Biology subjects

Brown, N. H.

Publications and source records attributed to Brown, N. H..

9 recordsLinked to original sources

bioRxiv2026

FlyAnatoView: Visualization of Drosophila Single-cell RNA-seq Datasets on Anatomographs

Single-cell RNA sequencing (scRNA-seq) is a powerful approach for measuring the transcriptomic landscape at single-cell resolution. However, while scRNA-seq data reveals what genes each cell expresses, they do not indicate where that cell was located within the tissue or organ, anatomically. We developed FlyAnatoView (https://www.flyrnai.org/tools/rna_seq_base/web/svg_files/), a resource that projects scRNA-seq data onto anatomical diagrams, restoring this missing spatial context.

bioinformatics↗

bioRxiv2026

Talin controls the spatial distribution of vinculin tension in focal adhesions

Cells transmit force between the extracellular matrix and the actin cytoskeleton through integrin adhesion complexes centred on talin and vinculin. Vinculin binds talin through -helical vinculin-binding sites (VBS) that are exposed when talin rod domains unfold under force. Dissecting the significance of this interaction has relied heavily on the A50I mutation in vinculin, which has been widely used as a talin-binding-null mutant. Here we show that although the A50I mutation abolishes binding to the -catenin VBS, it retains nanomolar-affinity binding to multiple talin VBS. We therefore designed an improved mutant, I12K/A50I, that eliminates this residual talin binding. Biochemical assays and single-molecule stretching experiments demonstrate that I12K/A50I VD1 fails to bind talin even when VBS are exposed by force. Using vinculin tension and conformation sensors, we show that talin binding is required for efficient recruitment of vinculin to focal adhesions and for establishing spatial gradients of vinculin tension. However, vinculin can still experience mechanical load in the absence of talin binding. These results demonstrate that A50I is not a talin-binding-null and reveal that, while talin is not required for vinculin loading, it is essential for organising the spatial distribution of mechanical load within adhesion complexes.

cell biology↗

bioRxiv2026

An Aggregation Prone Region (APR) in talin controls talin self-interactions to regulate integrin adhesion complex dynamics

The tight regulation of the integrin family of extracellular matrix (ECM) receptors is essential for the coordination of most cellular processes. Talin regulates integrin function by binding to the cytoplasmic tail of the integrin beta subunit. Two integrin-binding sites (IBS) in talin have been reported, one in the talin head domain (IBS1), and a second in the C-terminal rod region of talin (IBS2), mapped to the R11-R12 domains. Whilst the structural details of integrin binding to IBS1 are well understood, IBS2s mode of binding integrin is less clear, and the biochemical details of this site have been elusive. Here we report that talin R11 contains a cryptic high affinity talin-binding site. We show that mechanical unfolding of R11 exposes an Aggregation Prone Region (APR) in Helix50 that oligomerizes with other talin R11s. Our data support a model whereby the process of mechanically unfolding one R11, exposes and maintains the APR in a high affinity conformation that drives unfolding of other R11 domains and oligomerisation. Atomic Force Microscopy confirms that the APR region alone forms micron long amyloid-like fibres, via a beta-sheet amyloid arrangement which can be abolished using a canonical "gatekeeper" point mutation, V2078K, that prevents APR interactions. Introducing this gatekeeper mutant in talin eliminates recruitment of talin IBS2 to integrin adhesion sites. The APR region in talin overlaps with a vinculin-binding site in Helix50, and we show a novel role for vinculin as a mechano-chaperone for talin, able to resolve the talin large species by binding to the VBS in Helix50. In light of this new information, we propose that the ability of R11-R12 to target to integrin adhesion complexes and enhance integrin activation can be better explained by a model where functional aggregation of talin serves to recruit and activate more talin molecules at the adhesion site leading to enhanced integrin activation. Whilst further work is required to fully exclude integrin binding to R11 we suggest that the name IBS2 might be a misnomer and propose the name Aggregation Prone Region 1 (APR1) for this talin oligomerisation motif.

