Biology subjects

Iliopoulou, M.

Publications and source records attributed to Iliopoulou, M..

7 recordsLinked to original sources

bioRxiv2024

Antigen mobility regulates the dynamics and precision of antigen capture in the B cell immune synapse

B cells discriminate antigens in immune synapses by capturing them from antigen-presenting cells. This discrimination relies on the application of mechanical force to B cell receptor (BCR)-antigen bonds, allowing B cells to selectively disrupt low-affinity interactions while internalizing high-affinity antigens. Using DNA-based tension sensors combined with high-resolution imaging, we demonstrate that the magnitude, location, and timing of forces within the immune synapse are influenced by the fluidity of the antigen-presenting membrane. Transitioning antigens from a high-mobility to a low-mobility substrate significantly increases the probability and speed of antigen extraction while also improving affinity discrimination. This shift in antigen mobility also reshapes the synapse architecture, altering spatial patterns of antigen uptake. Despite these adaptations, B cells maintain consistent levels of proximal and downstream signaling pathway activation regardless of antigen mobility. They also efficiently transport internalized antigens to major histocompatibility complex class II (MHCII)-positive compartments for processing. These results demonstrate that B cells mount effective responses to antigens across diverse physical environments, though the characteristics of that environment may influence the speed and accuracy of B cell adaptation during an immune response.

immunology↗

bioRxiv2022

Subcapsular sinus macrophage sensing of extracellular matrix rigidity alters membrane topography and immune complex mobility

Subcapsular sinus macrophages (SSMs) play a key role in immune defence by forming immunological barriers that control the transport of pathogens from lymph into lymph node follicles. SSMs participate in antibody responses by presenting antigens directly to naive B cells and by supplying antigens to follicular dendritic cells to propagate germinal centre reactions. Despite the prominent roles that SSMs play during immune responses, little is known about their cell biology because they are technically challenging to isolate and study in vitro. Here, we used multi-colour fluorescence microscopy to identify lymph nodederived SSMs in culture. We focused on the role of SSMs as antigen-presenting cells and found that their actin cytoskeleton regulates the spatial organisation and mobility of immune complexes displayed on the cell surface. Moreover, we determined that SSMs are mechanosensitive cells that respond to changes in extracellular matrix (ECM) rigidity by altering the architecture of the actin cytoskeleton, leading to changes in cell morphology, membrane topography, and immune complex mobility. Our results reveal a new mechanism regulating physical aspects of antigen presentation by antigen-presenting cells, which may have implications for B cell activation and antibody responses.

immunology↗

bioRxiv2022

Live Cell Multicolour Lifetime Imaging Using Genetically Encodable Fluorophores

Nine fluorescent proteins (FPs) simultaneous imaging has been demonstrated in a single acquisition using fluorescence lifetime imaging microscopy (FLIM) combined with pulsed interleaved excitation (PIE) for three laser lines. We also show how to unmix spectrally similar FPs in a pixel-by-pixel manner with an analytical non-fitting solution.

biophysics↗

bioRxiv2021

Immune-evasion of KRAS-mutant lung adenocarcinoma mediated by cAMP response element-binding protein

cAMP response element-binding protein (CREB) mediates proliferative and inflammatory gene transcription in neurodegeneration and cancer, but its role in malignant immune-evasion of lung adenocarcinoma (LUAD) is unknown. We show that human LUAD of smokers are frequently altered along the CREB pathway and we employ mouse models to discover that KRAS-mutant LUAD co- opt CREB to evade immune rejection by tumoricidal neutrophils. For this, KRAS- driven CREB activation suppresses CXC-chemokine expression and prevents recruitment of CXCR1+ neutrophils. CREB1 is shown to be pro-tumorigenic in five different LUAD models, a function that is dependent on host CXCR1. Pharmacologic CREB blockade prevents tumor growth and restores neutrophil recruitment only when initiated before immune-evasion of KRAS-mutant LUAD. CREB and CXCR1 expression in human LUAD are compartmentalized to tumor and stromal cells, respectively, while CREB-regulated genes and neutrophils impact survival. In summary, CREB-mediated immune evasion of KRAS-mutant LUAD relies on signaling to neutrophil CXCR1 and is actionable.

