We provide a detailed protocol for the mass culturing of primary cells dissociated from Drosophila embryos. The advantage of this protocol over others is that we have optimized it for a robust large-scale performance that is suitable for screening. More importantly, we further present conditions to treat these cells with double stranded (ds) RNAs for gene knockdown. Efficient RNAi in Drosophila primary cells is accomplished by simply bathing the cells in dsRNA-containing culture medium. This method provides the basis for functional genomic screens in differentiated cells, such as neurons and muscles, using RNAi or small molecules. The entire protocol takes approximately 14 d, whereas the preparation of primary cells from Drosophila embryos only requires 2-4 h.

To identify Huntington's Disease therapeutics, we conducted high-content small molecule and RNAi suppressor screens using a Drosophila primary neural culture Huntingtin model. Drosophila primary neurons offer a sensitive readout for neurotoxicty, as their neurites develop dysmorphic features in the presence of mutant polyglutamine-expanded Huntingtin compared to nonpathogenic Huntingtin. By tracking the subcellular distribution of mRFP-tagged pathogenic Huntingtin and assaying neurite branch morphology via live-imaging, we identified suppressors that could reduce Huntingtin aggregation and/or prevent the formation of dystrophic neurites. The custom algorithms we used to quantify neurite morphologies in complex cultures provide a useful tool for future high-content screening approaches focused on neurodegenerative disease models. Compounds previously found to be effective aggregation inhibitors in mammalian systems were also effective in Drosophila primary cultures, suggesting translational capacity between these models. However, we did not observe a direct correlation between the ability of a compound or gene knockdown to suppress aggregate formation and its ability to rescue dysmorphic neurites. Only a subset of aggregation inhibitors could revert dysmorphic cellular profiles. We identified lkb1, an upstream kinase in the mTOR/Insulin pathway, and four novel drugs, Camptothecin, OH-Camptothecin, 18β-Glycyrrhetinic acid, and Carbenoxolone, that were strong suppressors of mutant Huntingtin-induced neurotoxicity. Huntingtin neurotoxicity suppressors identified through our screen also restored viability in an in vivo Drosophila Huntington's Disease model, making them attractive candidates for further therapeutic evaluation.

The Hippo pathway controls metazoan organ growth by regulating cell proliferation and apoptosis. Many components have been identified, but our knowledge of the composition and structure of this pathway is still incomplete. Using existing pathway components as baits, we generated by mass spectrometry a high-confidence Drosophila Hippo protein-protein interaction network (Hippo-PPIN) consisting of 153 proteins and 204 interactions. Depletion of 67% of the proteins by RNA interference regulated the transcriptional coactivator Yorkie (Yki) either positively or negatively. We selected for further characterization a new member of the alpha-arrestin family, Leash, and show that it promotes degradation of Yki through the lysosomal pathway. Given the importance of the Hippo pathway in tumor development, the Hippo-PPIN will contribute to our understanding of this network in both normal growth and cancer.

The cytokine-activated Janus kinase (JAK)/signal transducer and activator of transcription (STAT) pathway plays an important role in the control of a wide variety of biological processes. When misregulated, JAK/STAT signaling is associated with various human diseases, such as immune disorders and tumorigenesis. To gain insights into the mechanisms by which JAK/STAT signaling participates in these diverse biological responses, we carried out a genome-wide RNA interference (RNAi) screen in cultured Drosophila cells. We identified 121 genes whose double-stranded RNA (dsRNA)-mediated knockdowns affected STAT92E activity. Of the 29 positive regulators, 13 are required for the tyrosine phosphorylation of STAT92E. Furthermore, we found that the Drosophila homologs of RanBP3 and RanBP10 are negative regulators of JAK/STAT signaling through their control of nucleocytoplasmic transport of STAT92E. In addition, we identified a key negative regulator of Drosophila JAK/STAT signaling, protein tyrosine phosphatase PTP61F, and showed that it is a transcriptional target of JAK/STAT signaling, thus revealing a novel negative feedback loop. Our study has uncovered many uncharacterized genes required for different steps of the JAK/STAT signaling pathway.

