Biology subjects

MacDonald, C.

Publications and source records attributed to MacDonald, C..

At least 19 recordsLinked to original sources

bioRxiv2026

Transcriptional responses of acute glucose deprivation reveal a role for Snf12 and Spt20 in metabolic adaptation during stress

The budding yeast Saccharomyces cerevisiae is a well-established model organism to study cellular stress response and underlying mechanistic regulation. Although glucose starvation fundamentally alters gene regulation and cell behaviour, inconsistent deprivation protocols often trigger gross morphological artefacts. These non-specific changes confound findings by activating pathways independently of true glucose-signalling mechanisms. Furthermore, a thorough transcriptomic profile of glucose starvation using non-confounding conditions remains lacking. Consequently, the precise transcriptional impact of losing key metabolic regulators that mediate adaptation to glucose starvation remains undefined. Here we have employed a refined glucose starvation protocol, utilising raffinose exchange, which shows induction of vast transcriptional stress response with minimal impact on cellular morphology confirmed by label-free imaging. Transcriptomic profiling revealed shifts in metabolic regulation, ATP turnover, and cell-to-cell communication as acute glucose deprivation driving cells towards oxidation-driven metabolism. Additionally, we characterise transcriptional alterations seen in deletion mutants of SNF12 and SPT20, known regulators of cellular metabolism, showing previously unappreciated transcriptional conservation, in part mimicking glucose starvation response. Finally, we identified cargo and stress-specific expression related to both eisosome components and surface transporters that are critical for metabolic adaptation. Overall, this dataset provides a comprehensive transcriptomic resource for dissecting stress signalling and driving novel hypothesis generation.

cell biology↗

bioRxiv2026

HaloUMI: Physics-informed analysis of inhibition halo assays

Quantification of microbial growth inhibition is central to assays from antibiotic susceptibility to antifungal sensitivity, yet existing approaches struggle with irregular inhibition zones and variation in microbial lawn density. Here, we present Halo Unbiased Measurement of growth Inhibition (HaloUMI), an open-source Python graphical interface for automated, high-throughput analysis of lawn-based microbial assays. HaloUMI integrates robust image processing with physics-informed modelling to quantify inhibition zones without assuming circular geometry, enabling analysis of uniform and irregular halo phenotypes. Using diffusion- and growth-based physical modelling, HaloUMI experimentally validates a correction for variation in microbial lawn density, a major source of assay variability that can confound quantitative comparison of inhibition phenotypes. Validation using simulations and yeast killer-toxin assays demonstrates precise, reproducible measurement across diverse conditions. HaloUMI is applicable to multiple assay formats, including microbial interaction, mating, and conventional disc-diffusion assays, providing an accessible and generalisable framework for quantitative analysis of microbial growth inhibition.

biophysics↗

bioRxiv2026

Ktd1 is a phospho-regulated member of the Dup240 family that mediates defence against killer toxin K28

The DUP240 gene family in Saccharomyces cerevisiae encodes ten proteins containing two transmembrane domains (TMDs). Despite decades of interest driven by their high sequence similarity, little functional information exists regarding whether Dup240 family members share redundant or distinct roles. In this study, we combined computational modelling, subcellular localisation, and functional assays across the family to identify shared and unique features. Computational modelling revealed that Ktd1 possesses a unique structural element adjacent to its TMD region. Out of six successfully localised family members, Ktd1 was the only protein predominantly targeted to the vacuolar membrane and the sole Dup240 required for defence against the K28 killer toxin. Computational predictions further indicated that Ktd1 undergoes extensive post-translational regulation, containing multiple validated phosphorylation sites. Screening potential regulatory kinases and phosphatases identified several enzymes required for K28 defence, which were independently validated using liquid-based toxin sensitivity assays. A multicopy suppressor screen demonstrated that KTD1 overexpression rescued K28 sensitivity across most enzyme mutant backgrounds, confirming Ktd1 acts downstream or in parallel to many factors. However, the phosphatase Sit4 and the kinase Hog1 scored as most likely co-factors in Ktd1 mediated defence. Live-cell fluorescence imaging of these two enzymes revealed no dramatic spatial re-localisation during K28 exposure, suggesting that phospho-dependent regulation of Ktd1-mediated defence may occur through transient signalling events. Together, these findings identify Ktd1 as the central effector of the Dup240 family in toxin defence and provide a mechanistic framework for understanding Dup240 regulation.

