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1.  Systemic Organ Wasting Induced by Localized Expression of the Secreted Insulin/IGF Antagonist ImpL2 
Developmental cell  2015;33(1):36-46.
SUMMARY
Organ wasting, related to changes in nutrition and metabolic activity of cells and tissues, is observed under conditions of starvation and in the context of diseases, including cancers. We have developed a model for organ wasting in adult Drosophila, whereby overproliferation induced by activation of Yorkie, the Yap1 oncogene ortholog, in intestinal stem cells leads to wasting of the ovary, fat body, and muscle. These organ-wasting phenotypes are associated with a reduction in systemic insulin/IGF signaling due to increased expression of the secreted insulin/IGF antagonist ImpL2 from the overproliferating gut. Strikingly, expression of rate-limiting glycolytic enzymes and central components of the insulin/IGF pathway is upregulated with activation of Yorkie in the gut, which may provide a mechanism for this overproliferating tissue to evade the effect of ImpL2. Altogether, our study provides insights into the mechanisms underlying organ-wasting phenotypes in Drosophila and how overproliferating tissues adapt to global changes in metabolism.
doi:10.1016/j.devcel.2015.02.012
PMCID: PMC4437243  PMID: 25850671
2.  Identification of potential drug targets for tuberous sclerosis complex by synthetic screens combining CRISPR-based knockouts with RNAi 
Science signaling  2015;8(393):rs9.
The tuberous sclerosis complex (TSC) family of tumor suppressors, TSC1 and TSC2, function together in an evolutionarily conserved protein complex that is a point of convergence for major cell signaling pathways that regulate mTOR complex 1 (mTORC1). Mutation or aberrant inhibition of the TSC complex is common in various human tumor syndromes and cancers. The discovery of novel therapeutic strategies to selectively target cells with functional loss of this complex is therefore of clinical relevance to patients with nonmalignant TSC and those with sporadic cancers. We developed a CRISPR-based method to generate homogeneous mutant Drosophila cell lines. By combining TSC1 or TSC2 mutant cell lines with RNAi screens against all kinases and phosphatases, we identified synthetic interactions with TSC1 and TSC2. Individual knockdown of three candidate genes (mRNA-cap, Pitslre, and CycT; orthologs of RNGTT, CDK11, and CCNT1 in humans) reduced the population growth rate of Drosophila cells lacking either TSC1 or TSC2 but not that of wild-type cells. Moreover, individual knockdown of these three genes had similar growth-inhibiting effects in mammalian TSC2-deficient cell lines, including human tumor-derived cells, illustrating the power of this cross-species screening strategy to identify potential drug targets.
doi:10.1126/scisignal.aab3729
PMCID: PMC4642709  PMID: 26350902
4.  A Cross-Species Study of PI3K Protein-Protein Interactions Reveals the Direct Interaction of P85 and SHP2 
Scientific Reports  2016;6:20471.
Using a series of immunoprecipitation (IP) – tandem mass spectrometry (LC-MS/MS) experiments and reciprocal BLAST, we conducted a fly-human cross-species comparison of the phosphoinositide-3-kinase (PI3K) interactome in a drosophila S2R+ cell line and several NSCLC and human multiple myeloma cell lines to identify conserved interacting proteins to PI3K, a critical signaling regulator of the AKT pathway. Using H929 human cancer cells and drosophila S2R+ cells, our data revealed an unexpected direct binding of Corkscrew, the drosophila ortholog of the non-receptor protein tyrosine phosphatase type II (SHP2) to the Pi3k21B (p60) regulatory subunit of PI3K (p50/p85 human ortholog) but no association with Pi3k92e, the human ortholog of the p110 catalytic subunit. The p85-SHP2 association was validated in human cell lines, and formed a ternary regulatory complex with GRB2-associated-binding protein 2 (GAB2). Validation experiments with knockdown of GAB2 and Far-Western blots proved the direct interaction of SHP2 with p85, independent of adaptor proteins and transfected FLAG-p85 provided evidence that SHP2 binding on p85 occurred on the SH2 domains. A disruption of the SHP2-p85 complex took place after insulin/IGF1 stimulation or imatinib treatment, suggesting that the direct SHP2-p85 interaction was both independent of AKT activation and positively regulates the ERK signaling pathway.
