Biology subjects

Leake, M. C.

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

51 records · Page 3Linked to original sources

bioRxiv2020

The Mechanism of Vesicle Solubilization by the Detergent Sodium Dodecyl Sulfate

Membrane solubilization by sodium dodecyl sulfate (SDS) is indispensable for many established biotech-nological applications, including viral inactivation and protein extraction. Although the ensemble thermo-dynamics have been thoroughly explored, the underlying molecular dynamics have remained inaccessible, owing to major limitations of traditional measurement tools. Here, we integrate multiple advanced biophysical approaches to gain multi-angle insight into the time-dependence and fundamental kinetic steps associated with the solubilization of single sub-micron sized vesicles in response to SDS. We find that the accumulation of SDS molecules on in-tact vesicles triggers biphasic solubilization kinetics comprising an initial vesicle expansion event followed by rapid lipid loss and micellization. Our findings support a general mechanism of detergent-induced membrane solubilization and we expect the framework of correlative biophysical technologies presented here will form a general platform for elucidating the complex kinetics of membrane perturbation induced by a wide variety of surfactants and disrupting agents.

biophysics↗

bioRxiv2020

A nucleoid-associated protein bends and bridges DNA in a multiplicity of topological states with varying specificity

Nucleoid-associated proteins (NAPs) are crucial in organizing prokaryotic DNA and regulating genes. Vital to these activities are complex nucleoprotein structures, however, how these form remains unclear. Integration host factor (IHF) is an Escherichia coli NAP that creates very sharp bends in DNA at sequences relevant to several functions including transcription and recombination, and is also responsible for general DNA compaction when bound non-specifically. We show that IHF-DNA structural multimodality is more elaborate than previously thought, and provide insights into how this drives mechanical switching towards strongly bent DNA. Using single-molecule atomic force microscopy and atomic molecular dynamics simulations we find three binding modes in roughly equal proportions: "associated" (73{degrees} of DNA bend), "half-wrapped" (107{degrees}) and "fully-wrapped" (147{degrees}), only the latter occurring with sequence specificity. We show IHF bridges two DNA double helices through non-specific recognition that gives IHF a stoichiometry greater than one and enables DNA mesh assembly. We observe that IHF-DNA structural multiplicity is driven through non-specific electrostatic interactions that we anticipate to be a general NAP feature for physical organization of chromosomes.

biophysics↗

bioRxiv2020

SerraNA: a program to infer elastic constants from local to global using nucleic acids simulation data

The resistance of DNA to stretch, twist and bend is broadly well estimated by experiments and is important for gene regulation and chromosome packing. However, their sequence-dependence and how bulk elastic constants emerge from local fluctuations is less understood. Here, we present SerraNA, which is an open software that calculates elastic parameters of double-stranded nucleic acids from dinucleotide length up to the whole molecule using ensembles from numerical simulations. The program reveals that global bendability emerge from local periodic bending angles in phase with the DNA helicoidal shape. We also apply SerraNA to the whole set of 136 tetra-bp combinations and we observe a high degree of sequence-dependence for all elastic parameters with differences over 200%. Tetramers with TA and CA base-pair steps are especially flexible, while tetramers containing AA and AT tend to be the most rigid. Our results thus suggest AT-rich motifs generate extreme mechanical properties depending of the exact sequence ordering, which seems critical for creating strong global bendability on longer sequences when phased properly. SerraNA is a tool to be applied in the next generation of interdisciplinary investigations to further understand what determines the elasticity of DNA. Graphical TOC Entry O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=81 SRC="FIGDIR/small/004945v2_ufig1.gif" ALT="Figure 1"> View larger version (18K): org.highwire.dtl.DTLVardef@18aa2ddorg.highwire.dtl.DTLVardef@ca5e02org.highwire.dtl.DTLVardef@1e1febeorg.highwire.dtl.DTLVardef@1b772b_HPS_FORMAT_FIGEXP M_FIG C_FIG

biophysics↗

bioRxiv2020

Correlating single-molecule characteristics of the yeast aquaglyceroporin Fps1 with environmental perturbations directly in living cells

