Biology subjects

Whelan, D. R.

Publications and source records attributed to Whelan, D. R..

5 recordsLinked to original sources

bioRxiv2023

Virally induced lipid droplets are a platform for innate immune signalling complexes

Lipid droplets (LDs) are upregulated by host cells in the face of pathogen infection, however, the reason for this phenomenon remains largely unknown. Here, we demonstrate that virally induced LDs house a distinct and dynamic proteome containing key antiviral signalling pathway members, including the essential pattern recognition receptor; RIG-I, key adaptor proteins; STAT1 and STAT2 and prominent interferon inducible proteins; viperin and MX1. Changes in the LD proteome were underpinned by specific key changes in the lipidome of virally driven LDs, particularity in the phospholipid membrane. Following virus infection, key antiviral proteins formed complex protein-protein interactions on the LD surface, positioning this organelle as a key antiviral signalling platform for the first time. It is clear that dynamic regulation of both the proteome and the lipidome of LDs occurs rapidly following viral infection towards the initiation of a successful innate immune response.

immunology↗

bioRxiv2020

Ultra-Low Colcemid Doses Induce Microtubule Dysfunction as Revealed by Super-Resolution Microscopy

Microtubule-interacting drugs, sometimes referred to as antimitotics, are used in cancer therapy to target and disrupt micro-tubules. However, their side effects require the development of safer drug regimens that still retain clinical efficacy. Currently, many questions remain regarding microtubule-interacting drugs at clinically relevant and ultra-low doses. Here, we use super-resolution microscopies (single molecule localization and optical fluctuation based) to reveal the initial microtubule dysfunctions caused by nanomolar concentrations of colcemid. Short exposure to 30 - 80 nM colcemid results in aberrant microtubule curvature while microtubule fragmentation is detected upon treatment with [≥]100 nM colcemid. Remarkably, even ultra-low doses (5 hours at <20 nM) led to subtle but significant microtubule architecture remodeling and suppression of microtubule dynamics. These challenges to microtubule function represent less severe precursor perturbations compared to the established antimitotic effects of microtubule-interacting drugs, and therefore offer potential for improved understanding and design of anti-cancer agents.

cell biology↗

bioRxiv2020

Intracellular Lipid Droplet Accumulation Occurs Early Following Viral Infection and Is Required for an Efficient Interferon Response

Lipid droplets (LDs) are increasingly recognized as critical organelles in signalling events, transient protein sequestration and inter-organelle interactions. However, the role LDs play in antiviral innate immune pathways remains unknown. Here we demonstrate that induction of LDs occurs as early as 2 hours post viral infection, is transient, and returns to basal levels by 72 hours. This phenomenon occurred following viral infections, both in vitro and in vivo. Virally driven LD induction was type-I interferon (IFN) independent, however, was dependent on EGFR engagement, offering an alternate mechanism of LD induction in comparison to our traditional understanding of their biogenesis. Additionally, LD induction corresponded with enhanced cellular type-I and -III IFN production in infected cells, with enhanced LD accumulation decreasing viral replication of both HSV-1 and Zika virus (ZIKV). Here, we demonstrate for the first time, that LDs play vital roles in facilitating the magnitude of the early antiviral immune response specifically through the enhanced modulation of IFN following viral infection, and control of viral replication. By identifying LDs as a critical signalling organelle, this data represents a paradigm shift in our understanding of the molecular mechanisms which coordinate an effective antiviral response.

microbiology↗

bioRxiv2020

Super-resolution visualization of distinct stalled and broken replication fork structures

Endogenous genotoxic stress occurs in healthy cells due to competition between DNA replication machinery, and transcription and topographic relaxation processes. This causes replication fork (RF) stalling and regression, which can further collapse to form single-ended double strand breaks (seDSBs). To avoid mutagenesis, these breaks require repair via Homologous Recombination (HR). Here we apply multicolor single molecule super resolution microscopy to visualize individual RFs under mild stress from the trapping of Topoisomerase I cleavage complexes, a damage induction which closely mimics endogenous replicative stress. We identify RAD51 and RAD52, alongside RECQ1, as the first responder proteins to stalled but unbroken forks, whereas Ku and MRE11 are initially recruited to seDSBs. Ku loads directly onto the DSB end whereas MRE11 associates with nascent DNA away from the break, and both proteins colocalize contemporaneously with a single seDSB. We are thus able to discern closely related RF stress motifs and their repair pathways in vivo, uncovering mechanistic insights into the nature of RF damage and repair.

genomics↗

bioRxiv2017

A Method for Quantifying Molecular Interactions Using Stochastic Modelling and Super-Resolution Microscopy

We introduce the Interaction Factor (IF), a measure for quantifying the interaction of molecular clusters in super-resolution microscopy images. The IF is robust in the sense that it is independent of cluster density, and it only depends on the extent of the pair-wise interaction between different types of molecular clusters in the image. The IF for a single or a collection of images is estimated by first using stochastic modelling where the locations of clusters in the images are repeatedly randomized to estimate the distribution of the overlaps between the clusters in the absence of interaction (IF=0). Second, an analytical form of the relationship between IF and the overlap (which has the random overlap as its only parameter) is used to estimate the IF for the experimentally observed overlap. The advantage of IF compared to conventional methods to quantify interaction in microscopy images is that it is insensitive to changing cluster density and is an absolute measure of interaction, making the interpretation of experiments easier. We validate the IF method by using both simulated and experimental data and provide an ImageJ plugin for determining the IF of an image.

bioinformatics↗