FlyBook review on RNAi screening in Drosophila cells and in vivo

Heigwer F, Port F, Boutros M. RNA Interference (RNAi) Screening in Drosophila. Genetics. 2018 Mar;208(3):853-874. PMID: 29487145.

From the abstract: "... RNA interference (RNAi) ... has had an important impact on identifying and characterizing gene function. First discovered in Caenorhabditis elegans, RNAi can be used to silence the expression of genes through introduction of exogenous double-stranded RNA into cells. In Drosophila, RNAi has been applied in cultured cells or in vivo to perturb the function of single genes or to systematically probe gene function on a genome-wide scale. In this review, we will describe the use of RNAi to study gene function in Drosophila with a particular focus on high-throughput screening methods applied in cultured cells. ..."

Single-molecule imaging study of a Wnt ligand using S2 and S2R+ Drosophila cultured cells

Lippert A, Janeczek AA, Fürstenberg A, Ponjavic A, Moerner WE, Nusse R, Helms JA, Evans ND, Lee SF. Single-Molecule Imaging of Wnt3A Protein Diffusion on Living Cell Membranes. Biophys J. 2017 Dec 19;113(12):2762-2767. PMID: 29262368.

Abstract: "Wnt proteins are secreted, hydrophobic, lipidated proteins found in all animals that play essential roles in development and disease. Lipid modification is thought to facilitate the interaction of the protein with its receptor, Frizzled, but may also regulate the transport of Wnt protein and its localization at the cell membrane. Here, by employing single-molecule fluorescence techniques, we show that Wnt proteins associate with and diffuse on the plasma membranes of living cells in the absence of any receptor binding. We find that labeled Wnt3A transiently and dynamically associates with the membranes of Drosophila Schneider 2 cells, diffuses with Brownian kinetics on flattened membranes and on cellular protrusions, and does not transfer between cells in close contact. In S2 receptor-plus (S2R+) cells, which express Frizzled receptors, membrane diffusion rate is reduced and membrane residency time is increased. These results provide direct evidence of Wnt3A interaction with living cell membranes, and represent, to our knowledge, a new system for investigating the dynamics of Wnt transport."

Boutros lab releases web app for screen image data analysis and visualization

http://biorxiv.org/content/early/2017/02/11/107821

Genome-wide screen in study of heterochromatin factor HP1a

The cell-based RNAi screen described in this study was done using a DRSC library and follow-up reagents. Congratulations, Joel and colleagues!

Swenson JM, Colmenares SU, Strom AR, Costes SV, Karpen GH. The composition and organization of Drosophila heterochromatin are heterogeneous and dynamic. Elife. 2016 Aug 11;5. pii: e16096. PMID: 27514026; PMCID: PMC4981497.

Abstract: "Heterochromatin is enriched for specific epigenetic factors including Heterochromatin Protein 1a (HP1a), and is essential for many organismal functions. To elucidate heterochromatin organization and regulation, we purified Drosophila melanogaster HP1a interactors, and performed a genome-wide RNAi screen to identify genes that impact HP1a levels or localization. The majority of the over four hundred putative HP1a interactors and regulators identified were previously unknown. We found that 13 of 16 tested candidates (83%) are required for gene silencing, providing a substantial increase in the number of identified components that impact heterochromatin properties. Surprisingly, image analysis revealed that although some HP1a interactors and regulators are broadly distributed within the heterochromatin domain, most localize to discrete subdomains that display dynamic localization patterns during the cell cycle. We conclude that heterochromatin composition and architecture is more spatially complex and dynamic than previously suggested, and propose that a network of subdomains regulates diverse heterochromatin functions."

Genome-wide cell-based screen related to microtubule bundling and lysosome motility

Jolly AL, Luan CH, Dusel BE, Dunne SF, Winding M, Dixit VJ, Robins C, Saluk JL, Logan DJ, Carpenter AE, Sharma M, Dean D, Cohen AR, Gelfand VI. A Genome-wide RNAi Screen for Microtubule Bundle Formation and Lysosome Motility Regulation in Drosophila S2 Cells. Cell Rep. 2016 Jan 26;14(3):611-20. PMID: 26774481.

Cell-based RNAi screen looks at TGF-beta signaling--methods-focused report

Chen X, Xu L. Genome-Wide RNAi Screening to Dissect the TGF-β Signal Transduction Pathway. Methods Mol Biol. 2016;1344:365-77. PMID: 26520138.

