Optogenetic tools for Drosophila S2 cells

Osswald M, Santos AF, Morais-de-Sá E. Light-Induced Protein Clustering for Optogenetic Interference and Protein Interaction Analysis in Drosophila S2 Cells. Biomolecules. 2019 Feb 12;9(2). pii: E61. doi: 10.3390/biom9020061. PubMed PMID: 30759894; PubMed Central PMCID: PMC6406598.

Abstract: "Drosophila Schneider 2 (S2) cells are a simple and powerful system commonly used in cell biology because they are well suited for high resolution microscopy and RNAi-mediated depletion. However, understanding dynamic processes, such as cell division, also requires methodology to interfere with protein function with high spatiotemporal control. In this research study, we report the adaptation of an optogenetic tool to Drosophila S2 cells. Light-activated reversible inhibition by assembled trap (LARIAT) relies on the rapid light-dependent heterodimerization between cryptochrome 2 (CRY2) and cryptochrome-interacting bHLH 1 (CIB1) to form large protein clusters. An anti-green fluorescent protein (GFP) nanobody fused with CRY2 allows this method to quickly trap any GFP-tagged protein in these light-induced protein clusters. We evaluated clustering kinetics in response to light for different LARIAT modules, and showed the ability of GFP-LARIAT to inactivate the mitotic protein Mps1 and to disrupt the membrane localization of the polarity regulator Lethal Giant Larvae (Lgl). Moreover, we validated light-induced co-clustering assays to assess protein-protein interactions in S2 cells. In conclusion, GFP-based LARIAT is a versatile tool to answer different biological questions, since it enables probing of dynamic processes and protein-protein interactions with high spatiotemporal resolution in Drosophila S2 cells."

Report of detection of miRNAs in cell-free media from cultured Drosophila cells

Van den Brande S, Gijbels M, Wynant N, Santos D, Mingels L, Gansemans Y, Van Nieuwerburgh F, Vanden Broeck J. The presence of extracellular microRNAs in the media of cultured Drosophila cells. Sci Rep. 2018 Nov 23;8(1):17312. PMID: 30470777.

Abstract: "While regulatory RNA pathways, such as RNAi, have commonly been described at an intracellular level, studies investigating extracellular RNA species in insects are lacking. In the present study, we demonstrate the presence of extracellular microRNAs (miRNAs) in the cell-free conditioned media of two Drosophila cell lines. More specifically, by means of quantitative real-time PCR (qRT-PCR), we analysed the presence of twelve miRNAs in extracellular vesicles (EVs) and in extracellular Argonaute-1 containing immunoprecipitates, obtained from the cell-free conditioned media of S2 and Cl.8 cell cultures. Next-generation RNA-sequencing data confirmed our qRT-PCR results and provided evidence for selective miRNA secretion in EVs. To our knowledge, this is the first time that miRNAs have been identified in the extracellular medium of cultured cells derived from insects, the most speciose group of animals."

Video report -- cell-based assay

Peters KA, Detmar E, Sepulveda L, Del Valle C, Valsquier R, Ritz A, Rogers SL, Applewhite DA. A Cell-based Assay to Investigate Non-muscle Myosin II Contractility via the Folded-gastrulation Signaling Pathway in Drosophila S2R+ Cells. J Vis Exp. 2018 Aug 19;(138). doi: 10.3791/58325. PubMed PMID: 30176023.

DRSC screen contributes to research report on signaling and lifespan

Sung EJ, Ryuda M, Matsumoto H, Uryu O, Ochiai M, Cook ME, Yi NY, Wang H, Putney JW, Bird GS, Shears SB, Hayakawa Y. Cytokine signaling through Drosophila Mthl10 ties lifespan to environmental stress. Proc Natl Acad Sci U S A. 2017 Dec 11. PMID: 29229844.