Off-target effects have been demonstrated to be a major source of false-positives in RNA interference (RNAi) high-throughput screens. In this study, we re-assess the previously published transcriptional reporter-based whole-genome RNAi screens for the Wingless and Hedgehog signaling pathways using second generation double-stranded RNA libraries. Furthermore, we investigate other factors that may influence the outcome of such screens, including cell-type specificity, robustness of reporters, and assay normalization, which determine the efficacy of RNAi-knockdown of target genes.

Lipid droplets are ubiquitous triglyceride and sterol ester storage organelles required for energy storage homeostasis and biosynthesis. Although little is known about lipid droplet formation and regulation, it is clear that members of the PAT (perilipin, adipocyte differentiation related protein, tail interacting protein of 47 kDa) protein family coat the droplet surface and mediate interactions with lipases that remobilize the stored lipids. We identified key Drosophila candidate genes for lipid droplet regulation by RNA interference (RNAi) screening with an image segmentation-based optical read-out system, and show that these regulatory functions are conserved in the mouse. Those include the vesicle-mediated Coat Protein Complex I (COPI) transport complex, which is required for limiting lipid storage. We found that COPI components regulate the PAT protein composition at the lipid droplet surface, and promote the association of adipocyte triglyceride lipase (ATGL) with the lipid droplet surface to mediate lipolysis. Two compounds known to inhibit COPI function, Exo1 and Brefeldin A, phenocopy COPI knockdowns. Furthermore, RNAi inhibition of ATGL and simultaneous drug treatment indicate that COPI and ATGL function in the same pathway. These data indicate that the COPI complex is an evolutionarily conserved regulator of lipid homeostasis, and highlight an interaction between vesicle transport systems and lipid droplets.

Hypoxia-inducible factors (HIFs) are a family of evolutionary conserved alpha-beta heterodimeric transcription factors that induce a wide range of genes in response to low oxygen tension. Molecular mechanisms that mediate oxygen-dependent HIF regulation operate at the level of the alpha subunit, controlling protein stability, subcellular localization, and transcriptional coactivator recruitment. We have conducted an unbiased genome-wide RNA interference (RNAi) screen in Drosophila cells aimed to the identification of genes required for HIF activity. After 3 rounds of selection, 30 genes emerged as critical HIF regulators in hypoxia, most of which had not been previously associated with HIF biology. The list of genes includes components of chromatin remodeling complexes, transcription elongation factors, and translational regulators. One remarkable hit was the argonaute 1 (ago1) gene, a central element of the microRNA (miRNA) translational silencing machinery. Further studies confirmed the physiological role of the miRNA machinery in HIF-dependent transcription. This study reveals the occurrence of novel mechanisms of HIF regulation, which might contribute to developing novel strategies for therapeutic intervention of HIF-related pathologies, including heart attack, cancer, and stroke.

The spontaneous and reversible formation of foci and filaments that contain proteins involved in different metabolic processes is common in both the nucleus and the cytoplasm. Stress granules (SGs) and processing bodies (PBs) belong to a novel family of cellular structures collectively known as mRNA silencing foci that harbour repressed mRNAs and their associated proteins. SGs and PBs are highly dynamic and they form upon stress and dissolve thus releasing the repressed mRNAs according to changes in cell physiology. In addition, aggregates containing abnormal proteins are frequent in neurodegenerative disorders. In spite of the growing relevance of these supramolecular aggregates to diverse cellular functions a reliable automated tool for their systematic analysis is lacking. Here we report a MATLAB Script termed BUHO for the high-throughput image analysis of cellular foci. We used BUHO to assess the number, size and distribution of distinct objects with minimal deviation from manually obtained parameters. BUHO successfully addressed the induction of both SGs and PBs in mammalian and insect cells exposed to different stress stimuli. We also used BUHO to assess the dynamics of specific mRNA-silencing foci termed Smaug 1 foci (S-foci) in primary neurons upon synaptic stimulation. Finally, we used BUHO to analyze the role of candidate genes on SG formation in an RNAi-based experiment. We found that FAK56D, GCN2 and PP1 govern SG formation. The role of PP1 is conserved in mammalian cells as judged by the effect of the PP1 inhibitor salubrinal, and involves dephosphorylation of the translation factor eIF2α. All these experiments were analyzed manually and by BUHO and the results differed in less than 5% of the average value. The automated analysis by this user-friendly method will allow high-throughput image processing in short times by providing a robust, flexible and reliable alternative to the laborious and sometimes unfeasible visual scrutiny.

