Biology subjects

Golding, M.

Publications and source records attributed to Golding, M..

4 recordsLinked to original sources

bioRxiv2026

Orphan GPR84 facilitates uropod de-adhesion to terminate leukocyte diapedesis during inflammation

Leukocyte migration through venular walls is an essential component of effective immunity. While the key molecular players driving the initial steps of this response are known, the terminating signals remain unclear. Here, we have identified a conserved role for GPR84 family GPCRs in successful completion of the final stages of leukocyte extravasation through acutely inflamed vessels. The integration of high resolution intravital imaging with cell-specific genetics revealed that genetic deficiency of GPR84 orthologues in mice and Drosophila, results in defective detachment of transmigrating immune cells from vessel walls. Mechanistically, transcriptomics revealed GPR84-deficient neutrophils exhibit defective actin cytoskeletal regulation and cellular adhesion/de-adhesion. Consistent with this, our fly-murine pipeline shows that GPR84 supports localized and dynamic Rho activation to enable detachment of the immune cell uropod from vessel exit sites. Moreover, pharmacological blockade of GPR84 signaling dampened immune cell migration in multiple murine acute inflammatory settings. Collectively, our findings present GPR84 as a novel physiological regulator of immune cell extravasation that is amenable to therapeutic targeting for modulating leukocyte infiltration into inflamed tissues. SummaryHere we identify the GPR84 family of GPCRs as key regulators of effective immune cell extravasation in vivo. Mechanistically, through integrating genetically tractable Drosophila and murine in vivo models, we show how leukocyte GPR84 supports dynamic Rho-dependent detachment of stretched uropods as these cells exit vessels.

immunology↗

bioRxiv2025

Force sensing by Piezo1 regulates endothelial secretory granule exocytosis

Endothelial cells (ECs) rapidly alter their phenotype to support haemostasis and inflammation through the exocytosis of specialised storage organelles, known as Weibel-Palade bodies (WPBs). This exocytic response is stimulated by a variety of physiological and pathological triggers which act through well-described transmembrane receptors. However, the influence of mechanical forces and how they shape this fundamental endothelial response remains relatively unexplored. Here we demonstrate that opening of the mechanosensitive cation channel Piezo1 on the EC surface triggers rapid WPB exocytosis. The dynamics and mechanisms were investigated using the Piezo1 agonist (Yoda1) and by modelling mechanical opening through EC-ICAM-1 ligation by THP-1 cells under low shear flow. In vitro, localised von Willebrand Factor (VWF) secretion occurred at THP-1 adhesion sites, suggesting a reciprocity of VWF release alongside leukocyte capture. In vivo, Yoda1 stimulation of tissues resulted in a dramatic increase in neutrophil and platelet adhesion to postcapillary venular walls, spatially aligned with cargo release (VWF). The rapid dynamics of this response was revealed by the application of high resolution confocal intravital microscopy to transgenic EGFP-Rab27a mice that exhibit fluorescent WPBs and neutrophils. These data reveal a previously unrecognised instigator of WPB exocytosis and uncover a mechanosensitive molecular pathway coupling inflammation with haemostasis. GRAPHICAL ABSTRACT O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=166 SRC="FIGDIR/small/678766v1_ufig1.gif" ALT="Figure 1"> View larger version (60K): org.highwire.dtl.DTLVardef@d8ed6org.highwire.dtl.DTLVardef@1cbc0corg.highwire.dtl.DTLVardef@7dd6a9org.highwire.dtl.DTLVardef@197f286_HPS_FORMAT_FIGEXP M_FIG Graphical abstract: Activation of Piezo1 by pharmacological agonism or leukocyte adherence to ICAM-1 under low flow promotes VWF secretion and platelet capture through a protein kinase C-dependent mechanism. C_FIG SUMMARYThis research describes how the mechanosensitive cation channel Piezo1 elicits the rapid exocytosis of endothelial secretory organelles, provoking platelet capture to sites of leukocyte adherence. The EGFP-Rab27a transgenic mouse demonstrates a technical advance, facilitating intravital imaging of endothelial secretory organelles.

cell biology↗

bioRxiv2025

Leveraging crystallographic fragment screening, algorithmic merging and digital chemistry to target NCS-1 protein-protein interactions for treatment of neurological disorders

Efficient drug discovery relies on workflows that integrate structural insights with rapid and cost-effective exploration of chemical space. Here, we present a data-driven fragment-based lead discovery approach to target Neuronal Calcium Sensor 1 (NCS-1) protein-protein interactions (PPIs). This study represents a complete implementation of a single high-value design-make-test-analyze cycle that directly yields compounds with micromolar affinity with the potential to modulate NCS-1 interactions with key targets, including the G-protein chaperone Ric-8A and the dopamine D2 and cannabinoid CB1 receptors. X-ray crystallographic fragment screening (CFS) revealed diverse interaction patterns within the NCS-1 hydrophobic crevice. Algorithmically guided fragment evolution and automated synthesis enabled the rapid generation of over 250 derivatives, with biophysical validation using LC-MS and Grating-coupled interferometry. Structural analyses highlighted key pharmacophores, with selected compounds exhibiting favorable drug-like properties and potential blood-brain barrier penetration, making them promising candidates for neurodegenerative and neurodevelopmental disorders. Our results demonstrate the feasibility of accelerated hit-to-lead development at synchrotrons, demonstrating a robust, scalable platform for PPI-targeting drug discovery. The generated chemically diverse scaffolds provide a strong foundation for future therapeutic optimization.

biophysics↗

bioRxiv2024

Identifying novel chemical matter against the Chikungunya virus nsP3 macrodomain through crystallographic fragment screening

Chikungunya virus (CHIKV) causes severe fever, rash and debilitating joint pain that can last for months 1,2or even years. Millions of people have been infected with CHIKV, mostly in low and middle-income countries, and the virus continues to spread into new areas due to the geographical expansion of its mosquito hosts. Its genome encodes a macrodomain, which functions as an ADP-ribosyl hydrolase, removing ADPr from viral and host-cell proteins interfering with the innate immune response. Mutational studies have shown that the CHIKV nsP3 macrodomain is necessary for viral replication, making it a potential target for the development of antiviral therapeutics. We, therefore, performed a high-throughput crystallographic fragment screen against the CHIKV nsP3 macrodomain, yielding 109 fragment hits covering the ADPr-binding site and two adjacent subsites that are absent in the homologous macrodomain of SARS-CoV-2 but may be present in other alphaviruses, such as Venezuelan equine encephalitis virus (VEEV) and eastern equine encephalitis virus (EEEV). Finally, a subset of overlapping fragments was used to manually design three fragment merges covering the adenine and oxyanion subsites. The rich dataset of chemical matter and structural information discovered from this fragment screen is publicly available and can be used as a starting point for developing a CHIKV nsP3 macrodomain inhibitor.

biochemistry↗