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Identification of a caspase-9 substrate and detection of its cleavage in programmed cell death during mouse
K Nakanishi1, M Maruyama, T Shibata
1Bioarchitect Research Group, RIKEN (Institute of Physical and Chemical Research), 2-1 Hirosawa, Wako, Saitama 351-0198, Japan.
Abstract:
The caspase family of proteases represents the main machinery by which apoptosis occurs. In vitro studies have revealed that upstream caspases are activated in response to apoptotic stimuli, and the active caspases in turn process downstream effector caspases that are involved in the destruction of cellular structure. Caspase-9 is an upstream caspase that can become active in response to cellular damage, including deprivation of growth factors and exposure to oxidative stress in vitro. Little is known, however, about how activation of caspase-9 is temporally and spatially regulated in vivo, e.g. during development. We have identified vimentin as the first example of a caspase-9 substrate that is not a downstream procaspase. Immunohistochemical analysis, using a specific antibody against the vimentin fragments generated by caspase-9, showed that caspase-9 cleaves vimentin in apoptotic cells in the embryonic nervous system and the interdigital regions. This result is consistent with observations that gene knockouts of caspase-9 and its activator, Apaf-1, result in developmental defects in these tissues. Our results show that the specific antibody is useful for in situ detection of caspase-9 activation in programmed cell death.
Insights
Caspase-9 cleaves vimentin, a non-procaspase protein, during apoptosis in the developing embryo. This finding aids in understanding programmed cell death regulation in vivo.
Area of Science:
- Cellular Biology
- Developmental Biology
- Biochemistry
Background:
- Apoptosis is executed by caspases, a protease family crucial for cellular structure destruction.
- Upstream caspases activate in response to apoptotic stimuli, processing downstream caspases.
- Caspase-9 activation in vivo during development remains poorly understood.
Purpose of the Study:
- To identify novel substrates of caspase-9 beyond procaspases.
- To investigate the in vivo spatiotemporal regulation of caspase-9 activation during embryonic development.
- To develop tools for detecting caspase-9 activity in situ.
Main Methods:
- Identification of vimentin as a direct caspase-9 substrate.
- Immunohistochemical analysis using a specific antibody against caspase-9-generated vimentin fragments.
- Examination of apoptotic cells in the embryonic nervous system and interdigital regions.
Main Results:
- Vimentin was identified as the first non-procaspase substrate of caspase-9.
- Caspase-9 was shown to cleave vimentin in apoptotic cells within the embryonic nervous system and interdigital regions.
- The observed cleavage patterns correlate with developmental defects in caspase-9 and Apaf-1 knockout models.
Conclusions:
- Vimentin is a novel in vivo substrate for caspase-9 during programmed cell death.
- The specific antibody against vimentin fragments is a valuable tool for detecting caspase-9 activation in situ.
- This research elucidates a new aspect of caspase-9 regulation in embryonic development.