title: Autonomous and non-autonomous roles of DNase II during cell death in C. elegans embryos.
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.
authors.affiliation: Department and Graduate Institute of Biomedical Sciences, College of Medicine, Chang Gung University, Taoyuan 33302, Taiwan.
authors.forename: Hsiang
authors.initials: H
authors.lastname: Yu
authors.affiliation: Department and Graduate Institute of Biomedical Sciences, College of Medicine, Chang Gung University, Taoyuan 33302, Taiwan Department of Molecular, Cellular and Developmental Biology, University of Colorado, Boulder 80302, U.S.A.
authors.forename: Huey-Jen
authors.initials: HJ
authors.lastname: Lai
authors.affiliation: Department and Graduate Institute of Biomedical Sciences, College of Medicine, Chang Gung University, Taoyuan 33302, Taiwan.
authors.forename: Tai-Wei
authors.initials: TW
authors.lastname: Lin
authors.affiliation: Department and Graduate Institute of Biomedical Sciences, College of Medicine, Chang Gung University, Taoyuan 33302, Taiwan losj@mail.cgu.edu.tw.
authors.forename: Szecheng J
authors.initials: SJ
authors.lastname: Lo
mesh_terms: D000818:Animals; D030801:Animals, Genetically Modified; D017209:Apoptosis; D001757:Blastomeres / Q000166:cytology / Q000378:metabolism; D017173:Caenorhabditis elegans / Q000166:cytology / Q000196:embryology* / Q000502:physiology; D029742:Caenorhabditis elegans Proteins / Q000235:genetics / Q000378:metabolism*; D016923:Cell Death / Q000502:physiology; D004625:Embryo, Nonmammalian / Q000166:cytology; D004706:Endodeoxyribonucleases / Q000235:genetics / Q000378:metabolism*; D005456:Fluorescent Dyes / Q000737:chemistry; D008565:Membrane Proteins / Q000235:genetics / Q000378:metabolism; D009154:Mutation
chemical_list: D029742:Caenorhabditis elegans Proteins; D005456:Fluorescent Dyes; D008565:Membrane Proteins; C422043:ced-1 protein, C elegans; D004706:Endodeoxyribonucleases; C019329:deoxyribonuclease II
keywords: Caenorhabditis elegans; DNase II; apoptosis; exocytosis; fluorescence probes; lysosome