doi:10.1038/srep20471
PMCID: PMC4738311  PMID: 26839216
5.  Recruitment of Scribble to the Synaptic Scaffolding Complex Requires GUK-holder, a Novel DLG Binding Protein 
Current biology : CB  2002;12(7):531-539.
Summary
Background
Membrane-associated guanylate kinases (MAGUKs), such as Discs-Large (DLG), play critical roles in synapse maturation by regulating the assembly of synaptic multiprotein complexes. Previous studies have revealed a genetic interaction between DLG and another PDZ scaffolding protein, SCRIBBLE (SCRIB), during the establishment of cell polarity in developing epithelia. A possible interaction between DLG and SCRIB at synaptic junctions has not yet been addressed. Likewise, the biochemical nature of this interaction remains elusive, raising questions regarding the mechanisms by which the actions of both proteins are coordinated.
Results
Here we report the isolation of a new DLG-interacting protein, GUK-holder, that interacts with the GUK domain of DLG and which is dynamically expressed during synaptic bouton budding. We also show that at Drosophila synapses DLG colocalizes with SCRIB and that this colocalization is likely to be mediated by direct interactions between GUKH and the PDZ2 domain of SCRIB. We show that DLG, GUKH, and SCRIB form a tripartite complex at synapses, in which DLG and GUKH are required for the proper synaptic localization of SCRIB.
Conclusions
Our results provide a mechanism by which developmentally important PDZ-mediated complexes are associated at the synapse.
PMCID: PMC4661175  PMID: 11937021
6.  Combining Genetic Perturbations and Proteomics to Examine Kinase-Phosphatase Networks in Drosophila Embryos 
Developmental cell  2014;31(1):114-127.
Summary
Connecting phosphorylation events to kinases and phosphatases is key to understanding the molecular organization and signaling dynamics of networks. We have generated a validated set of transgenic RNA-interference reagents for knockdown and characterization of all protein kinases and phosphatases present during early Drosophila melanogaster development. These genetic tools enable collection of sufficient quantities of embryos depleted of single gene products for proteomics. As a demonstration of an application of the collection, we have used multiplexed isobaric-labeling for quantitative proteomics to derive global phosphorylation signatures associated with kinase-depleted embryos, in order to systematically link phosphosites with relevant kinases. We demonstrate how this strategy uncovers kinase consensus motifs and prioritizes phosphoproteins for kinase target validation. We validate this approach by providing auxiliary evidence for Wee kinase-directed regulation of the chromatin regulator Stonewall. Further, we show how correlative phosphorylation at the site level can indicate function, as exemplified by Sterile20-like kinase-dependent regulation of Stat92E.
doi:10.1016/j.devcel.2014.07.027
PMCID: PMC4208667  PMID: 25284370
8.  Spatial and temporal organization of signaling pathways 
Trends in biochemical sciences  2014;39(10):457-464.
The development and maintenance of the many different cell types in metazoan organisms requires robust and diverse intercellular communication mechanisms. Relatively few such signaling pathways have been identified, leading to the question of how such a broad diversity of output is generated from relatively simple signals. Recent studies have revealed complex mechanisms integrating temporal and spatial information to generate diversity in signaling pathway output. We review some general principles of signaling pathways, focusing on transcriptional outputs in Drosophila. We consider the role of spatial and temporal aspects of different transduction pathways and then discuss how recently developed tools and approaches are helping to dissect the complex mechanisms linking pathway stimulation to output.
doi:10.1016/j.tibs.2014.07.008
PMCID: PMC4477539  PMID: 25155749
signaling pathways; signaling dynamics; crosstalk
9.  A transgenic resource for conditional competitive inhibition of conserved Drosophila microRNAs 
Nature communications  2015;6:7279.
