Limits...
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.

Show MeSH
Nuclear activation of caspase-3 precedes apoptotic nuclear changes. (A) Ratio images and phase-contrast images of NLS-SCAT–expressing cells. HeLa cells were transfected with 0.5 μg pcDNA-SCAT3. Imaging analysis was started 18 h after transfection. (B) Venus/ECFP emission ratio changes of individual cells examined in A.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC2172647&req=5

fig5: Nuclear activation of caspase-3 precedes apoptotic nuclear changes. (A) Ratio images and phase-contrast images of NLS-SCAT–expressing cells. HeLa cells were transfected with 0.5 μg pcDNA-SCAT3. Imaging analysis was started 18 h after transfection. (B) Venus/ECFP emission ratio changes of individual cells examined in A.

Mentions: We constructed and expressed NLS-SCAT3, in which an NLS was placed at the COOH terminus (Fig. 5 A). We monitored the nuclear morphology by phase-contrast images at the same time. We observed that in the presence of TNF-α/CHX, caspase-3 was activated in the nucleus. Interestingly, the simultaneous observation of morphology and fluorescence revealed that the nuclear activation of caspase-3 preceded the apoptotic nuclear morphological changes, such as nuclear fragmentation. In addition, we observed the leakage of NLS-SCAT3 protein from the nucleus into the cytosol when the nuclear morphological changes had been completed (Fig. 5 A). We also found that, as observed in the cytosol, the nuclear activation of caspase-3 was a rapid process, reaching its maximum in 11.6 ± 3.7 min (n = 17; Fig. 5 B).


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)

Nuclear activation of caspase-3 precedes apoptotic nuclear changes. (A) Ratio images and phase-contrast images of NLS-SCAT–expressing cells. HeLa cells were transfected with 0.5 μg pcDNA-SCAT3. Imaging analysis was started 18 h after transfection. (B) Venus/ECFP emission ratio changes of individual cells examined in A.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2172647&req=5

fig5: Nuclear activation of caspase-3 precedes apoptotic nuclear changes. (A) Ratio images and phase-contrast images of NLS-SCAT–expressing cells. HeLa cells were transfected with 0.5 μg pcDNA-SCAT3. Imaging analysis was started 18 h after transfection. (B) Venus/ECFP emission ratio changes of individual cells examined in A.
Mentions: We constructed and expressed NLS-SCAT3, in which an NLS was placed at the COOH terminus (Fig. 5 A). We monitored the nuclear morphology by phase-contrast images at the same time. We observed that in the presence of TNF-α/CHX, caspase-3 was activated in the nucleus. Interestingly, the simultaneous observation of morphology and fluorescence revealed that the nuclear activation of caspase-3 preceded the apoptotic nuclear morphological changes, such as nuclear fragmentation. In addition, we observed the leakage of NLS-SCAT3 protein from the nucleus into the cytosol when the nuclear morphological changes had been completed (Fig. 5 A). We also found that, as observed in the cytosol, the nuclear activation of caspase-3 was a rapid process, reaching its maximum in 11.6 ± 3.7 min (n = 17; Fig. 5 B).

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