Drosophila phosphopantothenoylcysteine synthetase is required for tissue morphogenesis during oogenesis

Background: Coenzyme A (CoA) is an essential metabolite, synthesized from vitamin B5 by the subsequent action of five enzymes: PANK, PPCS, PPCDC, PPAT and DPCK. Mutations in Drosophila dPPCS disrupt female fecundity and in this study we analyzed the female sterile phenotype of dPPCS mutants in detail. Results: We demonstrate that dPPCS is required for various processes that occur during oogenesis including chorion patterning. Our analysis demonstrates that a mutation in dPPCS disrupts the organization of the somatic and germ line cells, affects F-actin organization and results in abnormal PtdIns(4,5)P2 localization. Improper cell organization coincides with aberrant localization of the membrane molecules Gurken (Grk) and Notch, whose activities are required for specification of the follicle cells that pattern the eggshell. Mutations in dPPCS also induce alterations in scutellar patterning and cause wing vein abnormalities. Interestingly, mutations in dPANK and dPPAT-DPCK result in similar patterning defects. Conclusion: Together, our results demonstrate that de novo CoA biosynthesis is required for proper tissue morphogenesis

Grp/DChk1 is required for G(2)-M checkpoint activation in Drosophila S2 cells, whereas Dmnk/DChk2 is dispensable

Cell-cycle checkpoints are signal-transduction pathways required to maintain genomic stability in dividing cells. Previously, it was reported that two kinases essential for checkpoint signalling, Chk1 and Chk2 are structurally conserved. In contrast to yeast, Xenopus and mammals, the Chk1- and Chk2-dependent pathways in Drosophila are not understood in detail. Here, we report the function of these checkpoint kinases, referred to as Grp/DChk1 and Dmnk/DChk2 in Drosophila Schneider's cells, and identify an upstream regulator as well as downstream targets of Grp/DChk1. First, we demonstrate that S2 cells are a suitable model for G(2)/M checkpoint studies. S2 cells display Grp/DChk1-dependent and Dmnk/DChk2-independent cell-cycle-checkpoint activation in response to hydroxyurea and ionizing radiation. S2 cells depleted for Grp/DChk1 using RNA interference enter mitosis in the presence of impaired DNA integrity, resulting in prolonged mitosis and mitotic catastrophe. Grp/DChk1 is phosphorylated in a Mei-41/DATR-dependent manner in response to hydroxyurea and ionizing radiation, indicating that Mei-41/ATR is an upstream component in the Grp/DChk1 DNA replication and DNA-damage-response pathways. The level of Cdc25(Stg) and phosphorylation status of Cdc2 are modulated in a Grp/DChk1-dependent manner in response to hydroxyurea and irradiation, indicating that these cell-cycle regulators are downstream targets of the Grp/DChk1-dependent DNA replication and DNA-damage responses. By contrast, depletion of Dmnk/DChk2 by RNA interference had little effect on checkpoint responses to hydroxyurea and irradiation. We conclude that Grp/DChk1, and not Dmnk/DChk2, is the main effector kinase involved in G2/M checkpoint control in Drosophila cells

Stwl Modifies Chromatin Compaction and Is Required to Maintain DNA Integrity in the Presence of Perturbed DNA Replication

Hydroxyurea, a well-known DNA replication inhibitor, induces cell cycle arrest and intact checkpoint functions are required to survive DNA replication stress induced by this genotoxic agent. Perturbed DNA synthesis also results in elevated levels of DNA damage. It is unclear how organisms prevent accumulation of this type of DNA damage that coincides with hampered DNA synthesis. Here, we report the identification of stonewall ( stwl) as a novel hydroxyurea-hypersensitive mutant. We demonstrate that Stwl is required to prevent accumulation of DNA damage induced by hydroxyurea; yet, Stwl is not involved in S/M checkpoint regulation. We show that Stwl is a heterochromatin-associated protein with transcription-repressing capacities. In stwl mutants, levels of trimethylated H3K27 and H3K9 ( two hallmarks of silent chromatin) are decreased. Our data provide evidence for a Stwl-dependent epigenetic mechanism that is involved in the maintenance of the normal balance between euchromatin and heterochromatin and that is required to prevent accumulation of DNA damage in the presence of DNA replication stress.</p

<it>Drosophila </it>phosphopantothenoylcysteine synthetase is required for tissue morphogenesis during oogenesis

Abstract Background Coenzyme A (CoA) is an essential metabolite, synthesized from vitamin B5 by the subsequent action of five enzymes: PANK, PPCS, PPCDC, PPAT and DPCK. Mutations in Drosophila dPPCS disrupt female fecundity and in this study we analyzed the female sterile phenotype of dPPCS mutants in detail. Results We demonstrate that dPPCS is required for various processes that occur during oogenesis including chorion patterning. Our analysis demonstrates that a mutation in dPPCS disrupts the organization of the somatic and germ line cells, affects F-actin organization and results in abnormal PtdIns(4,5)P2 localization. Improper cell organization coincides with aberrant localization of the membrane molecules Gurken (Grk) and Notch, whose activities are required for specification of the follicle cells that pattern the eggshell. Mutations in dPPCS also induce alterations in scutellar patterning and cause wing vein abnormalities. Interestingly, mutations in dPANK and dPPAT-DPCK result in similar patterning defects. Conclusion Together, our results demonstrate that de novo CoA biosynthesis is required for proper tissue morphogenesis.</p

