Features of programmed cell death in intact Xenopus oocytes and early embryos revealed by near-infrared fluorescence and real-time monitoring.
Factors influencing apoptosis of vertebrate eggs and early embryos have been studied in cell-free systems and in intact embryos by analyzing individual apoptotic regulators or caspase activation in static samples. A novel method for monitoring caspase activity in living Xenopus oocytes and early embryos is described here. The approach, using microinjection of a near-infrared caspase substrate that emits fluorescence only after its proteolytic cleavage by active effector caspases, has enabled the elucidation of otherwise cryptic aspects of apoptotic regulation. In particular, we show that brief caspase activity (10 min) is sufficient to cause apoptotic death in this system. We illustrate a cytochrome c dose threshold in the oocyte, which is lowered by Smac, a protein that binds thereby neutralizing the inhibitor of apoptosis proteins. We show that meiotic oocytes develop resistance to cytochrome c, and that the eventual death of oocytes arrested in meiosis is caspase-independent. Finally, data acquired through imaging caspase activity in the Xenopus embryo suggest that apoptosis in very early development is not cell-autonomous. These studies both validate this assay as a useful tool for apoptosis research and reveal subtleties in the cell death program during early development. Moreover, this method offers a potentially valuable screening modality for identifying novel apoptotic regulators.
Johnson
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Department of Pharmacology and Cancer Biology, Duke University Medical Center, Durham, NC 27710, USA.
Freel
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Kornbluth
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D000818:Animals; D017209:Apoptosis; D053470:Caspases, Effector / Q000378:metabolism; D045304:Cytochromes c / Q000378:metabolism; D004625:Embryo, Nonmammalian / Q000166:cytology / Q000201:enzymology*; D031541:Fluorescence Resonance Energy Transfer; D005456:Fluorescent Dyes / Q000378:metabolism; D007211:Indoles / Q000008:administration & dosage; D051034:Inhibitor of Apoptosis Proteins / Q000378:metabolism; D008845:Microinjections; D024101:Mitochondrial Proteins / Q000378:metabolism; D009865:Oocytes / Q000166:cytology / Q000201:enzymology* / Q000378:metabolism; D019265:Spectroscopy, Near-Infrared; D029867:Xenopus Proteins / Q000378:metabolism; D014982:Xenopus laevis
C520098:DIABLO protein, Xenopus; D005456:Fluorescent Dyes; C427728:IRDye800; D007211:Indoles; D051034:Inhibitor of Apoptosis Proteins; D024101:Mitochondrial Proteins; D029867:Xenopus Proteins; D045304:Cytochromes c; D053470:Caspases, Effector