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The major human pathogen Staphylococcus aureus forms biofilms comprising of a fibrin network that increases attachment to surfaces and shields bacteria from the immune system. It secretes two coagulases, Coagulase (Coa) and von Willebrand factor binding protein (vWbp), which hijack the host coagulation cascade and trigger the formation of this fibrin clot. However, it is unclear how Coa and vWbp contribute differently to the localisation and dynamics of clot assembly in growing biofilms.Here, we address this question using high-precision time-resolved confocal microscopy of fluorescent fibrin to establish the spatiotemporal dynamics of fibrin clot formation in functional biofilms. We also use fluorescent fusion proteins to visualise the locations of Coa and vWbp in biofilms using both confocal laser scanning and high resolution highly inclined and laminated optical sheet microscopy. We visualise and quantify the spatiotemporal dynamics of fibrin production during initiation of biofilms in plasma amended with fluorescently labelled fibrinogen.We find that human serum stimulates coagulase production, and that Coa and vWbp loosely associate to the bacterial cell surface. Coa localises to cell surfaces to produce a surface-attached fibrin pseudocapsule but can diffuse from cells to produce matrix-associated fibrin. vWbp produces matrix-associated fibrin in the absence of Coa, and furthermore accelerates pseudocapsule production when Coa is present. Finally, we observe that fibrin production varies across the biofilm. A sub-population of non-dividing cells does not produce any pseudocapsule but remains within the protective extended fibrin network, which could be important for the persistence of S. aureus biofilm infections as antibiotics are more effective against actively growing cells.Our findings indicate a more cooperative role between Coa and vWbp in building fibrin networks than previously thought, and a bet-hedging cell strategy where some cells produce biofilm matrix while others do not, but instead assume a dormant phenotype that could be associated with antibiotic tolerance.

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 microscopy. 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.

Summary: Fluorescent biosensors are powerful tools allowing the concentration of metabolites and small molecules, and other properties such as pH and molecular crowding to be measured inside live single cells. The technology has been hampered by lack of simple software to identify cells and quantify biosensor signals in single cells. We have developed a new software package, FRETzel, to address this gap and demonstrate its use by measuring insulin-stimulated glucose uptake in individual fat cells of varying sizes for the first time. Our results support the long-standing hypothesis that larger fat cells are less sensitive to insulin than smaller ones, a finding that has important implications for the battle against type 2 diabetes. FRETzel has been optimized using the messy and crowded environment of cultured adipocytes, demonstrating its utility for quantification of FRET biosensors in a wide range of other cell types, including fibroblasts and yeast via a simple user-friendly quantitative interface.

This paper examines how smallholder farmers are differentially affected by agricultural expansion in their communities and landscapes. Contributing to the debate on intensification versus expansion, and implications for sustainability, we employ mixed methods research with smallholders in four communities in Ghana and Ethiopia to explore the impact of agricultural expansion among different social groups (men, women, the young, older, the poor and rich community members) across different timescales. Surveys were conducted with 200 households per community on livelihoods, land management practices and involvement in agricultural expansion. Focus group discussions were conducted with different categories of farming households to support the initial surveys.Results indicate that agricultural expansion may have both negative and positive effects on livelihood outcomes depending on timescale, participation in expansion, choices of other households, and individual roles in the household. Short-term wins are likely to result in losses in the long-term due to changing conditions. Households that have not expanded may lose benefits such as food and income from nature, due to the externalities resulting from the activities of expanders.

Here we use singe-molecule optical proteomics and computational analysis of live cell bacterial images, using millisecond super-resolved tracking and quantification of fluorescently labelled protein SpoIIE in single live Bacillus subtilis bacteria to understand its crucial role in cell development. Asymmetric cell division during sporulation in Bacillus subtilis presents a model system for studying cell development. SpoIIE is a key integral membrane protein phosphatase that couples morphological development to differential gene expression. However, the basic mechanisms behind its operation remain unclear due to limitations of traditional tools and technologies. We instead used advanced single-molecule imaging of fluorescently tagged SpoIIE in real time on living cells to reveal vital changes to the patterns of expression, localization, mobility and stoichiometry as cells undergo asymmetric cell division then engulfment of the smaller forespore by the larger mother cell. We find, unexpectedly, that SpoIIE forms tetramers capable of cell- and stage-dependent clustering, its copy number rising to ~ 700 molecules as sporulation progresses. We observed that slow moving SpoIIE clusters initially located at septa are released as mobile clusters at the forespore pole as phosphatase activity is manifested and compartment-specific RNA polymerase sigma factor, σF, becomes active. Our findings reveal that information captured in its quaternary organization enables one protein to perform multiple functions, extending an important paradigm for regulatory proteins in cells. Our findings more generally demonstrate the utility of rapid live cell single-molecule optical proteomics for enabling mechanistic insight into the complex processes of cell development during the cell cycle.

We present a novel experimental setup in which magnetic and optical tweezers are combined for torque and force transduction onto single filamentous molecules in a transverse configuration to allow simultaneous mechanical measurement and manipulation. Previously we have developed a super-resolution imaging module which, in conjunction with advanced imaging techniques such as Blinking assisted Localisation Microscopy (BaLM), achieves localisation precision of single fluorescent dye molecules bound to DNA of ~30 nm along the contour of the molecule; our work here describes developments in producing a system which combines tweezing and super-resolution fluorescence imaging. The instrument also features an acousto-optic deflector that temporally divides the laser beam to form multiple traps for high throughput statistics collection. Our motivation for developing the new tool is to enable direct observation of detailed molecular topological transformation and protein binding event localisation in a stretching/twisting mechanical assay that previously could hitherto only be deduced indirectly from the end-to-end length variation of DNA. Our approach is simple and robust enough for reproduction in the lab without the requirement of precise hardware engineering, yet is capable of unveiling the elastic and dynamic properties of filamentous molecules that have been hidden using traditional tools.