Related Experiment Videos
Caspase-3 controls both cytoplasmic and nuclear events associated with Fas-mediated apoptosis in vivo
T S Zheng1, S F Schlosser, T Dao
1Section of Immunobiology, Yale University School of Medicine, New Haven, CT 06520, USA.
Abstract:
Both caspase-1- and caspase-3-like activities are required for Fas-mediated apoptosis. However, the role of caspase-1 and caspase-3 in mediating Fas-induced cell death is not clear. We assessed the contributions of these caspases to Fas signaling in hepatocyte cell death in vitro. Although wild-type, caspase-1(-/-), and caspase-3(-/-) hepatocytes were killed at a similar rate when cocultured with FasL expressing NIH 3T3 cells, caspase-3(-/-) hepatocytes displayed drastically different morphological changes as well as significantly delayed DNA fragmentation. For both wild-type and caspase-1(-/-) apoptotic hepatocytes, typical apoptotic features such as cytoplasmic blebbing and nuclear fragmentation were seen within 6 hr, but neither event was observed for caspase-3(-/-) hepatocytes. We extended these studies to thymocytes and found that apoptotic caspase-3(-/-) thymocytes exhibited similar "abnormal" morphological changes and delayed DNA fragmentation observed in hepatocytes. Furthermore, the cleavage of various caspase substrates implicated in mediating apoptotic events, including gelsolin, fodrin, laminB, and DFF45/ICAD, was delayed or absent. The altered cleavage of these key substrates is likely responsible for the aberrant apoptosis observed in both hepatocytes and thymocytes deficient in caspase-3.
Insights
Caspase-3 is crucial for typical apoptosis morphology and DNA fragmentation during Fas-mediated cell death. While caspase-1 is not essential, caspase-3 deficiency significantly delays these key apoptotic events in hepatocytes and thymocytes.
Area of Science:
- Cell Biology
- Molecular Biology
- Immunology
Background:
- Fas-mediated apoptosis is a critical process in regulating cell death.
- Both caspase-1 and caspase-3 activities are implicated in Fas-induced apoptosis, but their specific roles remain unclear.
- Understanding the precise contribution of each caspase is vital for comprehending cell death pathways.
Purpose of the Study:
- To investigate the distinct roles of caspase-1 and caspase-3 in Fas-mediated apoptosis.
- To elucidate the contribution of these caspases to Fas signaling in hepatocyte cell death in vitro.
- To analyze the impact of caspase deficiency on apoptotic morphology and substrate cleavage.
Main Methods:
- Coculture of wild-type, caspase-1(-/-), and caspase-3(-/-) hepatocytes with FasL-expressing cells.
- Microscopic observation of apoptotic morphological changes (blebbing, nuclear fragmentation).
- Assessment of DNA fragmentation and cleavage of known caspase substrates (gelsolin, fodrin, laminB, DFF45/ICAD).
Main Results:
- Hepatocytes deficient in caspase-3 showed significantly delayed DNA fragmentation and altered morphology compared to wild-type and caspase-1(-/-) cells.
- Typical apoptotic features were observed within 6 hours in wild-type and caspase-1(-/-) cells but were absent in caspase-3(-/-) cells.
- Cleavage of key caspase substrates was delayed or absent in caspase-3(-/-) hepatocytes and thymocytes, leading to aberrant apoptosis.
Conclusions:
- Caspase-3 plays a critical role in executing the morphological and DNA fragmentation aspects of Fas-mediated apoptosis.
- Caspase-1 is not essential for these specific apoptotic events.
- Deficiency in caspase-3 leads to abnormal apoptosis due to impaired cleavage of essential substrates, affecting both hepatocytes and thymocytes.