Although the impact of microRNAs (miRNAs) in development and disease is well established, understanding the function of individual miRNAs remains challenging. Development of competitive inhibitor molecules such as miRNA sponges has allowed the community to address individual miRNA function in vivo. However, the application of these loss-of-function strategies has been limited. Here we offer a comprehensive library of 141 conditional miRNA sponges targeting well-conserved miRNAs in Drosophila. Ubiquitous miRNA sponge delivery and consequent systemic miRNA inhibition uncovers a relatively small number of miRNA families underlying viability and gross morphogenesis, with false discovery rates in the 4–8% range. In contrast, tissue-specific silencing of muscle-enriched miRNAs reveals a surprisingly large number of novel miRNA contributions to the maintenance of adult indirect flight muscle structure and function. A strong correlation between miRNA abundance and physiological relevance is not observed, underscoring the importance of unbiased screens when assessing the contributions of miRNAs to complex biological processes.
doi:10.1038/ncomms8279
PMCID: PMC4471878  PMID: 26081261
10.  A transgenic resource for conditional competitive inhibition of conserved Drosophila microRNAs 
Nature Communications  2015;6:7279.
Although the impact of microRNAs (miRNAs) in development and disease is well established, understanding the function of individual miRNAs remains challenging. Development of competitive inhibitor molecules such as miRNA sponges has allowed the community to address individual miRNA function in vivo. However, the application of these loss-of-function strategies has been limited. Here we offer a comprehensive library of 141 conditional miRNA sponges targeting well-conserved miRNAs in Drosophila. Ubiquitous miRNA sponge delivery and consequent systemic miRNA inhibition uncovers a relatively small number of miRNA families underlying viability and gross morphogenesis, with false discovery rates in the 4–8% range. In contrast, tissue-specific silencing of muscle-enriched miRNAs reveals a surprisingly large number of novel miRNA contributions to the maintenance of adult indirect flight muscle structure and function. A strong correlation between miRNA abundance and physiological relevance is not observed, underscoring the importance of unbiased screens when assessing the contributions of miRNAs to complex biological processes.
Transgenic RNA ‘sponges' that inhibit specific microRNAs are valuable tools for functional studies. Fulga et al. present a library of conditional microRNA sponges in Drosophila, and reveal widespread involvement of microRNAs in the maintenance of adult muscle structure and function.
doi:10.1038/ncomms8279
PMCID: PMC4471878  PMID: 26081261
11.  Reagent and Data Resources for Investigation of RNA Binding Protein Functions in Drosophila melanogaster Cultured Cells 
G3: Genes|Genomes|Genetics  2015;5(9):1919-1924.
RNA binding proteins (RBPs) are involved in many cellular functions. To facilitate functional characterization of RBPs, we generated an RNA interference (RNAi) library for Drosophila cell-based screens comprising reagents targeting known or putative RBPs. To test the quality of the library and provide a baseline analysis of the effects of the RNAi reagents on viability, we screened the library using a total ATP assay and high-throughput imaging in Drosophila S2R+ cultured cells. The results are consistent with production of a high-quality library that will be useful for functional genomics studies using other assays. Altogether, we provide resources in the form of an initial curated list of Drosophila RBPs; an RNAi screening library we expect to be used with additional assays that address more specific biological questions; and total ATP and image data useful for comparison of those additional assay results with fundamental information such as effects of a given reagent in the library on cell viability. Importantly, we make the baseline data, including more than 200,000 images, easily accessible online.
doi:10.1534/g3.115.019364
PMCID: PMC4555228  PMID: 26199285
Drosophila; RNA binding protein; RNAi; data sharing; high-throughput screen
12.  GLAD: an Online Database of Gene List Annotation for Drosophila 
Journal of Genomics  2015;3:75-81.
