Biology subjects

Adamson, A. D.

Publications and source records attributed to Adamson, A. D..

15 recordsLinked to original sources

bioRxiv2026

In vivo cellular localisation and nanoscale organisation of NLRP3 inflammasomes

The NLRP3 inflammasome is a critical regulator of inflammation, yet the localisation, organisation, and cellular sources of endogenous NLRP3 inflammasomes remain incompletely understood. Here, we generated NLRP3-mScarlet-I endogenous reporter mice enabling visualisation of NLRP3 at physiological levels in primary cells and in vivo. We show that activated NLRP3 associated with PI4P-positive membranes from multiple organelles, supporting a model where diverse membrane platforms act as a scaffold to nucleate inflammasome assembly. Super-resolution imaging revealed that NLRP3 and ASC occupy distinct nanoscale architectures within the inflammasome, with NLRP3 displaying marked structural heterogeneity and stimulus-dependent organisation. Unexpectedly, circulating monocytes and neutrophils, rather than tissue-resident populations, emerged as the dominant NLRP3-expressing cells in vivo which rapidly infiltrated tissues following systemic inflammation, highlighting an underappreciated cellular source of rapid inflammasome-driven responses. These findings reveal previously unrecognised insights into inflammasome organisation and localisation, establishing a powerful resource for investigating endogenous NLRP3 biology in health and disease.

immunology↗

bioRxiv2025

Oscillatory Co-expression of HES1 and HES5 Enables a Hybrid State in a Bistable Transcription Factor Regulatory Motif

Many cell fate decisions in the developing neural tube are directed by cross-repressive transcription factor (TF) motifs that generate bistability, enforcing expression of one dominant TF. However, evidence of hybrid states, where cells co-express opposing fate determinants, challenges this model. We hypothesised that oscillatory expression enables co-existence of cross-repressive TFs within single cells, allowing hybrid states in bistable motifs. To test this, we focused on HES1 and HES5, oscillatory, cross-repressive TFs that regulate neural progenitor maintenance and are expressed in adjacent dorsoventral domains in the developing spinal cord. Using live-cell imaging of fluorescent reporters and computational modelling, we show that HES1 and HES5 co-express and oscillate in-phase within single cells. Differences in protein stability result in distinct free-running periodicity, but co-expression results in entrainment and phase-locking. Modulating cross-repression strength and/or abundance shifts the system towards bistability and dominance of a single TF oscillator. Consistent with this, we observe progressive separation of the HES expression domains in vivo, through a decrease in oscillatory co-expression. Our findings provide a mechanism for hybrid states to emerge in a developmental bistable motif.

developmental biology↗

bioRxiv2025

Increased in vivo transduction of AAV-9 cargo in Alport podocytes

Alport syndrome is a rare genetic disorder characterized by progressive kidney disease, hearing loss, and eye abnormalities. Gene therapy for Alport syndrome has not yet been realized due to technical challenges, including effective transduction of target cells. In this study, we established a quantitative testing platform using podocytes in culture, ex vivo glomeruli, and a mouse model of Alport syndrome to evaluate the transduction efficacy of adeno-associated Virus-9 (AAV9) as a gene delivery vehicle. We compared transduction levels of AAV9-GFP vectors between healthy and Alport podocytes in culture, revealing that both cell types exhibited similar transduction rates. We then incubated ex vivo glomeruli with AAV9-GFP and found enhanced transduction in Alport compared to wild type podocytes. Finally in mice following a peripheral intravenous injection of AAV9-GFP we found a striking increase in transduction in Alport podocytes suggesting that the pathological environment may facilitate higher penetration of the vector. These findings underscore the potential of AAV9 for effective gene delivery in the context of Alport syndrome, providing a foundation for future therapeutic strategies aimed at correcting the underlying genetic defects. Plain Language SummaryThis study highlights the clinical significance of AAV9 as a potential gene delivery vehicle for Alport syndrome treatment. Our findings demonstrate that Alport podocytes exhibit enhanced transduction levels in ex vivo and in vivo environments compared to healthy podocytes. This suggests that AAV9 could effectively target podocytes in Alport syndrome, paving the way for innovative gene therapy strategies that may improve patient outcomes and quality of life.

cell biology↗

bioRxiv2025

Generation of a Ym1 Deficient Mouse utilising CRISPR-Cas9 in CB6 Embryos

Chitinase-like proteins (CLPs) are of wide interest due to their significant roles during both biological and pathological processes. Human CLPs such as YKL-40 have been suggested as biomarkers of disease severity in many conditions. Murine CLPs include Brp39, Ym1, and Ym2 and these are similarly upregulated in multiple mouse models of pathology. Investigation of these molecules, particularly Ym1 and Ym2, is plagued by complexity in the genomic locus due to recent gene duplication events in the C57BL/6 strain. Using a novel CRISPR-Cas9 targeting approach involving CB6 mixed background embryos, we generated a Ym1 deficient mouse. Validation using flow cytometry, ELISA, and immunofluorescence confirmed no expression of mature Ym1 protein with no alteration in the expression of related chitinases/CLP genes including Chia and Chil4. This new transgenic mouse line will be key for investigating CLP functions and the genetic approach utilised may provide a useful strategy for other genes which show differences between inbred mouse strains.