A crucial aim upon completion of whole genome sequences is the functional analysis of all predicted genes. We have applied a high-throughput RNA-interference (RNAi) screen of 19,470 double-stranded (ds) RNAs in cultured cells to characterize the function of nearly all (91%) predicted Drosophila genes in cell growth and viability. We found 438 dsRNAs that identified essential genes, among which 80% lacked mutant alleles. A quantitative assay of cell number was applied to identify genes of known and uncharacterized functions. In particular, we demonstrate a role for the homolog of a mammalian acute myeloid leukemia gene (AML1) in cell survival. Such a systematic screen for cell phenotypes, such as cell viability, can thus be effective in characterizing functionally related genes on a genome-wide scale.

Receptor tyrosine kinase (RTK) signalling through extracellular-signal-regulated kinases (ERKs) has pivotal roles during metazoan development, underlying processes as diverse as fate determination, differentiation, proliferation, survival, migration and growth. Abnormal RTK/ERK signalling has been extensively documented to contribute to developmental disorders and disease, most notably in oncogenic transformation by mutant RTKs or downstream pathway components such as Ras and Raf. Although the core RTK/ERK signalling cassette has been characterized by decades of research using mammalian cell culture and forward genetic screens in model organisms, signal propagation through this pathway is probably regulated by a larger network of moderate, context-specific proteins. The genes encoding these proteins may not have been discovered through traditional screens owing, in particular, to the requirement for visible phenotypes. To obtain a global view of RTK/ERK signalling, we performed an unbiased, RNA interference (RNAi), genome-wide, high-throughput screen in Drosophila cells using a novel, quantitative, cellular assay monitoring ERK activation. Here we show that ERK pathway output integrates a wide array of conserved cellular processes. Further analysis of selected components-in multiple cell types with different RTK ligands and oncogenic stimuli-validates and classifies 331 pathway regulators. The relevance of these genes is highlighted by our isolation of a Ste20-like kinase and a PPM-family phosphatase that seem to regulate RTK/ERK signalling in vivo and in mammalian cells. Novel regulators that modulate specific pathway outputs may be selective targets for drug discovery.

Circadian clocks regulate many different physiological processes and synchronize these to environmental light:dark cycles. In Drosophila, light is transmitted to the clock by a circadian blue light photoreceptor CRYPTOCHROME (CRY). In response to light, CRY promotes the degradation of the circadian clock protein TIMELESS (TIM) and then is itself degraded. To identify novel genes involved in circadian entrainment, we performed an unbiased genome-wide screen in Drosophila cells using a sensitive and quantitative assay that measures light-induced degradation of CRY. We systematically knocked down the expression of approximately 21,000 genes and identified those that regulate CRY stability. These genes include ubiquitin ligases, signal transduction molecules, and redox molecules. Many of the genes identified in the screen are specific for CRY degradation and do not affect degradation of the TIM protein in response to light, suggesting that, for the most part, these two pathways are distinct. We further validated the effect of three candidate genes on CRY stability in vivo by assaying flies mutant for each of these genes. This work identifies a novel regulatory network involved in light-dependent CRY degradation and demonstrates the power of a genome-wide RNAi approach for understanding circadian biology.

Protein aggregates are a common pathological feature of most neurodegenerative diseases (NDs). Understanding their formation and regulation will help clarify their controversial roles in disease pathogenesis. To date, there have been few systematic studies of aggregates formation in Drosophila, a model organism that has been applied extensively in modeling NDs and screening for toxicity modifiers. We generated transgenic fly lines that express enhanced-GFP-tagged mutant Huntingtin (Htt) fragments with different lengths of polyglutamine (polyQ) tract and showed that these Htt mutants develop protein aggregates in a polyQ-length- and age-dependent manner in Drosophila. To identify central regulators of protein aggregation, we further generated stable Drosophila cell lines expressing these Htt mutants and also established a cell-based quantitative assay that allows automated measurement of aggregates within cells. We then performed a genomewide RNA interference screen for regulators of mutant Htt aggregation and isolated 126 genes involved in diverse cellular processes. Interestingly, although our screen focused only on mutant Htt aggregation, several of the identified candidates were known previously as toxicity modifiers of NDs. Moreover, modulating the in vivo activity of hsp110 (CG6603) or tra1, two hits from the screen, affects neurodegeneration in a dose-dependent manner in a Drosophila model of Huntington's disease. Thus, other aggregates regulators isolated in our screen may identify additional genes involved in the protein-folding pathway and neurotoxicity.