cancer biology↗

bioRxiv2020

KRAS signalling in malignant pleural mesothelioma

Malignant pleural mesothelioma (MPM) arises from mesothelial cells lining the pleural cavity of asbestos-exposed individuals and rapidly leads to the development of pleural effusion and death. MPM harbours loss-of-function mutations in genes like BAP1, NF2, CDKN2A, and TP53, but isolated deletion of these genes alone in mice does not cause MPM and mouse models of the disease are sparse. Here we show that a significant proportion of human MPM harbour point mutations and copy number alterations in the KRAS proto-oncogene. These mutations are likely pathogenic, since ectopic expression of mutant KRASG12D in the pleural mesothelium of conditional mice causes MPM. Murine MPM cell lines derived from these tumours carry the initiating KRASG12D lesions, secondary Bap1 alterations, and human MPM-like gene expression profiles. Moreover, they are transplantable and actionable by KRAS inhibition. Our results indicate that KRAS mutations likely play an important and underestimated role in MPM, which warrants further exploration.

physiology↗

bioRxiv2019

Single-cell glycolytic activity regulates membrane tension 1 and HIV-1 fusion

There has been resurgence in determining the role of host metabolism in viral infection yet deciphering how the metabolic state of single cells affects viral entry and fusion remains unknown. Here, we have developed a novel assay multiplexing genetically encoded biosensors with single virus tracking (SVT) to evaluate the influence of global metabolic processes on the success rate of virus entry in single cells. We found that cells with a lower ATP:ADP ratio prior to virus addition were less permissive to virus fusion and infection. These results indicated a relationship between host metabolic state and the likelihood for virus-cell fusion to occur. SVT revealed that HIV-1 viruses were arrested at hemifusion in glycolytically-inactive cells. Interestingly, cells acutely treated with glycolysis inhibitor 2-deoxyglucose (2-DG) become resistant to virus infection and also display less surface membrane cholesterol. Addition of cholesterol in these in glycolytically-inactive cells rescued the virus entry block at hemifusion and enabled completion of HIV-1 fusion. Further investigation with FRET-based membrane tension and membrane-order reporters revealed a link between host cell glycolytic activity and host membrane order and tension. Indeed, cells treated with 2-DG possessed lower plasma membrane lipid order and higher tension values, respectively. Our novel imaging approach that combines lifetime imaging (FLIM) and SVT revealed not only changes in plasma membrane tension at the point of viral fusion, but also that HIV is less likely to enter cells at areas of higher membrane tension. We therefore have identified a connection between host cell glycolytic activity and membrane tension that influences HIV-1 fusion in real-time at the single-virus fusion level in live cells. As glycolytic activity sets membrane tension levels by altering cellular cholesterol surface levels, our results suggest additional previously unknown benefits of cholesterol-lowering medication in HIV-1 infection.

immunology↗

bioRxiv2019

Robin Hood: non-fitting, non-smoothing image detrending for bleaching correction

Recent advances in protein labelling--gene tagging with CRIPSR-Cas9--have made it possible to label proteins of interest endogenously. This represents a major breakthrough in the field of quantitative microscopy, especially when quantifying protein-protein interactions. This is because over-expression of labelled proteins may cause a distortion in localization, function and perhaps artificially force protein-protein interactions due to crowding effects. A microscopy technique that is particularly well suited to detect protein interactions with low photon budgets is number and brightness (N&B). Detrending (removal of global trends in data) is a necessary pre-processing step to N&B calculations, but all current detrending methods perform poorly at low intensities. Here, we present the Robin Hood automatic detrending algorithm which performs well at low intensities, evaluating it with simulated and low photon budget live cell images. RH is available as an ImageJ plugin and as an R package.\n\nSTATEMENT OF SIGNIFICANCEFluorescence microscopy in general and Fluorescence fluctuation methods in particular are very much dependent on detrend algorithms and so far, the user needs to decide an arbitrary number to correct for bleaching when using the box plot or the running average approach. Here, we have developed a tool available to everybody as an ImageJ plugin that is automatic, user-free and able to correct bleached images with very low photon counts.

biophysics↗