Simon J Davis

University of Oxford, Oxford, England, United Kingdom

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Publications (111)1136.69 Total impact

  • Michael L Dustin · Simon J Davis
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    ABSTRACT: Phagocytosis is key for many organismal functions. In a recent issue of Cell, Freeman et al. (2016) demonstrate a feed-forward signaling mechanism wherein F-actin and integrin receptors drive contact formation between phagocytes and antibody-coated solid particles, signaling their engulfment. This mechanism translates nanoscale proximity effects into wider self-propagating signals.
    No preview · Article · Jan 2016 · Developmental Cell
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    ABSTRACT: Detecting intracellular calcium signaling with fluorescent calcium indicator dyes is often coupled with microscopy techniques to follow the activation state of non-excitable cells, including lymphocytes. However, the analysis of global intracellular calcium responses both at the single-cell level and in large ensembles simultaneously has yet to be automated. Here, we present a new software package, CalQuo (Calcium Quantification), which allows the automated analysis and simultaneous monitoring of global fluorescent calcium reporter-based signaling responses in up to 1000 single cells per experiment, at temporal resolutions of sub-seconds to seconds. CalQuo quantifies the number and fraction of responding cells, the temporal dependence of calcium signaling and provides global and individual calcium-reporter fluorescence intensity profiles. We demonstrate the utility of the new method by comparing the calcium-based signaling responses of genetically manipulated human lymphocytic cell lines.
    Full-text · Article · Nov 2015 · Scientific Reports
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    ABSTRACT: The extent to which Rhodopsin family G-protein-coupled receptors (GPCRs) form invariant oligomers is contentious. Recent single-molecule fluorescence imaging studies mostly argue against the existence of constitutive receptor dimers and instead suggest that GPCRs only dimerize transiently, if at all. However, whether or not even transient dimers exist is not always clear due to difficulties in unambiguously distinguishing genuine interactions from chance colocalizations, particularly with respect to short-lived events. Previous single-molecule studies have depended critically on calculations of chance colocalization rates and/or comparison with unfixed control proteins whose diffusional behavior may or may not differ from that of the test receptor. Here, we describe a single-molecule imaging assay that 1) utilizes comparisons with well-characterized control proteins, i.e., the monomer CD86 and the homodimer CD28, and 2) relies on cell fixation to limit artifacts arising from differences in the distribution and diffusion of test proteins versus these controls. The improved assay reliably reports the stoichiometry of the Glutamate-family GPCR dimer, γ-amino butyric acid receptor b2, whereas two Rhodopsin-family GPCRs, β2-adrenergic receptor and mCannR2, exhibit colocalization levels comparable to those of CD86 monomers, strengthening the case against invariant GPCR oligomerization.
    Preview · Article · Nov 2015 · Biophysical Journal
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    ABSTRACT: Far-field optical microscopy using focused light is an important tool in a number of scientific disciplines including chemical, (bio)physical and biomedical research, particularly with respect to the study of living cells and organisms. Unfortunately, the applicability of the optical microscope is limited, since the diffraction of light imposes limitations on the spatial resolution of the image. Consequently the details of, for example, cellular protein distributions, can be visualized only to a certain extent. Fortunately, recent years have witnessed the development of 'super-resolution' far-field optical microscopy (nanoscopy) techniques such as stimulated emission depletion (STED), ground state depletion (GSD), reversible saturated optical (fluorescence) transitions (RESOLFT), photoactivation localization microscopy (PALM), stochastic optical reconstruction microscopy (STORM), structured illumination microscopy (SIM) or saturated structured illumination microscopy (SSIM), all in one way or another addressing the problem of the limited spatial resolution of far-field optical microscopy. While SIM achieves a two-fold improvement in spatial resolution compared to conventional optical microscopy, STED, RESOLFT, PALM/STORM, or SSIM have all gone beyond, pushing the limits of optical image resolution to the nanometer scale. Consequently, all super-resolution techniques open new avenues of biomedical research. Because the field is so young, the potential capabilities of different super-resolution microscopy approaches have yet to be fully explored, and uncertainties remain when considering the best choice of methodology. Thus, even for experts, the road to the future is sometimes shrouded in mist. The super-resolution optical microscopy roadmap of Journal of Physics D: Applied Physics addresses this need for clarity. It provides guidance to the outstanding questions through a collection of short review articles from experts in the field, giving a thorough discussion on the concepts underlying super-resolution optical microscopy, the potential of different approaches, the importance of label optimization (such as reversible photoswitchable proteins) and applications in which these methods will have a significant impact. Mark Bates, Christian Eggeling
    Full-text · Article · Oct 2015 · Journal of Physics D Applied Physics
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    ABSTRACT: Some of the most important and interesting molecules in metazoan biology are glycoproteins. The importance of the carbohydrate component of these structures is often revealed by the disease phenotypes that manifest when the biosynthesis of particular glycoforms is disrupted. On the other hand, the presence of large amounts of carbohydrate can often hinder the structural and functional analysis of glycoproteins. There are often good reasons, therefore, for wanting to engineer and predefine the N-glycans present on glycoproteins, e.g., in order to characterize the functions of the glycans or facilitate their subsequent removal. Here, we describe in detail two distinct ways in which to usefully interfere with oligosaccharide processing, one involving the use of specific processing inhibitors, and the other the selection of cell lines mutated at gene loci that control oligosaccharide processing, using cytotoxic lectins. Both approaches have the capacity for controlled, radical alteration of oligosaccharide processing in eukaryotic cells used for heterologous protein expression, and have great utility in the structural analysis of glycoproteins.
    No preview · Article · Jun 2015 · Methods in molecular biology (Clifton, N.J.)
  • Erdinc Sezgin · Simon J. Davis · Christian Eggeling