cell biology↗

bioRxiv2023

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 statementComprehensive 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.

bioinformatics↗

bioRxiv2023

Actin-Driven Nanotopography Enhances Integrin Molecular Clutch in Developing Tissue

Morphogenesis requires building stable macromolecular structures from highly dynamic proteins. Muscles are anchored by long-lasting integrin adhesions to resist contractile force. However, the mechanisms governing integrin diffusion, immobilization, and activation within developing tissues remain elusive. Here, we show that actin polymerisation-driven membrane protrusions form nanotopographies that enable strong adhesion at Drosophila muscle attachment sites (MAS). Super-resolution microscopy revealed that integrins assemble adhesive belts around Arp2/3-dependent actin protrusions, forming invadosome-like structures with membrane nanotopographies. Single protein tracking showed that, during MAS development, integrins became immobile and confined within diffusion traps formed by the membrane nanotopographies. Actin filaments also displayed restricted motion and confinement, indicating strong mechanical connection with integrins. Using isolated muscles cells, we show that substrate nanotopography, rather than rigidity, drives adhesion maturation by regulating actin protrusion, integrin diffusion and immobilization. These results thus demonstrate that actin-polymerisation driven membrane protrusions are essential for the formation of strong integrin adhesions sites in the developing embryo, and highlight the important contribution of geometry to morphogenesis.

cell biology↗

bioRxiv2023

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.

bioinformatics↗

bioRxiv2021

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 (L-R) expression. Recently, data generated from single cell RNA sequencing (scRNA-seq) have enabled L-R 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 L-R pairs for the major fly signaling pathways and developed FlyPhoneDB, a quantification algorithm that calculates interaction scores to predict L-R interactions between cells. At the FlyPhoneDB user interface, results are presented in a variety of tabular and graphical formats to facilitate biological interpretation. To demonstrate that FlyPhoneDB can effectively identify active ligands and receptors to uncover cell-cell communication events, we applied FlyPhoneDB to Drosophila scRNA-seq 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 scRNA-seq data in Drosophila.

bioinformatics↗

bioRxiv2020

Integrin-mediated adhesion in the unicellular holozoan Capsaspora owczarzaki

In animals, cell-matrix adhesion is mainly mediated by integrins and their associated proteins. Comparative genomic analyses have shown that core components of the integrin adhesome pre-date the emergence of animals, 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. Using an adhesion assay, we show that C. owczarzaki adheres to surfaces using actin-dependent filopodia. We show that integrin {beta}2 and its associated protein vinculin localise as distinct patches in the filopodia. We also demonstrate that substrate adhesion and integrin localisation are enhanced by the ligand fibronectin. Finally, using a specific antibody for Integrin {beta}2, we inhibited cell adhesion to a fibronectin-coated surface. Our results show that adhesion to the substrate in C. owczarzaki is mediated by integrins. This suggests that integrin-mediated adhesion pre-dates the emergence of animals.

evolutionary biology↗

bioRxiv2018

Integrin signaling downregulates filopodia in muscle-tendon attachment

Cells in developing tissues need to sense their environment for their accurate targeting to specific destinations. This occurs in developing muscles, which need to attach to their respective tendon cell 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 PS2 and {beta}PS increased filopodia number and length at stages when they are normally suppressed. Conversely, inducing integrin signaling, achieved by expression of constitutively dimerised {beta}PS cytoplasmic domain (di{beta}), prematurely suppressed filopodia. We discovered that the integrin signal is transmitted through the ArfGAP and scaffolding protein Git (G-protein receptor coupled interacting protein) and its downstream kinase Pak (p21-activated kinase). Absence of these proteins causes profuse filopodia formation and prevents filopodial inhibition by di{beta}. Thus, integrin signaling switches off the exploratory behaviour of myotubes seeking tendons, enabling the actin machinery to focus on forming a strong attachment and assembling the contractile apparatus.

developmental biology↗