Centrosomes split in the presence of impaired DNA integrity during mitosis

A well-established function of centrosomes is their role in accomplishing a successful mitosis that gives rise to a pair of identical daughter cells. We recently showed that DNA replication defects and DNA damage in Drosophila embryos trigger centrosomal changes, but it remained unclear whether comparable centrosomal responses can be provoked in somatic mammalian cells. To investigate the centrosomal organization in the presence of impaired DNA integrity, live and ultrastructural analysis was performed on �-tubulin–GFP and EGFP–�-tubulin–expressing Chinese hamster ovary cells. We have shown that during mitosis in the presence of incompletely replicated or damaged DNA, centrosomes split into fractions containing only one centriole. This results in the formation of multipolar spindles with extra centrosome-like structures. Despite the extra centrosomes and the multipolarity of the spindles, cells do exit from mitosis, resulting in severe division errors. Our data provide evidence of a novel mechanism showing how numerous centrosomes and spindle defects can arise and how this can lead to the formation of aneuploid cells

A long-term flow cytometry assay to analyze the role of specific genes of Drosophila melanogaster S2 cells in surviving genotoxic stress

Drosophila S2 cells are easy to manipulate and culture and are a versatile model system for high-throughput screens such as genome-wide siRNA screens to find genes involved in stress or therapy resistance or for screening through large compound libraries to identify cytotoxins. Clonogenic assays are considered the gold-standard to investigate the cytotoxicity of specific treatments or to compare the sensitivity of various cell types for a specific treatment. However, this assay cannot be used for Drosophila S2 cells as they are virtually unable to grow in distinct colonies. We designed a novel fluorescence-based flow cytometry assay to study long-term proliferation of S2 cells under various conditions and in the presence of specific gene products or after downregulation of specific gene products. Here we validate this assay and we used this novel method to investigate the role of checkpoint genes grapes/Dchk1 and DmChk2 in cell survival responses. Our data demonstrate that Grapes/Dchk1 but not DmChk2 is required to survive hydroxyurea. Our assay will be of use to investigate the long-term effects of various treatments in S2 cells and to evaluate the role of specific proteins therein. 2008 International Society for Advancement of Cytometry

Contribution of Growth and Cell Cycle Checkpoints to Radiation Survival in Drosophila

Cell cycle checkpoints contribute to survival after exposure to ionizing radiation (IR) by arresting the cell cycle and permitting repair. As such, yeast and mammalian cells lacking checkpoints are more sensitive to killing by IR. We reported previously that Drosophila larvae mutant for grp (encoding a homolog of Chk1) survive IR as well as wild type despite being deficient in cell cycle checkpoints. This discrepancy could be due to differences either among species or between unicellular and multicellular systems. Here, we provide evidence that Grapes is needed for survival of Drosophila S2 cells after exposure to similar doses of IR, suggesting that multicellular organisms may utilize checkpoint-independent mechanisms to survive irradiation. The dispensability of checkpoints in multicellular organisms could be due to replacement of damaged cells by regeneration through increased nutritional uptake and compensatory proliferation. In support of this idea, we find that inhibition of nutritional uptake (by starvation or onset of pupariation) or inhibition of growth factor signaling and downstream targets (by mutations in cdk4, chico, or dmyc) reduced the radiation survival of larvae. Further, some of these treatments are more detrimental for grp mutants, suggesting that the need for compensatory proliferation is greater for checkpoint mutants. The difference in survival of grp and wild-type larvae allowed us to screen for small molecules that act as genotype-specific radiation sensitizers in a multicellular context. A pilot screen of a small molecule library from the National Cancer Institute yielded known and approved radio-sensitizing anticancer drugs. Since radiation is a common treatment option for human cancers, we propose that Drosophila may be used as an in vivo screening tool for genotype-specific drugs that enhance the effect of radiation therapy

De novo CoA biosynthesis is required to maintain DNA integrity during development of the Drosophila nervous system

In a forward genetic screen in Drosophila melanogaster , aimed to identify genes required for normal locomotor function, we isolated dPPCS (the second enzyme of the Coenzyme A biosynthesis pathway). The entire Drosophila CoA synthesis route was dissected, annotated and additional CoA mutants were obtained (dPANK/fumble ) or generated (dPPAT-DPCK ). Drosophila CoA mutants suffer from neurodegeneration, altered lipid homeostasis and the larval brains display increased apoptosis. Also, de novo CoA biosynthesis is required to maintain DNA integrity during the development of the central nervous system. In humans, mutations in the PANK2 gene, the first enzyme in the CoA synthesis route, are associated with pantothenate kinase-associated neurodegeneration. Currently, the pathogenesis of this neurodegenerative disease is poorly understood. We provide the first comprehensive analysis of the physiological implications of mutations in the entire CoA biosynthesis route in an animal model system. Surprisingly, our findings reveal a major role of this conserved pathway in maintaining DNA and cellular integrity, explaining how impaired CoA synthesis during CNS development can elicit a neurodegenerative phenotype