Biology subjects

Leake, M. C.

Publications and source records attributed to Leake, M. C..

At least 19 recordsLinked to original sources

bioRxiv2026

MaCADEMIA: a software toolbox for single-molecule fluorescence image quantification

Single-molecule localisation microscopy (SMLM) is a powerful tool in molecular and cellular biology and part of the broad umbrella of super-resolution microscopy. A distinct feature of SMLM, is its capacity for deep quantification, beyond just spatial information, to copy number, stoichiometry, molecular interaction and dynamics in living cells. The hardware required is increasingly straightforward to implement. However, complexity remains in the image analysis where much remains fragmented across specialised software tools. Here, we present MaCADEMIA (Matlab Computational Analysis of Diffusion, Emitter Molecular Intensity and Abundance), a simple yet powerful toolbox of MATLAB functions and associated documentation to enable straightforward extraction of quantitative single-molecule information. MaCADEMIA performs ensemble particle detection achieving localisation accuracies down to 0.5 pixels Root mean squared error (RMSE) across wide signal-to-noise (SNR) ranges on standardised particle tracking benchmarks, consistently matching or exceeding the performance of 14 leading methods. Our integrated workflow enables molecular counting and stoichiometry determination through stepwise photobleaching analysis, mobility measurement via mean squared displacement, colocalisaton analysis and absolute protein copy number quantification using three-dimensional cellular models.

bioinformatics↗

bioRxiv2026

Oncogenic ESR1 fusions form chromatin-anchored condensates that remodel nuclear architecture

Estrogen receptor (ESR1) fusions are recurrent drivers of endocrine-resistant breast cancer, yet their mechanisms of action remain poorly understood. Here, we show that ESR1-e6 fusion proteins, including ESR1-DAB2, ESR1-SOX9, and ESR1-YAP1, assemble into nuclear condensates across multiple cell types. Sequence analysis reveals that these fusions combine chromatin-targeting domains with increased intrinsic disorder, promoting multivalent interactions and phase separation. Live-cell imaging, fluorescence recovery after photobleaching, and mechanical perturbation demonstrate that condensates exhibit liquid-like properties, but progressively mature into stable, viscoelastic assemblies constrained by chromatin. Disruption of DNA binding abolishes condensate formation, indicating that chromatin engagement is essential for their assembly and organisation. ESR1 fusion condensates localise to chromatin-depleted regions, reduce nuclear stiffness, and promote chromatin decompaction, consistent with active remodelling of nuclear architecture. We propose that ESR1 fusions exploit phase separation to generate chromatin-anchored assemblies that reorganise the nuclear microenvironment, providing a physical framework through which ESR1 fusions may support sustained transcriptional activity, endocrine resistance and tumour progression.

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

DNA gyrase in live bacteria forms liquid condensates through weak multivalent bonding of excess GyrB

Type IIA bacterial topoisomerase DNA gyrase, a GyrA/GyrB heterotetramer, has crucial roles maintaining transcription and DNA replication by relaxing positive DNA supercoils through introducing negative supercoils. However, rates of gyrase-catalysed supercoiling in vitro cannot explain much higher rates required in vivo. To address this puzzle, we used high-speed single-molecule fluorescence imaging of GyrA/GyrB reporters in live Escherichia coli, indicating that cells contain [~]40% more GyrB than GyrA expressed in a diffuse pool or in clusters whose mobility depends on whether they are bound to DNA. Unexpectedly, we discovered that clusters are non-stoichiometric containing [~]150% more GyrB than GyrA, significantly greater than the cellular average, with fluorescence recovery after photobleaching revealing that clustered GyrA and GyrB behave as a liquid whose abundance can be increased by applying gyrase-targeting antibiotics. Structural docking indicates that the liquid state is stabilised through excess GyrB progressively binding to existing clusters via weak, multivalent interactions. By operating in liquid condensates, A2B2 that dissociates from DNA can rebind rapidly instead of diffusing away, increasing enzyme processivity to enable multiple rounds of catalysis that can keep pace with transcription and DNA replication in vivo. This demonstrates a new role for condensates of overcoming kinetic limitations imposed by diffusion. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=158 SRC="FIGDIR/small/744598v1_ufig1.gif" ALT="Figure 1"> View larger version (53K): org.highwire.dtl.DTLVardef@1514d4aorg.highwire.dtl.DTLVardef@a95ceeorg.highwire.dtl.DTLVardef@11417daorg.highwire.dtl.DTLVardef@2a2856_HPS_FORMAT_FIGEXP M_FIG C_FIG

