A new experimental evidence-weighted signaling pathway resource in FlyBase.

Research in model organisms is central to the characterization of signaling pathways in multicellular organisms. Here, we present the comprehensive and systematic curation of 17 Drosophila signaling pathways using the Gene Ontology framework to establish a dynamic resource that has been incorporated into FlyBase, providing visualization and data integration tools to aid research projects. By restricting to experimental evidence reported in the research literature and quantifying the amount of such evidence for each gene in a pathway, we captured the landscape of empirical knowledge of signaling pathways in Drosophila.

A new experimental evidence-weighted signaling pathway resource in FlyBase.

Research in model organisms is central to the characterization of signaling pathways in multicellular organisms. Here, we present the systematic curation of 17 Drosophila signaling pathways using the Gene Ontology framework to establish a comprehensive and dynamic resource that has been incorporated into FlyBase, providing visualization and data integration tools to aid research projects. By restricting to experimental evidence reported in the research literature and quantifying the amount of such evidence for each gene in a pathway, we captured the landscape of empirical knowledge of signaling pathways in Drosophila . Summary statement: Comprehensive curation of Drosophila signaling pathways and new visual displays of the pathways provides a new FlyBase resource for researchers, and new insights into signaling pathway architecture.

PANGEA: a new gene set enrichment tool for Drosophila and common research organisms.

Gene set enrichment analysis (GSEA) plays an important role in large-scale data analysis, helping scientists discover the underlying biological patterns over-represented in a gene list resulting from, for example, an 'omics' study. Gene Ontology (GO) annotation is the most frequently used classification mechanism for gene set definition. Here we present a new GSEA tool, PANGEA (PAthway, Network and Gene-set Enrichment Analysis; https://www.flyrnai.org/tools/pangea/), developed to allow a more flexible and configurable approach to data analysis using a variety of classification sets. PANGEA allows GO analysis to be performed on different sets of GO annotations, for example excluding high-throughput studies. Beyond GO, gene sets for pathway annotation and protein complex data from various resources as well as expression and disease annotation from the Alliance of Genome Resources (Alliance). In addition, visualizations of results are enhanced by providing an option to view network of gene set to gene relationships. The tool also allows comparison of multiple input gene lists and accompanying visualisation tools for quick and easy comparison. This new tool will facilitate GSEA for Drosophila and other major model organisms based on high-quality annotated information available for these species.

PANGEA: A New Gene Set Enrichment Tool for Drosophila and Common Research Organisms.

Gene set enrichment analysis (GSEA) plays an important role in large-scale data analysis, helping scientists discover the underlying biological patterns over-represented in a gene list resulting from, for example, an 'omics' study. Gene Ontology (GO) annotation is the most frequently used classification mechanism for gene set definition. Here we present a new GSEA tool, PANGEA (PAthway, Network and Gene-set Enrichment Analysis; https://www.flyrnai.org/tools/pangea/ ), developed to allow a more flexible and configurable approach to data analysis using a variety of classification sets. PANGEA allows GO analysis to be performed on different sets of GO annotations, for example excluding high-throughput studies. Beyond GO, gene sets for pathway annotation and protein complex data from various resources as well as expression and disease annotation from the Alliance of Genome Resources (Alliance). In addition, visualisations of results are enhanced by providing an option to view network of gene set to gene relationships. The tool also allows comparison of multiple input gene lists and accompanying visualisation tools for quick and easy comparison. This new tool will facilitate GSEA for Drosophila and other major model organisms based on high-quality annotated information available for these species.

FlyPhoneDB: an integrated web-based resource for cell-cell communication prediction in Drosophila.

Multicellular organisms rely on cell-cell communication to exchange information necessary for developmental processes and metabolic homeostasis. Cell-cell communication pathways can be inferred from transcriptomic datasets based on ligand-receptor expression. Recently, data generated from single-cell RNA sequencing have enabled ligand-receptor interaction predictions at an unprecedented resolution. While computational methods are available to infer cell-cell communication in vertebrates such a tool does not yet exist for Drosophila. Here, we generated a high-confidence list of ligand-receptor pairs for the major fly signaling pathways and developed FlyPhoneDB, a quantification algorithm that calculates interaction scores to predict ligand-receptor interactions between cells. At the FlyPhoneDB user interface, results are presented in a variety of tabular and graphical formats to facilitate biological interpretation. To illustrate that FlyPhoneDB can effectively identify active ligands and receptors to uncover cell-cell communication events, we applied FlyPhoneDB to Drosophila single-cell RNA sequencing data sets from adult midgut, abdomen, and blood, and demonstrate that FlyPhoneDB can readily identify previously characterized cell-cell communication pathways. Altogether, FlyPhoneDB is an easy-to-use framework that can be used to predict cell-cell communication between cell types from single-cell RNA sequencing data in Drosophila.