We present a resource of high quality lists of functionally related Drosophila genes, e.g. based on protein domains (kinases, transcription factors, etc.) or cellular function (e.g. autophagy, signal transduction). To establish these lists, we relied on different inputs, including curation from databases or the literature and mapping from other species. Moreover, as an added curation and quality control step, we asked experts in relevant fields to review many of the lists. The resource is available online for scientists to search and view, and is editable based on community input. Annotation of gene groups is an ongoing effort and scientific need will typically drive decisions regarding which gene lists to pursue. We anticipate that the number of lists will increase over time; that the composition of some lists will grow and/or change over time as new information becomes available; and that the lists will benefit the scientific community, e.g. at experimental design and data analysis stages. Based on this, we present an easily updatable online database, available at www.flyrnai.org/glad, at which gene group lists can be viewed, searched and downloaded.
doi:10.7150/jgen.12863
PMCID: PMC4495321  PMID: 26157507
GLAD; Drosophila; genes
13.  Inducing RNAi in Drosophila Cells by Transfection with dsRNA 
Cold Spring Harbor protocols  2014;2014(5):10.1101/pdb.prot080747 pdb.prot080747.
In Drosophila cells, RNA interference (RNAi) can be triggered by synthetic long double-stranded RNAs (dsRNAs). For many Drosophila cell lines and cell types, passive dsRNA uptake is inefficient. More complete silencing responses can often be obtained in Drosophila S2 cells using transfection, perhaps because higher levels of intracellular dsRNA are achieved. In this protocol, S2 cells are transfected with dsRNA using QIAGEN’s Effectene reagent, which has proven to be reliable for many investigators. A plasmid DNA can also be included in the transfection mix to provide additional functionality. The plasmid DNA can encode, for example, a reporter of the activity of a pathway or specific transcription factor, or a marker that allows visualization of some cellular behavior or structure. It is also useful to include a plasmid that encodes a fluorescent protein simply to monitor transfection efficiency.
doi:10.1101/pdb.prot080747
PMCID: PMC4465107  PMID: 24786505
14.  Receptor Tyrosine Kinases in Drosophila Development 
Tyrosine phosphorylation plays a significant role in a wide range of cellular processes. The Drosophila genome encodes more than 20 receptor tyrosine kinases and extensive studies in the past 20 years have illustrated their diverse roles and complex signaling mechanisms. Although some receptor tyrosine kinases have highly specific functions, others strikingly are used in rather ubiquitous manners. Receptor tyrosine kinases regulate a broad expanse of processes, ranging from cell survival and proliferation to differentiation and patterning. Remarkably, different receptor tyrosine kinases share many of the same effectors and their hierarchical organization is retained in disparate biological contexts. In this comprehensive review, we summarize what is known regarding each receptor tyrosine kinase during Drosophila development. Astonishingly, very little is known for approximately half of all Drosophila receptor tyrosine kinases.
About 20 RTKs are encoded by the Drosophila genome, most of which have a mammalian counterpart. They provide a means of communication between different tissues and cell types that leads to a robust and reproducible developmental program.
doi:10.1101/cshperspect.a009050
PMCID: PMC3660834  PMID: 23732470
15.  A sharp end to sugary Wingless travels 
The Journal of Cell Biology  2014;206(7):819-821.
Drosophila melanogaster follicle stem cells are controlled by Wingless (Wg) ligands secreted 50 µm away, raising the question of how long-distance Wg spreading occurs. In this issue of JCB, Wang and Page-McCaw (2014. J. Cell Biol. http://dx.doi.org/10.1083/jcb.201403084) demonstrate a potential mechanism by which the heparan sulfate proteoglycan Dally-like (Dlp) promotes Wg travel, whereas matrix Mmp2 (Metalloproteinase 2) impedes it by inactivating Dlp.
doi:10.1083/jcb.201408115
PMCID: PMC4178964  PMID: 25267292
16.  A Systems-Level Interrogation Identifies Regulators of Drosophila Blood Cell Number and Survival 
PLoS Genetics  2015;11(3):e1005056.