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

Single-molecule insights into DNA gyrase in live bacteria

Molecular motors travelling along DNA introduce positive supercoils that present as barriers to replication leading to genome instability. To counter these, bacterial cells express DNA gyrase, a topoisomerase that introduces negative supercoils. While much is known about DNA gyrase from genetic and in vitro biochemical studies, the spatiotemporal dynamics of this enzyme remain a mystery. Only recently have we been able to observe the in vivo spatiotemporal dynamics down to single molecule level using advanced super-resolution microscopy techniques. We used Slimfield microscopy, a cutting-edge molecule microscopy technique to address the gap in our knowledge. We analysed a dual fluorescently labelled Escherichia coli strain expressing the replisome marker DnaN-mCherry along with mYPet-GyrB as the enzyme marker. We performed sequential Slimfield microscopy of the labelled proteins from the same strain and analysed in vivo GyrB dynamics in live E. coli cells in relation to the replisome. We find that the majority of replisomes are associated with GyrB. Inhibition of gyrase activity reduces the proportion of replisomes associated with GyrB. Interestingly, GyrB behaviour is distinct from that observed for GyrA in a previous study. Our results reveal the previously unknown dynamics of GyrB inside living bacterial cells highlighting the advantages of in vivo single molecule investigations. Our findings also demonstrate the importance of analysing all subunits of a functional enzyme complex to gain comprehensive understanding of its in vivo mechanisms. This study demonstrates the utility of single-molecule super-resolved microscopy as a valuable underpinning technology to understand in vivo behaviour of biomedically important molecules. Our insights will help impact discovery and development of novel antibiotics that interfere with gyrase function, thus contributing to tackling the growing problem of antimicrobial resistance.

biophysics↗

bioRxiv2025

The molecular architecture of the aquaglyceroporin gating system in Saccharomyces cerevisiae

Aquaglyceroporins have been extensively studied in multiple systems. However, the molecular architecture and hence the precise mechanisms of action remain unknow. The yeast aquaglyceroporin Fps1 plays the main role in controlling cellular turgor. It is regulated by the two Rgc proteins which activate the Fps1 channel. To date, the molecular architecture and dynamics of the Fps1-Rgc gating system is unknown. We employed dual-coloured super-resolution millisecond fluorescence microscopy with a single-molecule sensitivity, to track Fps1 and Rgc2 in real time. Unlike previously believed, our data shows that Rgc2 mainly operates as a homodimer. At the same time, Rgc1, the seemingly redundant regulatory ortholog of Rgc2, has a clear effect on the architecture, colocalisation behaviour and molecular dynamics of the Fps1 and Rgc2 together. Moreover, Rgc1 seems to promote Rgc2 stability in response to hydrogen peroxide stress. Together, our results provide novel insights into the mechanism behind aquaglyceroporins regulation. ImportanceThe components of the Fps1 aquaglyceroporin gating system is conserved in Fungi and present in both bakers yeast Saccharomyces cerevisiae as well as common pathogenic included in the WHO fungal priority pathogens list: Candida albicans, Candida glabrata, Aspergillus fumigatus, Pichia kudriavzevii. For instance, in C. glabrata, the orthologs of both Fps1 and Rgc2 are involved in key pathogenic processes related to acetate tolerance, phagocytosis by macrophages, resistance to antifungal agents and overall drug uptake and efflux. As the Fps1 gating system is not present in metazoans, it constitutes an attractive drug target for antifungal agents. Thus, understanding the architecture and the finer mechanisms of the Fps1 pore regulation is a stepping stone in development of effective antifungal treatment and targeted antifungal drugs.

molecular biology↗

bioRxiv2025

Detergent-Triggered Membrane Remodelling Monitored via Intramembrane Fluorescence De-Quenching

