32 research outputs found

    The Drosophila IKK-related kinase (Ik2) and Spindle-F proteins are part of a complex that regulates cytoskeleton organization during oogenesis

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    <p>Abstract</p> <p>Background</p> <p>IkappaB kinases (IKKs) regulate the activity of Rel/NF-kappaB transcription factors by targeting their inhibitory partner proteins, IkappaBs, for degradation. The <it>Drosophila </it>genome encodes two members of the IKK family. Whereas the first is a kinase essential for activation of the NF-kappaB pathway, the latter does not act as IkappaB kinase. Instead, recent findings indicate that Ik2 regulates F-actin assembly by mediating the function of nonapoptotic caspases via degradation of DIAP1. Also, it has been suggested that <it>ik2 </it>regulates interactions between the minus ends of the microtubules and the actin-rich cortex in the oocyte. Since <it>spn-F </it>mutants display oocyte defects similar to those of <it>ik2 </it>mutant, we decided to investigate whether Spn-F could be a direct regulatory target of Ik2.</p> <p>Results</p> <p>We found that Ik2 binds physically to Spn-F, biomolecular interaction analysis of Spn-F and Ik2 demonstrating that both proteins bind directly and form a complex. We showed that Ik2 phosphorylates Spn-F and demonstrated that this phosphorylation does not lead to Spn-F degradation. Ik2 is localized to the anterior ring of the oocyte and to punctate structures in the nurse cells together with Spn-F protein, and both proteins are mutually required for their localization.</p> <p>Conclusion</p> <p>We conclude that Ik2 and Spn-F form a complex, which regulates cytoskeleton organization during <it>Drosophila </it>oogenesis and in which Spn-F is the direct regulatory target for Ik2. Interestingly, Ik2 in this complex does not function as a typical IKK in that it does not direct SpnF for degradation following phosphorylation.</p

    Localization of the Drosophila Rad9 Protein to the Nuclear Membrane Is Regulated by the C-Terminal Region and Is Affected in the Meiotic Checkpoint

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    Rad9, Rad1, and Hus1 (9-1-1) are part of the DNA integrity checkpoint control system. It was shown previously that the C-terminal end of the human Rad9 protein, which contains a nuclear localization sequence (NLS) nearby, is critical for the nuclear transport of Rad1 and Hus1. In this study, we show that in Drosophila, Hus1 is found in the cytoplasm, Rad1 is found throughout the entire cell and that Rad9 (DmRad9) is a nuclear protein. More specifically, DmRad9 exists in two alternatively spliced forms, DmRad9A and DmRad9B, where DmRad9B is localized at the cell nucleus, and DmRad9A is found on the nuclear membrane both in Drosophila tissues and also when expressed in mammalian cells. Whereas both alternatively spliced forms of DmRad9 contain a common NLS near the C terminus, the 32 C-terminal residues of DmRad9A, specific to this alternative splice form, are required for targeting the protein to the nuclear membrane. We further show that activation of a meiotic checkpoint by a DNA repair gene defect but not defects in the anchoring of meiotic chromosomes to the oocyte nuclear envelope upon ectopic expression of non-phosphorylatable Barrier to Autointegration Factor (BAF) dramatically affects DmRad9A localization. Thus, by studying the localization pattern of DmRad9, our study reveals that the DmRad9A C-terminal region targets the protein to the nuclear membrane, where it might play a role in response to the activation of the meiotic checkpoint

    Sequestration of Highly Expressed mRNAs in Cytoplasmic Granules, P-Bodies, and Stress Granules Enhances Cell Viability

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    Transcriptome analyses indicate that a core 10%–15% of the yeast genome is modulated by a variety of different stresses. However, not all the induced genes undergo translation, and null mutants of many induced genes do not show elevated sensitivity to the particular stress. Elucidation of the RNA lifecycle reveals accumulation of non-translating mRNAs in cytoplasmic granules, P-bodies, and stress granules for future regulation. P-bodies contain enzymes for mRNA degradation; under stress conditions mRNAs may be transferred to stress granules for storage and return to translation. Protein degradation by the ubiquitin-proteasome system is elevated by stress; and here we analyzed the steady state levels, decay, and subcellular localization of the mRNA of the gene encoding the F-box protein, UFO1, that is induced by stress. Using the MS2L mRNA reporter system UFO1 mRNA was observed in granules that colocalized with P-bodies and stress granules. These P-bodies stored diverse mRNAs. Granules of two mRNAs transported prior to translation, ASH1-MS2L and OXA1-MS2L, docked with P-bodies. HSP12 mRNA that gave rise to highly elevated protein levels was not observed in granules under these stress conditions. ecd3, pat1 double mutants that are defective in P-body formation were sensitive to mRNAs expressed ectopically from strong promoters. These highly expressed mRNAs showed elevated translation compared with wild-type cells, and the viability of the mutants was strongly reduced. ecd3, pat1 mutants also exhibited increased sensitivity to different stresses. Our interpretation is that sequestration of highly expressed mRNAs in P-bodies is essential for viability. Storage of mRNAs for future regulation may contribute to the discrepancy between the steady state levels of many stress-induced mRNAs and their proteins. Sorting of mRNAs for future translation or decay by individual cells could generate potentially different phenotypes in a genetically identical population and enhance its ability to withstand stress

