Drosophila (fly)

Image of green fluorescence in GFP-tagged Drosophila cultured cells

New cell lines & new understandings using cutting-edge techniques

February 2, 2024

As a facility that supports large-scale screens in Drosophila and other insect cell lines, we get excited about reports of new Drosophila cell lines and related info.

We'd like to highlight two recent papers.

One report, a collaboration between Amanda Simcox's group, the DGRC, and our group here at the DRSC, describes new cell lines made in Amanda's group and characterized in a collaboration of the three groups. Muscle cells that pulse? Yes. That and other exciting new cell lines are reported in the publication below, and the cells are available at the DGRC...

Read more about New cell lines & new understandings using cutting-edge techniques
Hong-Wen Tang, Kerstin Spirohn, Yanhui Hu, Tong Hao, István A Kovács, Yue Gao, Richard Binari, Donghui Yang-Zhou, Kenneth H Wan, Joel S Bader, Dawit Balcha, Wenting Bian, Benjamin W Booth, Atina G Coté, Steffi de Rouck, Alice Desbuleux, Kah Yong Goh, Dae-Kyum Kim, Jennifer J Knapp, Wen Xing Lee, Irma Lemmens, Cathleen Li, Mian Li, Roujia Li, Hyobin Julianne Lim, Yifang Liu, Katja Luck, Dylan Markey, Carl Pollis, Sudharshan Rangarajan, Jonathan Rodiger, Sadie Schlabach, Yun Shen, Dayag Sheykhkarimli, Bridget TeeKing, Frederick P. Roth, Jan Tavernier, Michael A Calderwood, David E Hill, Susan E Celniker, Marc Vidal, Norbert Perrimon, and Stephanie E. Mohr. 2023. “Next-generation large-scale binary protein interaction network for Drosophila melanogaster,” 14, 1, Pp. 2162. Publisher's VersionAbstract
Generating reference maps of interactome networks illuminates genetic studies by providing a protein-centric approach to finding new components of existing pathways, complexes, and processes. We apply state-of-the-art methods to identify binary protein-protein interactions (PPIs) for Drosophila melanogaster. Four all-by-all yeast two-hybrid (Y2H) screens of > 10,000 Drosophila proteins result in the ‘FlyBi’ dataset of 8723 PPIs among 2939 proteins. Testing subsets of data from FlyBi and previous PPI studies using an orthogonal assay allows for normalization of data quality; subsequent integration of FlyBi and previous data results in an expanded binary Drosophila reference interaction network, DroRI, comprising 17,232 interactions among 6511 proteins. We use FlyBi data to generate an autophagy network, then validate in vivo using autophagy-related assays. The deformed wings (dwg) gene encodes a protein that is both a regulator and a target of autophagy. Altogether, these resources provide a foundation for building new hypotheses regarding protein networks and function.
Nikki Coleman-Gosser, Yanhui Hu, Shiva Raghuvanshi, Shane Stitzinger, Weihang Chen, Arthur Luhur, Daniel Mariyappa, Molly Josifov, Andrew Zelhof, Stephanie E Mohr, Norbert Perrimon, and Amanda Simcox. 2023. “Continuous muscle, glial, epithelial, neuronal, and hemocyte cell lines for research.” Elife, 12.Abstract

Expression of activated Ras, Ras, provides cultured cells with a proliferation and survival advantage that simplifies the generation of continuous cell lines. Here, we used lineage-restricted Ras expression to generate continuous cell lines of muscle, glial, and epithelial cell type. Additionally, cell lines with neuronal and hemocyte characteristics were isolated by cloning from cell cultures established with broad Ras expression. Differentiation with the hormone ecdysone caused maturation of cells from mesoderm lines into active muscle tissue and enhanced dendritic features in neuronal-like lines. Transcriptome analysis showed expression of key cell-type-specific genes and the expected alignment with single-cell sequencing and in situ data. Overall, the technique has produced in vitro cell models with characteristics of glia, epithelium, muscle, nerve, and hemocyte. The cells and associated data are available from the Genomic Resource Center.

