Biology subjects

Gilmore, A. P.

Publications and source records attributed to Gilmore, A. P..

10 recordsLinked to original sources

bioRxiv2025

Multiplexed proximity labelling proteomics identifies a non-canonical Bcl-2 family interactome associated with apoptotic priming

Bcl-2 family proteins govern the intrinsic apoptotic pathway by regulating mitochondrial outer membrane permeabilisation (MOMP), releasing apoptogenic factors into the cytosol. How close a cell is to MOMP, termed mitochondrial priming, is determined by the interactions between different Bcl-2 proteins at the outer mitochondrial membrane (OMM). However, Bcl-2 proteins can also drive diverse processes that do not result in cell death, such as incomplete MOMP, sublethal caspase activation, pro-inflammatory signalling and regulation of cellular metabolism. To understand the wider Bcl-2 family interactome and how it might be involved in these processes, we undertook an unbiased proteomic BioID screen, using both pro- and anti-apoptotic Bcl-2 family members as bait proteins. We found that most high-confidence potential interactions in non-apoptotic cells were outside the canonical Bcl-2 family interactome. Analysis of how this interactome changed in response to either BH3-mimetics or full-length BH3-only proteins revealed dynamic changes at multiple organelles and membrane contact sites in response to altered mitochondrial priming. These findings underscore the complex and varied interactions of the Bcl-2 family proteins, expanding their scope and function beyond the frequently studies intra-family interactions.

cell biology↗

bioRxiv2025

BioBatchNet: A Dual-Encoder Framework for Robust Batch Effect Correction in Imaging Mass Cytometry

MotivationImaging Mass Cytometry (IMC) is a cutting-edge technology for analysing spatially resolved protein expression at the single-cell level. However, its downstream analyses are often hindered by batch effects, which introduce systematic biases and obscure true biological variations. Existing correction methods, largely developed for scRNA-seq data, struggle to achieve precise control, leading to either over-correction by removing critical biological information, or under-correction by leaving residual batch effects. Moreover, these methods face challenges in adapting to IMC data due to differences in data characteristics. Furthermore, IMC data often feature imbalanced and overlapping cell populations, complicating clustering and downstream analysis. These challenges underscore the need for a robust and controllable batch effect correction approach tailored to IMC data. ResultsWe present BioBatchNet, a dual-encoder framework utilising adversarial training to explicitly disentangle batch-specific and biological signals. BioBatchNet enables controllable batch effect correction, effectively balancing correction with the preservation of biological variation. We evaluated BioBatchNet on three IMC datasets, where it outperformed seven benchmarking methods with robustness in both correction and biological signal conservation. Additionally, we developed a Constrained Pairwise Clustering (CPC) method, which employs constrained pairs to improve clustering performance, even in datasets with imbalanced and overlapping cell populations. To validate its generalisability, BioBatchNet was also applied to four scRNA-seq datasets, where it delivered competitive performance compared to eight typical methods. These results demonstrate BioBatchNets generalisability and robustness in correcting batch effects across diverse single-cell datasets and underscore its potential for large-scale biological analyses.

bioinformatics↗

bioRxiv2024

A stretchable and biomimetic polyurethane membrane for lung alveolar in vitro modelling

The lung alveolus constitutes a morphologically and mechanistically complex tissue that is constantly subjected to cyclic tension and exhibits unique elastic properties. Available materials used to mimic alveolar tissue often lack biomimicry and the mechanical properties required for cyclic tension. Here, we report a fully synthetic fibrous polyurethane scaffold that approximates tissue stiffness, is elastic under breathing simulations and supports long-term culture of alveolar epithelial-like cells. Using electrospinning a fibrous membrane of tuneable thickness, set fibre diameter and small pore size is prepared. When subjected to cyclic uniaxial tension the material retains its elasticity at both low and high frequency mimicking human and mouse breathing. Thanks to the small pore size, lung alveolar cells can be cultured on its apical surface forming an epithelial monolayer. This monolayer can be maintained long term (at least 15 days) and in an air-liquid interface. In the latter conditions, cells differentiate and exhibit expression of surfactant protein A, a constituent of the surfactant layer that plays a key role in lung physiology. Owing to its lung-mimicking characteristics, the electrospun membrane holds the potential to be adapted for breathing lung models.

bioengineering↗

bioRxiv2023

Optimising a self-assembling peptide hydrogel as a Matrigel alternative for 3-dimensional mammary epithelial cell culture

