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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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Properties of SCAT3 and CY3. pH resistance (A) and Cl− resistance (B) of SCAT3 compared with CY3. HeLa cells transfected with pcDNA-SCAT3 or pCFP-DEVD-YFP were lysed with 0.5% Triton X-100–containing buffer. The 530/475-nm emission ratio was determined with a 435-nm excitation wavelength. The data represent results from three independent experiments. Error bars indicate SD. (C and D) The stability of SCAT compared with CY3 in living single cells. The imaging analysis was started on exposing HeLa cells to 1 μM STS at 18 h after transfection. To compare SCAT3 and CY3, the emission ratio was normalized by defining the baseline ratio before the FRET disruption as 1. Arrowheads indicate the time cells first showed the early apoptotic cell death morphology, including membrane blebbing and cell shrinkage. (E) Western blot analysis of SCAT3- or CY3-expressing HeLa cells exposed by STS. (F) The emission intensity profiles of EYFP and ECFP in CY3 (DEVG)-expressing HeLa cells. Cells 1 and 2 are the same as those shown in D. Arrows indicate the time of the emission ratio decrease in CY3 (DEVG) in D.
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fig2: Properties of SCAT3 and CY3. pH resistance (A) and Cl− resistance (B) of SCAT3 compared with CY3. HeLa cells transfected with pcDNA-SCAT3 or pCFP-DEVD-YFP were lysed with 0.5% Triton X-100–containing buffer. The 530/475-nm emission ratio was determined with a 435-nm excitation wavelength. The data represent results from three independent experiments. Error bars indicate SD. (C and D) The stability of SCAT compared with CY3 in living single cells. The imaging analysis was started on exposing HeLa cells to 1 μM STS at 18 h after transfection. To compare SCAT3 and CY3, the emission ratio was normalized by defining the baseline ratio before the FRET disruption as 1. Arrowheads indicate the time cells first showed the early apoptotic cell death morphology, including membrane blebbing and cell shrinkage. (E) Western blot analysis of SCAT3- or CY3-expressing HeLa cells exposed by STS. (F) The emission intensity profiles of EYFP and ECFP in CY3 (DEVG)-expressing HeLa cells. Cells 1 and 2 are the same as those shown in D. Arrows indicate the time of the emission ratio decrease in CY3 (DEVG) in D.

Mentions: Our initial goal was to generate an indicator for caspase activation under physiological conditions, such as in a developing embryo. It is important that the indicator does not detect any signals except for caspase activation. We noticed that CY3 was significantly sensitive to changes in the concentration of both H+ and Cl− (Fig. 2, A and B) in vitro. For example, acidification from pH 7.5 to 7.0 decreased the emission ratio of CY3 by 26%. Under the same conditions, only a 0.01% decrease was observed for SCAT3. Therefore, SCAT3 was highly stable under these conditions compared with CY3.


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)

Properties of SCAT3 and CY3. pH resistance (A) and Cl− resistance (B) of SCAT3 compared with CY3. HeLa cells transfected with pcDNA-SCAT3 or pCFP-DEVD-YFP were lysed with 0.5% Triton X-100–containing buffer. The 530/475-nm emission ratio was determined with a 435-nm excitation wavelength. The data represent results from three independent experiments. Error bars indicate SD. (C and D) The stability of SCAT compared with CY3 in living single cells. The imaging analysis was started on exposing HeLa cells to 1 μM STS at 18 h after transfection. To compare SCAT3 and CY3, the emission ratio was normalized by defining the baseline ratio before the FRET disruption as 1. Arrowheads indicate the time cells first showed the early apoptotic cell death morphology, including membrane blebbing and cell shrinkage. (E) Western blot analysis of SCAT3- or CY3-expressing HeLa cells exposed by STS. (F) The emission intensity profiles of EYFP and ECFP in CY3 (DEVG)-expressing HeLa cells. Cells 1 and 2 are the same as those shown in D. Arrows indicate the time of the emission ratio decrease in CY3 (DEVG) in D.
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Related In: Results  -  Collection

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fig2: Properties of SCAT3 and CY3. pH resistance (A) and Cl− resistance (B) of SCAT3 compared with CY3. HeLa cells transfected with pcDNA-SCAT3 or pCFP-DEVD-YFP were lysed with 0.5% Triton X-100–containing buffer. The 530/475-nm emission ratio was determined with a 435-nm excitation wavelength. The data represent results from three independent experiments. Error bars indicate SD. (C and D) The stability of SCAT compared with CY3 in living single cells. The imaging analysis was started on exposing HeLa cells to 1 μM STS at 18 h after transfection. To compare SCAT3 and CY3, the emission ratio was normalized by defining the baseline ratio before the FRET disruption as 1. Arrowheads indicate the time cells first showed the early apoptotic cell death morphology, including membrane blebbing and cell shrinkage. (E) Western blot analysis of SCAT3- or CY3-expressing HeLa cells exposed by STS. (F) The emission intensity profiles of EYFP and ECFP in CY3 (DEVG)-expressing HeLa cells. Cells 1 and 2 are the same as those shown in D. Arrows indicate the time of the emission ratio decrease in CY3 (DEVG) in D.
Mentions: Our initial goal was to generate an indicator for caspase activation under physiological conditions, such as in a developing embryo. It is important that the indicator does not detect any signals except for caspase activation. We noticed that CY3 was significantly sensitive to changes in the concentration of both H+ and Cl− (Fig. 2, A and B) in vitro. For example, acidification from pH 7.5 to 7.0 decreased the emission ratio of CY3 by 26%. Under the same conditions, only a 0.01% decrease was observed for SCAT3. Therefore, SCAT3 was highly stable under these conditions compared with CY3.

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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