    Expression of the Drosophila melanogaster GADD45 Homolog (CG11086) Affects Egg Asymmetric Development That Is Mediated by the c-Jun N-Terminal Kinase Pathway

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    The mammalian GADD45 (growth arrest and DNA-damage inducible) gene family is composed of three highly homologous small, acidic, nuclear proteins: GADD45α, GADD45β, and GADD45γ. GADD45 proteins are involved in important processes such as regulation of DNA repair, cell cycle control, and apoptosis. Annotation of the Drosophila melanogaster genome revealed that it contains a single GADD45-like protein (CG11086; D-GADD45). We found that, as its mammalian homologs, D-GADD45 is a nuclear protein; however, D-GADD45 expression is not elevated following exposure to genotoxic and nongenotoxic agents in Schneider cells and in adult flies. We showed that the D-GADD45 transcript increased following immune response activation, consistent with previous microarray findings. Since upregulation of GADD45 proteins has been characterized as an important cellular response to genotoxic and nongenotoxic agents, we aimed to characterize the effect of D-GADD45 overexpression on D. melanogaster development. Overexpression of D-GADD45 in various tissues led to different phenotypic responses. Specifically, in the somatic follicle cells overexpression caused apoptosis, while overexpression in the germline affected the dorsal–ventral polarity of the eggshell and disrupted the localization of anterior–posterior polarity determinants. In this article we focused on the role of D-GADD45 overexpression in the germline and found that D-GADD45 caused dorsalization of the eggshell. Since mammalian GADD45 proteins are activators of the c-Jun N-terminal kinase (JNK)/p38 mitogen-activated protein kinase (MAPK) signaling pathways, we tested for a genetic interaction in D. melanogaster. We found that eggshell polarity defects caused by D-GADD45 overexpression were dominantly suppressed by mutations in the JNK pathway, suggesting that the JNK pathway has a novel, D-GADD45-mediated, function in the Drosophila germline

    Localization of the <i>Drosophila</i> Rad9, Hus1 and Rad1 proteins in S<sub>2</sub>R+ and follicle cells.

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    <p>A–G, Confocal images of S<sub>2</sub>R+ cells, I–K, Confocal images of follicle cells from egg chambers. (A) S<sub>2</sub>R+ cells expressi ng HA-DmHus1 and stained with anti-HA antibodies in red. (B) S<sub>2</sub>R+ cells expressing GFP-DmRad1. (C) S<sub>2</sub>R+ cells expressing GFP-DmRad9A. (F) S<sub>2</sub>R+ cells expressing DmRad9B-GFP. (D) and (G) Staining with anti-lamin antibodies, which mark the nuclear membrane, in red. (E and H) are merged image of (C with differential interference contrast (DIC) image) and (F with a DIC image), respectively. (I) Egg chamber from HA-DmHus1::<i>CY2Gal4</i> transgenic flies. (J) Egg chamber from GFP-DmRad1::<i>CY2Gal4</i> transgenic flies. (K) Egg chamber from FLAG-DmRad9A::<i>CY2Gal4</i> transgenic flies. In both S<sub>2</sub>R+ and follicle cells, DmHus1 is found in the cytoplasm, DmRad1 is found throughout the cell and Dm DmRad9A is localized to the nuclear membrane. DmRad9B is localized to the nucleus in S<sub>2</sub>R+ cells.</p

    Localization of the <i>Drosophila</i> Rad9, Hus1 and Rad1 proteins in mammalian

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    <p>Human Embryonic Kidney 293 <b>(HEK293).</b> Confocal images of cells expressing (A) GFP-DmHus1, (D) GFP-DmRad1, and (G) GFP-DmRad9A. (B, E and H) Antibody staining of the NUP 414 protein, which recognizes several nucleoporins. (C, F and I) are merged images of (A–B), (D–E), and (G–H), respectively.</p

    Identification of the DmRad9A nuclear localization signal.

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    <p>Confocal images of S<sub>2</sub>R+ cells expressing DmRad9A mutated in suspected NLS sequences. (A) DmRad9A mutated at position 287 – 289 (NLS1). (D) DmRad9A mutated Position 300–302 (NLS2). (G) DmRad9A mutated Position 314–316 (NLS3). (B, E and H) stained with anti-lamin antibodies, which mark the nuclear membrane, in red. (C) Merged image of (A) and (B). (F) Merged image of (D) and (E). (I) Merged image of (G) and (H).</p
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