Detergent-induced membrane solubilization is important for several biotechnological applications including membrane protein isolation, cell lysis and virus inactivation. The thermodynamic details of the underlying process have been previously examined, but the mechanistic details remain largely underexplored owing in part, to a lack of suitable technologies capable of assessing nanoscopic membrane disruption events. Key open questions include: how do detergents remodel the membrane structure at sub-solubilizing concentrations? And what is the sequence of morphological transitions that lead up to solubilization? Here, we introduce a single-colour assay based on the fluorescence de-quenching of membrane-integrated fluorophores as a sensitive and generalizable tool to probe nanoscale membrane remodelling events induced by detergents. We demonstrate, using fluorescence spectroscopy and time-correlated single photon counting, that the widely used detergent Triton X-100 triggers substantial morphological changes at concentrations below its critical micellar concentration. Moreover, by taking advantage of single vesicle fluorescence lifetime imaging and scanning electron microscopy, we reveal that the swelling step involves a morphological transition from spherical vesicles to toroidal structures, providing direct evidence for detergent-driven membrane reorganization prior to solubilization. Our findings support and refine a multistep model of detergent-induced membrane solubilization, positioning fluorescence de-quenching as a tool for detecting conformational intermediates. We show that the fluorescence de-quenching approach performs robustly across multiple cyanine-based probes and experimental conditions and its nanoscale sensitivity provides a platform from which to interrogate membrane perturbations induced by a wide variety of molecular disruptors, including those with important biomedical significance.

biophysics↗

bioRxiv2025

A DNA scaffold approach facilitates 5' labelling of the SARS-CoV-2 RNA pseudoknot for smFRET investigation

Single-molecule Forster resonance energy transfer (smFRET) studies of highly structured RNA molecules are often frustrated by issues with efficient dye conjugation. Here, we develop a DNA scaffold-based labelling strategy, and apply it to the frameshift-stimulating RNA pseudoknot from the SARS-CoV-2 genome. We prepare FRET-active reporters containing both Cy3 (donor) and Cy5 (acceptor) molecules and conduct measurements on freely diffusing single molecules, enabling the evaluation of conformational heterogeneity via smFRET population distributions. We identify that freely diffusing pseudoknots, modified at the base of stem 1, display a broad range of NaCl-dependent FRET states in solution, consistent with conformational freedom that extends beyond the static X-ray and cryo-EM structures. This work is a proof-of-principle demonstration of the feasibility of our DNA scaffold approach in enabling smFRET studies on this important class of biomolecule. Together, this work outlines new biochemical and biophysical approaches towards the study of RNA conformational dynamics in pseudoknots, riboswitches and other structured RNA elements.

biophysics↗

bioRxiv2025

HU searches and binds specific DNA via a multi-step process combining weak electrostatic binding, protein reorientation and DNA flexibility

Despite their limited resolution, coarse-grained simulations have been the chosen method to obtain mechanistic information regarding the process of protein binding to DNA. Here, we demonstrate that state-of-the-art atomic simulations can capture binding events and provide new molecular insight. By using the bacterial model protein HU, we link existing crystal structures to the search and recognition states. We find that weak contacts mediated by only a few positively charged residues enable initial binding, followed by facilitated diffusion and transient rolling on DNA. Extended arms in the HU structure serve as antennae to search for DNA, permitting intrasegmental hops and intersegmental jumps. The transition to specific binding only occurs at the DNA target site, helped by its bendability, indicating a "concerted" binding mechanism between the protein and DNA. We observe a lack of direct binding to the most positively charged area, which is hidden behind HUs arms and defines the recognition state. This prevents the protein from being trapped in random DNA. Instead, the multi-step binding process found here ensures that the high-affinity complex only occurs at the target position. We anticipate that other DNA-binding proteins with multiple surface-charged regions and DNA-bend induction capability might follow a similar strategy.

biophysics↗

bioRxiv2025

A new protein-dependent riboswitch activates ribosomal frameshifting

Programmed -1 ribosomal frameshifting (PRF) is a translational control mechanism used by RNA viruses to regulate the relative abundance of proteins encoded in different reading frames. Cardioviruses exhibit the highest known PRF efficiency, with ~85% of ribosomes shifting into the -1 frame. This unusual event requires an interaction between the viral 2A protein and a stimulatory element in the RNA genome, but the basis for protein-dependence is unclear. To address this, here we investigate structure and dynamics of the PRF signal in Theiler's murine encephalitis virus (TMEV). By combining X-ray crystallography, small angle X-ray scattering (SAXS) and single-molecule fluorescence resonance energy transfer (smFRET), we show that 2A binding switches the RNA from a stem-loop conformation into a pseudoknot, and we demonstrate that pseudoknot formation is essential for efficient PRF in vitro and in cells. Together, these findings illustrate how the cardiovirus PRF element behaves as a protein-dependent riboswitch, defining the molecular mechanism by which frameshifting is conditionally activated.

