Biology subjects

Wollman, A. J.

Publications and source records attributed to Wollman, A. J..

9 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↗

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↗

bioRxiv2024

Flight-Scope: microscopy with microfluidics in microgravity

With the European Space Agency (ESA) and NASA working to return humans to the moon and onwards to Mars, it has never been more important to study the impact of altered gravity conditions on biological organisms. These include astronauts but also useful micro-organisms they may bring with them to produce food, medicine, and other useful compounds by synthetic biology. Parabolic flights are one of the most accessible microgravity research platforms but present their own challenges: relatively short periods of altered gravity ([~]20s) and aircraft vibration. Live-imaging is necessary in these altered-gravity conditions to readout any real-time phenotypes. Here we present Flight-Scope, a new microscopy and microfluidics platform to study dynamic cellular processes during the short, altered gravity periods on parabolic flights. We demonstrated Flight-Scopes capability by performing live and dynamic imaging of fluorescent glucose uptake by yeast, S. cerevisiae, on board an ESA parabolic flight. Flight-Scope operated well in this challenging environment, opening the way for future microgravity experiments on biological organisms.

biophysics↗

bioRxiv2021

Membraneless organelles formed by liquid-liquid phase separation increase bacterial fitness

Liquid-liquid phase separation is emerging as a crucial phenomenon in several fundamental cell processes. A range of eukaryotic systems exhibit liquid condensates. However, their function in bacteria, which in general lack membrane-bound compartments, remains less clear. Here, we used high-resolution optical microscopy to observe single bacterial aggresomes, nanostructured intracellular assemblies of proteins, to undercover their role in cell stress. We find that proteins inside aggresomes are mobile and undergo dynamic turnover, consistent with a liquid state. Our observations are in quantitative agreement with phase-separated liquid droplet formation driven by interacting proteins under thermal equilibrium that nucleate following diffusive collisions in the cytoplasm. We have discovered aggresomes in multiple species of bacteria, and show that these emergent, metastable liquid-structured protein assemblies increase bacterial fitness by enabling cells to tolerate environmental stresses. One Sentence SummaryBacteria use subcellular proteinaceous liquid droplets to survive stress

biophysics↗

bioRxiv2021

PySTACHIO: Python Single-molecule TrAcking stoiCHiometry Intensity and simulatiOn, a flexible, extensible, beginner-friendly and optimized program for analysis of single-molecule microscopy data

As camera pixel arrays have grown larger and faster, and optical microscopy techniques ever more refined, there has been an explosion in the quantity of data acquired during routine light microcopy. At the single-molecule level, analysis involves multiple steps and can rapidly become computationally expensive, in some cases intractable on office workstations. Complex bespoke software can present high activation barriers to entry for new users. Here, we redevelop our quantitative single-molecule analysis routines into an optimized and extensible Python program, with GUI and command-line implementations to facilitate use on local machines and remote clusters, by beginners and advanced users alike. We demonstrate that its performance is on par with previous MATLAB implementations but runs an order of magnitude faster. We tested it against challenge data and demonstrate its performance is comparable to state-of-the-art analysis platforms. We show the code can extract fluorescence intensity values for single reporter dye molecules and, using these, estimate molecular stoichiometries and cellular copy numbers of fluorescently-labeled biomolecules. It can evaluate 2D diffusion coefficients for the characteristically short single-particle tracking data. To facilitate benchmarking we include data simulation routines to compare different analysis programs. Finally, we show that it works with 2-color data and enables colocalization analysis based on overlap integration, to infer interactions between differently labelled biomolecules. By making this freely available we aim to make complex light microscopy single-molecule analysis more democratized. HighlightsO_LIWe present PySTACHIO, a refined version of our spot tracking algorithm C_LIO_LIWe demonstrate highly improved performance over previous MATLAB versions C_LIO_LIPySTACHIO can accurately estimate stoichiometries and 2D diffusion coefficients C_LIO_LIPerformance is comparable to state-of-the-art packages on challenge data C_LIO_LIPySTACHIO has both GUI and command line interfaces and can be hosted as a web app C_LI Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=77 SRC="FIGDIR/small/435952v3_ufig1.gif" ALT="Figure 1"> View larger version (27K): org.highwire.dtl.DTLVardef@17cc45dorg.highwire.dtl.DTLVardef@3fb66eorg.highwire.dtl.DTLVardef@e20360org.highwire.dtl.DTLVardef@4ba47d_HPS_FORMAT_FIGEXP M_FIG C_FIG

biophysics↗

bioRxiv2021

Tetrameric UvrD helicase is located at the E. coli replisome due to frequent replication blocks

