In Vivo Imaging of Hierarchical Spatiotemporal Activation of Caspase-8 during Apoptosis
| Autor: | Kumiko Chiba, Katsuya Kominami, Jun Nishimura, Yaeta Endo, Kazuhiro Sakamaki, Noboru Manabe, Tatsuya Sawasaki, Jun Nakabayashi, Takeharu Nagai, Hideo Yokota, Yuki Tsujimura, Masateru Tsuchimochi, Kenta Yashima, Koji Koyamada, Yasuhiro Sunaga, Atsushi Miyawaki |
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| Jazyk: | angličtina |
| Rok vydání: | 2012 |
| Předmět: |
Fluorescence-lifetime imaging microscopy
Recombinant Fusion Proteins Molecular Sequence Data lcsh:Medicine Caspase 3 Apoptosis Biosensing Techniques Caspase 8 Transfection Biochemistry Molecular Genetics Single-cell analysis Molecular Cell Biology Genetics Escherichia coli Fluorescence Resonance Energy Transfer Signaling in Cellular Processes Humans Gene Regulation Amino Acid Sequence lcsh:Science Biology Caspase Apoptotic Signaling Cascade Apoptotic Signaling Enzyme Precursors Multidisciplinary biology Cell Death Chemistry lcsh:R Signaling Cascades Cell biology Enzymes Molecular Imaging Enzyme Activation Förster resonance energy transfer HEK293 Cells Gene Expression Regulation biology.protein lcsh:Q Signal transduction Single-Cell Analysis Research Article Signal Transduction HeLa Cells |
| Zdroj: | PLoS ONE PLoS ONE, Vol 7, Iss 11, p e50218 (2012) |
| ISSN: | 1932-6203 |
| Popis: | [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. |
| Databáze: | OpenAIRE |
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