Biology subjects

Milburn, A.

Publications and source records attributed to Milburn, A..

6 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

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↗

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

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↗