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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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An improved indicator for activated caspases: SCAT. (A) Schematic representation of SCAT. (B) Linker sequences of the SCAT family of indicators. (C) Spectral analysis of SCAT3 in cells exposed to TNF-α/CHX. HeLa cells in 100-mm dishes were transfected with 6 μg pcDNA-SCAT3. 18 h after transfection, the cells were exposed to CHX or TNF-α/CHX and incubated for 6 h. The cell suspensions were then subjected to spectral analysis. (D) Changes in the emission ratio (530/475 nm) of SCAT3 compared with CY3 in cells treated with TNF-α/CHX. The cell suspensions were prepared as described above. The 530/475-nm emission ratio was measured with an excitation wavelength at 435 nm. The data represent results from three independent experiments. Error bars indicate SD.
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fig1: An improved indicator for activated caspases: SCAT. (A) Schematic representation of SCAT. (B) Linker sequences of the SCAT family of indicators. (C) Spectral analysis of SCAT3 in cells exposed to TNF-α/CHX. HeLa cells in 100-mm dishes were transfected with 6 μg pcDNA-SCAT3. 18 h after transfection, the cells were exposed to CHX or TNF-α/CHX and incubated for 6 h. The cell suspensions were then subjected to spectral analysis. (D) Changes in the emission ratio (530/475 nm) of SCAT3 compared with CY3 in cells treated with TNF-α/CHX. The cell suspensions were prepared as described above. The 530/475-nm emission ratio was measured with an excitation wavelength at 435 nm. The data represent results from three independent experiments. Error bars indicate SD.

Mentions: We generated a hybrid protein, ECFP-Venus, with an 18-amino acid linkage. The linking sequences contained an optimum sequence for caspase cleavage, which was used in a previous report (Tyas et al., 2000). Caspase activity is detected from the FRET from ECFP to Venus; thus, cleavage of the linker peptide by activated caspases abolishes the FRET (Fig. 1 A). We named this indicator SCAT (a sensor for activated caspases based on FRET). We constructed two SCATs, SCAT3 and SCAT9, which contained the DEVD (SCAT3) or LEHD (SCAT9) sequence in the linker region, which is mainly cleaved by caspase-3 or caspase-9, respectively (Thornberry et al., 1997; Fig. 1 B). We constructed SCAT3 (DEVG) for control experiments; it contained a glycine substituted for a critical aspartic acid as the fourth residue of the cleavage site. To detect the nuclear activation of caspase-3, we also constructed nuclear localization signal (NLS)-SCAT3.


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)

An improved indicator for activated caspases: SCAT. (A) Schematic representation of SCAT. (B) Linker sequences of the SCAT family of indicators. (C) Spectral analysis of SCAT3 in cells exposed to TNF-α/CHX. HeLa cells in 100-mm dishes were transfected with 6 μg pcDNA-SCAT3. 18 h after transfection, the cells were exposed to CHX or TNF-α/CHX and incubated for 6 h. The cell suspensions were then subjected to spectral analysis. (D) Changes in the emission ratio (530/475 nm) of SCAT3 compared with CY3 in cells treated with TNF-α/CHX. The cell suspensions were prepared as described above. The 530/475-nm emission ratio was measured with an excitation wavelength at 435 nm. The data represent results from three independent experiments. Error bars indicate SD.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2172647&req=5

fig1: An improved indicator for activated caspases: SCAT. (A) Schematic representation of SCAT. (B) Linker sequences of the SCAT family of indicators. (C) Spectral analysis of SCAT3 in cells exposed to TNF-α/CHX. HeLa cells in 100-mm dishes were transfected with 6 μg pcDNA-SCAT3. 18 h after transfection, the cells were exposed to CHX or TNF-α/CHX and incubated for 6 h. The cell suspensions were then subjected to spectral analysis. (D) Changes in the emission ratio (530/475 nm) of SCAT3 compared with CY3 in cells treated with TNF-α/CHX. The cell suspensions were prepared as described above. The 530/475-nm emission ratio was measured with an excitation wavelength at 435 nm. The data represent results from three independent experiments. Error bars indicate SD.
Mentions: We generated a hybrid protein, ECFP-Venus, with an 18-amino acid linkage. The linking sequences contained an optimum sequence for caspase cleavage, which was used in a previous report (Tyas et al., 2000). Caspase activity is detected from the FRET from ECFP to Venus; thus, cleavage of the linker peptide by activated caspases abolishes the FRET (Fig. 1 A). We named this indicator SCAT (a sensor for activated caspases based on FRET). We constructed two SCATs, SCAT3 and SCAT9, which contained the DEVD (SCAT3) or LEHD (SCAT9) sequence in the linker region, which is mainly cleaved by caspase-3 or caspase-9, respectively (Thornberry et al., 1997; Fig. 1 B). We constructed SCAT3 (DEVG) for control experiments; it contained a glycine substituted for a critical aspartic acid as the fourth residue of the cleavage site. To detect the nuclear activation of caspase-3, we also constructed nuclear localization signal (NLS)-SCAT3.

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.

Show MeSH
Related in: MedlinePlus