molecular biology↗

bioRxiv2025

Macrophage-derived reactive oxygen species promote Salmonella aggresome formation contributing to bacterial antibiotic persistence

In this study, we reveal that macrophage-derived reactive oxygen species (ROS) can trigger the rapid formation of Salmonella aggresomes, which substantially contribute to the increased frequency of persisters induced by phagocytosis. Salmonella containing aggresomes exhibited a dormant phenotype characterized by reduced adenosine triphosphate (ATP) levels and decreased metabolic activity. Furthermore, these dormant bacteria showed upregulated expression of Salmonella pathogenicity island 1 (SPI-1)-encoded type III secretion system (T3SS)-related genes, followed by later expression of SPI-2 T3SS-related genes when macrophages ROS production declined. Our results demonstrate that Salmonella containing aggresomes can enter a dormant state to escape antibiotic attack, while crucially maintaining the ability to resuscitate when the stress environment is improved. Research on bacterial aggresomes could potentially provide therapeutic strategies to combat bacterial antibiotic persistence. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=122 SRC="FIGDIR/small/657582v1_ufig1.gif" ALT="Figure 1"> View larger version (55K): org.highwire.dtl.DTLVardef@7e5f1forg.highwire.dtl.DTLVardef@970a39org.highwire.dtl.DTLVardef@204449org.highwire.dtl.DTLVardef@632c47_HPS_FORMAT_FIGEXP M_FIG C_FIG

biophysics↗

bioRxiv2024

Rep structures can be tuned by ionicity via metastable intermediates in the absence of DNA

DNA helicases undergo conformational changes; however, their structural dynamics are poorly understood. Here, we study single molecules of superfamily 1A DNA helicase Rep, which undergo conformational transitions during bacterial DNA replication, repair and recombination. We use time-correlated single-photon counting (TCSPC), fluorescence correlation spectroscopy (FCS), rapid single-molecule Forster resonance energy transfer (smFRET), Anti-Brownian ELectrokinetic (ABEL) trapping and molecular dynamics simulations (MDS) to provide unparalleled temporal and spatial resolution of Reps domain movements. We detect four states revealing two hitherto hidden intermediates (S2, S3), between the open (S1) and closed (S4) structures, whose stability is salt dependent. Reps open-to-closed switch involves multiple changes to all four subdomains 1A, 1B, 2A and 2B along the S1[->]S2[->]S3[->]S4 transitional pathway comprising an initial truncated swing of 2B which then rolls across the 1B surface, following by combined rotations of 1B, 2A and 2B. High forward and reverse rates for S1[->]S2 suggest that 1B may act to frustrate 2B movement to prevent premature Rep closure in the absence of DNA. These observations support a more general binding model for accessory DNA helicases that utilises conformational plasticity to explore a multiplicity of structures whose landscape can be tuned by salt prior to locking-in upon DNA binding.

biophysics↗

bioRxiv2024

SlimVar: rapid in vivo single-molecule tracking of chromatin regulators in plants

Epigenetic regulation occurs over many rounds of cell division in higher organisms. However, visualisation of the regulators in vivo is limited by imaging dynamic molecules deep in tissue. We report a technology--Variable-angle Slimfield microscopy (SlimVar)-- that enables tracking of single fluorescent reporters to 30 {micro}m depth through multiple Arabidopsis thaliana root tip cell layers. SlimVar uses rapid photobleaching to resolve tracked particles to molecular steps in intensity. By modifying widefield microscopy to minimise optical aberrations and robustly post-process few-photon signals, SlimVar mitigates performance losses at depth. We use SlimVar to quantify chromatin-protein assemblies in nuclei, finding that two homologous proteins key to epigenetic switching at FLOWERING LOCUS C (FLC) --cold-induced VERNALISATION INSENSITIVE3 (VIN3) and constitutively expressed VERNALISATION 5 (VRN5)--exhibit dynamic assemblies during FLC silencing. Upon cold exposure, the number of assembly molecules increases up to 100% to a median of [~]20 molecules. Larger VRN5 assemblies preferentially colocalise with an FLC lacO transgenic reporter during prolonged cold and persist after return to warmth. Our findings support a hybrid model of epigenetic memory in which nucleation of histone trimethylation is assisted by dynamic protein assemblies over extended durations. SlimVar offers molecular insights into proteins expressed at physiological levels in tissues.