genetics↗

bioRxiv2025

Colonic inflammation modulates the intestinal circadian landscape

Inflammatory bowel disease (IBD) is a chronic inflammatory condition that carries significant morbidity. The circadian (24h) clock regulates many aspects of immunity and circadian disruption is implicated in inflammatory disease. We examined the impact of acute dextran sulphate sodium-induced colitis on the intestinal circadian landscape and investigated consequences of intestinal epithelial cell (IEC) clock disruption on immune function in health and under inflammatory stress. Within the inflamed colon, we observed attenuated IEC clock function. Furthermore, de novo oscillations in numbers of colonic regulatory T cells emerged, associated with diurnal variation in proliferation and activation markers. IEC-specific deletion of core clock gene Bmal1 drove alterations in the rhythmic colonic transcriptome (impacting key immune pathways) and marked damping of microbiota rhythmicity. IEC-specific Bmal1 deletion did not impact colitis severity. These results highlight the impact of colonic inflammation on circadian processes and suggest that circadian logic could be applied in IBD treatment.

immunology↗

bioRxiv2024

Mis-regulation of GSK-3β causes axonal microtubule curling through Shot and Tau

Glycogen Synthase Kinase 3{beta} (GSK-3{beta}) is a key coordinator of neuronal development and maintenance; hyperactive GSK-3{beta} is linked to neurodevelopmental and -degenerative diseases and therefore a promising therapeutic target. In neurons, GSK-3{beta} coordinates the cytoskeleton by phosphorylating microtubule-binding proteins. In this study, we found that tight regulation of GSK-3{beta} kinase activity is required for the maintenance of parallel microtubule bundles in Drosophila and rat axons. Up- or down-regulation of GSK-3{beta} led to axons forming pathological swellings in which microtubule bundles disintegrated into disorganised, curled microtubules. We identified the microtubule bundling proteins Shot and Tau as key GSK-3{beta} targets and found that GSK-3{beta} exerted its regulatory effect on microtubule bundling through them. GSK-3{beta} regulates the ability of Shot and Tau to attach to microtubules and/or the plus-end protein Eb1. Mis-regulation of GSK-3{beta} leads to the loss of Eb1-Shot-mediated guidance of polymerising microtubules into parallel bundles, thus causing disorganisation. We propose microtubule disorganisation as a new explanation for how GSK-3{beta} hyperactivity leads to neurodegeneration and why global inhibition of GSK-3{beta} has not been successful in clinical trials for neuronal disorders.

cell biology↗

bioRxiv2024

The level of HAND1 controls the specification of multipotent cardiac and extraembryonic progenitors

Diverse sets of progenitors contribute to the development of the embryonic heart, but the mechanisms of their specification have remained elusive. Here, using a human pluripotent stem cell (hPSC) model, we deciphered cardiac and non-cardiac lineage trajectories in differentiation and identified transcription factors underpinning cell specification, identity and function. We discovered a concentration-dependent, fate determining function in mesodermal progenitors for the basic helix-loop-helix transcription factor HAND1 and uncovered its gene regulatory network. At low level, HAND1 directs differentiation towards multipotent juxta-cardiac field progenitors able to make cardiomyocytes and epicardial cells, whereas at high level it promotes the development of extraembryonic mesoderm. Importantly, HAND1-low progenitors can be propagated in their multipotent state. This detailed mechanistic insight into human development will accelerate the delivery of effective disease modelling, including for congenital heart disease, and cell therapy-based regenerative medicine.

developmental biology↗

bioRxiv2024

Collagen fibril formation at the plasma membrane occurs independently from collagen secretion

Collagen fibrils are the primary supporting scaffold of vertebrate tissues but how they are assembled is unclear. Here, using CRISPR-tagging of type I collagen and SILAC labelling, we elucidate the cellular mechanism for the spatiotemporal assembly of collagen fibrils, in cultured fibroblasts. Our findings reveal multifaceted trafficking of collagen, including constitutive secretion, intracellular pooling, and plasma membrane-directed fibrillogenesis. Notably, we differentiate the processes of collagen secretion and fibril assembly and identify the crucial involvement of endocytosis in regulating fibril formation. By employing Col1a1 knockout fibroblasts we demonstrate the incorporation of exogenous collagen into nucleation sites at the plasma membrane through these recycling mechanisms. Our study sheds light on the assembly process and its regulation in health and disease. Mass spectrometry data are available via ProteomeXchange with identifier PXD036794.