From the abstract: "A systems-level understanding of cytokine-mediated, intertissue signaling is one of the keys to developing fundamental insight into the links between aging and inflammation. Here, we employed Drosophila, a routine model for analysis of cytokine signaling pathways in higher animals, to identify a receptor for the growth-blocking peptide (GBP) cytokine. Having previously established that the phospholipase C/Ca2+ signaling pathway mediates innate immune responses to GBP, we conducted a dsRNA library screen for genes that modulate Ca2+ mobilization in Drosophila S3 cells. A hitherto orphan G protein coupled receptor, Methuselah-like receptor-10 (Mthl10), was a significant hit. Secondary screening confirmed specific binding of fluorophore-tagged GBP to both S3 cells and recombinant Mthl10-ectodomain. We discovered that the metabolic, immunological, and stress-protecting roles of GBP all interconnect through Mthl10. This we established by Mthl10 knockdown in three fly model systems: in hemocyte-like Drosophila S2 cells, Mthl10 knockdown decreases GBP-mediated innate immune responses; in larvae, Mthl10 knockdown decreases expression of antimicrobial peptides in response to low temperature; in adult flies, Mthl10 knockdown increases mortality rate following infection with Micrococcus luteus and reduces GBP-mediated secretion of insulin-like peptides. ... We describe how our data offer opportunities for further molecular interrogation of yin and yang between homeostasis and longevity."

Genome-wide fly cell RNAi screen related to Wolbachia infection

White PM, Serbus LR, Debec A, Codina A, Bray W, Guichet A, Lokey RS, Sullivan W. Reliance of Wolbachia on High Rates of Host Proteolysis Revealed by a Genome-Wide RNAi Screen of Drosophila Cells. Genetics. 2017 Feb 3. pii: genetics.116.198903. PMID: 28159754.

From the abstract: "Wolbachia are gram-negative, obligate, intracellular bacteria carried by a majority of insect species worldwide. Here we use a Wolbachia-infected Drosophila cell line and genome-wide RNAi screening to identify host factors that influence Wolbachia titer. .. we identified 36 candidate genes that dramatically reduced Wolbachia titer and 41 that increased Wolbachia titer. ... knockdown of 7 genes in the host ubiquitin and proteolysis pathways significantly reduced Wolbachia titer. To test the in vivo relevance of these results, we found that drug and mutant inhibition of proteolysis reduced levels of Wolbachia in the Drosophila oocyte ... Given Wolbachia lack essential amino acid biosynthetic pathways, the reliance of Wolbachia on high rates of host proteolysis via ubiquitination and the ERAD pathways may be a key mechanism for provisioning Wolbachia with amino acids. In addition, the reliance of Wolbachia on the ERAD pathway and disruption of ER morphology suggests a previously unsuspected mechanism for Wolbachia's potent ability to prevent RNA virus replication."

Methods publications relevant to cell and in vivo RNAi

Methods in Molecular Biology has recently published papers relevant to Drosophila cell culture, cell-based RNAi, and in vivo RNAi.

Debec A, Megraw TL, Guichet A. Methods to Establish Drosophila Cell Lines. Methods Mol Biol. 2016;1478:333-351. PubMed PMID: 27730593. 

Billmann M, Boutros M. Methods for High-Throughput RNAi Screening in Drosophila Cells. Methods Mol Biol. 2016;1478:95-116. PubMed PMID: 27730577.

Kaya-Çopur A, Schnorrer F. A Guide to Genome-Wide In Vivo RNAi Applications in Drosophila. Methods Mol Biol. 2016;1478:117-143. PubMed PMID: 27730578.

Gene editing in fly cells--new report from Kunzelmann et al. in G3

Kunzelmann S, Böttcher R, Schmidts I, Förstemann K. A Comprehensive Toolbox for Genome Editing in Cultured Drosophila melanogaster Cells. G3 (Bethesda). 2016 Jun 1;6(6):1777-85. PMID: 27172193

From the abstract: "... Following up on our initial publication, we now describe a considerably simplified, more efficient, and readily scalable experimental workflow for PCR-based genome editing in cultured Drosophila melanogaster cells. Our analysis at the act5C locus suggests that PCR-based homology arms of 60 bp are sufficient to reach targeting efficiencies of up to 80% after selection; extension to 80 bp (PCR) or 500 bp (targeting vector) did not further improve the yield. We have expanded our targeting system to N-terminal epitope tags; this also allows the generation of cell populations with heterologous expression control of the tagged locus via the copper-inducible mtnDE promoter. We present detailed, quantitative data on editing efficiencies for several genomic loci that may serve as positive controls or benchmarks in other laboratories. ..."