From the abstract: "... Here, we describe a protocol for image-based whole-genome RNAi screening aimed at identifying molecules required for TGF-β signaling into the nucleus. Using this protocol we examined 90% of annotated Drosophila open reading frames (ORF) individually and successfully uncovered several novel factors serving critical roles in the TGF-β pathway. Thus cell-based high-throughput functional genomics can uncover new mechanistic insights on signaling pathways beyond what the classical genetics had revealed."

Methods review on genome-wide cell-based screens to identify centrosome components

Dobbelaere J. Genome-wide RNAi screens in S2 cells to identify centrosome components. Methods Cell Biol. 2015;129:279-300. PMID: 26175444.

From the abstract: "... In this paper, we present detailed instructions for designing, performing, and analyzing a genome-wide screen in Drosophila tissue culture cells to identify centrosome components using a microscopy-based approach. "

Methods paper--RNAi screening and the centrosome

Dobbelaere J. Genome-wide RNAi screens in S2 cells to identify centrosome components. Methods Cell Biol. 2015;129:279-300.PMID: 26175444.

From the abstract: "... Genome-wide RNA interference (RNAi) allows for comprehensive screening ... Drosophila tissue culture cells provide an attractive model for such screens. ... Drosophila centrosomes are similar to their human counterparts, but less complex. Thus, all major centrosome components are conserved and fewer redundancies apply ... In this paper, we present detailed instructions for designing, performing, and analyzing a genome-wide screen in Drosophila tissue culture cells to identify centrosome components using a microscopy-based approach."

Actin in the nucleus -- genome-wide cell-based RNAi screen

Excited to see this report on a screen performed by J. Dopie at the DRSC!

Dopie J, Rajakylä EK, Joensuu MS, Huet G, Ferrantelli E, Xie T, Jäälinoja H, Jokitalo E, Vartiainen MK. Genome-wide RNAi screen for nuclear actin reveals a network of cofilin regulators. J Cell Sci. 2015 May 28. pii: jcs.169441. PMID: 26021350.

From the abstract: "Nuclear actin plays an important role in many processes that regulate gene expression. ... Here we have performed a genome-wide RNAi screen in Drosophila cells to identify proteins that influence either nuclear polymerization or import of actin. We validate 19 factors as specific hits, and show that Chinmo/Bach2, SNF4Aγ/Prkag1 and Rab18 play a role in nuclear localization of actin in both fly and mammalian cells. We identify several novel regulators of cofilin activity, and characterize modulators of both cofilin kinases and phosphatase. ... Our screen therefore reveals novel aspects of actin regulation and links nuclear actin to many cellular processes."

Defining the cell shape space

Sailem H, Bousgouni V, Cooper S, Bakal C. Cross-talk between Rho and Rac GTPases drives deterministic exploration of cellular shape space and morphological heterogeneity. Open Biol. 2014 Jan 22;4:130132. PMID: 24451547; PubMed Central PMCID: PMC3909273.

  From the abstract: "... We first exploit the shape diversity generated by systematic RNAi screening and comprehensively define the shape space a migratory cell explores. ... We validate the predictions made by our model using live-cell imaging. Our work explains how cross-talk between Rho and Rac can generate different cell shapes, and thus morphological heterogeneity, in genetically identical populations."

Localization of CPTI collection of YFP-trap proteins

Lye CM, Naylor HW, Sanson B. Subcellular localisations of the CPTI collection of YFP-tagged proteins in Drosophila embryos. Development. 2014 Oct;141(20):4006-17. PMID: 25294944.

From the abstract:  "... The Cambridge Protein Trap Consortium generated, via piggyBac transposition, over 600 novel YFP-trap proteins tagging just under 400 Drosophila loci. Here, we characterise the subcellular localisations and expression patterns of these insertions, called the CPTI lines, in Drosophila embryos. ..."

Drosophila cell-based genome-wide screen related to Parkinson's disease

Ivatt RM, Sanchez-Martinez A, Godena VK, Brown S, Ziviani E, Whitworth AJ. Genome-wide RNAi screen identifies the Parkinson disease GWAS risk locus SREBF1 as a regulator of mitophagy. Proc Natl Acad Sci U S A. 2014 Jun 10;111(23):8494-9. PMID: 24912190; PubMed Central PMCID: PMC4060696.  

From the abstract: "... We undertook a genome-wide RNAi screen as an unbiased approach to identify genes regulating the PINK1/Parkin pathway. We identified several genes that have a conserved function in promoting mitochondrial translocation of Parkin and subsequent mitophagy, most notably sterol regulatory element binding transcription factor 1 (SREBF1), F-box and WD40 domain protein 7 (FBXW7), and other components of the lipogenesis pathway. ..."