Membrane proteins play key roles at the interface between the cell and its environment by mediating selective import and export of molecules via plasma membrane channels. Despite a multitude of studies on transmembrane channels, understanding of their dynamics directly within living systems is limited. To address this, we correlated molecular scale information from living cells with real time changes to their microenvironment. We employed super-resolved millisecond fluorescence microscopy with a single-molecule sensitivity, to track labelled molecules of interest in real time. We use as example the aquaglyceroporin Fps1 in the yeast Saccharomyces cerevisiae to dissect and correlate its stoichiometry and molecular turnover kinetics with various extracellular conditions. In this way we shed new light on aspects of architecture and dynamics of glycerol-permeable plasma membrane channels.

biophysics↗

bioRxiv2020

A glucose starvation response governs endocytic trafficking and eisosomal retention of surface cargoes

Eukaryotic cells adapt their metabolism to the extracellular environment. Downregulation of surface cargo proteins in response to nutrient stress reduces the burden of anabolic processes whilst elevating catabolic production in the lysosome. We use yeast to show that glucose starvation triggers a transcriptional response that simultaneously increases internalisation from the plasma membrane whilst supressing recycling from endosomes back to the surface. Nuclear export of the Mig1 transcriptional repressor in response to glucose starvation increases levels of the Yap1801/02 clathrin adaptors, which is sufficient to increase cargo internalisation. We also show Gpa1, which coordinates endosomal lipid homeostasis, is required for surface recycling of cargo. Nuclear translocation of Mig1 increases GPA2 levels and inhibits recycling, potentially by diverting Gpa1 to the surface and interfering with its endosomal role in recycling. Finally, we show glucose starvation results in transcriptional upregulation of many eisosomal factors, which serve to sequester a portion of nutrient transporters to persist the starvation period and maximise nutrient uptake upon return to replete conditions. This latter mechanism provides a physiological benefit for cells to rapidly recover from glucose starvation. Collectively, this remodelling of the surface protein landscape during glucose starvation calibrates metabolism to available nutrients.

cell biology↗

bioRxiv2019

Biophysical characterization of DNA origami nanostructures reveals inaccessibility to intercalation binding sites

Intercalation of drug molecules into synthetic DNA nanostructures formed through self-assembled origami has been postulated as a valuable future method for targeted drug delivery. This is due to the excellent biocompatibility of synthetic DNA nanostructures, and high potential for flexible programmability including facile drug release into or near to target cells. Such favourable properties may enable high initial loading and efficient release for a predictable number of drug molecules per nanostructure carrier, important for efficient delivery of safe and effective drug doses to minimise non-specific release away from target cells. However, basic questions remain as to how intercalation-mediated loading depends on the DNA carrier structure. Here we use the interaction of dyes YOYO-1 and acridine orange with a tightly-packed 2D DNA origami tile as a simple model system to investigate intercalation-mediated loading. We employed multiple biophysical techniques including single-molecule fluorescence microscopy, atomic force microscopy, gel electrophoresis and controllable damage using low temperature plasma on synthetic DNA origami samples. Our results indicate that not all potential DNA binding sites are accessible for dye intercalation, which has implications for future DNA nanostructures designed for targeted drug delivery.

biophysics↗

bioRxiv2019

Unveiling the multi-step solubilization mechanism of single vesicles by detergents.

The solubilization of membranes by detergents is critical for many technological applications and has become widely used in biochemistry research to induce cell rupture, extract cell constituents, and to purify, reconstitute and crystallize membrane proteins. The thermodynamic details of solubilization have been extensively investigated, but the kinetic aspects remain poorly understood. Here we used a combination of single-vesicle Forster resonance energy transfer (svFRET), fluorescence correlation spectroscopy and quartz-crystal microbalance with dissipation monitoring to access the real-time kinetics and elementary solubilization steps of sub-micron sized vesicles, which are inaccessible by conventional diffraction-limited optical methods. Real-time injection of a non-ionic detergent, Triton X, induced biphasic solubilization kinetics of surface-immobilized vesicles labelled with the Dil/DiD FRET pair. The nanoscale sensitivity accessible by svFRET allowed us to unambiguously assign each kinetic step to distortions of the vesicle structure comprising an initial fast vesicle-swelling event followed by slow lipid loss and micellization. We expect the svFRET platform to be applicable beyond the sub-micron sizes studied here and become a unique tool to unravel the complex kinetics of detergent-lipid interactions.