cell biology↗

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↗

bioRxiv2026

Ubiquitinome dynamics and regulation by DUB2 during Leishmania differentiation

Leishmaniasis is caused by Leishmania parasites, which undergo cellular adaptation when transitioning from the insect stage (promastigote) to the mammalian stage (amastigote). While the ubiquitin-proteasome system (UPS) is vital for life cycle progression, global ubiquitination dynamics have remained unmapped. We established a quantitative ubiquitinomics workflow for Leishmania mexicana, identifying over 9,100 ubiquitination sites across 38% of the proteome, revealing thousands of stage-specific regulatory events. Promastigote-enriched sites associate with cell motility, while amastigote-enriched sites link to metabolism and glycosome organization. We identified extensive ubiquitination on UPS components, including the essential virulence factor deubiquitinase 2 (DUB2). Using inducible gene deletion and XL-BioID proximitomics, we identified 111 potential DUB2 substrates. High-confidence substrates include the E2 conjugating enzyme UBC2, which is required for differentiation, and SUMO, a critical regulator of ubiquitin crosstalk. The discovery of UBC2 as a substrate of DUB2 directly links ubiquitination with promastigote to amastigote differentiation. Our findings provide a comprehensive map of the Leishmania ubiquitinome and demonstrate that DUB2 acts as a pleiotropic regulator controlling post-translational modifications of essential proteins involved in life cycle progression.

microbiology↗

bioRxiv2025

Causal modelling reveals lower coral genetic diversity in more heterogenous reef environments

Conserving genetic diversity is crucial for maintaining species evolutionary potential and resilience to environmental change. Yet, directly measuring genetic diversity across large spatial scales remains resource-intensive and impractical for conservation planning. Identifying reliable, accessible, and cost-effective spatial predictors of genetic diversity would significantly enhance our ability to incorporate genetic diversity into conservation efforts. Caribbean coral reefs represent a compelling system for investigating such predictors, as these ecosystems face unprecedented threats from disease outbreaks and climate change. Here, we investigate the utility of several environmental proxies as surrogates for genetic diversity, which may be useful for conservation planning in resource- or data-poor regions. To achieve this goal, we used reduced-representation genomic sequencing to estimate intraspecific genetic diversity, and in doing so, identified two genetically distinct and sympatric cryptic coral species in the Agaricia tenuifolia species complex. We employed a causal inference approach to assess the relationships between genetic alpha diversity and habitat area and connectivity, habitat heterogeneity, and temperature. Statistical investigations revealed that increased habitat heterogeneity, derived from remotely sensed habitat maps, was associated with lower genetic diversity within coral populations, suggesting that habitat characteristics promoting species diversity may not maintain high genetic diversity within a single species. Habitat area and connectivity, and temperature have predominantly negative effects on genetic diversity with coral habitat area and connectivity showing markedly contrasting responses in two cryptic taxa. Thus, our findings reveal that context matters greatly, as results differ between cryptic species, across variables, and among spatial scales.

evolutionary biology↗

bioRxiv2025

Uptake mechanisms and physiological effects of furanic compounds from the Maillard reaction in budding yeast

Maillard reaction products (MRPs) are formed during the thermal processing of foods and exhibit important sensory attributes. Furanic compounds are a subset of MRPs commonly found in food products that are toxic to eukarytoic cells, although the mechanisms of toxicity are poorly understood. We used budding yeast to explore uptake mechanisms of common furanic compounds: 5-hydroxymethylfurfural (HMF), furfural (FUR), and 2-Furyl methyl ketone (FMK). Titrations of each furanic compound were used to identify concentrations that have an inhibitory effect on growth. We identified HMF as a potential substrate of the Pdr5 multidrug resistance pump and linked HMF and FUR toxicity to surface nutrient transporter levels. Live cell imaging shows that HMF disrupts mitochondria whilst FUR affects the endolysosomal system. Results indicate these furanic compounds may have distinct uptake, efflux, and toxicity mechanisms. As many of these cellular components are conserved throughout evolution, this work could shed light on the metabolism of toxic compounds commonly found within animal food sources.

cell biology↗

bioRxiv2024

Killer Toxin K28 resistance in yeast relies on COG complex mediated trafficking of the defence factor Ktd1

