Biology subjects

Pike, J. A.

Publications and source records attributed to Pike, J. A..

14 recordsLinked to original sources

bioRxiv2026

PBF/PTTG1IP coordinates focal adhesion formation, polarity and cell motility

Directional cell migration requires cells to sense extracellular cues and coordinate adhesion, cytoskeletal remodelling and polarity, yet the molecular regulators that integrate these events remain incompletely defined. PTTG1-binding factor (PBF/PTTG1IP) is a transmembrane glycoprotein extensively characterised in pathological overexpression and cancer models, where elevated expression promotes tumourigenic cellular phenotypes. However, its endogenous physiological function remains poorly understood. Here, separate enrichment analyses of transcriptomic and phosphoproteomic profiles from PBF-overexpressing cells collectively highlighted cell adhesion, extracellular matrix organisation, cytoskeletal regulation and cell motility as major PBF-associated programmes. Using a novel Pbf knockout mouse model, we show that primary Pbf-KO mouse embryonic fibroblasts exhibit impaired migration and invasion. PBF loss reduced fibronectin adhesion, disrupted focal adhesion number, size and distribution, attenuated FAK Tyr397 phosphorylation and delayed early adhesion formation. Live-cell imaging and Golgi orientation assays further revealed altered actin organisation and impaired front-rear polarity in Pbf-KO cells. Key phenotypes were conserved in CRISPR-Cas9 PBF-KO human thyroid cancer cells, while targeted PBF re-expression restored migration in Pbf-KO MEFs. We thus identify PBF as an endogenous regulator of cell adhesion, polarity and directional motility, providing a framework for understanding how pathological PBF overexpression may promote invasive behaviour in cancer.

cell biology↗

bioRxiv2024

Remote force modulation of the T-cell receptor reveals an NFAT-threshold for CD4+ T cell activation

Abstract/SummaryMechano-modulation of cell surface proteins to influence cell activation has been shown as a promising new advanced therapy for regenerative medicine applications. These strategies rely on the manipulation of mechanosensitive cell surface receptors to initiate intracellular signal transduction. The cell surface receptor of T lymphocytes (TCR), which recognises peptide-MHC molecules central to driving the adaptive immune response, has recently been suggested to be mechano-responsive. Despite this advance, little is known as to whether the TCR can be mechanically modulated to achieve TCR signalling and subsequent T cell activation, and whether these characteristics can be exploited for immunotherapies. Here, we describe a magnetic particle-based platform for mechanical modulation of the TCR and outline how this platform can be utilised to achieve CD4+ T cell activation. We demonstrate that mechanical manipulation of the TCR induces cell surface clustering of the TCR and downstream TCR signalling, leading to eventual TCR downregulation and T cell activation. We investigate the temporal relationship between mechanical modulation of the TCR and subsequent T cell activation, hereby identifying that accumulation of signalling events within the NFAT-pathway is required to reach the threshold required for CD4+ T cell activation, outlining an axis which controls the CD4+ T cell response to external mechanical cues. These findings identify how CD4+ T cells can modulate their function in response to such cues, whilst also outlining a remote-magnetic particle-based platform that may be used for the control of T cell responses.

immunology↗

bioRxiv2024

Expansion microscopy allows quantitative characterisation of structural organisation of platelet aggregates

1Current microscopy approaches applied to platelet aggregates in both haemostatic and thrombotic settings indicate their structure has important implications in efficient haemostasis and in clinical treatment of thrombosis. However, current fluorescence microscopy approaches are not amenable to volumetric imaging of platelet aggregate structures. This is largely due to the small size of individual platelets and the tight packing of platelets within aggregates, resulting in optical opacity. Here we demonstrate that expansion microscopy, applied to platelet aggregates, can reveal multi-scale information about the structure of platelet aggregates. We produced volumetric images at nanoscale resolution of >700 platelet aggregates under normal and perturbed conditions, stained for cytoskeletal and membrane components. We demonstrate our custom analysis workflow provides quantitative description of platelet numbers, volumes and morphology within entire platelet aggregates. Additionally, we quantitatively describe subcellular organisation of F-actin. By comparing these measurements following treatment with the actin inhibitors, cytochalasin D and latrunculin A, we can robustly detect structural disruptions in platelet aggregates. Together these data provide a workflow to qualitatively and quantitatively describe the architecture of platelet aggregates at a range of scales (whole aggregates down to sub-cellular features within individual platelets).

