Features of programmed cell death in intact Xenopus oocytes and early embryos revealed by near-infrared fluorescence and real-time monitoring.
Abstract
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.
Type
Journal articleSubject
AnimalsApoptosis
Caspases, Effector
Cytochromes c
Embryo, Nonmammalian
Fluorescence Resonance Energy Transfer
Fluorescent Dyes
Indoles
Inhibitor of Apoptosis Proteins
Microinjections
Mitochondrial Proteins
Oocytes
Spectroscopy, Near-Infrared
Xenopus Proteins
Xenopus laevis
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https://hdl.handle.net/10161/8380Published Version (Please cite this version)
10.1038/cdd.2009.120Publication Info
Johnson, CE; Freel, CD; & Kornbluth, S (2010). Features of programmed cell death in intact Xenopus oocytes and early embryos revealed
by near-infrared fluorescence and real-time monitoring. Cell Death Differ, 17(1). pp. 170-179. 10.1038/cdd.2009.120. Retrieved from https://hdl.handle.net/10161/8380.This is constructed from limited available data and may be imprecise. To cite this
article, please review & use the official citation provided by the journal.
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Show full item recordScholars@Duke
Christopher Freel
Assoc VP, Research
Sally A. Kornbluth
Jo Rae Wright University Distinguished Professor Emerita
Our lab studies the regulation of complex cellular processes, including cell cycle
progression and programmed cell death (apoptosis). These tightly orchestrated processes
are critical for appropriate cell proliferation and cell death, and when they go awry
can result in cancer and degenerative disorders. Within these larger fields, we have
focused on understanding the cellular mechanisms that prevent the onset of mitosis
prior to the completion of DNA replication, the process
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