In multicellular organisms, cell number is typically determined by a balance of intracellular signals that positively and negatively regulate cell survival and proliferation. Dissecting these signaling networks facilitates the understanding of normal development and tumorigenesis. Here, we study signaling by the Drosophila PDGF/VEGF Receptor (Pvr) in embryonic blood cells (hemocytes) and in the related cell line Kc as a model for the requirement of PDGF/VEGF receptors in vertebrate cell survival and proliferation. The system allows the investigation of downstream and parallel signaling networks, based on the ability of Pvr to activate Ras/Erk, Akt/TOR, and yet-uncharacterized signaling pathway/s, which redundantly mediate cell survival and contribute to proliferation. Using Kc cells, we performed a genome wide RNAi screen for regulators of cell number in a sensitized, Pvr deficient background. We identified the receptor tyrosine kinase (RTK) Insulin-like receptor (InR) as a major Pvr Enhancer, and the nuclear hormone receptors Ecdysone receptor (EcR) and ultraspiracle (usp), corresponding to mammalian Retinoid X Receptor (RXR), as Pvr Suppressors. In vivo analysis in the Drosophila embryo revealed a previously unrecognized role for EcR to promote apoptotic death of embryonic blood cells, which is balanced with pro-survival signaling by Pvr and InR. Phosphoproteomic analysis demonstrates distinct modes of cell number regulation by EcR and RTK signaling. We define common phosphorylation targets of Pvr and InR that include regulators of cell survival, and unique targets responsible for specialized receptor functions. Interestingly, our analysis reveals that the selection of phosphorylation targets by signaling receptors shows qualitative changes depending on the signaling status of the cell, which may have wide-reaching implications for other cell regulatory systems.
Author Summary
Signaling networks that drive cell survival and proliferation regulate cell number in development and disease. We use a simple Drosophila model of cell number control, which centers on PDGF/VEGF receptor signaling. Performing a genome-wide RNAi screen under Pvr-sensitized conditions, we identify regulators of cell number that have not been found in conventional screens. Validation by in vivo genetics reveals previously unrecognized roles for EcR and InR in the balance of cell survival in the Drosophila embryo. Phosphoproteomic analysis demonstrates distinct mechanisms of cell survival regulation by EcR and receptor tyrosine kinase signaling. It further identifies common phosphorylation targets of Pvr and InR including regulators of cell survival, and receptor-specific phosphorylation targets mediating unique functions of Pvr and InR. Importantly, the study provides precedence that the selection of phosphorylation targets by signaling receptors can change with the signaling status of the cell, which may have wide-reaching implications for other cell regulatory systems.
doi:10.1371/journal.pgen.1005056
PMCID: PMC4352040  PMID: 25749252
17.  RNAi screening comes of age: improved techniques and complementary approaches 
Gene silencing through sequence-specific targeting of mRNAs by RNAi has enabled genome-wide functional screens in cultured cells and in vivo in model organisms. These screens have resulted in the identification of new cellular pathways and potential drug targets. Considerable progress has been made to improve the quality of RNAi screen data through the development of new experimental and bioinformatics approaches. The recent availability of genome-editing strategies, such as the CRISPR (clustered regularly interspaced short palindromic repeats)-Cas9 system, when combined with RNAi, could lead to further improvements in screen data quality and follow-up experiments, thus promoting our understanding of gene function and gene regulatory networks.
doi:10.1038/nrm3860
PMCID: PMC4204798  PMID: 25145850
18.  Diversity and dynamics of the Drosophila transcriptome 
Nature  2014;512(7515):393-399.