cell biology↗

bioRxiv2023

CD163 and Tim-4 identify resident intestinal macrophages across sub-tissular regions that are spatially regulated by TGF-β

In bodily organs, macrophages are localised in poorly understood tissular and sub-tissular niches associated with defined macrophage ontogeny and activity. In the intestine, a paradigm is emerging that long-lived macrophages are dominantly present in the muscular layer, while highly monocyte-replenished populations are found in the lamina propria beneath the epithelial barrier. Whether longevity is restricted in such a simplified manner has not been well explored. Moreover, the impact of specific gut-associated factors on long-lived macrophage functionality and niche occupancy is unknown. We generated sc-RNA-Seq data from wild-type and Ccr2-/- mice to identify phenotypic features of long-lived macrophage populations in distinct intestinal niches and identified CD163 as a useful marker to distinguish submucosal/muscularis (S/M) from lamina propria (LP) macrophages. Challenging the emerging paradigm, long-lived macrophages, identified by Tim-4 expression, were found in the LP and S/M. Long-lived LP macrophages are restrained in their response to proinflammatory stimulation compared to short-lived populations in the same location, and to the long-lived population within the S/M. Employing a novel Timd4creTgfbr2fl/fl mouse line we demonstrate distinct functions of TGF-{beta} on long-lived macrophages in these two compartments. Importantly, in Timd4creTgfbr2fl/fl mice, zonation of CD163+ macrophages in the S/M was lost, suggesting TGF-{beta} plays an unappreciated role in positioning of macrophages in the tissue. These data highlight the importance of considering ontogeny and niche when assessing the action of key intestinal regulatory signals.

immunology↗

bioRxiv2023

Allergen-induced airway matrix remodelling in mice can be prevented or reversed by targeting chitinase-like proteins.

Chitinase-like proteins (CLPs) are biomarkers of inflammation and airway remodelling in asthma, yet their direct contribution towards disease pathogenesis is unknown. Using a mouse model of allergen-induced type 2/type 17 airway inflammation we sought to directly investigate the role of the murine CLPs Ym1 and Ym2 during chronic lung pathology. Data demonstrated distinct chronic inflammatory roles for Ym2, IL-13, and IL-17a signalling pathways. Notably, only CLPs were key for initiating the pathogenic accumulation and re-organisation of the pulmonary extracellular matrix (ECM) environment. Furthermore, inhibition of CLPs after chronic pathology developed, reversed airway remodelling independently of chronic inflammation. These studies disentangle chronic IL-13 and IL-17a signalling from the development of allergic airway remodelling and instead highlight a central role for CLPs, which provides new avenues to therapeutically target aberrant ECM accumulation.

immunology↗

bioRxiv2023

Overexpression of IkappaBalpha modulates NF-kappaB activation of inflammatory target gene expression

Cells respond to inflammatory stimuli such as cytokines by activation of the nuclear factor-{kappa}B (NF-{kappa}B) signalling pathway, resulting in oscillatory translocation of the transcription factor p65 between nucleus and cytoplasm to mediate immune response. We investigate the relationship between p65 and inhibitor-{kappa}B (I{kappa}B) protein levels and dynamic properties of the system, and how this interaction impacts on the expression of key inflammatory genes. Using bacterial artificial chromosomes, we developed new cell models of I{kappa}B-eGFP protein overexpression in a native genomic context. We find that cells with high levels of the negative regulator I{kappa}B remain responsive to inflammatory stimuli and maintain dynamics for both p65 and I{kappa}B. In contrast, canonical target gene expression is dramatically reduced by overexpression of I{kappa}B, but can be partially rescued by overexpression of p65. Treatment with leptomycin B to promote nuclear accumulation of I{kappa}B also suppresses canonical target gene expression, suggesting a mechanism in which nuclear I{kappa}B accumulation prevents productive p65 interaction with promoter binding sites. This causes reduced target promoter binding and gene transcription, which we validate by chromatin immune precipitation and in primary cells. Overall, we show how inflammatory gene transcription is modulated by the expression levels of both I{kappa}B and p65, and that transcription can be partially decoupled from p65 protein dynamics. This results in an anti-inflammatory effect on transcription, demonstrating a broad mechanism to modulate the strength of inflammatory response.

cell biology↗

bioRxiv2021

DExCon, DExogron, LUXon: on-demand expression control of endogenous genes reveals differential dynamics of Rab11 family members

