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Autonomous and non-autonomous roles of DNase II during cell death in C. elegans embryos
Hsiang Yu1, Huey-Jen Lai2, Tai-Wei Lin1
1Department and Graduate Institute of Biomedical Sciences, College of Medicine, Chang Gung University, Taoyuan 33302, Taiwan.
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.
Insights
A new method, topoisomerase ligation fluorescence probes (ToLFP), visualizes DNA fragments from DNase II during apoptosis in C. elegans. This reveals DNase II acts both autonomously within dying cells and non-autonomously, with NUC-1 being the primary enzyme involved.
Area of Science:
- Cell biology
- Developmental biology
- Genetics
Background:
- Apoptosis involves DNA fragmentation executed either autonomously within dying cells or non-autonomously by engulfing cells.
- The TUNEL assay detects DNA fragments from caspase-associated DNase (CAD) but not DNase II.
- DNase II plays a role in DNA degradation during apoptosis, but its precise modes of action remain unclear.
Purpose of the Study:
- To develop a novel method, topoisomerase ligation fluorescence probes (ToLFP), for visualizing DNA fragments generated by DNase II.
- To investigate the autonomous and non-autonomous modes of DNase II activity during apoptosis in Caenorhabditis elegans embryos.
- To elucidate the specific roles of DNase II enzymes, particularly NUC-1, in apoptotic DNA degradation.
Main Methods:
- Development and application of the topoisomerase ligation fluorescence probes (ToLFP) assay.
- Analysis of DNA fragmentation in wild-type (N2), ced-1, and various DNase II mutant (nuc-1, crn-6, crn-7) C. elegans embryos.
- Utilizing nuc-1-rescuing transgenic worms expressing NUC-1 fusion proteins to study its mode of action.
Main Results:
- The ToLFP method successfully visualized DNase II-generated DNA fragments in C. elegans embryos.
- Demonstrated autonomous DNase II activity in dying cells of ced-1 embryos, which lack engulfment.
- Quantified a higher ratio of non-autonomous to autonomous DNase II activity in wild-type embryos (~3-7).
- Confirmed NUC-1 as the major DNase II, with nuc-1 mutants showing significantly reduced ToLFP signals.
- Showed that ectopically expressed NUC-1 can degrade apoptotic DNA in a non-autonomous, secretion-dependent manner.
Conclusions:
- The ToLFP assay is a valuable tool for differentiating autonomous and non-autonomous DNase II activity in apoptosis.
- DNase II, primarily NUC-1, acts both within dying cells and is secreted to degrade DNA in neighboring cells.
- This study provides new insights into the mechanisms of apoptotic DNA degradation and the spatial regulation of DNase II function.
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