Besides its essential and well established role as a component of the cytoskeleton, actin is also present in the cell nucleus, where it has been linked to many processes that control gene expression. For example, nuclear actin regulates the activity of specific transcription factors, associates with all three RNA polymerases, and is a component of many chromatin remodelling complexes. Despite the fact that two export receptors, Crm1 and exportin 6, have been linked to nuclear export of actin, the mechanism by which actin enters the nucleus to elicit these essential functions has not been determined. It is also unclear whether actin is actively exchanged between the nucleus and the cytoplasm, and whether this connection has any functional significance for the cell. By applying a variety of live-cell imaging techniques we revealed that actin constantly shuttles in and out of the nucleus. The fast transport rates, which depend on the availability of actin monomers, suggest an active transport mechanism in both directions. Importantly, we identified importin 9 as the nuclear import factor for actin. Furthermore, our RNAi experiments showed that the active maintenance of nuclear actin levels by importin 9 is required for maximal transcriptional activity. Measurements of nuclear export rates and depletion studies also clarified that nuclear export of actin is mediated by exportin 6, and not by Crm1. These results demonstrate that cytoplasmic and nuclear actin pools are dynamically connected and identify the nuclear import and export mechanisms of actin.

Chromatin insulators of higher eukaryotes functionally divide the genome into active and inactive domains. Furthermore, insulators regulate enhancer/promoter communication, which is evident from the Drosophila bithorax locus in which a multitude of regulatory elements control segment specific gene activity. Centrosomal protein 190 (CP190) is targeted to insulators by CTCF or other insulator DNA-binding factors. Chromatin analyses revealed that insulators are characterized by open and nucleosome depleted regions. Here, we wanted to identify chromatin modification and remodelling factors required for an enhancer blocking function. We used the well-studied Fab-8 insulator of the bithorax locus to apply a genome-wide RNAi screen for factors that contribute to the enhancer blocking function of CTCF and CP190. Among 78 genes required for optimal Fab-8 mediated enhancer blocking, all four components of the NURF complex as well as several subunits of the dREAM complex were most evident. Mass spectrometric analyses of CTCF or CP190 bound proteins as well as immune precipitation confirmed NURF and dREAM binding. Both co-localise with most CP190 binding sites in the genome and chromatin immune precipitation showed that CP190 recruits NURF and dREAM. Nucleosome occupancy and histone H3 binding analyses revealed that CP190 mediated NURF binding results in nucleosomal depletion at CP190 binding sites. Thus, we conclude that CP190 binding to CTCF or to other DNA binding insulator factors mediates recruitment of NURF and dREAM. Furthermore, the enhancer blocking function of insulators is associated with nucleosomal depletion and requires NURF and dREAM.

Yeast genetics and in vitro biochemical analysis have identified numerous genes involved in protein secretion. As compared with yeast, however, the metazoan secretory pathway is more complex and many mechanisms that regulate organization of the Golgi apparatus remain poorly characterized. We performed a genome-wide RNA-mediated interference screen in a Drosophila cell line to identify genes required for constitutive protein secretion. We then classified the genes on the basis of the effect of their depletion on organization of the Golgi membranes. Here we show that depletion of class A genes redistributes Golgi membranes into the endoplasmic reticulum, depletion of class B genes leads to Golgi fragmentation, depletion of class C genes leads to aggregation of Golgi membranes, and depletion of class D genes causes no obvious change. Of the 20 new gene products characterized so far, several localize to the Golgi membranes and the endoplasmic reticulum.