RNAi screening, double RNAi and multi parametric image assays used to build map of cell cycle regulators

Billmann M, Horn T, Fischer B, Sandmann T, Huber W, Boutros M. A genetic interaction map of cell cycle regulators. Mol Biol Cell. 2016 Feb 24. PMID: 26912791.

From the abstract: "Cell based RNAi is a powerful approach to screen for modulators of many cellular processes. However, resulting candidate gene lists from cell-based assays comprise diverse effectors, both direct and indirect, and further dissecting their functions can be challenging. Here, we screened a genome-wide RNAi library for modulators of mitosis and cytokinesis in Drosophila S2 cells. ... We then characterized ∼300 candidate modifiers further by genetic interaction analysis using double RNAi and a multiparametric, imaging-based assay. ... Our results show that the combination of genome-scale RNAi screening and genetic interaction analysis using process-directed phenotypes provides a powerful two-step approach to assign components to specific pathways and complexes."

RNA pol II Cdk12 identified as an Nrf2 target via RNAi screening in S2 cells

Li X, Chatterjee N, Spirohn K, Boutros M, Bohmann D. Cdk12 Is A Gene-Selective RNA Polymerase II Kinase That Regulates a Subset of the Transcriptome, Including Nrf2 Target Genes. Sci Rep. 2016 Feb 25;6:21455. PMID: 26911346.

From the abstract: "The Nrf2 transcription factor is well conserved throughout metazoan evolution and serves as a central regulator of adaptive cellular responses to oxidative stress. We carried out an RNAi screen in Drosophila S2 cells to better understand the regulatory mechanisms governing Nrf2 target gene expression. This paper describes the identification and characterization of the RNA polymerase II (Pol II) kinase Cdk12 as a factor that is required for Nrf2 target gene expression in cell culture and in vivo. ... We suggest that Cdk12 acts as a gene-selective Pol II kinase that engages a global shift in gene expression to switch cells from a metabolically active state to "stress-defence mode" when challenged by external stress."

Cultured Drosophila cells 'humanized' for drug studies--video methods paper

Generating a "Humanized" Drosophila S2 Cell Line Sensitive to Pharmacological Inhibition of Kinesin-5. https://t.co/Cykmii1hlq

— flypapers (@fly_papers) February 11, 2016

Analysis of steroid hormone signaling in 41 Drosophila fly cell lines

Stoiber M, Celniker S, Cherbas L, Brown B, Cherbas P. Diverse Hormone Response Networks in 41 Independent Drosophila Cell Lines. G3 (Bethesda). 2016 Jan 15. pii: g3.115.023366. PMID: 26772746.

From the abstract: "Steroid hormones induce cascades of gene activation and repression with transformative effects on cell fate. Steroid transduction plays a major role in the development and physiology of nearly all metazoan species, and in the progression of the most common forms of cancer. Despite the paramount importance of steroids in developmental and translational biology, a complete map of transcriptional response has not been developed for any hormone. In the case of 20-hydroxyecdysone (ecdysone) in Drosophila melanogaster, these trajectories range from apoptosis to immortalization. We mapped the ecdysone transduction network in a cohort of 41 cell lines, the largest such atlas yet assembled. ... This atlas of steroid response reveals organizing principles of gene regulation by a model type II nuclear receptor and lays the foundation for comprehensive and predictive understanding of the ecdysone transduction network in the fruit fly."