cell biology↗

bioRxiv2024

Nano-org, a functional resource for single-molecule localisation microscopy data

We present a publicly accessible, curated, and functional resource, termed "nano-org", containing single-molecule localisation microscopy (SMLM) data representing the nanoscale distributions of proteins in cells. Nano-org is searchable by comparing the statistical similarity of the datasets it contains. This unique functionality allows the resource to be used to understand the relationships of nanoscale architectures between proteins, cell types or conditions, enabling a new field of spatial nano-omics.

biophysics↗

bioRxiv2023

A new paradigm of intracrine free fatty acid receptor 4 signaling at lipid droplets

G protein-coupled receptors (GPCRs), once thought to be active exclusively at the plasma membrane, have been shown to signal from multiple intracellular membrane compartments, including endosomes and the Golgi. However, the potential occurrence and functional relevance of intracellular signaling for the emerging family of metabolite-sensing GPCRs is largely unknown. Here, we used live-cell imaging, bioluminescence resonance energy transfer (BRET) measurements, and functional readouts to investigate signal compartmentalization of the free fatty acid receptor 4 (FFA4), a prototypical metabolite-sensing GPCR that is activated by medium- and long-chain free fatty acids (FFAs). Unexpectedly, we show that FFA4 largely resides on intracellular membranes that are intimately associated with lipid droplets in adipocytes. Upon lipolysis induction, the released FFAs rapidly bind to and activate this intracellular pool of FFA4, leading to local Gi/o coupling and inhibition of cAMP production in the vicinity of lipid droplets. This provides a spatiotemporally confined negative feedback mechanism allowing individual lipid droplets to rapidly adjust their lipolysis rate. Our results reveal a novel intracrine signaling modality by a prototypical metabolite-sensing GPCR and identify a new lipid-droplet-associated signaling hub implicated in the rapid regulation of lipid metabolism, with important implications for adipocyte physiology and pharmacology.

pharmacology and toxicology↗

bioRxiv2022

Male sex hormone and reduced plakoglobin jointly impair atrial conduction and cardiac sodium currents

Androgenic anabolic steroids (AAS) are commonly abused by young men. Male sex associates with earlier manifestation of common and rare cardiac conditions including atrial fibrillation and arrhythmogenic right ventricular cardiomyopathy (ARVC). Clinical data suggest an atrial involvement in ARVC. The disease is caused by desmosomal gene defects such as reduced plakoglobin expression. Analysis of clinical records from 146 ARVC patients identified male preponderance and increased prevalence of atrial arrhythmias in patients with definite ARVC. Definite patients displayed ECG changes suggesting atrial remodelling. To study mechanisms of atrial remodelling due to desmosomal vulnerability and AAS, young adult male mice, heterozygously deficient for plakoglobin (Plako+/-) and wildtype (WT) littermates, were chronically exposed to 5-dihydrotestosterone (DHT) or placebo. DHT increased atrial expression of pro-hypertrophic, fibrotic and inflammatory transcripts. DHT caused atrial conduction slowing, decreased peak sodium current density, reduced action potential amplitude and lowered the peak depolarisation rate in Plako+/- but not WT atria. Super-resolution microscopy revealed a reduction in Nav1.5 clustering in Plako+/- atrial cardiomyocytes following DHT exposure. These data reveal that AAS combined with plakoglobin deficiency cause pathological atrial electrical remodelling in young male hearts. AAS abuse may increase the risk of atrial myopathy in males with desmosomal gene variants.

molecular biology↗

bioRxiv2021

A framework for evaluating the performance of SMLM cluster analysis algorithms

Single molecule localisation microscopy (SMLM) generates data in the form of Cartesian coordinates of localised fluorophores. Cluster analysis is an attractive route for extracting biologically meaningful information from such data and has been widely applied. Despite the range of developed cluster analysis algorithms, there exists no consensus framework for the evaluation of their performance. Here, we use a systematic approach based on two metrics, the Adjusted Rand Index (ARI) and Intersection over Union (IoU), to score the success of clustering algorithms in diverse simulated clustering scenarios mimicking experimental data. We demonstrate the framework using three analysis algorithms: DBSCAN, ToMATo and KDE, show how to deduce optimal analysis parameters and how they are affected by fluorophore multiple blinking. We propose that these standard conditions and metrics become the basis for future analysis algorithm development and evaluation.

