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Role of factors downstream of caspases in nuclear disassembly during apoptotic execution
K Samejima1, P Villa, W C Earnshaw
1Institute of Cell and Molecular Biology, University of Edinburgh, UK.
Abstract:
We used cytoplasmic extracts from chicken DU249 cells at various stages along the apoptotic pathway to analyse the events of apoptotic execution. So-called S/M extracts from morphologically normal 'committed-stage' cells induce apoptotic morphology and DNA cleavage in substrate nuclei. These apoptotic changes appear to require the function of multiple caspases (cysteine aspartases, a specialized class of proteases) acting in parallel. Extracts from 'execution-stage' apoptotic cells induce apoptotic events in added nuclei in a caspase-independent manner. Biochemical fractionation of these extracts reveals that a column fraction enriched in endogenous active caspases is unable to induce DNA fragmentation or chromatin condensation in substrate nuclei, whereas a caspase-depleted fraction induces both changes. 'Execution-stage' extracts contain an ICAD/DFF45-inhibitable nuclease resembling CAD, plus another activity that is required for the apoptotic chromatin condensation. 'Committed-stage' S/M extracts lack these downstream activities. These observations reveal that caspases act in an executive fashion, serving to activate downstream factors that disassemble the nucleus rather than disassembling it themselves. They also suggest that activation of the downstream factors (rather than the caspases) is the critical event that occurs at the transition from the latent to the execution phase of apoptosis.
Insights
Cellular extracts reveal that caspases (cysteine aspartases) activate downstream factors for apoptosis, rather than directly causing nuclear disassembly. This activation is key to the transition from latent to execution phases of programmed cell death.
Area of Science:
- Cell Biology
- Molecular Biology
- Biochemistry
Background:
- Apoptosis is a tightly regulated process involving distinct execution phases.
- Caspases (cysteine aspartases) are key proteases in apoptosis, but their precise role in nuclear dismantling is debated.
Purpose of the Study:
- To investigate the molecular mechanisms underlying the execution phase of apoptosis.
- To determine whether caspases directly dismantle the nucleus or activate downstream effectors.
Main Methods:
- Utilized cytoplasmic extracts from chicken DU249 cells at different apoptotic stages.
- Performed biochemical fractionation of extracts.
- Assessed the ability of extracts and fractions to induce apoptotic morphology and DNA cleavage in substrate nuclei.
Main Results:
- 'Committed-stage' extracts induced apoptosis requiring caspases.
- 'Execution-stage' extracts induced apoptosis independently of caspases.
- Caspase-depleted fractions from 'execution-stage' extracts contained nucleases and chromatin condensation activities, while caspase-enriched fractions did not.
Conclusions:
- Caspases act upstream, activating downstream factors for nuclear disassembly during apoptosis.
- The activation of these downstream factors, not caspases themselves, marks the critical transition to the apoptotic execution phase.