A/B toxins are a diverse family of protein toxins that enter host cells via endocytosis and induce cell death. In yeast, the A/B toxin K28 is internalised to endosomes of susceptible yeast, before following the retrograde trafficking pathway and ultimately triggering cell cycle arrest. The endolysosomal defence factor Ktd1 protects against K28, but its regulation remains unclear. Cog7, a subunit of the conserved oligomeric Golgi (COG) tethering complex, has been implicated in K28 defence, though the mechanism is unknown. We developed a high throughput K28 sensitivity assay and bespoke analysis package to show that all lobe B COG subunits (Cog5 - 8) are required for K28 resistance. Although the COG complex modulates glycosylation of the surface molecules required to bind extracellular K28, our experiments reveal that the hypersensitivity of cog mutants is primarily explained by defects in Ktd1 trafficking. Ktd1 mis-localisation in cog mutants is reminiscent to disruptions in Snc1, a surface cargo that recycles multiple times via the Golgi. This work suggests not only that the COG complex is responsible for the precise trafficking Ktd1 required to mediate toxin defence, but that Ktd1 may survey endolysosomal compartments for internalised K28. This work underpins the importance of Ktd1 in defence against the A/B toxin K28, and implies various membrane trafficking regulators might influence toxin effects in other eukaryotic systems.

cell biology↗

bioRxiv2024

Extracellular vesicle bioactivity and potential clinical utility is determined by mesenchymal stromal cell clonal subtype

Mesenchymal stromal cells (MSCs) are a promising source of therapeutic extracellular vesicles (EVs), however it is not clear how heterogeneity within a non-clonal MSC population will affect the collective EV pool. Here we used immortalised clonal MSC lines, termed Y201 and Y202, to examine how MSC phenotype influences EV character and function. Although morphologically similar, Y201 EVs were more abundant in EV biomarkers versus Y202 EVs, with an enhanced miRNA and proteomic content, predicted to contribute to an elaborate EV corona particularly abundant in RGD-containing proteins fibronectin and MFG-E8. We demonstrated that Y201 EVs, but not Y202 EVs, significantly increased the proliferation of articular chondrocytes and that the proliferative effect was mediated at least in part via an RGD (integrin)-FAK-ERK1/2 axis. Both Y201 and Y202 EV subsets significantly reduced proliferative index scores of activated T cells. However, only Y201 EVs, not Y202 EVs, suppressed disease activity compared to controls in different in vivo models of inflammatory peritonitis and arthritis. EVs released by closely related MSC subtypes within the same heterogeneous population differ significantly in terms of cargo abundance, bioactivity, and pre-clinical in vivo efficacy. Analysis of defined EV subsets will aid mechanistic understanding and prioritisation for EV therapeutics.

cell biology↗

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

iPAR: a new reporter for eukaryotic cytoplasmic protein aggregation

BackgroundCells employ myriad regulatory mechanisms to maintain protein homeostasis, termed proteostasis, to ensure correct cellular function. Dysregulation of proteostasis, which is often induced by physiological stress and ageing, often results in Protein Aggregation in cells. These aggregated structures can perturb normal physiological function, compromising cell integrity and viability, a prime example being early onset of several neurodegenerative diseases. Understanding aggregate dynamics in vivo is therefore of strong interest for biomedicine and pharmacology. However, factors involved in formation, distribution and clearance of intracellular aggregates are not fully understood. MethodsHere, we report an improved methodology for production of fluorescent aggregates in model budding yeast which can be detected, tracked and quantified using fluorescence microscopy in live cells. This new openly-available technology, iPAR (inducible Protein Aggregation Reporter), involves monomeric fluorescent protein reporters fused to a {Delta}ssCPY* aggregation biomarker, with expression controlled under the copper-regulated CUP1 promoter. ResultsMonomeric tags overcome challenges associated with non-physiological reporter aggregation, whilst CUP1 provides more precise control of protein production. We show that iPAR and the associated bioimaging methodology enables quantitative study of cytoplasmic aggregate kinetics and inheritance features in vivo. We demonstrate that iPAR can be used with traditional epifluorescence and confocal microscopy as well as single-molecule precise Slimfield millisecond microscopy. Our results indicate that cytoplasmic aggregates are mobile and contain a broad range of number of iPAR molecules, from tens to several hundred per aggregate, whose mean value increases with extracellular hyperosmotic stress. DiscussionTime lapse imaging shows that although larger iPAR aggregates associate with nuclear and vacuolar compartments, and for the first time we show directly that these proteotoxic accumulations are not inherited by daughter cells, unlike nuclei and vacuoles. If suitably adapted, iPAR offers new potential for studying diseases relating to protein oligomerization processes in other model cellular systems.