Three-dimensional (3D) organoid models have been instrumental in understanding molecular mechanisms responsible for many cellular processes and diseases. However, established organic biomaterial scaffolds used for 3D hydrogel cultures, such as Matrigel, are biochemically complex and display significant batch variability, limiting reproducibility in experiments. Recently, there has been significant progress in the development of synthetic hydrogels for in vitro cell culture that are reproducible, mechanically tuneable, and biocompatible. Self-assembling peptide hydrogels (SAPHs) are synthetic biomaterials that can be engineered to be compatible with 3D cell culture. Here we investigate the ability of PeptiGel(R) SAPHs to model the mammary epithelial cell (MEC) microenvironment in vitro. The positively charged PeptiGel(R)Alpha4 supported MEC viability, but did not promote formation of polarised acini. Modifying the stiffness of PeptiGel(R) Alpha4 stimulated changes in MEC viability and changes in protein expression associated with altered MEC function, but did not fully recapitulate the morphologies of MECs grown in Matrigel. To supply the appropriate biochemical signals for MEC organoids, we supplemented PeptiGels(R) with laminin. Laminin was found to require negatively charged PeptiGel(R) Alpha7 for functionality, but was then able to provide appropriate signals for correct MEC polarisation and expression of characteristic proteins. Thus, optimisation of SAPH composition and mechanics allows tuning to support tissue-specific organoids.

cell biology↗

bioRxiv2023

HER2 overexpression initiates breast tumorigenesis non-cell-autonomously by inducing oxidative stress in the tissue microenvironment

HER2 is considered as a driver oncogene responsible for the HER2+ subtype of breast cancer. However, it is still unclear how HER2 induces the oncogenic transformation of breast cancer stem cells (BCSCs) and initiates tumorigenesis during premalignant stage breast cancer. Here, we used clinical samples and mouse models of HER2+ breast cancer to demonstrate that neither BCSCs nor their cell-of-origin express HER2/Neu in early-stage breast tumors. Instead, our results demonstrate that Neu overexpression results in the transformation of BCSCs in a non-cell-autonomous manner via triggering DNA damage and somatic mutagenesis in their Neu-negative cell-of-origin. This is caused by the increased oxidative stress in the tissue microenvironment generated by altered energy metabolism and increased reactive oxygen species levels in Neu-overexpressing mammary ducts. Therefore, our findings illustrate a previously unrecognized mechanism of HER2-induced breast tumor initiation in vivo with potential impacts on future preventive treatments for HER2+ premalignant breast cancer.

cancer biology↗

bioRxiv2022

Novel Laser Capture Microdissection-Proteomic Analysis Identifies Spatially Distinct Extracellular Matrix Signatures in the Core and Infiltrating Edge of Human Glioblastoma.

BackgroundGlioblastoma is the most common and aggressive primary brain tumour in adults. Hallmarks of glioblastoma include its intra-tumoural heterogeneity and extensive invasion of the surrounding brain. Glioblastoma is known to remodel the extracellular matrix (ECM) of the brain, resulting in altered mechanical properties and the establishment of a tumour-promoting microenvironment. How changes in the expression and spatial distribution of ECM constituents within glioblastoma contribute to invasion and disease progression is still unclear. MethodsHere we report on a protocol for laser-capture microdissection coupled with mass spectrometry (LCM-proteomics) that allowed a spatially resolved and unbiased analysis of the regional ECM proteome (matrisome) in formalin-fixed and paraffin-embedded (FFPE) samples of human glioblastoma. We investigated five molecularly characterised hemispheric adult glioblastomas where the brain/tumour interface and tumour epicentre were represented in the surgical specimens and snap-frozen tissue was available. LCM-proteomic analysis was validated with immunohistochemistry. ResultsLCM-proteomics identified 53 matrisome proteins in FFPE tissue, demonstrating comparable performance with conventional analysis of snap-frozen tissue. The analysis revealed distinct matrisome components in the brain/tumour interface versus the tumour epicentre. Guided by data from LCM-proteomic analysis, immunostaining for tenascin-R confirmed greater staining in the brain/tumour interface, whilst expression of fibronectin was higher in the tumour epicentre. ConclusionThe protocol described in this work allowed for accurate, spatially resolved analysis of ECM in FFPE tissues, with performance comparable to analysis of snap-frozen tissue. While the focus for this work was on the regional ECM composition of glioblastoma, we found that the LCM-proteomics protocol is also applicable to the study of the wider proteome, and represents a valuable tool for investigating tumour/tissue heterogeneity. This protocol opens the possibility to apply LCM-proteomics to retrospective studies with the advantage of accessing clinical history and follow-up information, providing a valuable resource for translational research in glioblastoma.

