Cultured Drosophila S2 Cells Research Articles

BHLH-ORANGE family genes regulate the expression of E-box clock genes in Drosophila

Drosophila has 13 basic helix-loop-helix-ORANGE (bHLH-O) family genes. One of the members, clockwork orange (cwo), which is the counterpart of mammalian clock genes Dec1 and Dec2, regulates the transcriptional feedback loops of circadian clock genes through binding to E-box sequences in target gene promoters. The goal of the current study was to determine the role of Drosophila bHLH-O proteins in circadian rhythms at the molecular and behavioral level. Promoter assays in cultured Drosophila S2 cells were carried out to investigate which of the known bHLH-O proteins directly regulates the transcription of clock genes. In addition to CWO, three other bHLH-O proteins, SIDE, Mβ and Mγ, suppressed E-box clock gene transcription in vitro. RNA interference (RNAi) was used to generate bHLH-O knockdown flies in pacemaker neurons, and then locomotor activity rhythm was measured. cwo knockdown flies exhibited a remarkable phenotype. To clarify the functional complementation in circadian regulation among CWO, SIDE, Mβ and Mγ, promoter activity in the presence of combinations of two bHLH-O genes and locomotor rhythm in double knockdown flies were examined. The results suggest that CWO predominantly acts as a key factor of circadian regulation both in vitro and in vivo.

The ADP-ribosylation factor 1 (Arf1) is involved in regulating copper uptake

Copper is a cofactor for many essential enzymes in aerobic organisms. When intracellular copper levels are elevated, the Menkes (ATP7A) P-Type ATPase traffics from the trans-Golgi network (TGN) towards the plasma membrane to facilitate copper efflux. The ADP-ribosylation factor 1 (Arf1) is required for maintenance of Golgi architecture and for vesicular trafficking, including the copper-responsive trafficking of ATP7A. Here we report an ATP7A-independent role of Arf1 in copper homeostasis. Whilst the loss of ATP7A function increased copper levels, RNA interference mediated Arf1 knockdown reduced copper accumulation in HeLa cells as well as in both wild-type and ATP7A-null cultured fibroblasts. Arf1 therefore affected copper levels independently of ATP7A mediated copper efflux. Knockdown of Arf79F, the Drosophila melanogasterArf1 orthologue, also reduced copper accumulation in cultured Drosophila S2 cells, indicating an evolutionarily conserved role for this protein in cellular copper homeostasis. Whereas severe Arf1 inhibition with brefeldin A caused fragmentation and dispersal of the TGN resident protein Golgin 97, the peri-nuclear localisation of the Golgin 97 was retained following Arf1 knockdown, consistent with a moderate reduction in Arf1 activity. Ctr1 levels at the plasma membrane of cultured fibroblast cells were reduced following Arf1 knockdown, indicating an Arf1-dependent trafficking pathway is required for correct distribution of this copper uptake protein. Arf1-dependent trafficking pathways are therefore required for optimal copper uptake efficiency in cultured human and Drosophila cells.

Identification and in vivo characterization of NvFP-7R, a developmentally regulated red fluorescent protein of Nematostella vectensis.

BackgroundIn recent years, the sea anemone Nematostella vectensis has emerged as a critical model organism for comparative genomics and developmental biology. Although Nematostella is a member of the anthozoan cnidarians (known for producing an abundance of diverse fluorescent proteins (FPs)), endogenous patterns of Nematostella fluorescence have not been described and putative FPs encoded by the genome have not been characterized.Methodology/Principal FindingsWe described the spatiotemporal expression of endogenous red fluorescence during Nematostella development. Spatially, there are two patterns of red fluorescence, both restricted to the oral endoderm in developing polyps. One pattern is found in long fluorescent domains associated with the eight mesenteries and the other is found in short fluorescent domains situated between tentacles. Temporally, the long domains appear simultaneously at the 12-tentacle stage. In contrast, the short domains arise progressively between the 12- and 16-tentacle stage. To determine the source of the red fluorescence, we used bioinformatic approaches to identify all possible putative Nematostella FPs and a Drosophila S2 cell culture assay to validate NvFP-7R, a novel red fluorescent protein. We report that both the mRNA expression pattern and spectral signature of purified NvFP-7R closely match that of the endogenous red fluorescence. Strikingly, the red fluorescent pattern of NvFP-7R exhibits asymmetric expression along the directive axis, indicating that the nvfp-7r locus senses the positional information of the body plan. At the tissue level, NvFP-7R exhibits an unexpected subcellular localization and a complex complementary expression pattern in apposed epithelia sheets comprising each endodermal mesentery.Conclusions/SignificanceThese experiments not only identify NvFP-7R as a novel red fluorescent protein that could be employed as a research tool; they also uncover an unexpected spatio-temporal complexity of gene expression in an adult cnidarian. Perhaps most importantly, our results define Nematostella as a new model organism for understanding the biological function of fluorescent proteins in vivo.