DNA replication in all organisms must overcome nucleoprotein blocks to complete genome duplication. Accessory replicative helicases in Escherichia coli, Rep and UvrD, help remove these blocks and aid the re-initiation of replication. Mechanistic details of Rep function have emerged from recent live cell studies; however, the division of UvrD functions between its activities in DNA repair and role as an accessory helicase remain unclear in live cells. By integrating super-resolved single-molecule fluorescence microscopy with biochemical analysis, we find that UvrD self-associates into tetrameric assemblies and, unlike Rep, is not recruited to a specific replisome protein despite being found at approximately 80% of replication forks. Instead, its colocation with forks is likely due to the very high frequency of replication blocks composed of DNA-bound proteins, including RNA polymerase and factors involved in repairing DNA damage. Deleting rep and DNA repair factor genes mutS and uvrA, and inhibiting transcription through RNA polymerase mutation and antibiotic inhibition, indicates that the level of UvrD at the fork is dependent on UvrDs function. Our findings show that UvrD is recruited to sites of nucleoprotein blocks via different mechanisms to Rep and plays a multi-faceted role in ensuring successful DNA replication.

biophysics↗

bioRxiv2021

Comparison of endogenously expressed fluorescent protein fusions behaviour for protein quality control and cellular aging research

The yeast Hsp104 protein disaggregase is often used as a reporter for misfolded or damaged protein aggregates and protein quality control and ageing research. Observing endogenously expressed Hsp104 fusions with fluorescent proteins is a popular approach to follow post stress protein aggregation, inclusion formation and disaggregation. Overall, such protein fusions used in molecular and microbiology research are often sparsely characterised. To address this issue, we performed a comparative assessment of Hsp104 fluorescent fusions function and behaviour. We provide experimental evidence that molecular behaviour may not only be altered by introducing a fluorescent protein tag but also varies depending on the fluorophore within the fusion. Although our findings are especially applicable to protein quality control and ageing research in yeast, similar effects and points may play a role in other eukaryotic systems.

molecular biology↗

bioRxiv2020

Correlative single-molecule fluorescence barcoding of gene regulation in Saccharomyces cerevisiae

Most cells adapt to their environment by switching combinations of genes on and off through a complex interplay of transcription factor proteins (TFs). The mechanisms by which TFs respond to signals, move into the nucleus and find specific binding sites in target genes is still largely unknown. Single-molecule fluorescence microscopes, which can image single TFs in live cells, have begun to elucidate the problem. Here, we show that different environmental signals, in this case carbon sources, yield a unique single-molecule fluorescence pattern of foci of a key metabolic regulating transcription factor, Mig1, in the nucleus of the budding yeast, Saccharomyces cerevisiae. This pattern serves as a barcode of the gene regulatory state of the cells which can be correlated with cell growth characteristics and other biological function. HighlightsO_LISingle-molecule microscopy of transcription factors in live yeast C_LIO_LIBarcoding single-molecule nuclear fluorescence C_LIO_LICorrelation with cell growth characteristics C_LIO_LIGrowth in different carbon sources C_LI

biophysics↗

bioRxiv2018

Stoichiometry and Mobility Switching of a Morphogenetic Protein in Live Differentiating Cells

Spore formation following asymmetric cell division in Bacillus subtilis offers a model system to study development, morphogenesis and signal transduction in more complex organisms. Extensive biochemical and genetic details of its sporulation factors are known, however, the molecular mechanisms by which asymmetry is generated remain unclear. A crucial membrane phosphatase, SpoIIE, couples gene regulation to morphology changes, but how it performs different functions dependent on cell stage is unknown. We addressed this puzzle using high-speed single-molecule fluorescence microscopy on live B. subtilis expressing genomically encoded SpoIIE fluorescent protein fusions during sporulation. Copy number analysis indicated a few tens of SpoIIE at sporulation onset increasing to 400-600 molecules per cell following asymmetric cell division with up to 30% greater proportion in the forespore, corresponding to a concentration enhancement in the smaller forespore sufficient for differential dephosphorylation of an anti-sigma factor antagonist and activation of the forespore specific transcription factor, {sigma}F. Step-wise photobleach analysis indicates that SpoIIE forms tetramers capable of reversible oligomerisation to form clusters correlated with stage-specific functions. Specifically, low mobility SpoIIE clusters which initially localize to the asymmetric septum are released as mobile SpoIIE clusters around the forespore when phosphatase activity is manifested. SpoIIE is subsequently recaptured at the septum in a SpoIIQ-dependent manner. After mother cell engulfment of the forespore, SpoIIE is released as a mix of higher mobility clusters and tetramers. Our findings suggest that additional information captured in the changing state of multimerization and mobility enable one protein to perform different roles at different cell stages.\n\nSignificance/impactCertain bacteria undergo sporulation involving cells dividing asymmetrically. A crucial protein SpoIIE facilitates this morphological asymmetry and directly links it to asymmetry in gene expression. Here, we used specialized light microscopy, capable of observing single molecules, plus biophysics, genetics and biochemical tools, to monitor SpoIIE in single living bacteria in real time, allowing us to count how many molecules are present in different cell regions, and how mobile they are. We find that SpoIIE clusters and moves depending on development stages, indicating that it has different roles depending on other binding proteins and their cellular locations. Our results suggest that changes in molecular stoichiometry and mobility may be used as switches in more complex cell processes.

microbiology↗