Drosophila cell team at the DGRC reports integrase-mediated cassette exchange approach to production of clonal transgenic fly cell lines

Lucy Cherbas1, Jennifer Hackney, Lei Gong, Claire Salzer, Eric Mauser, Dayu Zhang and Peter Cherbas. Tools for Targeted Genome Engineering of Established Drosophila Cell Lines.
Early online in G3.

From the abstract: "We describe an adaptation of ΦC31 integrase-mediated targeted cassette exchange for use in Drosophila cell lines. Single copies of an attP-bounded docking platform carrying a GFP-expression marker, with or without insulator elements flanking the attP sites, were inserted by P-element transformation into the Kc167 and Sg4 cell lines; each of the resulting docking site lines carries a single mapped copy of one of the docking platforms. ... We describe procedures for isolating cells carrying the substitutions ... When compared with clonal lines made by traditional transformation methods ... targeted insertion lines give more uniform expression, lower basal expression and higher induction ratios. Targeted substitution, though intricate, affords results that should greatly improve comparative expression assays – a major emphasis of cell-based studies." 

DGRC team reports on targeted insertion approach to modifying Drosophila cultured cell lines

Lucy Cherbas, Jennifer Hackney, Lei Gong, Claire Salzer, Eric Mauser, Dayu Zhang and Peter Cherbas. 2015. Tools for Targeted Genome Engineering of Established Drosophila Cell Lines. Early online at Genetics.

From the abstract: "We describe an adaptation of φC31 integrase-mediated targeted cassette exchange for use in Drosophila cell lines. ... We demonstrated the technology by integrating a cassette containing a Cu⁺⁺-inducible mCherry marker, and we report the expression properties of those lines. When compared with clonal lines made by traditional transformation methods, which lead to the illegitimate insertion of tandem arrays, targeted insertion lines give more uniform expression, lower basal expression and higher induction ratios. Targeted substitution, though intricate, affords results that should greatly improve comparative expression assays – a major emphasis of cell-based studies."

New at the DRSC website--protocols for single-cell cloning and stable transfection of Drosophila cells

Two new step-by-step protocols available at the DRSC website:

• Single-cell cloning of Drosophila cells
• Making stably transfected Drosophila cells

And check out these related publications and resources:

Housden BE, Lin S, Perrimon N. Cas9-based genome editing in Drosophila. Methods Enzymol. 2014;546:415-39. PMID: 25398351.

Housden BE, Valvezan AJ, Kelley C, Sopko R, Hu Y, Roesel C,
Lin S, Buckner M, Tao R, Yilmazel B, Mohr SE, Manning BD, Perrimon N. Identification of potential drug targets for tuberous sclerosis complex by synthetic screens combining CRISPR-based knockouts with RNAi. Sci Signal. 2015 Sep 8;8(393):rs9. PMID: 26350902.

Santos MG, Jorge SA, Brillet K, Pereira CA. Improving heterologous protein expression in transfected Drosophila S2 cells as assessed by EGFP expression. Cytotechnology. 2007 May;54(1):15-24. PMID: 19003014; PMCID: PMC2267513.

Have a qPCR machine but no software for high-resolution melt analysis following CRISPR modification? Check out the HRMA online tool.

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. "

CRISPR + RNAi screening in Drosophila cells points to potential new drug targets for treatment of tuberous sclerosis complex (TSC)

Housden BE, Valvezan AJ, Kelley C, Sopko R, Hu Y, Roesel C, Lin S, Buckner M, Tao R, Yilmazel B, Mohr SE, Manning BD, Perrimon N. Identification of potential drug targets for tuberous sclerosis complex by synthetic screens combining CRISPR-based knockouts with RNAi. Sci Signal. 2015 Sep 8;8(393):rs9. PMID: 26350902.

From the abstract: "The tuberous sclerosis complex (TSC) family of tumor suppressors, TSC1 and TSC2, function together in an evolutionarily conserved protein complex ... 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 ... 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 ... reduced the population growth rate of Drosophila cells lacking either TSC1 or TSC2 but not that of wild-type cells ... [and] 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."

DRSC publishes RBP library resource including 'baseline data' available lots of ways--including interactive!