CRISPR technology has made generation of gene knockouts widely achievable in cells. However, once inactivated, their reactivation remains difficult, especially in diploid cells. Here, we present DExCon (Doxycycline-mediated endogenous gene Expression Control), DExogron (DExCon combined with auxin-mediated targeted protein degradation) and LUXon (light responsive DExCon), approaches which combine one-step CRISPR-Cas9 mediated targeted knock-in of fluorescent proteins with an advanced Tet-inducible TRE3GS promoter. These approaches combine blockade of active gene transcription with the ability to reactivate transcription on demand, including activation of silenced genes. Systematic control can be exerted using doxycycline or spatiotemporally by light, and we demonstrate functional knockout/rescue in the closely related Rab11 family of vesicle trafficking regulators. Fluorescent protein knock-in results in bright signals compatible with low-light live microscopy from monoallelic modification, the potential to simultaneously image different alleles of the same gene and bypasses the need to work with clones. Protein levels are easily tunable to correspond with endogenous expression through cell sorting (DExCon), timing of light illumination (LUXon) or by exposing cells to different levels of auxin (DExogron). Furthermore, our approach allowed us to quantify previously unforeseen differences in vesicle dynamics, expression kinetics and protein stability among highly similar endogenous Rab11 family members and their colocalization in triple knock-in cells. GRAPHICAL ABSTRACT O_FIG_DISPLAY_L [Figure 1] M_FIG_DISPLAY C_FIG_DISPLAY IN BRIEFWe describe development of DExCon, LUXon and DExogron approaches, where a single CRIPR/Cas9-mediated gene editing event can block endogenous gene expression, with the ability to reactivate expression encoded such that even silent genes can be expressed. Expression can be controlled systematically using doxycycline, or spatiotemporally by light, allowing fluorescent tagging of endogenous proteins and quantification of expression kinetics, protein dynamics and stability for highly similar genes such as members of the Rab11 family.

molecular biology↗

bioRxiv2021

NLRP3 activation in response to disrupted endocytic traffic

Inflammation driven by the NLRP3 inflammasome is coordinated through multiple signaling pathways and with a poorly defined regulation by sub-cellular organelles. Here, we tested the hypothesis that NLRP3 senses disrupted endosome trafficking to trigger inflammasome formation and inflammatory cytokine secretion. NLRP3-activating stimuli disrupted endosome trafficking and triggered localization of NLRP3 to vesicles positive for endosome markers and the inositol lipid PtdIns4P. Chemical disruption of endosome trafficking sensitized macrophages to the NLRP3 activator imiquimod driving enhanced inflammasome activation and cytokine secretion. Together these data suggest that NLRP3 is capable of sensing disruptions in the trafficking of endosomal cargoes, and that this may explain in part the spatial activation of the NLRP3 inflammasome complex. These data highlight new mechanisms amenable for the therapeutic targeting of NLRP3. One-Sentence SummaryNLRP3 senses disruptions in endosome trafficking to trigger the formation of an inflammasome complex and initiate an inflammatory response.

immunology↗

bioRxiv2021

HES1 protein oscillations are necessary for neural stem cells to exit from quiescence

Quiescence is a dynamic process of reversible cell-cycle arrest. High-level sustained expression of the HES1 transcriptional repressor, which oscillates with an ultradian periodicity in proliferative neural stem cells (NSCs), is thought to mediate quiescence. However, it is not known whether this is due to a change in levels or in dynamics. Here, we induce quiescence in NSCs with BMP4, which does not increase HES1 level, and we find that HES1 continues to oscillate. To assess the role of HES1 dynamics, we express sustained HES1 under a moderate-strength promoter, which overrides the endogenous oscillations while maintaining the total HES1 level within physiological range. We find that sustained HES1 does not affect proliferation or entry into quiescence, however, exit from quiescence is impeded. Thus, oscillatory expression of HES1 is specifically required for NSCs to exit quiescence, a finding of potential importance for controlling reactivation of stem cells in tissue regeneration and cancer.

cell biology↗

bioRxiv2020

LRRC8A regulates hypotonicity-induced NLRP3 inflammasome activation

The NLRP3 inflammasome is a multi-molecular protein complex that converts inactive cytokine precursors into active forms of IL-1{beta} and IL-18. The NLRP3 inflammasome is frequently associated with the damaging inflammation of non-communicable disease states and is considered a therapeutic target. However, there is much regarding the mechanism of NLRP3 activation that remains unknown. Chloride efflux is suggested as an important step in NLRP3 activation, but the identity of which chloride channels are involved is still unknown. We used chemical, biochemical, and genetic approaches to establish the importance of Cl- channels in the regulation of NLRP3 activation. Specifically we identify LRRC8A, an essential component of volume-regulated anion channels (VRAC), as a vital regulator of hypotonicity-induced, but not DAMP-induced, NLRP3 inflammasome activation. Although LRRC8A was dispensable for canonical DAMP-dependent NLRP3 activation, this was still sensitive to Cl- channel inhibitors, suggesting there are additional and specific Cl- sensing and regulating mechanisms controlling NLRP3.

immunology↗