Quantitative analysis of pathways controlling extrinsic apoptosis in single cells
John G Albeck1, John M Burke, Bree B Aldridge
1Department of Systems Biology, Harvard Medical School, WAB Room 438, 200 Longwood Avenue, Boston, MA 02115, USA.
Abstract:
Apoptosis in response to TRAIL or TNF requires the activation of initiator caspases, which then activate the effector caspases that dismantle cells and cause death. However, little is known about the dynamics and regulatory logic linking initiators and effectors. Using a combination of live-cell reporters, flow cytometry, and immunoblotting, we find that initiator caspases are active during the long and variable delay that precedes mitochondrial outer membrane permeabilization (MOMP) and effector caspase activation. When combined with a mathematical model of core apoptosis pathways, experimental perturbation of regulatory links between initiator and effector caspases reveals that XIAP and proteasome-dependent degradation of effector caspases are important in restraining activity during the pre-MOMP delay. We identify conditions in which restraint is impaired, creating a physiologically indeterminate state of partial cell death with the potential to generate genomic instability. Together, these findings provide a quantitative picture of caspase regulatory networks and their failure modes.
Insights
Investigating apoptosis, this study reveals initiator caspases remain active during the delay before cell death. XIAP and proteasome activity restrain this, and their failure can cause genomic instability.
Area of Science:
- Cellular biology
- Molecular biology
- Biochemistry
Background:
- Apoptosis, or programmed cell death, is triggered by initiator caspases activating effector caspases.
- The precise dynamics and regulatory mechanisms linking initiator and effector caspases remain poorly understood.
Purpose of the Study:
- To elucidate the regulatory logic and dynamics of caspase activation during apoptosis.
- To identify factors that control the delay between initiator and effector caspase activation.
- To investigate the consequences of impaired caspase regulation.
Main Methods:
- Live-cell reporters to monitor caspase activity in real-time.
- Flow cytometry and immunoblotting for quantitative analysis.
- Mathematical modeling of apoptosis pathways.
- Experimental perturbation of caspase regulatory links.
Main Results:
- Initiator caspases show prolonged activity during the pre-MOMP (mitochondrial outer membrane permeabilization) delay.
- XIAP (X-linked inhibitor of apoptosis protein) and proteasome-dependent degradation restrain effector caspase activity.
- Impaired restraint leads to a partial cell death state with potential for genomic instability.
Conclusions:
- Provides a quantitative understanding of caspase regulatory networks in apoptosis.
- Highlights the role of XIAP and proteasomal degradation in controlling the apoptotic timeline.
- Identifies failure modes in caspase regulation that can lead to genomic instability.
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