Baolong Xia, Raghuvir Viswanatha, Yanhui Hu, Stephanie E Mohr, and Norbert Perrimon. 2023. “Pooled genome-wide CRISPR activation screening for rapamycin resistance genes in cells.” Elife, 12.Abstract

Loss-of-function and gain-of-function genetic perturbations provide valuable insights into gene function. In cells, while genome-wide loss-of-function screens have been extensively used to reveal mechanisms of a variety of biological processes, approaches for performing genome-wide gain-of-function screens are still lacking. Here, we describe a pooled CRISPR activation (CRISPRa) screening platform in cells and apply this method to both focused and genome-wide screens to identify rapamycin resistance genes. The screens identified three genes as novel rapamycin resistance genes: a member of the SLC16 family of monocarboxylate transporters (), a member of the lipocalin protein family (), and a zinc finger C2H2 transcription factor (). Mechanistically, we demonstrate that overexpression activates the RTK-Akt-mTOR signaling pathway and that activation of insulin receptor (InR) by requires cholesterol and clathrin-coated pits at the cell membrane. This study establishes a novel platform for functional genetic studies in cells.

Figure 1 from Xu, Kim et al 2022 in eLife

Light up your flies! Reagent and protocol information for application of our NanoTag system for epitope tagging in Drosophila

March 23, 2023

One of the areas of interest for our technology development group is nanobodies. These small, single-chain antibodies are particularly attractive for Drosophila research as in addition to being used for standard immune-technologies such as immunoblots and immunostaining of tissues, they can also be expressed in vivo as fusions to fluorescent proteins (‘chromobodies’) or functional domains (e.g., for degradation or re-localization).

Past applications of this technology in Drosophila relied on availability of nanobodies targeting a specific protein or use of nanobodies targeting...

