Biology subjects

Perrimon, N.

Publications and source records attributed to Perrimon, N..

12 recordsLinked to original sources

bioRxiv2018

A cell atlas of the adult Drosophila midgut

Studies of the adult Drosophila midgut have provided a number of insights on cell type diversity, stem cell regeneration, tissue homeostasis and cell fate decision. Advances in single-cell RNA sequencing (scRNA-seq) provide opportunities to identify new cell types and molecular features. We used inDrop to characterize the transcriptome of midgut epithelial cells and identified 12 distinct clusters representing intestinal stem cells (ISCs), enteroblasts (EBs), enteroendocrine cells (EEs), enterocytes (ECs) from different regions, and cardia. This unbiased approach recovered 90% of the known ISCs/EBs markers, highlighting the high quality of the dataset. Gene set enrichment analysis in conjunction with electron micrographs revealed that ISCs are enriched in free ribosomes and possess mitochondria with fewer cristae. We demonstrate that a subset of EEs in the middle region of the midgut expresses the progenitor marker esg and that individual EEs are capable of expressing up to 4 different gut hormone peptides. We also show that the transcription factor klumpfuss (klu) is expressed in EBs and functions to suppress EE formation. Lastly, we provide a web-based resource for visualization of gene expression in single cells. Altogether, our study provides a comprehensive resource for addressing novel functions of genes in the midgut epithelium.

genetics↗

bioRxiv2018

Conserved phosphorylation hotspots in eukaryotic protein domain families

Protein phosphorylation is the best characterized post-translational modification that regulates almost all cellular processes through diverse mechanisms such as changing protein conformations, interactions, and localization. While the inventory for phosphorylation sites across different species has rapidly expanded, their functional role remains poorly investigated. Here, we have combined 537,321 phosphosites from 40 eukaryotic species to identify highly conserved phosphorylation \"hotspot\" regions within domain families. Mapping these regions onto structural data revealed that they are often found at interfaces, near catalytic residues and tend to harbor functionally important phosphosites. Notably, functional studies of a phospho-deficient mutant in the C-terminal hotspot region within the Ribosomal S11 domain in the yeast ribosomal protein uS11 showed cold-sensitive phenotype and impaired 20S pre-rRNA processing. Altogether, our study identified phosphorylation hotspots for 162 protein domains suggestive of an ancient role for the control of diverse eukaryotic domain families.

cell biology↗

bioRxiv2018

iProteinDB: an integrative database of Drosophila post-translational modifications

Post-translational modification (PTM) serves as a regulatory mechanism for protein function, influencing stability, protein interactions, activity and localization, and is critical in many signaling pathways. The best characterized PTM is phosphorylation, whereby a phosphate is added to an acceptor residue, commonly serine, threonine and tyrosine. As proteins are often phosphorylated at multiple sites, identifying those sites that are important for function is a challenging problem. Considering that many phosphorylation sites may be non-functional, prioritizing evolutionarily conserved phosphosites provides a general strategy to identify the putative functional sites with regards to regulation and function. To facilitate the identification of conserved phosphosites, we generated a large-scale phosphoproteomics dataset from Drosophila embryos collected from six closely-related species. We built iProteinDB (https://www.flyrnai.org/tools/iproteindb/), a resource integrating these data with other high-throughput PTM datasets, including vertebrates, and manually curated information for Drosophila. At iProteinDB, scientists can view the PTM landscape for any Drosophila protein and identify predicted functional phosphosites based on a comparative analysis of data from closely-related Drosophila species. Further, iProteinDB enables comparison of PTM data from Drosophila to that of orthologous proteins from other model organisms, including human, mouse, rat, Xenopus laevis, Danio rerio, and Caenorhabditis elegans.

bioinformatics↗

bioRxiv2018

ovoD co-selection: a method for enriching CRISPR/Cas9-edited alleles in Drosophila

