SUMMARYThe nucleolus is a subnuclear factory, the activity of which is required beyond ribosome biogenesis for the regulation of cell growth, death and proliferation. In both Drosophila and mammalian cells, the activity of the nucleolus is regulated by the protooncogene Myc. Myc induces the transcription of genes required for ribosome biogenesis and the synthesis of rRNA by RNA polymerase I, a nucleolar event that is rate limiting for cell growth. Here, we show that the fruit fly Nol12 homologue Viriato is a key determinant of nucleolar architecture that is required for tissue growth and cell survival during Drosophila development. We further show that viriato expression is controlled by Drosophila Myc (dMyc), and that the ability of dMyc to stimulate nucleolar and cellular growth depends on viriato expression. Therefore, viriato acts downstream of dMyc to ensure a coordinated nucleolar response to dMyc-induced growth and, thereby, normal organ development. KEY WORDS: Drosophila, Cell proliferation, Cell growth, dMyc (Diminutive), Viriato, Nol12The Drosophila Nol12 homologue viriato is a dMyc target that regulates nucleolar architecture and is required for dMyc-stimulated cell growth HumanNOL12-GFP constructs were verified by DNA sequencing. Transgenic lines were generated by standard germline transformation methods and four independent lines were analysed. The Ubi-Gal4 driver was used to drive low-level expression of Vito-GFP in salivary glands, and ey-Gal4 was used to drive hNOL12-GFP expression during eye development and in salivary glands. ey-Gal4 drives 'leaky' Gal4 expression in the salivary glands. Mitotic recombinationMitotic recombination was induced using the Flp/FRT method. vito knockdown clones, or clones overexpressing dMyc alone or together with vitoRNAi, were induced by heat shock (1 hour at 37°C) at 48 ± 4 hours after egg laying (AEL) and dissected at 118 ± 4 hours AEL in larvae of the genotype y w hsflp/+; act>y+>Gal4, UAS-GFP/UAS-vitoRNAi, y w hsflp/+; act>y+>Gal4, UAS-GFP/+; UAS-dMyc/+ and y w hsflp/+; act>y+>Gal4, UAS-GFP/UAS-vitoRNAi; UAS-dMyc/+. ImmunostainingEye-antennal imaginal discs were prepared for immunohistochemistry using standard protocols. Primary antibodies used were: rabbit anti-cleaved Caspase-3 at 1:200 (Cell Signaling), mouse anti-Armadillo N27A1 at 1:100 [Developmental Studies Hybridoma Bank (DSHB)], mouse anti--galactosidase at 1:1000 (Promega, #Z3783), rat anti-Elav 7E8A10 at 1:100 (DSHB), mouse anti-Lamin ADL101 at 1:1 (DSHB), and rabbit antiFibrillarin at 1:250 (Abcam, #ab5821). Appropriate Alexa Fluorconjugated secondary antibodies were from Molecular Probes. Images were obtained with the Leica SP2 confocal system and processed with Adobe Photoshop. In situ hybridisationA digoxigenin (DIG)-labelled vito antisense RNA probe was synthesized by in vitro transcription with T7 RNA polymerase and DIG-UTP after linearisation of cDNA LD10447 with NotI and were used for whole-mount in situ hybridisation of fixed larvae. A sense RNA probe was used as a negative control. The DIG...
Immune checkpoint therapies target tumor antigen-specific T cells, but less is known about their effects on natural killer (NK) cells, which help control metastasis. In studying the development of lung metastases, we found that NK cells lose their cytotoxic capacity and acquire a molecular signature defined by the expression of coinhibitory receptors. In an effort to overcome this suppressive mechanism, we evaluated NK cell responses to the immunostimulatory cytokine IL12. Exposure to IL12 rescued the cytotoxicity of NK cells but also led to the emergence of an immature NK cell population that expressed high levels of the coinhibitory molecules PD-1, Lag-3, and TIGIT, thereby limiting NK cell-mediated control of pulmonary metastases. Notably, checkpoint blockade therapy synergized with IL12 to fully enable tumor control by NK cells, demonstrating that checkpoint blockers are not only applicable to enhance T cell-mediated immunotherapy, but also to restore the tumor-suppressive capacity of NK cells. .
Drosophila Decapentaplegic (Dpp), a member of the BMP2/4 class of the TGF-βs, is required for organ growth, patterning and differentiation. However, much remains to be understood about the mechanisms acting downstream of these multiple roles. Here we investigate this issue during the development of the Drosophila eye. We have previously identified viriato (vito) as a dMyc-target gene encoding a nucleolar protein that is required for proper tissue growth in the developing eye. By carrying out a targeted in vivo double-RNAi screen to identify genes and pathways functioning with Vito during eye development, we found a strong genetic interaction between vito and members of the Dpp signaling pathway including the TGF-β receptors tkv (type I), put (type II), and the co-Smad medea (med). Analyzing the expression of the Dpp receptor Tkv and the activation pattern of the pathway's transducer, p-Mad, we found that vito is required for a correct signal transduction in Dpp-receiving cells. Overall, we validate the use of double RNAi to find specific genetic interactions and, in particular, we uncover a link between the Dpp pathway and Vito, a nucleolar component. vito would act genetically downstream of Dpp, playing an important role in maintaining a sufficient level of Dpp activity for the promotion of eye disc growth and regulation of photoreceptor differentiation in eye development.
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