biophysics↗

bioRxiv2021

Imaging Nanoscale Nuclear Structures with Expansion Microscopy

Commonly applied super-resolution light microscopies have provided insight into subcellular processes at the nanoscale. However, imaging depth, speed, throughput and cost remain significant challenges, reducing the numbers of three-dimensional, nanoscale processes that can be investigated and the number of laboratories able to undertake such analysis. Expansion microscopy solves many of these limitations but its application to imaging nuclear processes has been constrained by concerns of unequal nuclear expansion. Here we demonstrate the conditions for isotropic expansion of the nucleus. Using DNA damage response proteins, BRCA1, 53BP1 and RAD51 as exemplars we quantitatively describe the three-dimensional nanoscale organisation of over 50,000 DNA damage response structures. We demonstrate the ability to assess chromatin regulated events and show the simultaneous assessment of four elements. This study thus provides the means by which expansion microscopy can contribute to the investigation of nanoscale nuclear processes.

molecular biology↗

bioRxiv2020

Immobilised collagen prevents shedding and induces sustained GPVI clustering and signalling in platelets.

Collagen, the most thrombogenic constituent of blood vessel walls, activates platelets through glycoprotein VI (GPVI). In suspension, following platelet activation by collagen, GPVI is cleaved by A Disintegrin And Metalloproteinase (ADAM)10 and ADAM17. In this study, we use single-molecule localization microscopy and a 2-level DBSCAN-based clustering tool to show that GPVI remains clustered along immobilised collagen fibres for at least 3 hours in the absence of significant shedding. Tyrosine phosphorylation of spleen tyrosine kinase (Syk) and Linker of Activated T cells (LAT), and elevation of intracellular Ca2+, are sustained over this period. Syk, but not Src kinase-dependent signalling is required to maintain clustering of the collagen integrin α2β1, whilst neither is required for GPVI. We propose that clustering of GPVI on immobilised collagen protects GPVI from shedding in order to maintain sustained Src and Syk-kinases dependent signalling, activation of integrin α2β1 and continued adhesion.Competing Interest StatementThe authors have declared no competing interest.View Full Text

cell biology↗

bioRxiv2020

An adaptable analysis workflow for characterization of platelet spreading and morphology

The assessment of platelet spreading through light microscopy, and the subsequent quantification of parameters such as surface area and circularity, is a key assay for many platelet biologists. Here we present an analysis workflow which robustly segments individual platelets to facilitate the analysis of large numbers of cells while minimising user bias. Image segmentation is performed by interactive learning and touching platelets are separated with an efficient semi-automated protocol. We also use machine learning methods to robustly automate the classification of platelets into different subtypes. These adaptable and reproducible workflows are made freely available and are implemented using the open source software KNIME and ilastik.

bioinformatics↗

bioRxiv2019

Post-translational polymodification of the C-terminal tail of TUBB1 regulates motor protein processivity in platelet production and function

In specialised cells, the expression of specific tubulin isoforms and their subsequent post-translational modifications drive and coordinate unique morphologies and behaviours. The mechanisms by which {beta}1 tubulin, the platelet and megakaryocyte (MK) lineage restricted tubulin isoform, drives platelet production and function remains poorly understood. We investigated the roles of two key post-translational polymodifications (polyglutamylation and polyglycylation) on these processes using a cohort of thrombocytopenic patients, human induced pluripotent stem cell (iPSC) derived MKs, and healthy human donor platelets. We find distinct patterns of polymodification in MKs and platelets, mediated by the antagonistic activities of the cell specific expression of Tubulin Tyrosine Ligase Like (TTLLs) and Cytosolic Carboxypeptidae (CCP) enzymes. The resulting microtubule patterning spatially regulates motor proteins to drive proplatelet formation in megakaryocytes, and the cytoskeletal reorganisation required for thrombus formation. This work is the first to show a reversible system of polymodification by which different cell specific functions are achieved. Key PointsO_LIThe platelet specific {beta}1 tubulin (encoded by TUBB1) is polymodified (polyglutamylated and polyglycylated) in platelet producing iPSC-derived megakaryocytes (MKs), this patterning spatially regulates motor proteins, and its disruption in TUBB1 patient variants results in a loss of platelet production. C_LIO_LIA system of reversible polymodifications mediated through the graded expression of modifying enzymes (TTLLs and CCPs) throughout MK maturation is required for proplatelet formation and subsequent platelet function. C_LI

cell biology↗

bioRxiv2018

frenchFISH: Poisson models for quantifying DNA copy-number from fluorescence in situ hybridisation of tissue sections