Screening for successful CRISPR/Cas9 editing events remains a time consuming technical bottleneck in the field of Drosophila genome editing. This step can be particularly laborious for events that do not cause a visible phenotype, or those which occur at relatively low frequency. A promising strategy to enrich for desired CRISPR events is to co-select for an independent CRISPR event that produces an easily detectable phenotype. Here, we describe a simple negative co-selection strategy involving CRISPR-editing of a dominant female sterile allele, ovoD1. In this system (\"ovoD co-selection\"), the only functional germ cells in injected females are those that have been edited at the ovoD1 locus, and thus 100% of the offspring of these flies have undergone editing of at least one locus. We demonstrate that ovoD co-selection can be used to enrich for knock-out mutagenesis via nonhomologous end-joining (NHEJ), and for knock-in alleles via homology-directed repair (HDR). Altogether, our results demonstrate that ovoD co-selection reduces the amount of screening necessary to isolate desired CRISPR events in Drosophila.

genetics↗

bioRxiv2018

Pooled genome-wide CRISPR screening for basal and context-specific fitness gene essentiality in Drosophila cells

Genome-wide screens in Drosophila cells have offered numerous insights into gene function, yet a major limitation has been the inability to stably deliver large multiplexed DNA libraries to cultured cells allowing barcoded pooled screens. Here, we developed a site-specific integration strategy for library delivery and performed a genome-wide CRISPR knockout screen in Drosophila S2R+ cells. Under basal growth conditions, 1235 genes were essential for cell fitness at a false-discovery rate of 5%, representing the highest-resolution fitness gene set yet assembled for Drosophila, including 407 genes which likely duplicated along the vertebrate lineage and whose orthologs were underrepresented in human CRISPR screens. We additionally performed context-specific fitness screens for resistance to or synergy with trametinib, a Ras/ERK/ETS inhibitor, or rapamycin, an mTOR inhibitor, and identified key regulators of each pathway. The results present a novel, scalable, and versatile platform for functional genomic screens in low-redundancy animal cells.

genetics↗

bioRxiv2018

Long-range Notch-mediated tissue patterning requires actomyosin contractility

Dynamic, actin-based protrusions function in cell-cell signaling in a variety of systems. In the dorsal thorax of the developing fly, basal protrusions enable non-neighboring epithelial cells to touch, extending the range over which Notch-mediated lateral inhibition can occur during bristle patterning. Given that actin-based cell protrusions can exert mechanical forces on their environment and Notch receptor activation is mechanically sensitive, how might cytoskeletal contractility contribute to Notch signaling? We identify a pool of basal non-muscle myosin II (myosin II) that regulates protrusion dynamics, promotes Notch signaling, and is required in signal sending and receiving cells for Notch-dependent patterning. We show that interactions between protrusions are extensive and subject to actomyosin contractility. The effects of reducing myosin II activity are more pronounced for protrusion-mediated signaling than for signaling at lateral cell contacts. Together, these results reveal a role for actomyosin contractility in Notch activation, signaling, and patterning in a developmental context.

developmental biology↗

bioRxiv2018

Next generation CRISPR/Cas9 transcriptional activation in Drosophila using flySAM

CRISPR/Cas9-based transcriptional activation (CRISPRa) has recently emerged as a powerful and scalable technique for systematic over-expression genetic analysis in Drosophila melanogaster. We present flySAM, a potent new tool for in vivo CRISPRa, which offers a major improvement over existing strategies in terms of effectiveness, scalability, and ease-of-use. flySAM outperforms existing in vivo CRISPRa strategies, and approximates phenotypes obtained using traditional Gal4-UAS over-expression. Further, because flySAM typically only requires a single sgRNA, it dramatically improves scalability. We use flySAM to demonstrate multiplexed CRISPRa, which has not been previously shown in vivo. In addition, we have simplified the experimental usage of flySAM by creating a single vector encoding both the UAS:Cas9-activator and the sgRNA, allowing for inducible CRISPRa in a single genetic cross. flySAM will thus replace previous CRISPRa strategies as the basis of our growing genome-wide transgenic over-expression resource, TRiP-OE.

genetics↗

bioRxiv2017

Directed evolution of TurboID for efficient proximity labeling in living cells and organisms