Talin in mechanotransduction and mechanomemory at a glance.

Talins are cytoskeletal linker proteins that consist of an N-terminal head domain, a flexible neck region and a C-terminal rod domain made of 13 helical bundles. The head domain binds integrin ß-subunit cytoplasmic tails, which triggers integrin conformational activation to increase affinity for extracellular matrix proteins. The rod domain links to actin filaments inside the cell to transmit mechanical loads and serves as a mechanosensitive signalling hub for the recruitment of many other proteins. The α-helical bundles function as force-dependent switches - proteins that interact with folded bundles are displaced when force induces unfolding, exposing previously cryptic binding sites for other ligands. This leads to the notion of a talin code. In this Cell Science at a Glance article and the accompanying poster, we propose that the multiple switches within the talin rod function to process and store time- and force-dependent mechanical and chemical information.

Integrin-Mediated Adhesion in the Unicellular Holozoan Capsaspora owczarzaki.

In animals, cell-matrix adhesions are essential for cell migration, tissue organization, and differentiation, which have central roles in embryonic development [1-6]. Integrins are the major cell surface adhesion receptors mediating cell-matrix adhesion in animals. They are heterodimeric transmembrane proteins that bind extracellular matrix (ECM) molecules on one side and connect to the actin cytoskeleton on the other [7]. Given the importance of integrin-mediated cell-matrix adhesion in development of multicellular animals, it is of interest to discover when and how this machinery arose during evolution. Comparative genomic analyses have shown that core components of the integrin adhesome pre-date the emergence of animals [8-11]; however, whether it mediates cell adhesion in non-metazoan taxa remains unknown. Here, we investigate cell-substrate adhesion in Capsaspora owczarzaki, the closest unicellular relative of animals with the most complete integrin adhesome [11, 12]. Previous work described that the life cycle of C. owczarzaki (hereafter, Capsaspora) includes three distinct life stages: adherent; cystic; and aggregative [13]. Using an adhesion assay, we show that, during the adherent life stage, C. owczarzaki adheres to surfaces using actin-dependent filopodia. We show that integrin ß2 and its associated protein vinculin localize as distinct patches in the filopodia. We also demonstrate that substrate adhesion and integrin localization are enhanced by mammalian fibronectin. Finally, using a specific antibody for integrin ß2, we inhibited cell adhesion to a fibronectin-coated surface. Our results suggest that adhesion to the substrate in C. owczarzaki is mediated by integrins. We thus propose that integrin-mediated adhesion pre-dates the emergence of animals.

Building a pipeline to solicit expert knowledge from the community to aid gene summary curation.

Brief summaries describing the function of each gene's product(s) are of great value to the research community, especially when interpreting genome-wide studies that reveal changes to hundreds of genes. However, manually writing such summaries, even for a single species, is a daunting task; for example, the Drosophila melanogaster genome contains almost 14 000 protein-coding genes. One solution is to use computational methods to generate summaries, but this often fails to capture the key functions or express them eloquently. Here, we describe how we solicited help from the research community to generate manually written summaries of D. melanogaster gene function. Based on the data within the FlyBase database, we developed a computational pipeline to identify researchers who have worked extensively on each gene. We e-mailed these researchers to ask them to draft a brief summary of the main function(s) of the gene's product, which we edited for consistency to produce a 'gene snapshot'. This approach yielded 1800 gene snapshot submissions within a 3-month period. We discuss the general utility of this strategy for other databases that capture data from the research literature. Database URL: https://flybase.org/.

Integrin intracellular machinery in action.

Integrin-mediated adhesion to the extracellular matrix involves a surprisingly large number of intracellular proteins, the integrin-associated proteins (IAPs), which are a fraction of the total integrin adhesome. In this review we discuss how genetic approaches have improved our understanding of how each IAP contributes to integrin function, especially in the context of building a functional organism during development. We then begin the process of assembling IAP roles together into an integrated mechanism.

Integrin signaling downregulates filopodia during muscle-tendon attachment.

Cells need to sense their environment to ensure accurate targeting to specific destinations. This occurs in developing muscles, which need to attach to tendon cells before muscle contractions can begin. Elongating myotube tips form filopodia, which are presumed to have sensory roles, and are later suppressed upon building the attachment site. Here, we use live imaging and quantitative image analysis of lateral transverse (LT) myotubes in Drosophila to show that filopodia suppression occurs as a result of integrin signaling. Loss of the integrin subunits αPS2 and ßPS (also known as If and Mys, respectively, in flies) increased filopodia number and length at stages when they are normally suppressed. Conversely, inducing integrin signaling, achieved by the expression of constitutively dimerised ßPS cytoplasmic domain (diß), prematurely suppressed filopodia. We discovered that the integrin signal is transmitted through the protein G protein-coupled receptor kinase interacting ArfGAP (Git) and its downstream kinase p21-activated kinase (Pak). Absence of these proteins causes profuse filopodia and prevents the filopodial inhibition mediated by diß. Thus, integrin signaling terminates the exploratory behavior of myotubes seeking tendons, enabling the actin machinery to focus on forming a strong attachment and assembling the contractile apparatus.

