ORCID Profile
0000-0002-6222-6297
Current Organisations
King's College London
,
Beijing Institute of Technology
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Publisher: Wiley
Date: 10-10-2013
DOI: 10.1111/IMR.12107
Abstract: Natural killer (NK) cells discriminate between healthy and unhealthy target cells through a balance of activating and inhibitory signals at direct intercellular contacts called immune synapses. Rearrangements in the cellular cytoskeleton have long been known to be critical in assembly of immune synapses. Here, through bringing together the vast literature on this subject, the number of different ways in which the cytoskeleton is important becomes evident. The dynamics of filamentous actin are critical in (i) creating the nanometer-scale organization of NK cell receptors, (ii) establishing cellular polarity, (iii) coordinating immune receptor and integrin-mediated signaling, and (iv) directing secretion of lytic granules and cytokines. The microtubule network also is important in the delivery of lytic granules and vesicles containing cytokines to the immune synapse. Together, these data establish that the cytoskeleton acts as a central regulator of this complex and dynamic process - and an enormous amount of NK cell biology is controlled through the cytoskeleton.
Publisher: Springer Science and Business Media LLC
Date: 12-05-2013
DOI: 10.1038/NI.2609
Abstract: The mechanisms by which Lat (a key adaptor in the T cell antigen receptor (TCR) signaling pathway) and the TCR come together after TCR triggering are not well understood. We investigate here the role of SNARE proteins, which are part of protein complexes involved in the docking, priming and fusion of vesicles with opposing membranes, in this process. Here we found, by silencing approaches and genetically modified mice, that the vesicular SNARE VAMP7 was required for the recruitment of Lat-containing vesicles to TCR-activation sites. Our results indicated that this did not involve fusion of Lat-containing vesicles with the plasma membrane. VAMP7, which localized together with Lat on the subsynaptic vesicles, controlled the phosphorylation of Lat, formation of the TCR-Lat-signaling complex and, ultimately, activation of T cells. Our findings suggest that the transport and docking of Lat-containing vesicles with target membranes containing TCRs regulates TCR-induced signaling.
Publisher: Bentham Science Publishers Ltd.
Date: 12-2008
Publisher: Elsevier BV
Date: 03-2016
Publisher: Wiley
Date: 17-06-2010
Abstract: The authors employed photoactivatable localization microscopy (PALM) and direct stochastic optical reconstruction microscopy (dSTORM) imaging and image analysis based on Ripley's K-function to quantify the distribution and heterogeneity of proteins at the cell plasma membrane. The membrane targeting sequence of the N-terminal region of the T cell receptor-pathway kinase Lck fused to the photo-convertible fluorescent protein tdEos (Lck(N10)-tdEos), clusters into sub-100 nm regions which cover approximately 7% of the cell surface. 2-channel PALM imaging of Lck(N10)-tdEos and the N-terminus of the kinase Src (Src(N15)-PS-CFP2) are demonstrated. Finally, T cell microclusters at the immune synapse are imaged at super-resolution using dSTORM, showing that conventional TIRF images contain unresolved, small clusters. These methods are generally applicable to other cell and fluorophore systems to quantify 2-D molecular clustering at nanometer scales.
Publisher: Frontiers Media SA
Date: 2012
Publisher: Public Library of Science (PLoS)
Date: 28-05-2008
Publisher: Oxford University Press (OUP)
Date: 20-01-2017
DOI: 10.1093/BIOINFORMATICS/BTX026
Abstract: Unlike conventional microscopy which produces pixelated images, SMLM produces data in the form of a list of localization coordinates—a spatial point pattern (SPP). Often, such SPPs are analyzed using cluster analysis algorithms to quantify molecular clustering within, for ex le, the plasma membrane. While SMLM cluster analysis is now well developed, techniques for analyzing fibrous structures remain poorly explored. Here, we demonstrate a statistical methodology, based on Ripley’s K-function to quantitatively assess fibrous structures in 2D SMLM datasets. Using simulated data, we present the underlying theory to describe fiber spatial arrangements and show how these descriptions can be quantitatively derived from pointillist datasets. We also demonstrate the techniques on experimental data acquired using the image reconstruction by integrating exchangeable single-molecule localization (IRIS) approach to SMLM, in the context of the fibrous actin meshwork at the T cell immunological synapse, whose structure has been shown to be important for T cell activation. Freely available on the web at github.com/RubyPeters/Angular-Ripleys-K. Implemented in MatLab. Supplementary data are available at Bioinformatics online.
Publisher: Wiley
Date: 20-01-2015
DOI: 10.1111/BPH.13022
Publisher: Springer Science and Business Media LLC
Date: 02-2023
Publisher: Elsevier BV
Date: 04-2017
Publisher: Oxford University Press (OUP)
Date: 03-03-2004
DOI: 10.1093/HMG/DDH113
Publisher: Springer Science and Business Media LLC
Date: 04-12-2012
DOI: 10.1038/NCOMMS2273
Abstract: Lipid microdomains are postulated to regulate many membrane-associated processes but have remained highly controversial. Here we provide the first direct evidence that the plasma membrane of intact, live cells is comprised of a sub-resolution mixture of approximately 76% ordered and 24% disordered lipid domains, which correspond to liquid-ordered and -disordered model membranes. These measurements were based on the unmixing of fluorescence lifetime decays (phasor analysis) obtained from environmentally sensitive membrane dyes that report the degree of lipid packing. Using the transmembrane protein Linker for Activation of T cells (LAT) as an ex le, we demonstrate that association with ordered domains retarded LAT diffusion and decreased clustering in meso-scaled protein domains as analysed by super-resolution microscopy. Our data therefore propose a membrane model in which the majority of the plasma membrane is covered by cholesterol-dependent, ordered lipid domains that contribute to the non-random distribution and diffusion of membrane constituents.
