Abstract
Apoptosis occurs through a tightly regulated cascade of caspase activation. In the context of extrinsic apoptosis, caspase-8 is activated by dimerization inside a death receptor complex, cleaved by auto-proteolysis and subsequently released into the cytosol. This fully processed form of caspase-8 is thought to cleave its substrates BID and caspase-3. To test if the release is required for substrate cleavage, we developed a novel approach based on localization probes to quantitatively characterize the spatial-temporal activity of caspases in living single cells. Our study reveals that caspase-8 is significantly more active at the plasma membrane than within the cytosol upon CD95 activation. This differential activity is controlled by the cleavage of caspase-8 prodomain. As a consequence, targeting of caspase-8 substrates to the plasma membrane can significantly accelerate cell death. Subcellular compartmentalization of caspase-8 activity may serve to restrict enzymatic activity before mitochondrial pathway activation and offers new possibilities to interfere with apoptotic sensitivity of the cells.
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Abbreviations
- DISC:
-
death-inducing signaling complex
- BID:
-
BH3-interacting death agonist
- MOMP:
-
mitochondrial outer membrane permeabilization
- NES:
-
nuclear export signal
- Myr–Palm:
-
myristoylation–palmitoylation
- ER:
-
endoplasmic reticulum
- LZ-sCD95L:
-
leucine zipper-soluble CD95 ligand
- mGFP:
-
monomeric green fluorescent protein
- YFP:
-
yellow fluorescent protein
- EBFP2:
-
enhanced blue fluorescent protein 2
- XIAP:
-
X-linked inhibitor of apoptosis protein
- FRET:
-
fluorescence resonance energy transfer
- DMEM:
-
Dulbecco’s modified eagle medium
- LSCM:
-
laser scanning confocal microscope
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Acknowledgements
We are grateful to Stefan Kallenberger, Dr Inna Lavrik, Prof Peter Krammer, Dr Nathan Brady, and Dr Stefan Legewie (DKFZ Heidelberg) for critical comments and discussion of this study. This work was supported by the Initiative and Networking Fund of the Helmholtz Association within the Helmholtz Alliance on Systems Biology/ SBCancer. We further acknowledge support by the BMBF funded ForSys centre Viroquant. JB received support by the Center for Modeling and Simulation in the Biosciences (BIOMS).
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Beaudouin, J., Liesche, C., Aschenbrenner, S. et al. Caspase-8 cleaves its substrates from the plasma membrane upon CD95-induced apoptosis. Cell Death Differ 20, 599–610 (2013). https://doi.org/10.1038/cdd.2012.156
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DOI: https://doi.org/10.1038/cdd.2012.156
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