Read more about Light up your flies! Reagent and protocol information for application of our NanoTag system for epitope tagging in Drosophila
Shue Chen, Leah F Rosin, Gianluca Pegoraro, Nellie Moshkovich, Patrick J Murphy, Guoyun Yu, and Elissa P Lei. 8/12/2022. “NURF301 contributes to gypsy chromatin insulator-mediated nuclear organization.” Nucleic Acids Res, 50, 14, Pp. 7906-7924.Abstract
Chromatin insulators are DNA-protein complexes that can prevent the spread of repressive chromatin and block communication between enhancers and promoters to regulate gene expression. In Drosophila, the gypsy chromatin insulator complex consists of three core proteins: CP190, Su(Hw), and Mod(mdg4)67.2. These factors concentrate at nuclear foci termed insulator bodies, and changes in insulator body localization have been observed in mutants defective for insulator function. Here, we identified NURF301/E(bx), a nucleosome remodeling factor, as a novel regulator of gypsy insulator body localization through a high-throughput RNAi imaging screen. NURF301 promotes gypsy-dependent insulator barrier activity and physically interacts with gypsy insulator proteins. Using ChIP-seq, we found that NURF301 co-localizes with insulator proteins genome-wide, and NURF301 promotes chromatin association of Su(Hw) and CP190 at gypsy insulator binding sites. These effects correlate with NURF301-dependent nucleosome repositioning. At the same time, CP190 and Su(Hw) both facilitate recruitment of NURF301 to chromatin. Finally, Oligopaint FISH combined with immunofluorescence revealed that NURF301 promotes 3D contact between insulator bodies and gypsy insulator DNA binding sites, and NURF301 is required for proper nuclear positioning of gypsy binding sites. Our data provide new insights into how a nucleosome remodeling factor and insulator proteins cooperatively contribute to nuclear organization.
Hans M Dalton, Raghuvir Viswanatha, Roderick Brathwaite, Jae Sophia Zuno, Alexys R Berman, Rebekah Rushforth, Stephanie E Mohr, Norbert Perrimon, and Clement Y Chow. 2022. “A genome-wide CRISPR screen identifies DPM1 as a modifier of DPAGT1 deficiency and ER stress.” PLoS Genet, 18, 9, Pp. e1010430.Abstract
Partial loss-of-function mutations in glycosylation pathways underlie a set of rare diseases called Congenital Disorders of Glycosylation (CDGs). In particular, DPAGT1-CDG is caused by mutations in the gene encoding the first step in N-glycosylation, DPAGT1, and this disorder currently lacks effective therapies. To identify potential therapeutic targets for DPAGT1-CDG, we performed CRISPR knockout screens in Drosophila cells for genes associated with better survival and glycoprotein levels under DPAGT1 inhibition. We identified hundreds of candidate genes that may be of therapeutic benefit. Intriguingly, inhibition of the mannosyltransferase Dpm1, or its downstream glycosylation pathways, could rescue two in vivo models of DPAGT1 inhibition and ER stress, even though impairment of these pathways alone usually causes CDGs. While both in vivo models ostensibly cause cellular stress (through DPAGT1 inhibition or a misfolded protein), we found a novel difference in fructose metabolism that may indicate glycolysis as a modulator of DPAGT1-CDG. Our results provide new therapeutic targets for DPAGT1-CDG, include the unique finding of Dpm1-related pathways rescuing DPAGT1 inhibition, and reveal a novel interaction between fructose metabolism and ER stress.
Ying Xu, Raghuvir Viswanatha, Oleg Sitsel, Daniel Roderer, Haifang Zhao, Christopher Ashwood, Cecilia Voelcker, Songhai Tian, Stefan Raunser, Norbert Perrimon, and Min Dong. 2022. “CRISPR screens in Drosophila cells identify Vsg as a Tc toxin receptor.” Nature, 610, 7931, Pp. 349-355.Abstract
Entomopathogenic nematodes are widely used as biopesticides1,2. Their insecticidal activity depends on symbiotic bacteria such as Photorhabdus luminescens, which produces toxin complex (Tc) toxins as major virulence factors3-6. No protein receptors are known for any Tc toxins, which limits our understanding of their specificity and pathogenesis. Here we use genome-wide CRISPR-Cas9-mediated knockout screening in Drosophila melanogaster S2R+ cells and identify Visgun (Vsg) as a receptor for an archetypal P. luminescens Tc toxin (pTc). The toxin recognizes the extracellular O-glycosylated mucin-like domain of Vsg that contains high-density repeats of proline, threonine and serine (HD-PTS). Vsg orthologues in mosquitoes and beetles contain HD-PTS and can function as pTc receptors, whereas orthologues without HD-PTS, such as moth and human versions, are not pTc receptors. Vsg is expressed in immune cells, including haemocytes and fat body cells. Haemocytes from Vsg knockout Drosophila are resistant to pTc and maintain phagocytosis in the presence of pTc, and their sensitivity to pTc is restored through the transgenic expression of mosquito Vsg. Last, Vsg knockout Drosophila show reduced bacterial loads and lethality from P. luminescens infection. Our findings identify a proteinaceous Tc toxin receptor, reveal how Tc toxins contribute to P. luminescens pathogenesis, and establish a genome-wide CRISPR screening approach for investigating insecticidal toxins and pathogens.
Jiunn Song, Arda Mizrak, Chia-Wei Lee, Marcelo Cicconet, Zon Weng Lai, Wei-Chun Tang, Chieh-Han Lu, Stephanie E. Mohr, Robert V. Farese, and Tobias C. Walther. 2022. “Identification of two pathways mediating protein targeting from ER to lipid droplets.” Nature Cell Biol. Publisher's VersionAbstract
