transfection. (C) Venus/ECFP emission ratio changes of individual cells examined in B. Arrowheads indicate the time cells first showed the early apoptotic cell death morphology, including membrane blebbing and cell shrinkage.

Mentions: Using SCAT3, we monitored the activation of caspase-3 (DEVDase) in living HeLa cells after TNF-α/CHX exposure. The cleavage of SCAT3, but not of SCAT3 (DEVG), after TNF-α/CHX treatment was observed by Western blot analysis (Fig. 4 A), and we could not detect a significant ratio change in the SCAT3 (DEVG) FRET in response to TNF-α/CHX (unpublished data). We found that once caspase-3 activation was initiated in the cytosol, it progressed very rapidly, reaching its maximum in 7.2 ± 1.8 min (n = 13; Fig. 4, B and C, and Video 1, available at http://www.jcb.org/cgi/content/full/jcb.200207111/DC1). These imaging analyses also enabled us to observe the activation of caspase-3 in the nucleus (see next paragraph).


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)

Single-cell imaging analysis of SCAT3-expressing living HeLa cells. (A) Western blot analysis of SCAT3-expressing HeLa cells exposed to TNF-α/CHX. HeLa cells in a 6-well plate were transfected with 1 μg pcDNA-SCAT3. Cells were exposed to 50 ng/ml TNF-α and 10 μg/ml CHX 18 h after transfection. The cells were then lysed with sample buffer at the indicated times. The lysates were examined by Western blotting using an anti-myc antibody. (B) Ratio images of the SCAT3- expressing cells. HeLa cells were transfected with 0.5 μg pcDNA-SCAT3. Imaging analysis was started 18 h after transfection. (C) Venus/ECFP emission ratio changes of individual cells examined in B. Arrowheads indicate the time cells first showed the early apoptotic cell death morphology, including membrane blebbing and cell shrinkage.
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Related In: Results  -  Collection

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fig4: Single-cell imaging analysis of SCAT3-expressing living HeLa cells. (A) Western blot analysis of SCAT3-expressing HeLa cells exposed to TNF-α/CHX. HeLa cells in a 6-well plate were transfected with 1 μg pcDNA-SCAT3. Cells were exposed to 50 ng/ml TNF-α and 10 μg/ml CHX 18 h after transfection. The cells were then lysed with sample buffer at the indicated times. The lysates were examined by Western blotting using an anti-myc antibody. (B) Ratio images of the SCAT3- expressing cells. HeLa cells were transfected with 0.5 μg pcDNA-SCAT3. Imaging analysis was started 18 h after transfection. (C) Venus/ECFP emission ratio changes of individual cells examined in B. Arrowheads indicate the time cells first showed the early apoptotic cell death morphology, including membrane blebbing and cell shrinkage.
Mentions: Using SCAT3, we monitored the activation of caspase-3 (DEVDase) in living HeLa cells after TNF-α/CHX exposure. The cleavage of SCAT3, but not of SCAT3 (DEVG), after TNF-α/CHX treatment was observed by Western blot analysis (Fig. 4 A), and we could not detect a significant ratio change in the SCAT3 (DEVG) FRET in response to TNF-α/CHX (unpublished data). We found that once caspase-3 activation was initiated in the cytosol, it progressed very rapidly, reaching its maximum in 7.2 ± 1.8 min (n = 13; Fig. 4, B and C, and Video 1, available at http://www.jcb.org/cgi/content/full/jcb.200207111/DC1). These imaging analyses also enabled us to observe the activation of caspase-3 in the nucleus (see next paragraph).

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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