Complementary transcriptomic, lipidomic, and targeted functional genetic analyses in cultured Drosophila cells highlight the role of glycerophospholipid metabolism in Flock House virus RNA replication

BackgroundCellular membranes are crucial host components utilized by positive-strand RNA viruses for replication of their genomes. Published studies have suggested that the synthesis and distribution of membrane lipids are particularly important for the assembly and function of positive-strand RNA virus replication complexes. However, the impact of specific lipid metabolism pathways in this process have not been well defined, nor have potential changes in lipid expression associated with positive-strand RNA virus replication been examined in detail.ResultsIn this study we used parallel and complementary global and targeted approaches to examine the impact of lipid metabolism on the replication of the well-studied model alphanodavirus Flock House virus (FHV). We found that FHV RNA replication in cultured Drosophila S2 cells stimulated the transcriptional upregulation of several lipid metabolism genes, and was also associated with increased phosphatidylcholine accumulation with preferential increases in lipid molecules with longer and unsaturated acyl chains. Furthermore, targeted RNA interference-mediated downregulation of candidate glycerophospholipid metabolism genes revealed a functional role of several genes in virus replication. In particular, we found that downregulation of Cct1 or Cct2, which encode essential enzymes for phosphatidylcholine biosynthesis, suppressed FHV RNA replication.ConclusionThese results indicate that glycerophospholipid metabolism, and in particular phosphatidylcholine biosynthesis, plays an important role in FHV RNA replication. Furthermore, they provide a framework in which to further explore the impact of specific steps in lipid metabolism on FHV replication, and potentially identify novel cellular targets for the development of drugs to inhibit positive-strand RNA viruses.

Drosophila endogenous small RNAs bind to Argonaute 2 in somatic cells

RNA silencing is a conserved mechanism in which small RNAs trigger various forms of sequence-specific gene silencing by guiding Argonaute complexes to target RNAs by means of base pairing. RNA silencing is thought to have evolved as a form of nucleic-acid-based immunity to inactivate viruses and transposable elements. Although the activity of transposable elements in animals has been thought largely to be restricted to the germ line, recent studies have shown that they may also actively transpose in somatic cells, creating somatic mosaicism in animals. In the Drosophila germ line, Piwi-interacting RNAs arise from repetitive intergenic elements including retrotransposons by a Dicer-independent pathway and function through the Piwi subfamily of Argonautes to ensure silencing of retrotransposons. Here we show that, in cultured Drosophila S2 cells, Argonaute 2 (AGO2), an AGO subfamily member of Argonautes, associates with endogenous small RNAs of 20-22 nucleotides in length, which we have collectively named endogenous short interfering RNAs (esiRNAs). esiRNAs can be divided into two groups: one that mainly corresponds to a subset of retrotransposons, and the other that arises from stem-loop structures. esiRNAs are produced in a Dicer-2-dependent manner from distinctive genomic loci, are modified at their 3' ends and can direct AGO2 to cleave target RNAs. Mutations in Dicer-2 caused an increase in retrotransposon transcripts. Together, our findings indicate that different types of small RNAs and Argonautes are used to repress retrotransposons in germline and somatic cells in Drosophila.

