Biology subjects

Campbell, S. G.

Publications and source records attributed to Campbell, S. G..

5 recordsLinked to original sources

bioRxiv2025

Desmoplakin loss leads to PKC-dependent insertion of series sarcomeres and contractile dysfunction in cardiomyocytes

BackgroundMutations in DSP, which encodes the protein desmoplakin, lead to cardiomyopathy with unusually high penetrance. Clinical features include ventricular tachyarrhythmias, fibro-fatty infiltration of both ventricles, and ultimately dilated cardiomyopathy. While some data have been gathered to explain the electrophysiological and contractile consequences of desmoplakin cardiomyopathy, a comprehensive mechanism linking DSP mutations to ventricular dilation and heart failure remains elusive. MethodsWe use iPSC-derived engineered heart tissue (EHT) bearing a functional desmoplakin haploinsufficiency to model the heart failure phenotype that occurs in desmoplakin cardiomyopathy. Functional haploinsufficiency is secondary to a missense mutation, R451G, that results in proteolytic degradation of desmoplakin with no detectable protein. We complement functional data obtained in tissue-engineered constructs with cell biology assays in 2D cardiomyocytes to glean insights into the mechanism and mechanobiology of desmoplakin cardiomyopathy. ResultsEngineered heart tissues harboring a desmoplakin insufficiency recapitulate a patient phenotype notable for hypocontractility and ventricular dilation. Surprisingly, DSP-mutant tissues exhibited a shortened resting sarcomere length that was dependent on protein kinase C activity. Concurrently, mechanical load on -catenin was increased, suggesting a mechanism by which desmosomal insufficiency redistributes force to adherens junctions. Excessive loading on adherens junctions may act as a stimulus for avid insertion of series sarcomeres, shortening the length per sarcomere, and resulting in a contractile deficit. PKC inhibition rescues shortened sarcomere length in DSP- mutant tissues, suggesting that it could be a target for future molecular therapies. ConclusionsOur study uncovers a novel mechanism underlying systolic dysfunction in desmoplakin cardiomyopathy. We not only recapitulate the disease phenotype, but we identify sarcomere length regulation through altered force transmission at the intercalated disc as a previously-unrecognized mechanism.

physiology↗

bioRxiv2024

GRASPS: a simple-to-operate translatome technology reveals omics-hidden disease-associated pathways in TDP-43-related amyotrophic lateral sclerosis

Transcriptomes and translatomes measure genome-wide levels of total and ribosome-associated RNAs. A few hundred translatomes were reported over >250,000 transcriptomes highlighting the challenges of identifying translating RNAs. Here, we used a human isogenic inducible model of TDP-43-linked amyotrophic lateral sclerosis, which exhibits altered expression of thousands of transcripts, as a paradigm for the direct comparison of whole-cell, cytoplasmic and translating RNAs, showing broad uncoupling and poor correlation between disease-altered transcripts. Moreover, based on precipitation of endogenous ribosomes, we developed GRASPS (Genome-wide RNA Analysis of Stalled Protein Synthesis), a simple-to-operate translatome technology. Remarkably, GRASPS identified three times more differentially-expressed transcripts with higher fold changes and statistical significance, providing unprecedented opportunities for data modeling at stringent filtering and discovery of previously omics-missed disease-relevant pathways, which functionally map on dense gene regulatory networks of protein-protein interactions. Based on its simplicity and robustness, GRASPS is widely applicable across disciplines in the biotechnologies and biomedical sciences.

molecular biology↗

bioRxiv2022

Optimisation of immunocytochemistry methodology for the detection of endogenous eIF2B localised foci

The multisubunit eukaryotic initiation factor 2B (eIF2B), a guanine nucleotide exchange factor (GEF) for eIF2, is an essential regulator of translation initiation. Activation of the cellular integrated stress response (ISR) by factors such as endoplasmic reticulum stress leads to phosphorylation of eIF2 and inhibition of eIF2B GEF activity. Cytoplasmic bodies containing eIF2B subunits, termed eIF2B bodies, have been shown to alter in subunit composition and fluorescence recovery after photobleaching activity in response to the ISR. Analysis of the subunit composition of endogenous eIF2B bodies is dependent on accurate detection of each protein in a cellular context via immunocytochemistry (ICC). We describe bioinformatic techniques to optimize the ICC detection of eIF2B foci in U373 cells. The screening of commercially available primary antibodies against predicted epitopes enhanced measurements of the number, size and fluorescence intensity of eIF2B bodies. A consistent and reproducible ICC analysis of endogenous eIF2B bodies will aid characterisation of eIF2B bodies during the ISR or under disease conditions. SummaryeIF2B is a housekeeping protein and localised eIF2B foci, named eIF2B bodies, can be detected through immunocytochemistry. Here, we discuss the use of immunoinformatics to optimise eIF2B localisation detection.

cell biology↗

bioRxiv2022

In vitro engineering of the lung alveolus

Therapeutic lung regeneration is predicated upon successful reconstitution of lung alveoli, the functional units of gas exchange. Here, we identify requisite multimodal cues that are critical to reconstructing the alveolar epithelium and alveoli in lung scaffolds. Alveolar reconstruction in vitro is divided into several distinct phases. In the first phase, endothelial cells coordinate with fibroblasts and select exogenous factors to promote alveolar scaffold population with surfactant-secreting alveolar epithelial type 2 cells (AEC2s). After formation of organized epithelial alveolar-like structures, subsequent withdrawal of Wnt and FGF agonism synergizes with tidal-level mechanical strain to induce differentiation of AEC2s to squamous type 1 AECs (AEC1s) in cultured alveoli, in situ. These results outline a rational strategy to engineer an alveolus of AEC2s and AEC1s contained within epithelial-mesenchymal-endothelial units, and reveal the critical interplay amongst biochemical, cellular, and mechanical niche cues within the reconstituting alveolus.

bioengineering↗

bioRxiv2022

Dysregulation of stress-induced translational control by Porphyromonas gingivalis in host cells

Periodontitis, a chronic inflammatory gum disease, is caused in part by the periodontopathogen Porphyromonas gingivalis. Infection triggers activation of host inflammatory responses which induce stresses such as oxidative stress. Under such conditions, cells can activate the Integrated Stress Response (ISR), a signalling cascade which functions to determine cellular fate, by either downregulating protein synthesis and initiating a stress-response gene expression program, or if stress cannot be overcome, initiating programmed cell death. Recent studies have implicated the ISR signalling in both host antimicrobial defences and within the pathomechanism of certain microbes. In this study, we investigated how P. gingivalis infection alters translation attenuation during oxidative stress-induced activation of the ISR pathway in oral epithelial cells. P. gingivalis infection alone did not result in ISR activation. In contrast, infection coupled with stress led to differential stress granule formation and composition, along with dysregulation of the microtubule network. Infection also heightened stress-induced translational repression, a response which could not be rescued by ISRIB, a potent ISR inhibitor. Heightened translational repression during stress was observed with both P. gingivalis conditioned media and outer membrane vesicles, implicating the role of a secretory factor, probably proteases known as gingipains, in this exacerbated translational repression. The effects of gingipain inhibitors and gingipains-deficient P. gingivalis mutants further confirmed these pathogen-specific proteases as the effector. Gingipains are known to degrade the mammalian target of rapamycin (mTOR) and these studies implicate the gingipain-mTOR axis as the effector of host translational dysregulation during stress.

microbiology↗