Novel functions for integrin-associated proteins revealed by analysis of myofibril attachment in Drosophila.

We use the myotendinous junction of Drosophila flight muscles to explore why many integrin associated proteins (IAPs) are needed and how their function is coordinated. These muscles revealed new functions for IAPs not required for viability: Focal Adhesion Kinase (FAK), RSU1, tensin and vinculin. Genetic interactions demonstrated a balance between positive and negative activities, with vinculin and tensin positively regulating adhesion, while FAK inhibits elevation of integrin activity by tensin, and RSU1 keeps PINCH activity in check. The molecular composition of myofibril termini resolves into 4 distinct layers, one of which is built by a mechanotransduction cascade: vinculin facilitates mechanical opening of filamin, which works with the Arp2/3 activator WASH to build an actin-rich layer positioned between integrins and the first sarcomere. Thus, integration of IAP activity is needed to build the complex architecture of the myotendinous junction, linking the membrane anchor to the sarcomere.

Talin - the master of integrin adhesions.

Talin has emerged as the key cytoplasmic protein that mediates integrin adhesion to the extracellular matrix. In this Review, we draw on experiments performed in mammalian cells in culture and Drosophila to present evidence that talin is the most important component of integrin adhesion complexes. We describe how the properties of this adaptor protein enable it to orchestrate integrin adhesions. Talin forms the core of integrin adhesion complexes by linking integrins directly to actin, increasing the affinity of integrin for ligands (integrin activation) and recruiting numerous proteins. It regulates the strength of integrin adhesion, senses matrix rigidity, increases focal adhesion size in response to force and serves as a platform for the building of the adhesion structure. Finally, the mechano-sensitive structure of talin provides a paradigm for how proteins transduce mechanical signals to chemical signals.

Diverse integrin adhesion stoichiometries caused by varied actomyosin activity.

Cells in an organism are subjected to numerous sources of external and internal forces, and are able to sense and respond to these forces. Integrin-mediated adhesion links the extracellular matrix outside cells to the cytoskeleton inside, and participates in sensing, transmitting and responding to forces. While integrin adhesion rapidly adapts to changes in forces in isolated migrating cells, it is not known whether similar or more complex responses occur within intact, developing tissues. Here, we studied changes in integrin adhesion composition upon different contractility conditions in Drosophila embryonic muscles. We discovered that all integrin adhesion components tested were still present at muscle attachment sites (MASs) when either cytoplasmic or muscle myosin II was genetically removed, suggesting a primary role of a developmental programme in the initial assembly of integrin adhesions. Contractility does, however, increase the levels of integrin adhesion components, suggesting a mechanism to balance the strength of muscle attachment to the force of muscle contraction. Perturbing contractility in distinct ways, by genetic removal of either cytoplasmic or muscle myosin II or eliminating muscle innervation, each caused unique alterations to the stoichiometry at MASs. This suggests that different integrin-associated proteins are added to counteract different kinds of force increase.

Drosophila vinculin is more harmful when hyperactive than absent, and can circumvent integrin to form adhesion complexes.

Vinculin is a highly conserved protein involved in cell adhesion and mechanotransduction, and both gain and loss of its activity causes defective cell behaviour. Here, we examine how altering vinculin activity perturbs integrin function within the context of Drosophila development. Whereas loss of vinculin produced relatively minor phenotypes, gain of vinculin activity, through a loss of head-tail autoinhibition, caused lethality. The minimal domain capable of inducing lethality is the talin-binding D1 domain, and this appears to require talin-binding activity, as lethality was suppressed by competition with single vinculin-binding sites from talin. Activated Drosophila vinculin triggered the formation of cytoplasmic adhesion complexes through the rod of talin, but independently of integrin. These complexes contain a subset of adhesion proteins but no longer link the membrane to actin. The negative effects of hyperactive vinculin were segregated into morphogenetic defects caused by its whole head domain and lethality caused by its D1 domain. These findings demonstrate the crucial importance of the tight control of the activity of vinculin.

Microtubule organization is determined by the shape of epithelial cells.