Akirins Clarify NF-κB Signaling

Once again, the fruit fly has proven an invaluable tool for revealing information about the immune responses involving the transcription factor nuclear factor κB (NF-κB), as Beutler and Moresco emphasize. Goto et al . performed a genome-wide RNA interference (RNAi) screen in cultured Drosophila S2 cells for genes required for NF-κB-dependent gene expression and found the gene CG8580 , which they renamed Akirin (for the Japanese akiraka ni suru, meaning "making things clear"). The authors determined that Drosophila has a single Akirin gene, which is essential for viability, and there are two in mammals ( Akirin1 , which is not essential for viability in mice, and Akirin2 , which is). Transfection experiments with tagged versions of the proteins indicated that Akirins are localized to the nucleus, and the nuclear localization signal is the only recognizable motif in these proteins. S2 cells in which Akirin was knocked down showed defective activation of gene expression in response to stimuli that activate the Imd pathway, which mediates immune response to Gram-negative bacteria through the activation of the NF-κB homolog Relish. In contrast, the response to signaling through the Toll pathway, which mediates the response to Gram-positive bacteria through the activation of the NF-κB homolog Dif was not affected. Epistasis experiments placed Akirin at the level of Relish in the Imd pathway. In flies, knockdown of Akirin led to decreased survival after Gram-negative bacterial infection. Analysis of Akirin1 or Akirin2 knockout mouse embryo fibroblasts (MEFs) suggested that Akirin2 was the homolog involved in NF-κB signaling downstream of Toll-like receptors (TLRs) or the tumor necrosis factor (TNF) receptor. Furthermore, the human Akirin2 protein rescued S2 cells from defective Imd signaling caused by knockdown of endogenous Akirin. MEFs deficient for Akirin2 showed impaired expression of a subset of NF-κB-regulated genes, yet stimulation of NF-κB DNA binding and degradation of the inhibitor protein IκBα was not impaired. These results suggest that in mice, as in the fly, Akirin2 acts at the same level as NF-κB, and it will be interesting to see whether these two proteins form a complex, as has been recently reported for ribosomal protein S3 and NF-κB (see related resource). A. Goto, K. Matsushita, V. Gesellchen, L. El Chamy, D. Kuttenkeuler, O. Takeuchi, J. A. Hoffmann, S. Akira, M. Boutros, J.-M. Reichhart, Akirins are highly conserved nuclear proteins required for NF-κB-dependent gene expression in drosophila and mice. Nat. Immunol. 9 , 97-104 (2008). [PubMed] B. Beutler, E. M. Y. Moresco, Akirins versus infection. Nat. Immunol. 9 , 7-9 (2008). [PubMed]

RNA Is an Integral Component of Chromatin that Contributes to Its Structural Organization

Chromatin structure is influenced by multiples factors, such as pH, temperature, nature and concentration of counterions, post-translational modifications of histones and binding of structural non-histone proteins. RNA is also known to contribute to the regulation of chromatin structure as chromatin-induced gene silencing was shown to depend on the RNAi machinery in S. pombe, plants and Drosophila. Moreover, both in Drosophila and mammals, dosage compensation requires the contribution of specific non-coding RNAs. However, whether RNA itself plays a direct structural role in chromatin is not known. Here, we report results that indicate a general structural role for RNA in eukaryotic chromatin. RNA is found associated to purified chromatin prepared from chicken liver, or cultured Drosophila S2 cells, and treatment with RNase A alters the structural properties of chromatin. Our results indicate that chromatin-associated RNAs, which account for 2%–5% of total chromatin-associated nucleic acids, are polyA− and show a size similar to that of the DNA contained in the corresponding chromatin fragments. Chromatin-associated RNA(s) are not likely to correspond to nascent transcripts as they are also found bound to chromatin when cells are treated with α-amanitin. After treatment with RNase A, chromatin fragments of molecular weight >3.000 bp of DNA showed reduced sedimentation through sucrose gradients and increased sensitivity to micrococcal nuclease digestion. This structural transition, which is observed both at euchromatic and heterochromatic regions, proceeds without loss of histone H1 or any significant change in core-histone composition and integrity.