Animal transcriptomes are dynamic, each cell type, tissue and organ system expressing an ensemble of transcript isoforms that give rise to substantial diversity. We identified new genes, transcripts, and proteins using poly(A)+ RNA sequence from Drosophila melanogaster cultured cell lines, dissected organ systems, and environmental perturbations. We found a small set of mostly neural-specific genes has the potential to encode thousands of transcripts each through extensive alternative promoter usage and RNA splicing. The magnitudes of splicing changes are larger between tissues than between developmental stages, and most sex-specific splicing is gonad-specific. Gonads express hundreds of previously unknown coding and long noncoding RNAs (lncRNAs) some of which are antisense to protein-coding genes and produce short regulatory RNAs. Furthermore, previously identified pervasive intergenic transcription occurs primarily within newly identified introns. The fly transcriptome is substantially more complex than previously recognized arising from combinatorial usage of promoters, splice sites, and polyadenylation sites.
doi:10.1038/nature12962
PMCID: PMC4152413  PMID: 24670639
19.  Comparative Analysis of the Transcriptome across Distant Species 
Gerstein, Mark B. | Rozowsky, Joel | Yan, Koon-Kiu | Wang, Daifeng | Cheng, Chao | Brown, James B. | Davis, Carrie A | Hillier, LaDeana | Sisu, Cristina | Li, Jingyi Jessica | Pei, Baikang | Harmanci, Arif O. | Duff, Michael O. | Djebali, Sarah | Alexander, Roger P. | Alver, Burak H. | Auerbach, Raymond | Bell, Kimberly | Bickel, Peter J. | Boeck, Max E. | Boley, Nathan P. | Booth, Benjamin W. | Cherbas, Lucy | Cherbas, Peter | Di, Chao | Dobin, Alex | Drenkow, Jorg | Ewing, Brent | Fang, Gang | Fastuca, Megan | Feingold, Elise A. | Frankish, Adam | Gao, Guanjun | Good, Peter J. | Guigó, Roderic | Hammonds, Ann | Harrow, Jen | Hoskins, Roger A. | Howald, Cédric | Hu, Long | Huang, Haiyan | Hubbard, Tim J. P. | Huynh, Chau | Jha, Sonali | Kasper, Dionna | Kato, Masaomi | Kaufman, Thomas C. | Kitchen, Robert R. | Ladewig, Erik | Lagarde, Julien | Lai, Eric | Leng, Jing | Lu, Zhi | MacCoss, Michael | May, Gemma | McWhirter, Rebecca | Merrihew, Gennifer | Miller, David M. | Mortazavi, Ali | Murad, Rabi | Oliver, Brian | Olson, Sara | Park, Peter J. | Pazin, Michael J. | Perrimon, Norbert | Pervouchine, Dmitri | Reinke, Valerie | Reymond, Alexandre | Robinson, Garrett | Samsonova, Anastasia | Saunders, Gary I. | Schlesinger, Felix | Sethi, Anurag | Slack, Frank J. | Spencer, William C. | Stoiber, Marcus H. | Strasbourger, Pnina | Tanzer, Andrea | Thompson, Owen A. | Wan, Kenneth H. | Wang, Guilin | Wang, Huaien | Watkins, Kathie L. | Wen, Jiayu | Wen, Kejia | Xue, Chenghai | Yang, Li | Yip, Kevin | Zaleski, Chris | Zhang, Yan | Zheng, Henry | Brenner, Steven E. | Graveley, Brenton R. | Celniker, Susan E. | Gingeras, Thomas R | Waterston, Robert
Nature  2014;512(7515):445-448.
doi:10.1038/nature13424
PMCID: PMC4155737  PMID: 25164755
20.  Regulators of Autophagosome Formation in Drosophila Muscles 
PLoS Genetics  2015;11(2):e1005006.