Genome-wide, high-content image-based screen preformed at DRSC--identification of regulators of organization of polycomb foci

Gonzalez I, Mateos-Langerak J, Thomas A, Cheutin T, Cavalli G. Identification of regulators of the three-dimensional polycomb organization by a microscopy-based genome-wide RNAi screen. Mol Cell. 2014 May 8;54(3):485-99. PMID: 24703951.  

From the abstract: "... Here, we report the results of a high-resolution microscopy genome-wide RNAi screen that identifies 129 genes that regulate the nuclear organization of Pc foci. Candidate genes include PcG components and chromatin factors, as well as many protein-modifying enzymes, including components of the SUMOylation pathway. ... suggesting that the dynamic regulation of Pc SUMOylation regulates PcG-mediated silencing by modulating the kinetics of Pc binding to chromatin as well as its ability to form Polycomb foci."

in vivo fly RNAi kinase and phosphatase screen looks at synapse

Bulat V, Rast M, Pielage J. Presynaptic CK2 promotes synapse organization and stability by targeting Ankyrin2. J Cell Biol. 2014 Jan 6;204(1):77-94. PMID: 24395637; PMCID: PMC3882785.

Spindle assembly screen of 96 candidate genes

Gallaud E, Caous R, Pascal A, Bazile F, Gagné JP, Huet S, Poirier GG, Chrétien D, Richard-Parpaillon L, Giet R. Ensconsin/Map7 promotes microtubule growth and centrosome separation in Drosophila neural stem cells. J Cell Biol. 2014 Mar 31;204(7):1111-21. PMID: 24687279.

From the abstract: "... To identify new mitotic spindle assembly regulators, we isolated 855 microtubule-associated proteins (MAPs) from Drosophila melanogaster mitotic or interphasic embryos. Using RNAi, we screened 96 poorly characterized genes in the Drosophila central nervous system to establish their possible role during spindle assembly. ..."

Review--RNAi screening and signaling networks

Evans L, Sailem H, Vargas PP, Bakal C. Inferring signalling networks from images. J Microsc. 2013 Oct;252(1):1-7. PMID: 23841886.

From the abstract:  "This review describes the methodology used to map signalling networks using data generated in the context of RNAi screens."

Single-cell analysis of S2R+ RNAi screen image data

Dey G, Gupta GD, Ramalingam B, Sathe M, Mayor S, Thattai M. Exploiting cell-to-cell variability to detect cellular perturbations. PLoS One. 2014 Mar 4;9(3):e90540. PMID: 24594940; PMCID: PMC3942435.

New cell image analysis tool reported

Tsygankov D, Bilancia CG, Vitriol EA, Hahn KM, Peifer M, Elston TC. CellGeo: A computational platform for the analysis of shape changes in cells with complex geometries. J Cell Biol. 2014 Feb 3;204(3):443-60. PMID: 24493591.

Shaken, then swirled.

On my desk to read today? This new report of a Drosophila cell-based RNAi screen for which the researchers used a mechanical assay--shake, swirl, shake--to interrogate cell-cell adhesion.

Toret CP, D'Ambrosio MV, Vale RD, Simon MA, Nelson WJ. A genome-wide screen identifies conserved protein hubs required for cadherin-mediated cell-cell adhesion. J Cell Biol. 2014 Jan 20;204(2):265-79. PMID: 24446484.

Genome-wide S2 cell screen looking at centrosome-independent mitotic spindle assembly

Moutinho-Pereira S, Stuurman N, Afonso O, Hornsveld M, Aguiar P, Goshima G, Vale RD, Maiato H. Genes involved in centrosome-independent mitotic spindle assembly in Drosophila S2 cells. Proc Natl Acad Sci U S A. 2013 Nov 19. PMID: 24255106.

From the abstract: "Animal mitotic spindle assembly relies on centrosome-dependent and centrosome-independent mechanisms, but their relative contributions remain unknown. Here, we investigated the molecular basis of the centrosome-independent spindle assembly pathway by performing a whole-genome RNAi screen in Drosophila S2 cells lacking functional centrosomes. This screen identified 197 genes involved in acentrosomal spindle assembly, eight of which had no previously described mitotic phenotypes and produced defective and/or short spindles. ... Overall, these findings establish the constitutive nature of a centrosome-independent spindle assembly program and how this program is adapted to the presence/absence of centrosomes in animal somatic cells."