Spatio-temporal activation of caspase revealed by indicator that is insensitive to environmental effects

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.

Adipocyte Death and Chronic Inflammation in Obesity

“…For example, Takemoto et al have developed SCAT3 technology to sense caspase 3 activity (30). Live imaging in transgenic mouse model expressing SCAT3 revealed well-coordinated apoptosis cell death during the process of neural tube closure (31).…”

Caspase-mediated changes in histone H1 in early apoptosis: prolonged caspase activation in developing olfactory sensory neurons

“…However, during the cell-death progression, H1-venus became localized to the periphery of the nucleus, whereas the AE-4 epitope of both the endogenous H1 and exogenous H1-venus became unrecognized (Figure 5e middle, stage 2-3; double arrowheads) or only weakly Caspase activation and H1 changes in real time revealed by an improved indicator for activated caspase-3, 'CNL-SCAT3'. To investigate the caspase activation and nuclear events in real time in detail, we developed CNL-SCAT3 (Caspase-dependent Nuclear Localization-SCAT3), an improved version of the fluorescence energy transfer 20 Nuclear export signal (NES) and nuclear localization signal (NLS) sequences were fused to the N-terminal and C-terminal ends of SCAT3, respectively, enabling us to monitor caspase activity by measuring not only the FRET from enhanced cyan fluorescent protein (ECFP) to venus, but also the subcellular localization of the venus signal (Figure 6a). First, we checked whether CNL-SCAT3 was cleaved in a caspase-dependent manner (Figure 6c).…”

“…CNL-SCAT3 was generated by adding the NES NSNELALKLAGLDINKTE of protein kinase inhibitor a to the N terminus of NLS-SCAT3 in the pcDNA3.1(À) vector. 20 Mice. C57BL/6 mice were purchased from CLEA Japan (Tokyo, Japan) and used for the immunohistochemistry experiments as previously described.…”

Caspase-9 Activation Revealed by Semaphorin 7A Cleavage Is Independent of Apoptosis in the Aged Olfactory Bulb

“…The in vitro cleavage assay was performed as described previously (Takemoto et al, 2003). Sema7A protein was prepared by in vitro transcription and translation using a TNT-coupled reticulocyte lysate system (Promega).…”