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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 caspase-9 activation using SCAT9. (A) Western blot analysis of SCAT9-expressing HeLa cells treated with TNF-α/CHX. HeLa cells cultured in a 6-well plate were transfected with 1 μg pcDNA-SCAT9. The cells were treated with 50 ng/ml TNF-α and 10 μg/ml CHX 18 h after the 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 SCAT9-expressing HeLa cells exposed to 50 ng/ml TNF-α and 10 μg/ml CHX. HeLa cells were transfected with 0.5 μg pcDNA-SCAT9. 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 early apoptotic cell death morphological changes, such as membrane blebbing and cell shrinkage. (D) Schematic representation of the activation profile of caspase-3 (DEVDase) and -9 (LEHDase). Arrows indicate the time point of each event.
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fig7: Single-cell imaging analysis of caspase-9 activation using SCAT9. (A) Western blot analysis of SCAT9-expressing HeLa cells treated with TNF-α/CHX. HeLa cells cultured in a 6-well plate were transfected with 1 μg pcDNA-SCAT9. The cells were treated with 50 ng/ml TNF-α and 10 μg/ml CHX 18 h after the 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 SCAT9-expressing HeLa cells exposed to 50 ng/ml TNF-α and 10 μg/ml CHX. HeLa cells were transfected with 0.5 μg pcDNA-SCAT9. 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 early apoptotic cell death morphological changes, such as membrane blebbing and cell shrinkage. (D) Schematic representation of the activation profile of caspase-3 (DEVDase) and -9 (LEHDase). Arrows indicate the time point of each event.

Mentions: SCAT9 was cleaved in HeLa cells after exposure to TNF-α/CHX (Fig. 7 A). Therefore, we used SCAT9 to monitor the caspase-9 activation in living HeLa cells (Fig. 7 B and Video 2). Activation was observed in the cytosol, but it was unclear in the nuclei. In contrast to the caspase-3 activation by TNF-α/CHX, the activation of caspase-9 was a very slow process that took at least 48 min (n = 13) to reach its maximum (see Fig. 7 C, where it took 77.5 min and 91.5 min in cell 1 and cell 2, respectively). However, the time from the initial caspase-9 activation to the formation of apoptotic morphological changes was 10.1 ± 2.1 min (n = 13), which was similar to the time course seen with caspase-3 (10.0 ± 2.1 min; n = 13). Therefore, these results indicate that the initiation of the caspase-3 and caspase-9 activation occurred at almost the same time, but caspase-3 and caspase-9 activation was completed before and after the apoptotic morphological changes, respectively (Fig. 7 D).


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 caspase-9 activation using SCAT9. (A) Western blot analysis of SCAT9-expressing HeLa cells treated with TNF-α/CHX. HeLa cells cultured in a 6-well plate were transfected with 1 μg pcDNA-SCAT9. The cells were treated with 50 ng/ml TNF-α and 10 μg/ml CHX 18 h after the 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 SCAT9-expressing HeLa cells exposed to 50 ng/ml TNF-α and 10 μg/ml CHX. HeLa cells were transfected with 0.5 μg pcDNA-SCAT9. 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 early apoptotic cell death morphological changes, such as membrane blebbing and cell shrinkage. (D) Schematic representation of the activation profile of caspase-3 (DEVDase) and -9 (LEHDase). Arrows indicate the time point of each event.
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fig7: Single-cell imaging analysis of caspase-9 activation using SCAT9. (A) Western blot analysis of SCAT9-expressing HeLa cells treated with TNF-α/CHX. HeLa cells cultured in a 6-well plate were transfected with 1 μg pcDNA-SCAT9. The cells were treated with 50 ng/ml TNF-α and 10 μg/ml CHX 18 h after the 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 SCAT9-expressing HeLa cells exposed to 50 ng/ml TNF-α and 10 μg/ml CHX. HeLa cells were transfected with 0.5 μg pcDNA-SCAT9. 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 early apoptotic cell death morphological changes, such as membrane blebbing and cell shrinkage. (D) Schematic representation of the activation profile of caspase-3 (DEVDase) and -9 (LEHDase). Arrows indicate the time point of each event.
Mentions: SCAT9 was cleaved in HeLa cells after exposure to TNF-α/CHX (Fig. 7 A). Therefore, we used SCAT9 to monitor the caspase-9 activation in living HeLa cells (Fig. 7 B and Video 2). Activation was observed in the cytosol, but it was unclear in the nuclei. In contrast to the caspase-3 activation by TNF-α/CHX, the activation of caspase-9 was a very slow process that took at least 48 min (n = 13) to reach its maximum (see Fig. 7 C, where it took 77.5 min and 91.5 min in cell 1 and cell 2, respectively). However, the time from the initial caspase-9 activation to the formation of apoptotic morphological changes was 10.1 ± 2.1 min (n = 13), which was similar to the time course seen with caspase-3 (10.0 ± 2.1 min; n = 13). Therefore, these results indicate that the initiation of the caspase-3 and caspase-9 activation occurred at almost the same time, but caspase-3 and caspase-9 activation was completed before and after the apoptotic morphological changes, respectively (Fig. 7 D).

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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