The Drosophila Class B Scavenger Receptor NinaD-I Is a Cell Surface Receptor Mediating Carotenoid Transport for Visual Chromophore Synthesis

The blind Drosophila mutant ninaD lacks the visual chromophore. Genetic evidence that the molecular basis is a defect in carotenoid uptake which causes vitamin A deficiency exists. The ninaD gene encodes a scavenger receptor that is significantly homologous in sequence with the mammalian scavenger receptors SR-BI (scavenger receptor class B type I) and CD36 (cluster determinant 36), yet NinaD has not been characterized in functional detail. Therefore, we established a Drosophila S2 cell culture system for biochemically characterizing the ninaD gene products. We show that the two splice variant isoforms encoded by ninaD exhibit different subcellular localizations. NinaD-I, the long protein variant, is localized at the plasma membrane, whereas the short variant, NinaD-II, is localized at intracellular membranes. Only NinaD-I could mediate the cellular uptake of carotenoids from micelles in this cell culture system. Carotenoid uptake was concentration-dependent and saturable. By in vivo analyses of different mutant and transgenic fly strains, we provide evidence of an essential role of NinaD-I in the absorption of dietary carotenoids to support visual chromophore synthesis. Moreover, our analyses suggest a role of NinaD-I in tocopherol metabolism. Even though Drosophila is a sterol auxotroph, we found no evidence of a contribution of NinaD-I to the uptake of these compounds. Together, our study establishes an evolutionarily conserved connection between class B scavenger receptors and the numerous functions of fat soluble vitamins in animal physiology.

The ultrastructure of the kinetochore and kinetochore fiber in Drosophila somatic cells

Drosophila melanogaster is a widely used model organism for the molecular dissection of mitosis in animals. However, despite the popularity of this system, no studies have been published on the ultrastructure of Drosophila kinetochores and kinetochore fibers (K-fibers) in somatic cells. To amend this situation, we used correlative light (LM) and electron microscopy (EM) to study kinetochores in cultured Drosophila S2 cells during metaphase, and after colchicine treatment to depolymerize all microtubules (MTs). We find that the structure of attached kinetochores in S2 cells is indistinct, consisting of an amorphous inner zone associated with a more electron-dense peripheral surface layer that is approximately 40-50 nm thick. On average, each S2 kinetochore binds 11+/-2 MTs, in contrast to the 4-6 MTs per kinetochore reported for Drosophila spermatocytes. Importantly, nearly all of the kinetochore MT plus ends terminate in the peripheral surface layer, which we argue is analogous to the outer plate in vertebrate kinetochores. Our structural observations provide important data for assessing the results of RNAi studies of mitosis, as well as for the development of mathematical modelling and computer simulation studies in Drosophila and related organisms.

Identification of the Drosophila skpA gene as a novel target of the transcription factor DREF

SKPa is component of a Drosophila SCF complex that functions in combination with the ubiquitin-conjugating enzyme UbcD1. skpA null mutation results in centrosome overduplication, unusual chromatin condensation, defective endoreduplication and cell-cycle progression. While the molecular mechanisms that regulate expression of the skpA gene are poorly understood, the DNA replication-related element (DRE) and the DRE-binding factor (DREF) play important roles in regulating proliferation-related genes in Drosophila and DRE (5′-TATCGATA) and DRE-like (5′-CATCGATT) sequences were here found to be involved in skpA promoter activity. Thus both luciferase transient expression assays in cultured Drosophila S2 cells using skpA promoter- luciferase fusion plasmids and anti-lacZ immunostaining of various tissues from transgenic third instar larvae carrying the skpA promoter- lacZ fusion genes provided supportive evidence. Furthermore, anti-SKPa immunostaining of eye imaginal discs from flies overexpressing DREF showed ectopic expression of protein in the region posterior to the morphogenetic furrow where DREF is overexpressed. Knockdown of DREF in some tissues where SKPa distribution is well known almost completely abrogated the skpA gene expression. These findings, taken together, indicate that the Drosophila skpA gene is a novel target of the transcription factor DREF.

