Biology subjects

Calder, G.

Publications and source records attributed to Calder, G..

10 recordsLinked to original sources

bioRxiv2026

Dynamic diffusion analysis of the yeast plasma membrane using Airyscan based microscopic techniques

The yeast plasma membrane (PM) is highly compartmentalised into distinct nanoscale domains. The mechanisms by which this organisation regulates surface proteins are not fully understood, and it remains unclear how different biophysical modalities capture diffusion kinetics across varying spatial scales. Using confocal microscopy and an Airyscan2 detector, we benchmarked two prominent techniques: Fluorescence Correlation Spectroscopy (FCS) via the Zeiss Dynamics Profiler and Fluorescence Recovery After Photobleaching (FRAP). We quantified the lateral diffusion of three functionally diverse GFP-tagged model proteins: the exocytic t-SNARE Sso2, the lipid-binding protein Pmp3, and the eisosome-associated protein Ycp4. While diffusion coefficients aligned tightly between both modalities for Pmp3 and Ycp4, Sso2 exhibited a stark 14-fold discrepancy, displaying drastically faster local mobility by FCS compared to macroscopic recovery by FRAP. High-resolution 3D Structured Illumination Microscopy (3D-SIM) shows that Sso2 is partitioned into regional subdomains, that occupy less PM area than the network-like localisation of Pmp3. Our findings suggest that FCS captures rapid, localised diffusion within these microenvironments, whereas FRAP measures highly restricted transit across domain boundaries. Ultimately, this work demonstrates that membrane diffusion coefficients cannot be interpreted in isolation and capturing true lateral mobility requires pairing kinetic measurements with super-resolution spatial mapping to decode complex membrane compartmentalisation.

cell biology↗

bioRxiv2025

Bi-directional highways and super-seeder tissues underpin parasite dissemination in experimental visceral leishmaniasis.

Visceral leishmaniasis (VL) is a life-threatening parasitic disease caused by Leishmania donovani and L. infantum. Although VL is characterised by organ-specific immunopathology and parasitism of the spleen, liver and bone marrow, other tissues may harbour parasites without overt pathology. The mechanisms governing patterns of within-host dissemination and tissue tropism are largely unknown. We used a barcoded library of isogenic L. donovani and an ecological analysis framework to evaluate parasite population diversity across tissues and to map parasite dissemination in a murine infection model. We reveal an unexpected high degree of inter-connectivity between parasite populations demonstrating: i) continual bi-directional dissemination between viscera and skin; ii) the existence of "super-seeder" sites that act as hubs fuelling systemic spread; and iii) rerouting of dissemination "highways" following immune perturbation. These findings change our understanding of L. donovani pathogenesis, providing critical insights into infection dynamics with direct implications for relapse, treatment failure and transmission.

microbiology↗

bioRxiv2025

Non-canonical inflammasome drives intrinsic anti-microbial responses in human Natural Killer (NK) cells.

Inflammasome pathways are critical for detecting pathogens and initiating inflammatory responses, yet how individual cell types configure these signalling platforms remains poorly understood. Here, we demonstrate that human Natural Killer cells possess distinct inflammasome machinery, lacking canonical components and innate sensors (NLRP3, NLRC4, TLR4) but harbour a functional non-canonical inflammasome (NCI). Using Salmonella enterica Typhimurium infection of primary human NK cells, we show that cytosolic bacterial access triggers caspase-4-dependent pyroptosis and IL-18 secretion, without canonical inflammasome activation or IL-1{beta} production. Strikingly, murine NK cells express inflammasome transcripts but lack functional caspase-11 protein and do not undergo pyroptosis upon infection, revealing species-specific post-transcriptional regulation. We further identify IL-12, but not IFN{gamma}, as a priming signal for NCI components in human NK cells, a functional distinction from macrophages and epithelial cells. Together, these findings reveal that human NK cells have evolved a specialised inflammasome with unique regulatory inputs, establishing these cells as autonomous sensors of intracellular Gram-negative infection.

immunology↗

bioRxiv2025

Ontogeny-independent expression of LPCAT2 in granuloma macrophages during experimental visceral leishmaniasis

