Measuring dynamics of caspase-8 activation in a single living HeLa cell during TNFalpha-induced apoptosis.
In this study, we reported the first measurement of the dynamics of activation of caspase-8 in a single living cell. This measurement was conducted using a specially developed molecular sensor based on the FRET (fluorescence resonance energy transfer) technique. This sensor was constructed by fusing a CFP (cyan fluorescent protein) and a YFP (yellow fluorescent protein) with a linker containing a tandem caspase-8-specific cleavage site. The change of the FRET ratio upon cleavage was larger than 4-fold. Using this sensor, we found that during TNFalpha-induced apoptosis, the activation of caspase-8 was a slower process than that of caspase-3, and it was initiated much earlier than the caspase-3 activation. Inhibition of caspase-9 delayed the full activation of caspase-3 but did not affect the dynamics of caspase-8. Results of these single-cell measurements suggested that caspase-3 was activated by caspase-8 through two parallel pathways during TNFalpha-induced apoptosis in HeLa cells.
Luo
Kathy Q
KQ
Department of Biology, The Hong Kong University of Science and Technology, Clear Water Bay, Hong Kong, China.
Yu
Vivian C
VC
Pu
Yongmei
Y
Chang
Donald C
DC
D000595:Amino Acid Sequence; D017209:Apoptosis; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics; D053148:Caspase 3; D053181:Caspase 8; D053453:Caspase 9; D020169:Caspases / Q000378:metabolism*; D004789:Enzyme Activation; D031541:Fluorescence Resonance Energy Transfer; D005456:Fluorescent Dyes / Q000737:chemistry; D049452:Green Fluorescent Proteins; D006367:HeLa Cells; D006801:Humans; D008164:Luminescent Proteins / Q000737:chemistry / Q000235:genetics; D008856:Microscopy, Fluorescence; D008954:Models, Biological; D008969:Molecular Sequence Data; D011993:Recombinant Fusion Proteins / Q000032:analysis; D015398:Signal Transduction; D014409:Tumor Necrosis Factor-alpha / Q000494:pharmacology
D001426:Bacterial Proteins; D005456:Fluorescent Dyes; D008164:Luminescent Proteins; D011993:Recombinant Fusion Proteins; D014409:Tumor Necrosis Factor-alpha; C054562:yellow fluorescent protein, Bacteria; D049452:Green Fluorescent Proteins; C505407:CASP3 protein, human; C505530:CASP8 protein, human; C506433:CASP9 protein, human; D053148:Caspase 3; D053181:Caspase 8; D053453:Caspase 9; D020169:Caspases