plant biology↗

bioRxiv2024

Bacterial stress granule protects mRNA through ribonucleases exclusion

Membraneless droplets formed through liquid-liquid phase separation of ribonucleoprotein particles contribute to mRNA storage in eukaryotic cells. How such aggresomes contribute to mRNA dynamics under stress, and their functional role, is less understood in bacteria. Here we used multiple approaches including live-cell imaging, polymer physics modelling and transcriptomics to show that prolonged stress leading to ATP depletion in Escherichia coli results in increased aggresome formation, compaction, and selective mRNA enrichment within these aggresomes. Longer transcripts accumulate more in aggresomes than in the cytosol. Mass spectrometry and mutagenesis studies showed that mRNA ribonucleases are excluded from aggresomes due to electrostatic repulsion arising from their negative surface charges. Experiments with fluorescent reporters and disruption of aggresome formation showed that mRNA storage within aggresomes promoted rapid translation reactivation and associated with reduced lag phases during growth after stress removal. Our findings suggest that mRNA storage within aggresomes confers an advantage for bacterial survival and recovery from stress.

biophysics↗

bioRxiv2024

A promiscuous mechanism to phase separate eukaryotic carbon fixation in the green lineage

CO2 fixation is commonly limited by inefficiency of the CO2-fixing enzyme Rubisco. Eukaryotic algae concentrate and fix CO2 in phase-separated condensates called pyrenoids, which complete up to one-third of global CO2 fixation. Condensation of Rubisco in pyrenoids is dependent on interaction with disordered linker proteins that show little conservation between species. We developed a sequence-independent bioinformatic pipeline to identify linker proteins in green algae. We report the linker from Chlorella and demonstrate that it binds a conserved site on the Rubisco large subunit. We show the Chlorella linker phase separates Chlamydomonas Rubisco and that despite their separation by [~]800 million years of evolution, the Chlorella linker can support the formation of a functional pyrenoid in Chlamydomonas. This cross-species reactivity extends to plants, with the Chlorella linker able to drive condensation of some native plant Rubiscos in vitro and in planta. Our results represent an exciting frontier for pyrenoid engineering in plants, which is modelled to increase crop yields.

plant biology↗

bioRxiv2024

Modular structure of RNA 3' processing condensates involving the Arabidopsis RNA binding protein FCA

Our understanding of the functional requirements underpinning biomolecular condensation in vivo is still relatively poor. The Arabidopsis RNA binding protein FCA is found in liquid-like nuclear condensates that function in transcription termination, promoting proximal polyadenylation at many targets in the Arabidopsis genome. To further understand the properties of these condensates in vivo we used single particle tracking experiments on FCA reporters stably expressed at endogenous levels in plant nuclei. These revealed FCA forms a core oligomer of [~]4 molecules that multimerizes into higher-order particles. This assembles into macromolecular condensates through the function of the coiled-coil protein FLL2, which is genetically required for FCA function. FLL2 predominately co-localizes with FCA in larger sized condensates. A missense mutation in the FCA RRM domain, also genetically required for FCA function, reduced both average FCA particle and condensate size but did not perturb the core oligomer. Our work points to a modular structure for FCA condensates involving multimerization of core oligomers that assemble into functional macromolecular condensates via associated RNA and FLL2 interactions.

molecular biology↗

bioRxiv2024

Functional specialization of Arabidopsis VEL polymerization domains in the switch to Polycomb silencing

Cold-induced epigenetic silencing of Arabidopsis FLOWERING LOCUS C (FLC) requires the Polycomb Repressive Complex 2 and accessory proteins VIN3 and VRN5. VIN3 and VRN5 interact via head-to-tail VEL polymerization domains, but how these functionally contribute to the switch to an epigenetically silenced state remains poorly understood. Here, we determine that VIN3 VEL polymerization involves higher order nuclear VIN3 assemblies in vivo, promotes strong chromatin association and efficient H3K27me3 nucleation. However, we also show that the polymerization domains of VIN3 and VRN5 are not equivalent: VRN5 VEL domain is not required for silencing despite its role in physically connecting VIN3 with the PRC2 complex and VRN5 VEL is unable to functionally replace VIN3 VEL in vivo. Both VIN3 and VRN5 homologs are present throughout angiosperm species, suggesting a functional requirement for maintaining different polymerization modalities. This work reveals distinct roles for multifunctional polymerization domains of Polycomb accessory proteins underpinning the onset of epigenetic silencing.

plant biology↗