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Regulation of Caenorhabditis elegans p53/CEP-1-dependent germ cell apoptosis by Ras/MAPK signaling.

ABSTRACT: Maintaining genome stability in the germline is thought to be an evolutionarily ancient role of the p53 family. The sole Caenorhabditis elegans p53 family member CEP-1 is required for apoptosis induction in meiotic, late-stage pachytene germ cells in response to DNA damage and meiotic recombination failure. In an unbiased genetic screen for negative regulators of CEP-1, we found that increased activation of the C. elegans ERK orthologue MPK-1, resulting from either loss of the lip-1 phosphatase or activation of let-60 Ras, results in enhanced cep-1-dependent DNA damage induced apoptosis. We further show that MPK-1 is required for DNA damage-induced germ cell apoptosis. We provide evidence that MPK-1 signaling regulates the apoptotic competency of germ cells by restricting CEP-1 protein expression to cells in late pachytene. Restricting CEP-1 expression to cells in late pachytene is thought to ensure that apoptosis doesn't occur in earlier-stage cells where meiotic recombination occurs. MPK-1 signaling regulates CEP-1 expression in part by regulating the levels of GLD-1, a translational repressor of CEP-1, but also via a GLD-1-independent mechanism. In addition, we show that MPK-1 is phosphorylated and activated upon ionising radiation (IR) in late pachytene germ cells and that MPK-1-dependent CEP-1 activation may be in part direct, as these two proteins interact in a yeast two-hybrid assay. In summary, we report our novel finding that MAP kinase signaling controls CEP-1-dependent apoptosis by several different pathways that converge on CEP-1. Since apoptosis is also restricted to pachytene stage cells in mammalian germlines, analogous mechanisms regulating p53 family members are likely to be conserved throughout evolution.

SUBMITTER: Rutkowski R

PROVIDER: S-EPMC3161941 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:Cell death plays a major role during C. elegans oogenesis, where over half of the oogenic germ cells die in a process termed physiological apoptosis. How germ cells are selected for physiological apoptosis, or instead become oocytes, is not understood. Most oocytes produce viable embryos when apoptosis is blocked, suggesting that physiological apoptosis does not function to cull defective germ cells. Instead, cells targeted for apoptosis may function as nurse cells; the germline is syncytial, and all germ cells appear to contribute cytoplasm to developing oocytes. C. elegans has been a leading model for the genetics and molecular biology of apoptosis and phagocytosis, but comparatively few studies have examined the cell biology of apoptotic cells. We used live imaging to identify and examine pre-apoptotic germ cells in the adult gonad. After initiating apoptosis, germ cells selectively export their mitochondria into the shared pool of syncytial cytoplasm; this transport appears to use the microtubule motor kinesin. The apoptotic cells then shrink as they expel most of their remaining cytoplasm, and close off from the syncytium. Shortly thereafter the apoptotic cells restructure their microtubule and actin cytoskeletons, possibly to maintain cell integrity; the microtubules form a novel, cortical array of stabilized microtubules, and actin and cofilin organize into giant cofilin-actin rods. We discovered that some apoptotic germ cells are binucleate; the binucleate germ cells can develop into binucleate oocytes in apoptosis-defective strains, and appear capable of producing triploid offspring. Our results suggest that the nuclear layer of the germline syncytium becomes folded during mitosis and growth, and that binucleate cells arise as the layer unfolds or everts; all of the binucleate cells are subsequently removed by apoptosis. These results show that physiological apoptosis targets at least two distinct populations of germ cells, and that the apoptosis machinery efficiently recognizes cells with two nuclei.

Project description:Reactive oxygen species (ROS) are signalling molecules whose study in intact organisms has been hampered by their potential toxicity. This has prevented a full understanding of their role in organismal processes such as development, aging and disease. In Caenorhabditis elegans, the development of the vulva is regulated by a signalling cascade that includes LET-60ras (homologue of mammalian Ras), MPK-1 (ERK1/2) and LIN-1 (an ETS transcription factor). We show that both mitochondrial and cytoplasmic ROS act on a gain-of-function (gf) mutant of the LET-60ras protein through a redox-sensitive cysteine (C118) previously identified in mammals. We show that the prooxidant paraquat as well as isp-1, nuo-6 and sod-2 mutants, which increase mitochondrial ROS, inhibit the activity of LET-60rasgf on vulval development. In contrast, the antioxidant NAC and loss of sod-1, both of which decrease cytoplasmic H202, enhance the activity of LET-60rasgf. CRISPR replacement of C118 with a non-oxidizable serine (C118S) stimulates LET-60rasgf activity, whereas replacement of C118 with aspartate (C118D), which mimics a strongly oxidised cysteine, inhibits LET-60rasgf. These data strongly suggest that C118 is oxidized by cytoplasmic H202 generated from dismutation of mitochondrial and/or cytoplasmic superoxide, and that this oxidation inhibits LET-60ras. This contrasts with results in cultured mammalian cells where it is mostly nitric oxide, which is not found in worms, that oxidizes C118 and activates Ras. Interestingly, PQ, NAC and the C118S mutation do not act on the phosphorylation of MPK-1, suggesting that oxidation of LET-60ras acts on an as yet uncharacterized MPK-1-independent pathway. We also show that elevated cytoplasmic superoxide promotes vulva formation independently of C118 of LET-60ras and downstream of LIN-1. Finally, we uncover a role for the NADPH oxidases (BLI-3 and DUOX-2) and their redox-sensitive activator CED-10rac in stimulating vulva development. Thus, there are at least three genetically separable pathways by which ROS regulates vulval development.

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Regulation of Caenorhabditis elegans p53/CEP-1-dependent germ cell apoptosis by Ras/MAPK signaling.

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