Observation and modeling of induction effect on human transferrin production from stably transfected Drosophila S2 cell culture

Human transferrin (hTf) is a serum glycoprotein involved in iron transport. We performed, for the first time, mathematical modeling of stably transfected insect Drosophila melanogaster S2 cell culture, a nonlytic plasmid-based system that secretes recombinant hTf under control of the copper sulfate-inducible Drosophila metallothionein promoter. Cell growth patterns at various inducer concentrations revealed that the specific growth rate of S2 cells was substantially reduced as the specific rate of recombinant hTf production increased, and recovered to some extent when recombinant hTf production was nearly stopped. Additionally, the time profiles of specific production rates exhibited a maximum in the early culture period. Longer times and lower values of the maximum specific production rate were observed at lower inducer concentrations, and shorter times and higher values at higher inducer levels. Although the proposed S2 cell culture model was slightly limited with regard to prediction of cell growth profile at the late stage, we ensured that it gave reasonable predictions of the dynamics of glucose consumption and recombinant hTf production and confirmed its validity through simulating other culture experiments under different conditions. The S2 model proposed in this study can contribute to the elucidation of cell culture dynamics, and optimization of each culture variable to enhance heterologous protein production.

Regions of Drosophila Notch That Contribute to Ligand Binding and the Modulatory Influence of Fringe

Two glycosyltransferases that transfer sugars to epidermal growth factor (EGF) domains, OFUT1 and Fringe, regulate Notch signaling. To characterize the impact of glycosylation at the 23 consensus O-fucose sites in Drosophila Notch, we conducted deletion mapping and site-specific mutagenesis and then assayed the binding of soluble forms of Notch to cell-surface ligands. Our results support the conclusion that EGF11 and EGF12 are essential for ligand binding, but indicate that other EGF domains also make substantial contributions to ligand binding. Characterization of Notch deletion constructs and O-fucose site mutants further revealed that no single site or region can account for the influence of Fringe on Notch-ligand binding. Additionally, we observed an influence of Fringe on a Notch fragment including only 4 of its 36 EGF domains (EGF10-13). Together, our observations imply that glycosylation influences Notch-ligand interactions through a distributive mechanism that involves local interactions with multiple EGF domains and led us to suggest a structural model for how Notch interacts with its ligands.

MAE Fine-Tunes the Response

The Drosophila Ets transcription factors Yan and pointed protein 2 (Pnt-P2) function antagonistically, downstream of receptor tyrosine kinase (RTK) signaling. Yan inhibits expression of target genes in undifferentiated cells. RTK activation of mitogen-activated protein kinase (MAPK) signaling leads to Yan phosphorylation and relieves Yan's transcriptional repression, allowing phosphorylated Pnt-P2 to activate this same set of genes, thereby promoting cell differentiation. Tootle et al. investigated the mechanisms by which MAPK regulates Yan and Pnt-P2 and uncovered a novel role for the protein modulator of the activity of Ets (MAE), which facilitates MAPK phosphorylation of Yan. Stimulating MAPK pathways with constitutively active Ras (Ras V12 ) in cultured Drosophila S2 cells led to export of Yan from the nucleus. Transgenic flies expressing Yan mutants that were not exported in response to MAPK signaling displayed phenotypes like those seen with constitutively active Yan. The authors used Yan deletion mutants to show that pointed domain (PD), a motif common to all three proteins that has been implicated in MAE binding to Yan and Pnt-P2, was necessary for regulation of Yan localization. Manipulation of mae expression in cells expressing Yan mutants indicated that MAE was required for export of Yan independently of its role in promoting Yan phosphorylation. MAE overexpression inhibited Yan-mediated transcriptional repression and, unexpectedly, inhibited Pnt-P2 transcriptional activation as well. The authors proposed that MAE participated in multiple steps in regulating Yan and Pnt-P2 activity to fine-tune the response to RTK signaling. T. L. Tootle, P. S. Lee, I. Rebay, CRM1-mediated nuclear export and regulated activity of the receptor tyrosine kinase antagonist YAN require specific interactions with MAE. Development 130 , 845-857 (2003). [Abstract] [Full Text]

Human kidney diamine oxidase: heterologous expression, purification, and characterization.

