Biology subjects

Wollman, A.

Publications and source records attributed to Wollman, A..

6 recordsLinked to original sources

bioRxiv2018

Single-molecule live cell imaging of Rep reveals the dynamic interplay between an accessory replicative helicase and the replisome

DNA replication requires strategies to cope with nucleoprotein barriers that impair the efficient translocation of the replisome. Biochemical and genetic studies indicate accessory helicases play essential roles in continuity of replication in the presence of nucleoprotein barriers, but how they operate in the native cellular environment is unclear. With high-speed single-molecule microscopy we determine the dynamic patterns of localization of genomically-encoded fluorescent protein constructs of the bacterial accessory helicase Rep and core replisome protein DnaQ in live E. coli cells. We demonstrate that Rep colocalizes with 70% of replication forks. Colocalisation is dependent upon interaction with replicative helicase DnaB, with an underlying hexameric stoichiometry of Rep indicating maximal occupancy of the single DnaB hexamer within the replisome. We find that Rep associates dynamically with the replisome with an average dwell time of 6.5 ms dependent on ATP hydrolysis, indicating rapid binding then translocation away from the fork. We also imaged the PriC replication restart factor given the known Rep-PriC functional interaction and observe Rep-replisome association is also dependent on the presence of PriC. Our findings suggest two Rep-replisome populations in vivo: one involving Rep continually associating with DnaB then translocating away to aid nucleoprotein barrier removal ahead of the fork, another assisting PriC-dependent reloading of DnaB if replisome progression fails. These new findings reveal how a single type of helicase is recruited to the replisome to provide two independent ways of underpinning replication of protein-bound DNA, a problem that all organisms face as they replicate their genomes.\n\nSignificance statementAll organisms face the challenge of proteins bound to DNA acting as barriers to prevent DNA replication. We have performed fluorescence imaging experiments on living bacteria to track the positions of the replication machinery, a protein called Rep which is involved in removing these barriers, and a protein called PriC believed to be involved with reloading the replication machinery if the original replication machinery breaks down. We find that Rep is very dynamic with continual binding and movement away from the replication machinery. Association with the replication machinery depends on both binding to the replication machinery directly and on PriC. Thus Rep can circumvent barriers in two independent ways: a strategy which may be relevant to all organisms.

biophysics↗

bioRxiv2018

EGF signalling in epithelial carcinoma cells utilizes preformed receptor homoclusters, with larger heteroclusters post activation

Epidermal growth factor (EGF) signalling regulates cell growth, differentiation and proliferation in epithelium and EGF receptor (EGFR) overexpression has been reported in several carcinoma types. Structural and biochemical evidence suggests EGF binding stimulates EGFR monomer-dimer transitions, activating downstream signalling. However, mechanistic details of ligand binding to functional receptors in live cells remain contentious. We report real time single-molecule TIRF of human epithelial carcinoma cells with negligible native EGFR expression, transfected with GFP-tagged EGFR, before and after receptor activation with TMR-labelled EGF ligand. Fluorescently labelled EGFR and EGF are simultaneously tracked to 40nm precision to explore stoichiometry and spatiotemporal dynamics upon EGF binding. Using inhibitors that block binding to EGFR directly, or indirectly through HER2, our results indicate that pre-activated EGFR consists of preformed homoclusters, while larger heteroclusters including HER2 form upon activation. The relative stoichiometry of EGFR to EGF after binding peaks at 2, indicating negative cooperativity of EGFR activation.

biophysics↗

bioRxiv2018

Staphylococcus aureus toxin LukSF dissociates from its membrane receptor target to enable renewed ligand sequestration

backgroundStaphylococcus aureus Panton Valentine Leukocidin (PVL) is a pore-forming toxin targeting the human C5a receptor (hC5aR), enabling this pathogen to battle the immune response by destroying phagocytes through targeted lysis. The mechanisms that contribute to rapid cell lysis are largely unexplored.\n\nResultsHere we show that cell lysis may be enabled by a process of toxins targeting receptor clusters and receptor recycling which allows multiple toxin pores to be formed close together. Using live cell single-molecule super-resolution imaging, Forster resonance energy transfer (FRET) and nanoscale total internal reflection fluorescence (TIRF) colocalization microscopy we visualized toxin pore formation in the presence of its natural docking ligand.\n\nConclusionsWe demonstrate disassociation of hC5aR from toxin complexes and simultaneous binding of new ligands. This effect may free mobile receptors to amplify hyper inflammatory reactions in early stages of microbial infections and have implications for several other similar bi-component toxins and the design of new antibiotics.

microbiology↗

bioRxiv2017

The Yeast Mig1 Transcriptional Repressor Is Dephosphorylated By Glucose-Dependent And Independent Mechanisms

Saccharomyces cerevisiae AMPK/Snf1 regulates glucose derepression of genes required for utilization of alternative carbon sources through the transcriptional repressor Mig1. It has been suggested that the Glc7-Reg1 phosphatase dephosphorylates Mig1. Here we report that Mig1 is dephosphorylated by Glc7-Reg1 in an apparently glucose-dependent mechanism but also by a mechanism independent of glucose and Glc7-Reg1. In addition to serine/threonine phosphatases another process including tyrosine phosphorylation seems crucial for Mig1 regulation. Taken together, Mig1 dephosphorylation appears to be controlled in a complex manner, in line with the importance for rapid and sensitive regulation upon altered glucose concentrations in the growth medium.

biochemistry↗

bioRxiv2017

Characterization Of The Stoichiometry Of The Complex Formed By Staphylococcal LukSF And Human C5aR Receptor In Living Cells

Staphylococcus aureus Panton Valentine Leukocidin (PVL) is a pore-forming toxin comprising protein subunits LukS and LukF. Binding of LukS to human C5a receptor (hC5aR) on leukocytes induces secondary binding of LukF and assembly of lytic complexes. Previous analysis suggests that PVL consists of 4-plus-4 LukS/LukF subunits but the exact stoichiometry between LukS, LukF and hC5aR is not yet known. In this study we determine the stoichiometry and spatiotemporal dynamics of functional LukS/LukF-hC5aR complexes in living eukaryotic cells. By using rapid total internal reflection fluorescence (TIRF) and single-molecule photobleaching analysis we found that tetrameric LukS-hC5aR complexes are formed within a cluster of receptors. Upon binding to hC5aR each LukS subunit binds LukF leading to lytic pore formation and simultaneous dissociation of receptors from the complex. Our findings corroborate a hetero-octamer model but provide a new view on the kinetics of crucial virulence factor assembly on integrated host cell membrane receptors.

biophysics↗

bioRxiv2017

Transcription factor clusters regulate genes in eukaryotic cells

Transcription is regulated through binding factors to gene promoters to activate or repress expression, however, the mechanisms by which factors find targets remain unclear. Using single-molecule fluorescence microscopy, we determined in vivo stoichiometry and spatiotemporal dynamics of a GFP tagged repressor, Mig1, from a paradigm signaling pathway of Saccharomyces cerevisiae. We find the repressor operates in clusters, which upon extracellular signal detection, translocate from the cytoplasm, bind to nuclear targets and turnover. Simulations of Mig1 configuration within a 3D yeast genome model combined with a promoter-specific, fluorescent translation reporter confirmed clusters are the functional unit of gene regulation. In vitro and structural analysis on reconstituted Mig1 suggests that clusters are stabilized by depletion forces between intrinsically disordered sequences. We observed similar clusters of a co-regulatory activator from a different pathway, supporting a generalized cluster model for transcription factors that reduces promoter search times through intersegment transfer while stabilizing gene expression.

biophysics↗