Given the diversity of autophagy targets and regulation, it is important to characterize autophagy in various cell types and conditions. We used a primary myocyte cell culture system to assay the role of putative autophagy regulators in the specific context of skeletal muscle. By treating the cultures with rapamycin (Rap) and chloroquine (CQ) we induced an autophagic response, fully suppressible by knockdown of core ATG genes. We screened D. melanogaster orthologs of a previously reported mammalian autophagy protein-protein interaction network, identifying several proteins required for autophagosome formation in muscle cells, including orthologs of the Rab regulators RabGap1 and Rab3Gap1. The screen also highlighted the critical roles of the proteasome and glycogen metabolism in regulating autophagy. Specifically, sustained proteasome inhibition inhibited autophagosome formation both in primary culture and larval skeletal muscle, even though autophagy normally acts to suppress ubiquitin aggregate formation in these tissues. In addition, analyses of glycogen metabolic genes in both primary cultured and larval muscles indicated that glycogen storage enhances the autophagic response to starvation, an important insight given the link between glycogen storage disorders, autophagy, and muscle function.
Author Summary
Since the identification of the core autophagy genes in yeast, tissue culture cell lines have been the primary tool to evaluate the role and regulation of autophagy in higher organisms. However, since autophagy is a tissue-specific, context dependent process, stable cell lines can only give a limited view of the autophagic process. Here, we focus on the role of putative autophagy regulators in the specific context of the skeletal muscle, which has one of the most robust autophagy responses in mammals. We describe a fruitfly model of autophagy for skeletal muscles that integrates rapid genetic screening in primary cultured cells with robust in vivo validation in the larval muscle. We screened a set of genes previously linked to the autophagy pathway in humans, and identified both positive and negative regulators of autophagy. Our observation that genes involved in sugar metabolism impact muscle autophagy has important implications for both skeletal and cardiac myopathies associated with aberrant sugar storage. Surprisingly, we found that the proteasome is required to maintain autophagy in the muscle, suggesting that therapeutic treatments aiming to induce autophagy by proteasome inhibition must be carefully calibrated to ensure that the opposite phenotype does not occur.
doi:10.1371/journal.pgen.1005006
PMCID: PMC4334200  PMID: 25692684
21.  Functional screening in Drosophila identifies Alzheimer's disease susceptibility genes and implicates Tau-mediated mechanisms 
Human Molecular Genetics  2013;23(4):870-877.
Using a Drosophila model of Alzheimer's disease (AD), we systematically evaluated 67 candidate genes based on AD-associated genomic loci (P < 10−4) from published human genome-wide association studies (GWAS). Genetic manipulation of 87 homologous fly genes was tested for modulation of neurotoxicity caused by human Tau, which forms neurofibrillary tangle pathology in AD. RNA interference (RNAi) targeting 9 genes enhanced Tau neurotoxicity, and in most cases reciprocal activation of gene expression suppressed Tau toxicity. Our screen implicates cindr, the fly ortholog of the human CD2AP AD susceptibility gene, as a modulator of Tau-mediated disease mechanisms. Importantly, we also identify the fly orthologs of FERMT2 and CELF1 as Tau modifiers, and these loci have been independently validated as AD susceptibility loci in the latest GWAS meta-analysis. Both CD2AP and FERMT2 have been previously implicated with roles in cell adhesion, and our screen additionally identifies a fly homolog of the human integrin adhesion receptors, ITGAM and ITGA9, as a modifier of Tau neurotoxicity. Our results highlight cell adhesion pathways as important in Tau toxicity and AD susceptibility and demonstrate the power of model organism genetic screens for the functional follow-up of human GWAS.
doi:10.1093/hmg/ddt478
PMCID: PMC3900103  PMID: 24067533
22.  Control of lipid metabolism by Tachykinin in Drosophila 
Cell reports  2014;9(1):40-47.