The DRSC is pleased to announce our new cell-based RNAi library targeting RNA Binding Proteins and corresponding 'baseline' data set. These data should be a helpful reference for anyone analyzing data from screens of the same library with more sophisticated assays.  

Big thanks to all the folks who worked hard to make this possible!

Mohr, Hu, Rudd, Buckner, Gilly, Foster, Sierzputowska, Comjean, Ye and Perrimon (2015) Reagent and Data Resources for Investigation of RNA Binding Protein Functions in Drosophila melanogaster Cultured Cells. Early online at G3 July 2015.

At a DRSC webpage we summarize ways to view the data.

Just one example--when you view the total ATP readout data at Plot.ly you can hover to see gene and reagent identifiers, zoom in on sub-sections of the graph, etc. The data are also available at the DRSC, at NCBI PubChem and no doubt will be imported by the folks at GenomeRNAi.

Report of 'systems-level interrogation' includes DRSC RNAi screen data, phosphoproteomics and in vivo analyses

Sopko R, Lin YB, Makhijani K, Alexander B, Perrimon N, Brückner K. A systems-level interrogation identifies regulators of Drosophila blood cell number and survival. PLoS Genet. 2015 Mar 6;11(3):e1005056. PMID: 25749252; PMCID: PMC4352040.

From the abstract: "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. ... 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 ... Ecdysone receptor (EcR) and ultraspiracle (usp) ... as Pvr Suppressors. ... Phosphoproteomic analysis demonstrates distinct modes of cell number regulation by EcR and RTK signaling. ... 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."

Thinking ahead--What could custom CRISPR engineered cell lines do for your research?

Open call to the Drosophila research community from the DRSC:

Since it was founded in 2003 by Prof. N. Perrimon, the Drosophila RNAi Screening Center (DRSC) has served as a technology transfer center, helping the Drosophila community-at-large gain access to leading-edge technologies such as genome-wide RNAi.

As readers of this blog are likely aware, we have been working to support technology transfer in many areas additional to RNAi, including in the area of CRISPR-Cas9 engineering. For example, we developed and made freely available a database of short guide RNAs, accompanying genome browser-based online user interface, and sgRNA efficiency prediction tool to help support sgRNA selection for CRISPR-Cas9 engineering in flies (see www.flyrnai.org/crispr2).

The DRSC will apply early next year (end of February 2015) for renewal of our NIH R01 grant funding, which makes it possible for us to provide all we do to the community.

One of the things we are beginning to do with community members, and would like to propose to expand and continue in the next funding cycle, is to build custom CRISPR-Cas9-modified cell lines. These can be of value for a wide range of studies, including but not limited to RNAi screens using custom engineered cells (e.g. knockout mutant cells for sensitized screens, endogenously tagged loci for reporter assays or to screen for disruption of sub-cellular localization).

In short, we need your help!

If custom engineered cells would help your research--i.e. if you can imagine turning to the DRSC to help support making and/or screening of specific custom lines for your research within the next, say, 1-3 years, and particularly if you're a US-based lab, we would appreciate if you'd please get in touch and be willing to write a letter of support for our renewal application. We are of course also interested to hear from folks who are planning to use our library resources for other types of screens in the next few years, and from others who are depending on continuity of online resources and/or research services at the DRSC. Ideas for additional tools or resources are also welcome.

Please contact, or have your PI contact, DRSC Director (and blog author) Dr. Stephanie Mohr.

Review and detailed protocols--CRISPR-Cas9 in flies and fly cells

Housden BE, Lin S, Perrimon N. Cas9-based genome editing in Drosophila. Methods Enzymol. 2014;546:415-39. PMID: 25398351.  

From the abstract: "... we first discuss some general design principles for genome engineering experiments in Drosophila and then present detailed protocols for the production of CRISPR reagents and screening strategies to detect successful genome modification events in both tissue culture cells and animals."

Includes helpful tables listing sgRNA design tools, relevant fly stocks and plasmids.