biophysics↗

bioRxiv2022

The phosphatase Glc7 controls eisosomal response to starvation via posttranslational modification of Pil1

The yeast plasma membrane (PM) is organised into specific subdomains that regulate surface membrane proteins. Surface transporters actively uptake nutrients in particular regions of the PM where they are also susceptible to substrate induced endocytosis. However, transporters also diffuse into distinct subdomains termed eisosomes, where they are protected from endocytosis. Although most nutrient transporter populations are downregulated in the vacuole following glucose starvation, a small pool is retained in eisosomes to provide efficient recovery from starvation. We find the core eisosome subunit Pil1, a Bin, Amphiphysin and Rvs (BAR) domain protein required for eisosome biogenesis, is phosphorylated primarily by the kinase Pkh2. In response to acute glucose starvation, Pil1 is rapidly dephosphorylated. Enzyme localisation and activity screens implicate the phosphatase Glc7 is the primary enzyme responsible for Pil1 dephosphorylation. Both depletion of GLC7 and phospho-ablative or phospho-mimetic mutations of Pil1 correlate with Pil1 phosphorylation status, failure to properly retain transporters in eisosomes, and results in defective starvation recovery. We propose precise posttranslational control of Pil1 modulates nutrient transporter retention within eisosomes depending on extracellular nutrient levels, to maximise recovery following starvation.

cell biology↗

bioRxiv2022

Correlating viscosity and molecular crowding with fluorescent nanobeads and molecular probes: in vitro and in vivo

In eukaryotes, intracellular physicochemical properties like macromolecular crowding and cytoplasmic viscoelasticity influence key processes such as metabolic activities, molecular diffusion, and protein folding. However, mapping crowding and viscoelasticity in living cells remains challenging. One approach uses passive rheology in which diffusion of exogenous fluorescent particles internalised in cells is tracked and physicochemical properties inferred from derived mean square displacement relations. Recently, the crGE2.3 Forster Resonance Energy Transfer (FRET) biosensor was developed to quantify crowding in cells, though it is unclear how this readout depends on viscoelasticity and the molecular weight of the crowder. Here, we present correlative, multidimensional data to explore diffusion and molecular crowding characteristics of molecular crowding agents using super-resolved fluorescence microscopy and ensemble time-resolved spectroscopy. We firstly characterise in vitro and then apply these insights to live cells of budding yeast Saccharomyces cerevisiae. It is to our knowledge the first time this has been attempted. We demonstrate that these are usable both in vitro and in the case of endogenously expressed sensors in live cells. Finally, we present a method to internalise fluorescent beads as in situ viscoelasticity markers in the cytoplasm of live yeast cells, and discuss limitations of this approach including impairment of cellular function.

biophysics↗

bioRxiv2022

Determining the optimal SARS-CoV-2 mRNA vaccine dosing interval for maximum immunogenicity

ObjectiveEmerging evidence indicates that longer SARS-CoV-2 vaccine dosing intervals results in an enhanced immune response. However, the optimal vaccine dosing interval for achieving maximum immunogenicity is unclear. MethodsThis study included samples from adult paramedics in Canada who received two doses of either BNT162b2 or mRNA-1273 vaccines and provided blood samples 6 months (170 to 190 days) after the first vaccine dose. The main exposure variable was vaccine dosing interval (days), categorized as "short" (first quartile), "moderate" (second quartile), "long" (third quartile), and "longest" interval (fourth quartile). The primary outcome was total spike antibody concentrations, measured using the Elecsys SARS-CoV-2 total antibody assay. Secondary outcomes included: spike and RBD IgG antibody concentrations, and inhibition of angiotensin-converting enzyme 2 (ACE-2) binding to wild-type spike protein and several different Delta variant spike proteins. We fit a multiple log-linear regression model to investigate the association between vaccine dosing intervals and the antibody concentrations. ResultsA total of 564 adult paramedics (mean age 40 years, SD=10) were included. Compared to "short interval" ([≤]30 days), higher dosing interval quartiles (moderate: 31-38 days; long: 39-73 days and longest: [≥]74 days) were all associated with increased Elescys spike total antibody concentration. Compared to the short interval, "long" and "longest" interval quartiles were associated with higher spike and RBD IgG antibody concentrations. Similarly, increasing dosing intervals increased inhibition of ACE-2 binding to viral spike protein, regardless of the vaccine type. ConclusionIncreased mRNA vaccine dosing intervals longer than 30 days result in higher levels of circulating antibodies and viral neutralization when assessed at 6 months.