Summary
The intestine is a key organ for lipid uptake and distribution, and abnormal intestinal lipid metabolism is associated with obesity and hyperlipidemia. Although multiple regulatory gut hormones secreted from enteroendocrine cells (EEs) regulate systemic lipid homeostasis, such as appetite control and energy balance in adipose tissue, their respective roles regarding lipid metabolism in the intestine are not well understood. We demonstrate that Tachykinins (TKs), one of the most abundant secreted peptides expressed in midgut EEs, regulate intestinal lipid production and subsequently control systemic lipid homeostasis in Drosophila, and that TKs repress lipogenesis in enterocytes (ECs) associated with the TKR99D receptor and PKA signaling. Interestingly, nutrient deprivation enhances the production of TKs in the midgut. Finally, unlike the physiological roles of TKs produced from the brain, gut-derived TKs do not affect behavior, thus demonstrating that gut TK hormones specifically regulate intestinal lipid metabolism without affecting neuronal functions.
doi:10.1016/j.celrep.2014.08.060
PMCID: PMC4325997  PMID: 25263556
23.  Visualizing and Manipulating Temporal Signaling Dynamics with Fluorescence-Based Tools 
Science signaling  2014;7(319):re1.
The use of genome-wide proteomic and RNA interference approaches has moved our understanding of signal transduction from linear pathways to highly integrated networks centered on core nodes. However, probing the dynamics of flow of information through such networks remains technically challenging. In particular, how the temporal dynamics of an individual pathway can elicit distinct outcomes in a single cell type and how multiple pathways may interact sequentially or synchronously to influence cell fate remain open questions in many contexts. The development of fluorescence-based reporters and optogenetic regulators of pathway activity enables the analysis of signaling in living cells and organisms with unprecedented spatiotemporal resolution and holds the promise of addressing these key questions. We present a brief overview of the evidence for the importance of temporal dynamics in cellular regulation, introduce these fluorescence-based tools, and highlight specific studies that leveraged these tools to probe the dynamics of information flow through signaling networks. In particular, we highlight two studies in Caenorhabditis elegans sensory neurons and cultured mammalian cells that demonstrate the importance of signal dynamics in determining cellular responses.
doi:10.1126/scisignal.2005077
PMCID: PMC4319366  PMID: 24692594
24.  Drosophila as a Model for Context-Dependent Tumorigenesis 
Journal of cellular physiology  2014;229(1):27-33.
Drosophila can exhibit classic hallmarks of cancer, such as evasion of apoptosis, sustained proliferation, metastasis, prolonged survival, genome instability, and metabolic reprogramming, when cancer-related genes are perturbed. In the last two decades, studies in flies have identified several tumor suppressor and oncogenes. However, the greatest strength of the fly lies in its ability to model cancer hallmarks in a variety of tissue types, which enables the study of context-dependent tumorigenesis. We review the organs and tissues that have been used to model tumor formation, and propose new strategies to maximize the potential of Drosophila in cancer research.
doi:10.1002/jcp.24427
PMCID: PMC4034382  PMID: 23836429
25.  A rapid genome-wide microRNA screen identifies miR-14 as a modulator of Hedgehog signaling 
Cell reports  2014;7(6):2066-2077.
Summary
MicroRNAs (miRNAs) are small noncoding RNAs that regulate gene expression by binding to sequences within the 3′UTR of mRNAs. Because miRNAs bind to short sequences with partial complementarity, target identification is challenging. To complement the existing target prediction algorithms, we devised a systematic “reverse approach” screening platform which allows the empirical prediction of miRNA-target interactions. Using Drosophila cells, we screened the 3′UTRs of the Hedgehog pathway genes against a genome-wide miRNA library and identified both predicted and many non-predicted miRNA-target interactions. We demonstrate that miR-14 is essential for maintaining the proper level of Hedgehog signaling activity by regulating its physiological target, hedgehog. Furthermore, elevated levels of miR-14 suppress Hedgehog signaling activity by co-targeting its apparent non-physiological targets, patched and smoothened. Altogether, our systematic screening platform is a powerful approach to identifying both physiological and apparent non-physiological targets of miRNAs, which are relevant in both normal and diseased tissues.
doi:10.1016/j.celrep.2014.05.025
PMCID: PMC4142207  PMID: 24931604

Results 1-25 (101)