Abstract
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 an in situ assay 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 in Caenorhabditis elegans embryos. ToLFP analysis provided the first demonstration of a cell autonomous mode of DNase II activity in dying cells in ced-1 embryos, which are defective in engulfing apoptotic bodies. Compared with the number of ToLFP signals between ced-1 and 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-6 and crn-7), nuc-1 embryos 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.