microbiology↗

bioRxiv2021

The Rpd3-complex regulates expression of multiple cell surface recycling factors in yeast

Intracellular trafficking pathways control residency and bioactivity of integral membrane proteins at the cell surface. Upon internalisation, surface cargo proteins can be delivered back to the plasma membrane via endosomal recycling pathways. Recycling is thought to be controlled at the metabolic and transcriptional level, but such mechanisms are not fully understood. In yeast, recycling of surface proteins can be triggered by cargo deubiquitination and a series of molecular factors have been implicated in this trafficking. In this study, we follow up on the observation that many subunits of the Rpd3 lysine deacetylase complex are required for recycling. We validate ten Rpd3-complex subunits in recycling using two distinct assays and developed tools to quantify both. Fluorescently labelled Rpd3 localises to the nucleus and complements recycling defects, which we hypothesised were mediated by modulated expression of Rpd3 target gene(s). Bioinformatics implicated 32 candidates that function downstream of Rpd3, which were over-expressed and assessed for capacity to suppress recycling defects of rpd3{Delta} cells. This effort yielded 3 hits: Sit4, Dit1 and Ldb7, which were validated with a lipid dye recycling assay. Additionally, the essential phosphatidylinositol-4-kinase Pik1 was shown to have a role in recycling. We propose recycling is governed by Rpd3 at the transcriptional level via multiple downstream target genes.

cell biology↗

bioRxiv2021

BugSplit: highly accurate taxonomic binning of metagenomic assemblies enables genome-resolved metagenomics

A large gap remains between sequencing a microbial community and characterizing all of the organisms inside of it. Here we develop a novel method to taxonomically bin metagenomic assemblies through alignment of contigs against a reference database. We show that this workflow, BugSplit, bins metagenome-assembled contigs to species with a 33% absolute improvement in F1-score when compared to alternative tools. We perform nanopore mNGS on patients with COVID-19, and using a reference database predating COVID-19, demonstrate that BugSplits taxonomic binning enables sensitive and specific detection of a novel coronavirus not possible with other approaches. When applied to nanopore mNGS data from cases of Klebsiella pneumoniae and Neisseria gonorrhoeae infection, BugSplits taxonomic binning accurately separates pathogen sequences from those of the host and microbiota, and unlocks the possibility of sequence typing, in silico serotyping, and antimicrobial resistance prediction of each organism within a sample. BugSplit is available at https://bugseq.com/academic.

bioinformatics↗

bioRxiv2021

Investigating molecular crowding during cell division in budding yeast with FRET

Cell division, aging, and stress recovery triggers spatial reorganization of cellular components in the cytoplasm, including membrane bound organelles, with molecular changes in their compositions and structures. However, it is not clear how these events are coordinated and how they integrate with regulation of molecular crowding. We use the budding yeast Saccharomyces cerevisiae as a model system to study these questions using recent progress in optical fluorescence microscopy and crowding sensing probe technology. We used a Forster Resonance Energy Transfer (FRET) based sensor, illuminated by confocal microscopy for high throughput analyses and Slimfield microscopy for single-molecule resolution, to quantify molecular crowding. We determine crowding in response to cellular growth of both mother and daughter cells, in addition to osmotic stress, and reveal hot spots of crowding across the bud neck in the burgeoning daughter cell. This crowding might be rationalized by the packing of inherited material, like the vacuole, from mother cells. We discuss recent advances in understanding the role of crowding in cellular regulation and key current challenges and conclude by presenting our recent advances in optimizing FRET-based measurements of crowding whilst simultaneously imaging a third color, which can be used as a marker that labels organelle membranes. Our approaches can be combined with synchronised cell populations to increase experimental throughput and correlate molecular crowding information with different stages in the cell cycle.

biophysics↗

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↗