Granulomas are organized inflammatory lesions that form in response to persistent stimuli such as infections. Murine infection with Leishmania donovani results in the formation of granulomas around infected Kupffer cells in the liver and serves as a well-defined model of immune granuloma formation. The formation and resolution of granulomatous inflammation requires dynamic shifts in immune cell activation states, imposing significant metabolic demands. As mediators of energy homeostasis and cell signaling, lipids and lipid metabolism play a key role in regulating immune cell function during inflammation and the response to infection. However, the extent to which alterations in lipids are spatially linked to altered immune cell transcription has yet to be resolved. In this study, we performed a multimodal imaging analysis combining MALDI mass spectrometry, spatial and single cell transcriptomics, proteomics of flow-sorted macrophages and histopathology of L. donovani induced hepatic granulomas. Using this spatially-integrated approach, we identified LPCAT2-mediated membrane re-modelling of myeloid cells as a novel feature of these granulomas. Our study provides new insights into local immunometabolic changes associated with granuloma formation and macrophage activation.

immunology↗

bioRxiv2025

Spatial profiling of central nervous system cellular markers in murine breast cancer to brain metastases

BackgroundBrain metastasis occurs in approximately 16% of metastatic breast cancer patients, and incidence is increasing. Patients with breast cancer brain metastases frequently develop neurological problems including cognitive impairments and seizures. We previously demonstrated accumulation of activated microglia around the tumour site in a breast cancer brain metastasis model, which co-localised with spontaneously occurring local field potential events that resembled interictal epileptic discharges. However, the mechanisms underlying these spatially restricted effects on neuronal excitability are poorly understood. MethodsTo better understand the underlying cellular changes and potential mechanisms, we used digital spatial profiling of brain cell subtype-specific markers to chart the spatial organisation of neurons, oligodendrocytes, astrocytes, and microglia in relation to metastatic lesion site. We developed a novel chordline-based approach to analyse these spatial changes in biomarker distribution. ResultsSeveral protein markers associated with proliferation (Ki67), astrocytes (GFAP) and microglia (CD11b, IBA1, CD45, MSR1) were upregulated in the tumour compared to healthy contralateral brain parenchyma. In contrast, some neuroglial markers (MAP2, Neurofilament light, NeuN, S100B and TMEM119) were lower in the lesion compared to normal tissue. ConclusionsOverall, the protein marker changes in the lesion are indicative of neuronal cell loss and changes in the immune microenvironment of the metastatic lesion involving activated microglia, astrocytes and recruitment of peripheral immune cells. Such cellular changes may contribute to the changes in electrical activity resulting from brain metastases observed in mice and patients.

neuroscience↗

bioRxiv2024

High resolution live cell imaging to define ultrastructural and dynamic features of the halotolerant yeast Debaryomyces hansenii

Although some budding yeasts have proved tractable and intensely studied models, others are more recalcitrant. Debaryomyces hansenii, an important yeast species in food and biotechnological industries with curious physiological characteristics, has proved difficult to manipulate genetically and remains poorly defined. To remedy this, we have combined live cell fluorescent dyes with high resolution imaging techniques to define the sub-cellular features of D. hansenii, such as the mitochondria, nuclei, vacuoles and the cell wall. Using these tools, we define biological processes like the cell cycle, organelle inheritance and different membrane trafficking pathways of D. hansenii for the first time. Beyond this, reagents designed to study Saccharomyces cerevisiae proteins were used to access proteomic information about D. hansenii. Finally, we optimised the use of label free holotomography to image yeast, defining the physical parameters and visualising sub-cellular features like membranes and vacuoles. Not only does this work shed light on D. hansenii but this combinatorial approach serves as a template for how other cell biological systems, which are not amenable to standard genetic procedures, can be studied.

cell biology↗

bioRxiv2024

CyanoTag: Discovery of protein function facilitated by high-throughput endogenous tagging in a photosynthetic prokaryote

Despite their fundamental importance to aquatic ecosystems, global carbon cycling and exciting potential in sustainable bioindustries, the genomes of photosynthetic prokaryotes still contain large numbers of uncharacterised protein-coding genes. Here we present a high-throughput approach for scarless endogenous fluorescent protein tagging in the model cyanobacterium Synechococcus elongatus PCC7942. From 400 targets we successfully endogenously tag over 330 proteins corresponding to >10% of the proteome. We demonstrate how this resource can be used at scale to determine subcellular localisation, track relative protein abundances and to elucidate protein-protein interaction networks. Our data has provided biological insights into a diverse range of processes - from photosynthesis to cell division. Of particular interest, our CyanoTag lines enabled us to visualise in real time the rapid condensation of Calvin Cycle proteins Prk and Gap2 within seconds of withdrawal of light, effectively switching off photosynthesis in the dark. These insights, CyanoTag cell lines, associated data and optimised methods are intended to be shared as a resource to facilitate further discoveries relevant to cyanobacteria and more broadly to all photosynthetic life.

