摘要：

The method of ToLFP (topoisomerase labelled fluorescence probes) is useful for detecting the DNA fragments generated by DNase II inCaenorhabditis elegansembryos. It reveals ~70% ToLFP signals in dying cells and 30% in engulfing cells during embryogenesis. Generation of DNA fragments is a hallmark of cell apoptosis and is executed within the dying cells (autonomous) or in the engulfing cells (non-autonomous). The TUNEL (terminal deoxynucleotidyl transferase dUTP nick end labelling) method is used as anin situassay of apoptosis by labelling DNA fragments generated by caspase-associated DNase (CAD), but not those by the downstream DNase II. In the present study, we report a method of ToLFP (topoisomerase ligation fluorescence probes) for directly visualizing DNA fragments generated by DNase II inCaenorhabditis elegansembryos. ToLFP analysis provided the first demonstration of a cell autonomous mode of DNase II activity in dying cells inced-1embryos, which are defective in engulfing apoptotic bodies. Compared with the number of ToLFP signals betweenced-1and wild-type (N2) embryos, a 30% increase in N2 embryos was found, suggesting that the ratio of non-autonomous and autonomous modes of DNase II was ~3–7. Among three DNase II mutant embryos (nuc-1,crn-6andcrn-7),nuc-1embryos exhibited the least number of ToLFP. The ToLFP results confirmed the previous findings that NUC-1 is the major DNase II for degrading apoptotic DNA. To further elucidate NUC-1′s mode of action,nuc-1-rescuing transgenic worms that ectopically express free or membrane-bound forms of NUC-1 fusion proteins were utilized. ToLFP analyses revealed that anteriorly expressed NUC-1 digests apoptotic DNA in posterior blastomeres in a non-autonomous and secretion-dependent manner. Collectively, we demonstrate that the ToLFP method can be used to differentiate the locations of blastomeres where DNase II acts autonomously or non-autonomously in degrading apoptotic DNA.

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