    No preview · Article · Jun 2015 · Cell
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    Erdinc Sezgin · Simon J Davis · Christian Eggeling
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    ABSTRACT: The existence, nature, and role of highly ordered membrane domains, often referred to as lipid rafts, have been highly debated by cell biologists for many years. In this issue, Raghupathy et al. describe molecular mechanisms leading to the formation of ordered lipid-protein clusters. Copyright © 2015 Elsevier Inc. All rights reserved.
    Full-text · Article · Apr 2015 · Cell
  • James H Felce · Rachel G Knox · Simon J Davis
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    ABSTRACT: We show that in conventional, competition-based bioluminescence resonance energy transfer (BRET) assays of membrane protein stoichiometry, the presence of competitors can alter tagged-protein density and artifactually reduce energy transfer efficiency. A well-characterized monomeric type I membrane protein, CD86, and two G protein-coupled receptors β2AR and mCannR2, all of which behave as dimers in these conventional assays, exhibit monomeric behavior in an improved competition-based type-3 BRET assay designed to circumvent such artifacts.
    No preview · Article · Jun 2014 · Biophysical Journal
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    Full-text · Dataset · Mar 2014
  • Michael L Dustin · Simon J Davis
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    ABSTRACT: Acute inhibition of the regulatory kinase Csk reveals additional checkpoints for full activation of thymocytes via the T cell antigen receptor.
    No preview · Article · Jan 2014 · Nature Immunology
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    ABSTRACT: The glutamine synthetase-based protein expression system is widely used in industry and academia for producing recombinant proteins but relies on the cloning of transfected cells, necessitating substantial investments in time and handling. We streamlined the production of protein-producing cultures of Chinese hamster ovary cells using this system by co-expressing green fluorescent protein from an internal ribosomal entry site and selecting for high green fluorescent protein-expressing cells using fluorescence-activated cell sorting. Whereas other expression systems utilizing green fluorescent protein and fluorescence-activated cell sorting-based selection have relied on two or more sorting steps, we obtained stable expression of a test protein at levels >50% of that of an "average" clone and ~40% that of the "best" clone following a single sorting step. Versus clone-based selection, the principal savings are in the number of handling steps (reduced by a third), handling time (reduced by 70%), and the time needed to produce protein-expressing cultures (reduced by ~3 weeks). Coupling the glutamine synthetase-based expression system with product-independent selection in this way also facilitated the production of a hard-to-assay protein. Utilizing just a single fluorescence-activated cell sorting-based selection step, the new streamlined implementation of the glutamine synthetase-based protein expression system offers protein yields sufficient for most research purposes, where <10 mg/L of protein expression is often required but relatively large numbers of constructs frequently need to be trialed.
    Full-text · Article · Sep 2013 · BMC Biotechnology
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    ABSTRACT: Single-particle tracking (SPT) is widely used to study processes from membrane receptor organization to the dynamics of RNAs in living cells. While single-dye labeling strategies have the benefit of being minimally invasive, this comes at the expense of data quality; typically a data set of short trajectories is obtained and analyzed by means of the mean square displacements (MSD) or the distribution of the particles' displacements in a set time interval (jump distance, JD). To evaluate the applicability of both approaches, a quantitative comparison of both methods under typically encountered