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Constitutive nuclear localization and initial cytoplasmic apoptotic activation of endogenous caspase-3 evidenced by
Olivier Ramuz1, Daniel Isnardon, Elisabeth Devilard
1INSERM U119, Service d'Anatomie et Cytologie Pathologiques, Laboratoire d'Immunologie des Tumeurs, Institut Paoli-Calmettes, IFR 57 and Université de la Méditerranée, Marseille, France. anapath@marseille.fnclcc.fr
Abstract:
The localization of caspases and their substrates in different cellular compartments may be one way to regulate apoptosis. Caspase-3-dependent proteolysis of inhibitor caspase-activated deoxyribonuclease (ICAD) activates caspase-activated deoxyribonuclease (CAD), which induces apoptotic internucleosomal DNA degradation. The nuclear localization of ICAD, pro- and active-caspase-3 molecules remains a controversial issue. Using a combination of immunodetection of endogenous molecules and confocal microscopy, we analysed the kinetics of the procaspase-3 and CAD activation induced by FAS triggering in Jurkat cells. Through a semi-quantitative image analysis, we showed a constitutive nuclear localization of pro-caspase 3 and ICAD in non-apoptotic cells. FAS stimulation induced 7A6 apoptotic antigen expression, which could be related to three different sequential patterns of nuclear chromatin organization. Active-caspase-3 first appeared in the cytoplasm and was next observed in the nucleus. Simultaneously, the amount of ICAD located in the nucleus decreased, whereas the amount of ICAD located in the cytoplasm remained unchanged. Thus, our experiments using in situ immunodetection of endogenous molecules show that the ICAD cleavage by the active-caspase-3 probably takes place in the nucleus. These results provide new perspectives about the subcellular compartmentation and traffic of caspases during the apoptotic process.
Insights
This study reveals that pro-caspase-3 and its inhibitor ICAD are constitutively nuclear. Upon FAS stimulation, active-caspase-3 enters the nucleus, leading to ICAD cleavage and apoptosis.
Area of Science:
- Cell Biology
- Molecular Biology
- Biochemistry
Background:
- Apoptosis regulation involves caspase localization and substrate interactions within cellular compartments.
- Caspase-3 activation cleaves ICAD, releasing CAD to degrade DNA during apoptosis.
- The nuclear presence of ICAD, pro-caspase-3, and active-caspase-3 is debated.
Purpose of the Study:
- To investigate the subcellular localization and kinetics of pro-caspase-3 and ICAD during FAS-induced apoptosis.
- To determine the site of ICAD cleavage by active-caspase-3 within the cell.
Main Methods:
- Utilized immunodetection of endogenous molecules and confocal microscopy in Jurkat cells.
- Employed semi-quantitative image analysis to assess protein localization and kinetics.
- Analyzed apoptotic antigen expression and chromatin organization changes post-FAS stimulation.
Main Results:
- Pro-caspase-3 and ICAD exhibit constitutive nuclear localization in non-apoptotic cells.
- FAS stimulation leads to sequential appearance of active-caspase-3 in cytoplasm then nucleus.
- Nuclear ICAD levels decrease as active-caspase-3 enters the nucleus, suggesting nuclear cleavage.
Conclusions:
- ICAD cleavage by active-caspase-3 likely occurs within the nucleus during apoptosis.
- Findings offer new insights into caspase subcellular compartmentalization and trafficking during apoptosis.