biophysics↗

bioRxiv2019

Targeting the trypanosome kinetochore with CLK1 protein kinase inhibitors

The kinetochore is a macromolecular structure that assembles on the centromeres of chromosomes and provides the major attachment point for spindle microtubules during mitosis. In Trypanosoma brucei the proteins that make up the kinetochore are highly divergent, with the inner kinetochore comprising at least 20 distinct and essential proteins (KKT1-20) that include four protein kinases, CLK1 (KKT10), CLK2 (KKT19), KKT2 and KKT3. We performed a phenotypic screen of T. brucei bloodstream forms with a Novartis kinase-focused inhibitor library, which identified a number of selective inhibitors with potent pan-kinetoplastid activity. Deconvolution of an amidobenzimidazole series using a selection of 37 T. brucei mutants that over-express known essential protein kinases identified CLK1 as the primary target. Biochemical studies show that the irreversible competitive inhibition of CLK1 is dependent on a Michael acceptor forming an irreversible bond with C215 in the ATP binding pocket, a residue that is not present in human CLK1, thereby providing selectivity. Chemical inhibition of CLK1 impairs inner kinetochore recruitment and compromises cell cycle progression, leading to cell death. We show that KKT2 is a substrate for CLK1 and identify phosphorylation of S508 to be essential for KKT2 function and for kinetochore assembly. We propose that CLK1 is part of a novel signalling cascade that controls kinetochore function via phosphorylation of the inner kinetochore protein kinase KKT2. This work highlights a novel drug target for trypanosomatid parasitic protozoa and a new chemical tool for investigating the function of their divergent kinetochores.

microbiology↗

bioRxiv2019

Single-organelle quantification reveals the stoichiometric and structural variability of carboxysomes dependent on the environment

The carboxysome is a complex, proteinaceous organelle that plays essential roles in carbon assimilation in cyanobacteria and chemoautotrophs. It comprises hundreds of protein homologs that self-assemble in space to form an icosahedral structure. Despite its significance in enhancing CO2 fixation and potentials in bioengineering applications, the formation of carboxysomes and their structural composition, stoichiometry and adaptation to cope with environmental changes remain unclear. Here we use live-cell single-molecule fluorescence microscopy, coupled with confocal and electron microscopy, to decipher the absolute protein stoichiometry and organizational variability of single {beta}-carboxysomes in the model cyanobacterium Synechococcus elongatus PCC7942. We determine the physiological abundance of individual building blocks within the icosahedral carboxysome. We further find that the protein stoichiometry, diameter, localization and mobility patterns of carboxysomes in cells depend sensitively on the microenvironmental levels of CO2 and light intensity during cell growth, revealing cellular strategies of dynamic regulation. These findings, also applicable to other bacterial microcompartments and macromolecular self-assembling systems, advance our knowledge of the principles that mediate carboxysome formation and structural modulation. It will empower rational design and construction of entire functional metabolic factories in heterologous organisms, for example crop plants, to boost photosynthesis and agricultural productivity.\n\nOne Sentence SummaryDetermination of absolute protein stoichiometry reveals the organizational variability of carboxysomes in response to microenvironmental changes\n\nThe authors responsible for distribution of materials integral to the findings presented in this article in accordance with the policy described in the Instructions for Author (www.plantcell.org) is: Lu-Ning Liu (luning.liu@liverpool.ac.uk).

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

Ultra-fast super-resolution imaging of biomolecular mobility in tissues

Super-resolution techniques have addressed many biological questions, yet molecular quantification at rapid timescales in live tissues remains challenging. We developed a light microscopy system capable of sub-millisecond sampling to characterize molecular diffusion in heterogeneous aqueous environments comparable to interstitial regions between cells in tissues. We demonstrate our technique with super-resolution tracking of fluorescently labelled chemokine molecules in a collagen matrix and ex vivo lymph node tissue sections, outperforming competing methods.

biophysics↗

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↗