Human kidney diamine oxidase has been overexpressed as a secreted enzyme under the control of a metallothionein promoter in Drosophila S2 cell culture. This represents the first heterologous overexpression and purification of a catalytically active, recombinant mammalian copper-containing amine oxidase. A rapid and highly efficient purification protocol using chromatography on heparin affinity, hydroxyapatite, and gel filtration media allows for the recovery of large quantities of the recombinant enzyme, which is judged to be greater than 98% homogenous by SDS/PAGE. The availability of large quantities of highly purified enzyme makes it now possible to investigate the spectroscopic, mechanistic, functional, and structural properties of this human enzyme at the molecular level. Visible absorption, circular dichroism, electron paramagnetic resonance, and resonance Raman spectroscopic results are presented. The recombinant enzyme contains the cofactors 2,4,5-trihydroxyphenylalaninequinone and copper at stoichiometries of up to 1.1 and 1.5 mol per mol homodimer, respectively. In addition, tightly bound and stoichiometric calcium ions were identified and proposed to occupy a second metal-binding site. The apparent molecular weight of the recombinant protein, determined by analytical ultracentrifugation, suggests 20-26% glycosylation by weight. Detailed kinetic studies indicate the preferred substrates (k(cat)/K(M)) of human diamine oxidase are, in order, histamine, 1-methylhistamine, and putrescine, with K(M) values of 2.8, 3.4, and 20 microM, respectively. These results, demonstrating the substrate preference for histamine and 1-methylhistamine, were unanticipated given the available literature. The pH dependence of k(cat) for putrescine oxidation gives two apparent p K(a) values at 6.0 and 8.2. Tissue-specific expression of the human diamine oxidase gene was investigated using an mRNA array. The relevance of this work to earlier work and the suggested physiological roles of the human enzyme are discussed.

The SREBP Pathway in Drosophila: Regulation by Palmitate, Not Sterols

In mammals, synthesis of cholesterol and unsaturated fatty acids is controlled by SREBPs, a family of membrane-bound transcription factors. Here, we show that the Drosophila genome encodes all components of the SREBP pathway, including a single SREBP (dSREBP), SREBP cleavage-activating protein (dSCAP), and the two proteases that process SREBP at sites 1 and 2 to release the nuclear fragment. In cultured Drosophila S2 cells, dSREBP is processed at sites 1 and 2, and the liberated fragment increases mRNAs encoding enzymes of fatty acid biosynthesis, but not sterol or isoprenoid biosynthesis. Processing requires dSCAP, but is not inhibited by sterols as in mammals. Instead, dSREBP processing is blocked by palmitic acid. These findings suggest that the ancestral SREBP pathway functions to maintain membrane integrity rather than to control cholesterol homeostasis.

Transcription activation by ultrabithorax Ib protein requires a predicted alpha-helical region.

Characterization of their transcription activation domains is critical to understanding functional specificity within the Hox family of proteins. However, few Hox activation domains have been identified and none characterized in detail. In this study, promotor-reporter assays in yeast and Drosophila S2 cell culture were used to refine the boundaries of the activation domain of the Drosophila Hox protein Ultrabithorax (Ubx) and to identify critical elements within this domain. We found that residues 159-242 were sufficient for 50% function, and full transactivation capacity was achieved with inclusion of additional N-terminal sequences. Activation domain sequence and placement relative to the homeodomain differ between Ubx and other Hox proteins, consistent with the possibility that diverse activation mechanisms contribute to functional distinctions in vivo. The essential residues 159-242 in the UbxIb activation domain are predicted to contain a beta-sheet segment followed by an alpha-helix. This putative alpha-helical region was established to be necessary, but not sufficient, for transcriptional activation. Disruption of the helix by proline substitutions abolished activation function, while alteration of side chains presented on the surface of this putative helix with alanine or lysine mutations had no significant effect on activity. Collectively, these data indicate that this secondary structural element is a key component in forming an effective activation domain in the UbxIb protein. Interestingly, the alpha-helix critical for transcriptional activation is found only for Ubx orthologs from flies and not other species. The mutant Ubx proteins generated in this study have potential applications in deciphering Hox functions in vivo.