Pathways localizing proteins to their sites of action are essential for eukaryotic cell organization and function. Although mechanisms of protein targeting to many organelles have been defined, how proteins, such as metabolic enzymes, target from the endoplasmic reticulum (ER) to cellular lipid droplets (LDs) is poorly understood. Here we identify two distinct pathways for ER-to-LD protein targeting: early targeting at LD formation sites during formation, and late targeting to mature LDs after their formation. Using systematic, unbiased approaches in Drosophila cells, we identified specific membrane-fusion machinery, including regulators, a tether and SNARE proteins, that are required for the late targeting pathway. Components of this fusion machinery localize to LD–ER interfaces and organize at ER exit sites. We identified multiple cargoes for early and late ER-to-LD targeting pathways. Our findings provide a model for how proteins target to LDs from the ER either during LD formation or by protein-catalysed formation of membrane bridges.
Ashley Mae Conard, Nathaniel Goodman, Yanhui Hu, Norbert Perrimon, Ritambhara Singh, Charles Lawrence, and Erica Larschan. 2021. “TIMEOR: a web-based tool to uncover temporal regulatory mechanisms from multi-omics data.” Nucleic Acids Res, 49, W1, Pp. W641-W653.Abstract
Uncovering how transcription factors regulate their targets at DNA, RNA and protein levels over time is critical to define gene regulatory networks (GRNs) and assign mechanisms in normal and diseased states. RNA-seq is a standard method measuring gene regulation using an established set of analysis stages. However, none of the currently available pipeline methods for interpreting ordered genomic data (in time or space) use time-series models to assign cause and effect relationships within GRNs, are adaptive to diverse experimental designs, or enable user interpretation through a web-based platform. Furthermore, methods integrating ordered RNA-seq data with protein-DNA binding data to distinguish direct from indirect interactions are urgently needed. We present TIMEOR (Trajectory Inference and Mechanism Exploration with Omics data in R), the first web-based and adaptive time-series multi-omics pipeline method which infers the relationship between gene regulatory events across time. TIMEOR addresses the critical need for methods to determine causal regulatory mechanism networks by leveraging time-series RNA-seq, motif analysis, protein-DNA binding data, and protein-protein interaction networks. TIMEOR's user-catered approach helps non-coders generate new hypotheses and validate known mechanisms. We used TIMEOR to identify a novel link between insulin stimulation and the circadian rhythm cycle. TIMEOR is available at https://github.com/ashleymaeconard/TIMEOR.git and http://timeor.brown.edu.
Jonathan Zirin, Justin Bosch, Raghuvir Viswanatha, Stephanie E Mohr, and Norbert Perrimon. 2022. “State-of-the-art CRISPR for in vivo and cell-based studies in Drosophila.” Trends Genet, 38, 5, Pp. 437-453.Abstract
For more than 100 years, the fruit fly, Drosophila melanogaster, has served as a powerful model organism for biological and biomedical research due to its many genetic and physiological similarities to humans and the availability of sophisticated technologies used to manipulate its genome and genes. The Drosophila research community quickly adopted CRISPR technologies and, in the 8 years since the first clustered regularly interspaced short palindromic repeats (CRISPR) publications in flies, has explored and innovated methods for mutagenesis, precise genome engineering, and beyond. Moreover, the short lifespan and ease of genetics have made Drosophila an ideal testing ground for in vivo applications and refinements of the rapidly evolving set of CRISPR-associated (CRISPR-Cas) tools. Here, we review innovations in delivery of CRISPR reagents, increased efficiency of cutting and homology-directed repair (HDR), and alternatives to standard Cas9-based approaches. While the focus is primarily on in vivo systems, we also describe the role of Drosophila cultured cells as both an indispensable first step in the process of assessing new CRISPR technologies and a platform for genome-wide CRISPR pooled screens.
Justin A. Bosch and Norbert Perrimon. 2022. “Prime Editing for Precise Genome Engineering in Drosophila.” In Drosophila: Methods and Protocols, edited by Christian Dahmann, Pp. 113 - 134. New York, NY: Springer US. Publisher's VersionAbstract
Editing the Drosophila genome is incredibly useful for gene functional analysis. However, compared to gene knockouts, precise gene editing is difficult to achieve. Prime editing, a recently described CRISPR/Cas9-based technique, has the potential to make precise editing simpler and faster, and produce less errors than traditional methods. Initially described in mammalian cells, prime editing is functional in Drosophila somatic and germ cells. Here, we outline steps to design, generate, and express prime editing components in transgenic flies. Furthermore, we highlight a crossing scheme to produce edited fly stocks in less than 3 months.
Decorative cartoon drawn with BioRender depicting DRSC-BTRR technology concepts

So you want to do a CRISPR pooled screen in insect cells? You can! Here's how

May 12, 2022

At the DRSC-BTRR, we've been doing a lot of pooled-format CRISPR knockout screens in Drosophila cells. We're finding the results to be robust and reproducible. And best of all, the results have been informative, providing insights into diverse areas of biology.

Thinking about how to do CRISPR knockout screens in cells is a little different from thinking about how to do a genetic or RNAi screen in vivo or doing an arrayed-format RNAi screen....

Read more about So you want to do a CRISPR pooled screen in insect cells? You can! Here's how

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