Interphase microtubule organization is critical for cell function and tissue architecture. In general, physical mechanisms are sufficient to drive microtubule organization in single cells, whereas cells within tissues are thought to utilize signalling mechanisms. By improving the imaging and quantitation of microtubule alignment within developing Drosophila embryos, here we demonstrate that microtubule alignment underneath the apical surface of epithelial cells follows cell shape. During development, epidermal cell elongation and microtubule alignment occur simultaneously, but by perturbing cell shape, we discover that microtubule organization responds to cell shape, rather than the converse. A simple set of microtubule behaviour rules is sufficient for a computer model to mimic the observed responses to changes in cell surface geometry. Moreover, we show that microtubules colliding with cell boundaries zip-up or depolymerize in an angle-dependent manner, as predicted by the model. Finally, we show microtubule alignment responds to cell shape in diverse epithelia.

Evidence for the mechanosensor function of filamin in tissue development.

Cells integrate mechanical properties of their surroundings to form multicellular, three-dimensional tissues of appropriate size and spatial organisation. Actin cytoskeleton-linked proteins such as talin, vinculin and filamin function as mechanosensors in cells, but it has yet to be tested whether the mechanosensitivity is important for their function in intact tissues. Here we tested, how filamin mechanosensing contributes to oogenesis in Drosophila. Mutations that require more or less force to open the mechanosensor region demonstrate that filamin mechanosensitivity is important for the maturation of actin-rich ring canals that are essential for Drosophila egg development. The open mutant was more tightly bound to the ring canal structure while the closed mutant dissociated more frequently. Thus, our results show that an appropriate level of mechanical sensitivity is required for filamins' function and dynamics during Drosophila egg growth and support the structure-based model in which the opening and closing of the mechanosensor region regulates filamin binding to cellular components.

Drosophila p120-catenin is crucial for endocytosis of the dynamic E-cadherin-Bazooka complex.

The intracellular functions of classical cadherins are mediated through the direct binding of two catenins: ß-catenin and p120-catenin (also known as CTNND1 in vertebrates, and p120ctn in Drosophila). Whereas ß-catenin is crucial for cadherin function, the role of p120-catenin is less clear and appears to vary between organisms. We show here that p120-catenin has a conserved role in regulating the endocytosis of cadherins, but that its ancestral role might have been to promote endocytosis, followed by the acquisition of a new inhibitory role in vertebrates. In Drosophila, p120-catenin facilitates endocytosis of the dynamic E-cadherin-Bazooka subcomplex, which is followed by its recycling. The absence of p120-catenin stabilises this subcomplex at the membrane, reducing the ability of cells to exchange neighbours in embryos and expanding cell-cell contacts in imaginal discs.

Alternative mechanisms for talin to mediate integrin function.

Cell-matrix adhesion is essential for building animals, promoting tissue cohesion, and enabling cells to migrate and resist mechanical force. Talin is an intracellular protein that is critical for linking integrin extracellular-matrix receptors to the actin cytoskeleton. A key question raised by structure-function studies is whether talin, which is critical for all integrin-mediated adhesion, acts in the same way in every context. We show that distinct combinations of talin domains are required for each of three different integrin functions during Drosophila development. The partial function of some mutant talins requires vinculin, indicating that recruitment of vinculin allows talin to duplicate its own activities. The different requirements are best explained by alternative mechanisms of talin function, with talin using one or both of its integrin-binding sites. We confirmed these alternatives by showing that the proximity between the second integrin-binding site and integrins differs, suggesting that talin adopts different orientations relative to integrins. Finally, we show that vinculin and actomyosin activity help change talin's orientation. These findings demonstrate that the mechanism of talin function differs in each developmental context examined. The different arrangements of the talin molecule relative to integrins suggest that talin is able to sense different force vectors, either parallel or perpendicular to the membrane. This provides a paradigm for proteins whose apparent uniform function is in fact achieved by a variety of distinct mechanisms involving different molecular architectures.

Anchors and signals: the diverse roles of integrins in development.

Integrins mediate cell adhesion by providing a link between the actin cytoskeleton and the extracellular matrix. As well as acting to anchor cells, integrin adhesions provide sensory input via mechanotransduction and synergism with signaling pathways, and provide the cell with the conditions necessary for differentiation in a permissive manner. In this review, we explore how integrins contribute to development, and what this tells us about how they work. From a signaling perspective, the influence of integrins on cell viability and fate is muted in a developmental context as compared to cell culture. Integrin phenotypes tend to arise from a failure of normally specified cells to create tissues properly, due to defective adhesion. The diversity of integrin functions in development shows how cell adhesion is continuously adjusted, both within and between animals, to fit developmental purpose.

The many faces of cell adhesion during Drosophila muscle development.

Muscle development involves a series of morphogenetic events including cell fusion, migration and epidermal attachment. At various points in this complex developmental program, regulation of muscle-muscle and muscle-epidermal adhesion is crucial. One of the best-characterised adhesion events is the formation of stable, integrin-based adhesions at the attachment sites formed between the ends of muscles and epidermal cells, but other adhesion mechanisms are involved in earlier stages. Here we review recent work from Drosophila on the role of adhesion during muscle development, situating integrin function within the wider developmental program.