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- Title
In Vivo Imaging of Hierarchical Spatiotemporal Activation of Caspase-8 during Apoptosis.
- Authors
Katsuya Kominami; Takeharu Nagai; Tatsuya Sawasaki; Yuki Tsujimura; Kenta Yashima; Yasuhiro Sunaga; Masateru Tsuchimochi; Jun Nishimura; Kumiko Chiba; Jun Nakabayashi; Koji Koyamada; Yaeta Endo; Hideo Yokota; Atsushi Miyawaki; Noboru Manabe; Kazuhiro Sakamaki
- Abstract
Background:Activation of caspases is crucial for the execution of apoptosis. Although the caspase cascade associated with activation of the initiator caspase-8 (CASP8) has been investigated in molecular and biochemical detail, the dynamics of CASP8 activation are not fully understood. Methodology/Principal Findings:We have established a biosensor based on fluorescence resonance energy transfer (FRET) for visualizing apoptotic signals associated with CASP8 activation at the single-cell level. Our dual FRET (dual-FRET) system, comprising a triple fusion fluorescent protein, enabled us to simultaneously monitor the activation of CASP8 and its downstream effector, caspase-3 (CASP3) in single live cells. With the dual-FRET-based biosensor, we detected distinct activation patterns of CASP8 and CASP3 in response to various apoptotic stimuli in mammalian cells, resulting in the positive feedback amplification of CASP8 activation. We reproduced these observations by in vitro reconstitution of the cascade, with a recombinant protein mixture that included procaspases. Furthermore, using a plasma membrane-bound FRET-based biosensor, we captured the spatiotemporal dynamics of CASP8 activation by the diffusion process, suggesting the focal activation of CASP8 is sufficient to propagate apoptotic signals through death receptors. Conclusions:Our new FRET-based system visualized the activation process of both initiator and effector caspases in a single apoptotic cell and also elucidated the necessity of an amplification loop for full activation of CASP8.
- Subjects
PROTEIN-tyrosine kinases; BREAST cancer research; CANCER cells; CELL adhesion; CANCER patients; TUMOR suppressor proteins
- Publication
PLoS ONE, 2012, Vol 7, Issue 11, p1
- ISSN
1932-6203
- Publication type
Article
- DOI
10.1371/journal.pone.0050218