cancer biology↗

bioRxiv2020

Increased microenvironment stiffness leads to altered aldehyde metabolism and DNA damage in mammary epithelial cells through a RhoA-dependent mechanism

Microenvironmental stiffness regulates the behaviour of both normal and cancer cells. In breast tissue, high mammographic density (HMD), which reflects greater organisation and stiffness of the periductal collagen, represents a significant risk factor for cancer. However, the mechanistic link between extracellular matrix (ECM) stiffness and increased risk of breast tumour initiation remains unclear. In particular, how increased ECM stiffness might promote genomic damage, leading to the acquisition of transforming mutations, remains to be determined. Here we determine that ECM stiffness induces changes in mammary epithelial cell (MEC) metabolism that drive genomic damage. Using a 3D-culture model, we demonstrate that genome-wide transcriptional changes in response to increased ECM stiffness impair the ability of MECs to remove reactive aldehyde species, resulting in greater accumulation of DNA damage in a RhoA-dependent manner. Together, our results provide a mechanistic link between increased ECM stiffness and the genomic damage required for breast cancer initiation.

developmental biology↗

bioRxiv2019

Bax mitochondrial residency is more critical than Bax oligomerization for apoptosis

The Bax protein plays an important effector role in apoptosis by forming pores in the mitochondrial outer membrane. While doing so, Bax forms higher-order oligomers in the membrane, but it remains unclear whether this oligomer formation is essential for pore formation. Using cell-free and cellular experimental systems, we investigated two Bax C-terminus mutants, T182I and G179P. Neither mutant formed large oligomers when activated in liposomes. Nevertheless, the G179P mutant could produce membrane pores, suggesting that large oligomers are not required for permeabilization. Surprisingly, however, when G179P was transduced into Bax/Bak double knockout mouse embryonic fibroblasts, it was purely cytoplasmic and failed to mediate cell death. T182I behaved in the opposite manner. When mixed with liposomes, T182I was inefficient in both membrane insertion and permeabilization. However, transduced into cells, BaxT182I resided constitutively in mitochondria, owing to its slow retrotranslocation and mediated apoptosis as efficiently as wild-type Bax. We conclude that Baxs mitochondrial residence (regulated by targeting and retrotranslocation) is more important for apoptosis than its efficiency of membrane insertion and higher-order oligomerization.

cell biology↗

bioRxiv2019

BioID based proteomic screen identifies VDAC2 as a coordinator of Bid dependent apoptotic priming during mitosis.

Apoptotic priming controls the commitment of cells to apoptosis by determining how close they lie to mitochondrial permeabilisation. Variations in priming are important for how both healthy and cancer cells respond to chemotherapeutic agents, but how this is dynamically coordinated by Bcl-2 proteins remains unclear. The Bcl-2 family protein Bid is phosphorylated when cells enter mitosis, increasing apoptotic priming and sensitivity to anti-mitotic drugs. Here we report an unbiased proximity biotinylation (BioID) screen to identify regulators of apoptotic priming in mitosis, using Bid as bait. The screen primarily identified proteins outside of the canonical Bid interactome. Specifically, we found that voltage-dependent anion-selective channel protein 2 (VDAC2) was required for Bid phosphorylation dependent changes in apoptotic priming during mitosis. Thus, we identify an additional layer of regulation upstream of known Bid interactions that control dynamic changes in apoptotic priming. These results highlight the importance of understanding the wider Bcl-2 family interactome and its role in regulating the temporal control of apoptotic priming.

cell biology↗

bioRxiv2018

Nuclear decoupling is part of a rapid protein-level cellular response to high-intensity mechanical loading

Our current understanding of cellular mechano-signalling is based on static models, which do not replicate the dynamics of living tissues. Here, we examined the time-dependent response of primary human mesenchymal stem cells (hMSCs) to cyclic tensile strain (CTS). At low-intensity strain (1 hour, 4% CTS at 1 Hz) morphological changes mimicked responses to increased substrate stiffness. As the strain regime was intensified (frequency increased to 5 Hz), we characterised rapid establishment of a broad, structured and reversible protein-level response, even as transcription was apparently downregulated. Protein abundance was quantified coincident with changes to protein conformation and post transcriptional modification. Furthermore, we characterised changes within the linker of nucleo- and cytoskeleton (LINC) complex of proteins that bridges the nuclear envelope, and specifically to levels of SUN domain-containing protein 2 (SUN2). The result of this regulation was to decouple mechano-transmission between the cytoskeleton and the nucleus, thus conferring protection to chromatin.

biophysics↗