experimental conditions is necessary. Here we use Monte Carlo simulations to systematically compare the accuracy of diffusion coefficients (D-values) obtained for three cases: one population of diffusing species, two populations with different D-values, and a population switching between two D-values. For the first case we find that the MSD gives more or equally accurate results than the JD analysis (relative errors of D-values <6%). If two diffusing species are present or a particle undergoes a motion change, the JD analysis successfully distinguishes both species (relative error <5%). Finally we apply the JD analysis to investigate the motion of endogenous LPS receptors in live macrophages before and after treatment with methyl-β-cyclodextrin and latrunculin B.
    Full-text · Article · May 2013 · PLoS ONE
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    ABSTRACT: Persistent viral infections and tumors often pose a challenge to the immune system by exposing T and B cells to heightened antigenic loads and/or diverse immunoregulatory machinery. Conse­ quently, lymphocytes exposed to these environ­ ments are stricken with a state of dysfunction commonly referred to as immune exhaustion (Wherry, 2011). The term exhaustion refers to a state of functional decline that occurs when lymphocytes are chronically exposed to an anti­ gen. During a persistent viral infection, this is operationally defined for T cells as a progressive loss in their ability to lyse target cells and pro­ duce important cytokines such as IFN­, TNF, and IL­2 (Ahmed and Oldstone, 1988; Zajac et al., 1998; Brooks et al., 2005; Wherry et al., 2003). Exhaustion can in some instances be followed by clonal deletion, resulting in the physical removal of antiviral cells from the im­ mune repertoire (Moskophidis et al., 1993). It is now widely accepted that immune exhaustion contributes to the persistence of many viruses as well as tumors and is maintained by negative im­ mune regulators such as PD­1 (Barber et al., 2006; Velu et al., 2009), IL­10 (Brooks et al., 2006b; Ejrnaes et al., 2006), and CTLA­4 (Kaufmann et al., 2007). Recent studies have shown that ther­ apeutic blockade of negative immune regulators can reverse immune exhaustion and promote clearance of both viruses and tumors (Kim and Ahmed, 2010). Immunoregulatory blockade can also be added to therapeutic vaccination regimens to improve their efficacy (Brooks et al., 2008; Ha et al., 2008). In general, immune regulators are CORRESPONDENCE Dorian B. McGavern:
    Full-text · Dataset · Mar 2013
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    ABSTRACT: Immune responses to persistent viral infections and cancer often fail because of intense regulation of antigen-specific T cells-a process referred to as immune exhaustion. The mechanisms that underlie the induction of exhaustion are not completely understood. To gain novel insights into this process, we simultaneously examined the dynamics of virus-specific CD8(+) and CD4(+) T cells in the living spleen by two-photon microscopy (TPM) during the establishment of an acute or persistent viral infection. We demonstrate that immune exhaustion during viral persistence maps anatomically to the splenic marginal zone/red pulp and is defined by prolonged motility paralysis of virus-specific CD8(+) and CD4(+) T cells. Unexpectedly, therapeutic blockade of PD-1-PD-L1 restored CD8(+) T cell motility within 30 min, despite the presence of high viral loads. This result was supported by planar bilayer data showing that PD-L1 localizes to the central supramolecular activation cluster, decreases antiviral CD8(+) T cell motility, and promotes stable immunological synapse formation. Restoration of T cell motility in vivo was followed by recovery of cell signaling and effector functions, which gave rise to a fatal disease mediated by IFN-γ. We conclude that motility paralysis is a manifestation of immune exhaustion induced by PD-1 that prevents antiviral CD8(+) T cells from performing their effector functions and subjects them to prolonged states of negative immune regulation.