microbiology↗

bioRxiv2024

Testing microbial biomining from asteroidal material onboard the International Space Station

Expanding human space exploration beyond Earths orbit necessitates efficient technologies for self-sustainable acquisition of local resources to overcome unviable resupply missions from Earth. Potential source of materials are asteroids, some of which contain valuable metals, such as platinum group elements. The BioAsteroid experiment, performed onboard the International Space Station, tested the use of microorganisms (bacteria and fungi) to carry out mining of useful elements from asteroidal material (L-chondrite) under microgravity, in support of a long-term human presence in space. The fungus Penicillium simplicissimum, enhanced the mean release of palladium, platinum and other elements from the meteorite material in microgravity, compared to non-biological leaching. However, there was large variability in the results. For many elements, non-biological leaching under microgravity was enhanced compared to terrestrial gravity, while bioleaching was unaffected. Metabolomics results revealed clear patterns that highlight the influence of space conditions on the microbial metabolism, particularly for P. simplicissimum. We identified the presence of carboxylic acids, and molecules of potential biomining and pharmaceutical interest, enhanced in microgravity. These results show a non-trivial effect of microgravity on bioleaching, highlighting the requirement of an optimal combination of microorganism(s), rock substrate, and conditions for successful biomining, both in space and Earth.

microbiology↗

bioRxiv2021

Recycling of nutrient transporters from yeast endosomes is regulated by ubiquitinated Ist1

Trafficking of cell surface membrane proteins to and from the plasma membrane impinges on myriad biological processes and ensures correct cellular function. Upon internalization, many surface proteins are recycled back to the plasma membrane. Although these endosomal trafficking pathways control surface protein activity, the precise regulatory features and division of labour between interconnected pathways is poorly defined. Furthermore, how well endosomal trafficking mechanisms are conserved is unclear. In yeast, we show cargo recycling back to the surface occurs through distinct pathways. In addition to retrograde recycling pathways via the late Golgi, used by synaptobrevins and driven by cargo ubiquitination, we find nutrient transporter recycling bypasses the Golgi in a pathway driven by cargo deubiquitination. Nutrient transporters rapidly internalize to, and recycle from, endosomes marked by Vps4 and the ESCRT-III associated factor Ist1. This compartment serves as both early and recycling endosome, implying these features are evolutionarily conserved. Ist1 has previously been implicated in recycling in yeast and other eukaryotes. We show Ist1 is ubiquitinated and this is required for proper endosomal recruitment and cargo recycling to the surface. Additionally, the ubiquitin-binding adaptor Npl4 and the essential ATPase Cdc48 are required for cargo recycling possibly through regulation of ubiquitinated Ist1. This collectively suggests mechanistic features of recycling from endosomes to the plasma membrane are also conserved.

cell biology↗

bioRxiv2021

Endosomal recycling to the surface mediated by Gpa1 and PI3-Kinase is inhibited by glucose starvation

Cell surface protein trafficking is regulated in response to nutrient availability, with multiple pathways directing surface membrane proteins to the lysosome for degradation in response to suboptimal extracellular nutrients. Internalised protein and lipid cargoes recycle back to the surface efficiently in glucose replete conditions, but this trafficking is attenuated following glucose starvation. We find cells with either reduced or hyperactive phosphatidylinositol 3-kinase (PI3K) activity are defective for recycling. Furthermore, we find the yeast G subunit Gpa1, an endosomal PI3K effector, is required for surface recycling of cargoes. Following glucose starvation, mRNA and protein levels of a distinct G subunit Gpa2 are elevated following nuclear translocation of Mig1, which inhibits recycling of various cargoes. As Gpa1 and Gpa2 interact at the surface where Gpa2 concentrates during glucose starvation, we propose this disrupts PI3K activity required for recycling, potentially diverting Gpa1 to the surface and interfering with its endosomal role in recycling. In support of this model, glucose starvation and over-expression of Gpa2 alters PI3K endosomal phosphoinositide production. Glucose deprivation therefore triggers a survival mechanism to increase retention of surface cargoes in endosomes and promote their lysosomal degradation.

cell biology↗