Chromosomal aberration and DNA copy number change are robust hallmarks of cancer. Imaging of spots generated using fluorescence in situ hybridisation (FISH) of locus specific probes is routinely used to detect copy number changes in tumour nuclei. However, it often does not perform well on solid tumour tissue sections, where partially represented or overlapping nuclei are common. To overcome these challenges, we have developed a computational approach called FrenchFISH, which comprises a nuclear volume correction method coupled with two types of Poisson models: either a Poisson model for improved manual spot counting without the need for control probes; or a homogenous Poisson Point Process model for automated spot counting. We benchmarked the performance of FrenchFISH against previous approaches in a controlled simulation scenario and exemplify its use in 12 ovarian cancer FFPE-tissue sections, for which we assess copy number alterations in three loci (c-Myc, hTERC and SE7). We show that FrenchFISH outperforms standard spot counting approaches and that the automated spot counting is significantly faster than manual without loss of performance. FrenchFISH is a general approach that can be used to enhance clinical diagnosis on sections of any tissue.\n\nAuthor summaryCancer genomes can look very chaotic, because cancer cells are unable to fully repair errors in DNA replication during cell division. While a healthy genome has two copies of every chromosome, in a cancer genome some pieces can be lost completely and others can appear in 50 copies. To diagnose cancers and to decide on the right therapeutic strategy for a patient, it can be very important to know how many copies of a particular piece of DNA exist in a cell. The standard technique used in the clinic to assess DNA copy number is called FISH, short for fluorescence in situ hybridisation. This technique uses fluorescent probes that bind to a DNA piece of interest and show up as glowing spots in a microscopic image. Counting the spots in an image is a labour-and time-intensive process that is generally done by well-trained experts. Here we present a statistical approach to automatically count FISH spots, which outperforms previously proposed methods, and has the potential to substantially speed up clinical diagnostics.

bioinformatics↗

bioRxiv2018

Optimised CRISPR-Cas9 mediated single molecule imaging for accurate quantification through endogenous expression.

The use of CRISPR-Cas9 genome editing to introduce endogenously expressed tags has the potential to address a number of the classical limitations of single molecule localisation microscopy. In this work we present the first systematic comparison of inserts introduced through CRISPR- knock in, with the aim of optimising this approach for single molecule imaging. We show that more highly monomeric and codon optimised variants of mEos result in improved expression at the TubA1B locus, despite the use of identical guides, homology templates, and selection strategies. We apply this approach to target the G protein-coupled receptor (GPCR) CXCR4 and show a further insert dependent effect on expression and protein function. Finally, we show that compared to over-expressed CXCR4, endogenously labelled samples allow for accurate single molecule quantification on ligand treatment. This suggests that despite the complications evident in CRISPR mediated labelling, the development of CRISPR-PALM has substantial quantitative benefits.

cell biology↗

bioRxiv2018

Topological data analysis quantifies biological nano-structure from single molecule localization microscopy

The study of complex molecular organisation and nano-structure by localization based microscopy is limited by the available analysis tools. We present a segmentation protocol which, through the application of persistence based clustering, is capable of probing densely packed structures which vary in scale. An increase in segmentation performance over state-of-the-art methods is demonstrated. Moreover we employ persistent homology to move beyond clustering, and quantify the topological structure within data. This provides new information about the preserved shapes formed by molecular architecture. Our methods are flexible and we demonstrate this by applying them to receptor clustering in platelets, nuclear pore components and endocytic proteins. Both 2D and 3D implementations are provided within RSMLM, an R package for pointillist based analysis and batch processing of localization microscopy data.

bioinformatics↗