title: In vivo imaging of hierarchical spatiotemporal activation of caspase-8 during apoptosis.
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.
  
  
  RESULTS
  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.
authors.affiliation: Department of Animal Development and Physiology, Kyoto University, Kyoto, Japan.
authors.forename: Katsuya
authors.initials: K
authors.lastname: Kominami
authors.forename: Takeharu
authors.initials: T
authors.lastname: Nagai
authors.forename: Tatsuya
authors.initials: T
authors.lastname: Sawasaki
authors.forename: Yuki
authors.initials: Y
authors.lastname: Tsujimura
authors.forename: Kenta
authors.initials: K
authors.lastname: Yashima
authors.forename: Yasuhiro
authors.initials: Y
authors.lastname: Sunaga
authors.forename: Masateru
authors.initials: M
authors.lastname: Tsuchimochi
authors.forename: Jun
authors.initials: J
authors.lastname: Nishimura
authors.forename: Kumiko
authors.initials: K
authors.lastname: Chiba
authors.forename: Jun
authors.initials: J
authors.lastname: Nakabayashi
authors.forename: Koji
authors.initials: K
authors.lastname: Koyamada
authors.forename: Yaeta
authors.initials: Y
authors.lastname: Endo
authors.forename: Hideo
authors.initials: H
authors.lastname: Yokota
authors.forename: Atsushi
authors.initials: A
authors.lastname: Miyawaki
authors.forename: Noboru
authors.initials: N
authors.lastname: Manabe
authors.forename: Kazuhiro
authors.initials: K
authors.lastname: Sakamaki
mesh_terms: D000595:Amino Acid Sequence; D017209:Apoptosis / Q000235:genetics*; D015374:Biosensing Techniques / Q000379:methods*; D053148:Caspase 3 / Q000235:genetics / Q000378:metabolism*; D053181:Caspase 8 / Q000235:genetics / Q000378:metabolism*; D004789:Enzyme Activation; D004792:Enzyme Precursors / Q000235:genetics / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics; D031541:Fluorescence Resonance Energy Transfer; D005786:Gene Expression Regulation; D057809:HEK293 Cells; D006367:HeLa Cells; D006801:Humans; D057054:Molecular Imaging; D008969:Molecular Sequence Data; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D015398:Signal Transduction; D059010:Single-Cell Analysis / Q000379:methods*; D014162:Transfection
chemical_list: D004792:Enzyme Precursors; D011993:Recombinant Fusion Proteins; D053148:Caspase 3; D053181:Caspase 8