Protein interaction networks and protein compartmentation underlie every signaling process and regulatory mechanism in cells. Recently, proximity labeling (PL) has emerged as a new approach to study the spatial and interaction characteristics of proteins in living cells. However, the two enzymes commonly used for PL come with tradeoffs - BioID is slow, requiring tagging times of 18-24 hours, while APEX peroxidase uses substrates that have limited cell permeability and high toxicity. To address these problems, we used yeast display-based directed evolution to engineer two mutants of biotin ligase, TurboID and miniTurbo, with much greater catalytic efficiency than BioID, and the ability to carry out PL in cells in much shorter time windows (as little as 10 minutes) with non-toxic and easily deliverable biotin. In addition to shortening PL time by 100-fold and increasing PL yield in cell culture, TurboID enabled biotin-based PL in new settings, including yeast, Drosophila, and C. elegans.

bioengineering↗

bioRxiv2017

Zinc detoxification: a functional genomics and transcriptomics analysis in Drosophila melanogaster cultured cells

Cells require some metals, such as zinc and manganese, but excess levels of these metals can be toxic. As a result, cells have evolved complex mechanisms for maintaining metal homeostasis and surviving metal intoxication. Here, we present the results of a large-scale functional genomic screen in Drosophila cultured cells for modifiers of zinc chloride toxicity, together with transcriptomics data for wildtype or genetically zinc-sensitized cells challenged with mild zinc chloride supplementation. Altogether, we identified 47 genes for which knockdown conferred sensitivity or resistance to toxic zinc or manganese chloride treatment, and more than 1800 putative zinc-responsive genes. Analysis of the omics data points to the relevance of ion transporters, glutathione-related factors, and conserved disease-associated genes in zinc detoxification. Specific genes identified in the zinc screen include orthologs of human disease-associated genes CTNS, PTPRN (also known as IA-2), and ATP13A2 (also known as PARK9). We show that knockdown of red dog mine (rdog; CG11897), a candidate zinc detoxification gene encoding an ABCC-type transporter family protein related to yeast cadmium factor (YCF1), confers sensitivity to zinc intoxication in cultured cells and that rdog is transcriptionally up-regulated in response to zinc stress. As there are many links between the biology of zinc and other metals and human health, the omics datasets presented here provide a resource that will allow researchers to explore metal biology in the context of diverse health-relevant processes.

genetics↗

bioRxiv2017

Variable dose analysis: A novel RNAi-based method for detection of synthetic lethal interactions

Synthetic sick or synthetic lethal (SS/L) screens are a powerful way to identify candidate drug targets to specifically kill tumor cells but such screens generally suffer from low reproducibility. We found that many SS/L interactions involve essential genes and are therefore detectable within a limited range of knockdown efficiency. Such interactions are often missed by overly efficient RNAi reagents. We therefore developed an assay that measures viability over a range of knockdown efficiency within a cell population. This method, called variable dose analysis (VDA), is highly sensitive to viability phenotypes and reproducibly detects SS/L interactions. We applied the VDA method to search for SS/L interactions with TSC1 and TSC2, the two tumor suppressors underlying tuberous sclerosis complex (TSC) and generated a SS/L network for TSC. Using this network, we identified four FDA-approved drugs that selectively affect viability of TSC deficient cells, representing promising candidates for repurposing to treat TSC-related tumors.

cancer biology↗

bioRxiv2017

Gene2Function: An Integrated Online Resource For Gene Function Discovery

One of the most powerful ways to develop hypotheses regarding biological functions of conserved genes in a given species, such as in humans, is to first look at what is known about function in another species. Model organism databases (MODs) and other resources are rich with functional information but difficult to mine. Gene2Function (G2F) addresses a broad need by integrating information about conserved genes in a single online resource.

bioinformatics↗

bioRxiv2017

TOWARDS A GLOBAL SUPPORT OF CORE DATA RESOURCES FOR THE LIFE SCIENCES

On November 18-19, 2016, the Human Frontier Science Program Organization (HFSPO) hosted a meeting of senior managers of key data resources and leaders of several major funding organizations to discuss the challenges associated with sustaining biological and biomedical (i.e., life sciences) data resources and associated infrastructure. A strong consensus emerged from the group that core data resources for the life sciences should be supported through a coordinated international effort(s) that better ensure long-term sustainability and that appropriately align funding with scientific impact. Ideally, funding for such data resources should allow for access at no charge, as is presently the usual (and preferred) mechanism. Below, the rationale for this vision is described, and some important considerations for developing a new international funding model to support core data resources for the life sciences are presented.

scientific communication and education↗