    Full-text · Article · Mar 2013 · Journal of Experimental Medicine
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    ABSTRACT: PD-1, a receptor expressed by T-cells, B-cells and monocytes, is a potent regulator of immune responses and a promising therapeutic target. The structure and interactions of human PD-1 are, however, incompletely characterized. We present the solution nuclear magnetic resonance (NMR)-based structure of the human PD-1 extracellular region and detailed analyses of its interactions with its ligands, PD-L1 and PD-L2. PD-1 has typical immunoglobulin superfamily topology, but differs at the edge of the GFCC' sheet which is flexible and completely lacks a C″ strand. Changes in PD-1 backbone NMR signals induced by ligand binding suggest that, whilst binding is centred on the GFCC' sheet, PD-1 is engaged by its two ligands differently and in ways incompletely explained by crystal structures of mouse PD-1/ligand complexes. The affinities of these interactions and that of PD-L1 with the costimulatory protein B7-1, measured using surface plasmon resonance, are significantly weaker than expected. The three- to four-fold greater affinity of PD-L2 versus PD-L1 for human PD-1 is principally due to the three-fold smaller dissociation rate for PD-L2 binding. Isothermal titration calorimetry revealed that the PD-1/PD-L1 interaction is entropically driven, whereas PD-1/PD-L2 binding has a large enthalpic component. Mathematical simulations based on the biophysical data and quantitative expression data suggest an unexpectedly limited contribution of PD-L2 to PD-1 ligation during interactions of activated T-cells with antigen presenting cells. These findings provide a rigorous structural and biophysical framework for interpreting the important functions of PD-1, and reveal that potent inhibitory signalling can be initiated by weakly interacting receptors.
    Full-text · Article · Feb 2013 · Journal of Biological Chemistry
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    ABSTRACT: Determining the organization of key molecules on the surface of live cells in two dimensions and how this changes during biological processes, such as signalling, is a major challenge in cell biology and requires methods with nanoscale spatial resolution and high temporal resolution. Here, we review biophysical tools, based on scanning ion conductance microscopy and single-molecule fluorescence and the combination of both of these methods, which have recently been developed to address these issues. We then give examples of how these methods have been be applied to provide new insights into cell membrane organization and function, and discuss some of the issues that will need to be addressed to further exploit these methods in the future.
    Full-text · Article · Feb 2013 · Philosophical Transactions of The Royal Society B Biological Sciences
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    Preview · Article · Jan 2013 · Biophysical Journal
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    Full-text · Article · Jan 2013 · Biophysical Journal
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    Preview · Article · Jan 2013 · Biophysical Journal
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    Full-text · Article · Jan 2013 · Biophysical Journal

Publication Stats

6k Citations
1,136.69 Total Impact Points

Institutions

  • 1992-2015
    • University of Oxford
      • • MRC Human Immunology Unit
      • • Nuffield Department of Clinical Medicine
      • • Division of Structural Biology (STRUBI)
      • • Sir William Dunn School of Pathology
      Oxford, England, United Kingdom
  • 2013
    • University of Cambridge
      Cambridge, England, United Kingdom
  • 1996-2013
    • Oxford University Hospitals NHS Trust
      • Nuffield Department of Medicine
      Oxford, England, United Kingdom
  • 2009
    • Institute of Genetics and Molecular Medicine
      Edinburgh, Scotland, United Kingdom
  • 2006
    • University of Porto
      Oporto, Porto, Portugal
  • 1994
    • Tufts University
      Бостон, Georgia, United States