Publisher: Elsevier
Date: 2012
Publisher: Wiley
Date: 07-11-2017
DOI: 10.1111/TRA.12532
Abstract: During an immune response, T cells survey antigen presenting cells for antigenic peptides via the formation of an interface known as an immunological synapse. Among the complex and dynamic biophysical phenomena occurring at this interface is the trafficking of sub-synaptic vesicles carrying a variety of proximal signalling molecules. Here, we show that rather than being a homogeneous population, these vesicles display a ersity of membrane lipid order profiles, as measured using the environmentally sensitive dye di-4-ANEPPDHQ and multi-spectral TIRF microscopy. Using live-cell imaging, vesicle tracking and a variety of small molecule drugs to manipulate components of the actin and tubulin cytoskeleton, we show that the membrane lipid order of these vesicles correlate with their dynamics. Furthermore, we show that the key proximal signalling molecule Linker for Activation of T cells (LAT) is enriched in specific vesicle populations as defined by their higher membrane order. These results imply that vesicle lipid order may represent a novel regulatory mechanism for the sorting and trafficking of signalling molecules at the immunological synapse, and, potentially, other cellular structures.
Publisher: Cold Spring Harbor Laboratory
Date: 14-09-2022
DOI: 10.1101/2022.09.12.507560
Abstract: Single-molecule localisation microscopy produces data in the form of point-clouds. Here, we present a tool for assessing the similarity of two such point-clouds, which, unlike measures such as co-localisation, is insensitive to differences that are not preserved between data sets. The presented method can determine whether two point-clouds were generated from the same conditions and can identify from which of two experimental conditions an unseen point-cloud was likely derived.
Publisher: Springer Science and Business Media LLC
Date: 05-06-2011
DOI: 10.1038/NI.2049
Abstract: Engaged T cell antigen receptors (TCRs) initiate signaling through the adaptor protein Lat. In quiescent T cells, Lat is segregated into clusters on the cell surface, which raises the question of how TCR triggering initiates signaling. Using super-resolution fluorescence microscopy, we found that pre-existing Lat domains were neither phosphorylated nor laterally transported to TCR activation sites, which suggested that these clusters do not participate in TCR signaling. Instead, TCR activation resulted in the recruitment and phosphorylation of Lat from subsynaptic vesicles. Studies of Lat mutants confirmed that recruitment preceded and was essential for phosphorylation and that both processes were independent of surface clustering of Lat. Our data suggest that TCR ligation preconditions the membrane for vesicle recruitment and bulk activation of the Lat signaling network.
Publisher: Springer Science and Business Media LLC
Date: 02-12-2012
DOI: 10.1038/NI.2488
Abstract: Phosphorylation of the T cell antigen receptor (TCR) by the tyrosine kinase Lck is an essential step in the activation of T cells. Because Lck is constitutively active, spatial organization may regulate TCR signaling. Here we found that Lck distributions on the molecular level were controlled by the conformational states of Lck, with the open, active conformation inducing clustering and the closed, inactive conformation preventing clustering. In contrast, association with lipid domains and protein networks were not sufficient or necessary for Lck clustering. Conformation-driven Lck clustering was highly dynamic, so that TCR triggering resulted in Lck clusters that contained phosphorylated TCRs but excluded the phosphatase CD45. Our data suggest that Lck conformational states represent an intrinsic mechanism for the intermolecular organization of early T cell signaling.
Publisher: Hindawi Limited
Date: 2011
DOI: 10.1155/2011/521863
Abstract: Many infectious agents utilize CD46 for infection of human cells, and therapeutic applications of CD46-binding viruses are now being explored. Besides mediating internalization to enable infection, binding to CD46 can directly alter immune function. In particular, ligation of CD46 by antibodies or by measles virus can prevent activation of T cells by altering T-cell polarity and consequently preventing the formation of an immunological synapse. Here, we define a mechanism by which CD46 reorients T-cell polarity to prevent T-cell receptor signaling in response to antigen presentation. We show that CD46 associates with lipid rafts upon ligation, and that this reduces recruitment of both lipid rafts and the microtubule organizing centre to the site of receptor cross-linking. These data combined indicate that polarization of T cells towards the site of CD46 ligation prevents formation of an immunological synapse, and this is associated with the ability of CD46 to recruit lipid rafts away from the site of TCR ligation.
Publisher: Elsevier BV
Date: 07-2013
Publisher: Frontiers Media SA
Date: 2012
Publisher: Wiley
Date: 14-06-2012
Abstract: Recently developed super-resolution microscopy techniques are changing our understanding of lipid rafts and membrane organisation in general. The lipid raft hypothesis postulates that cholesterol can drive the formation of ordered domains within the plasma membrane of cells, which may serve as platforms for cell signalling and membrane trafficking. There is now a wealth of evidence for these domains. However, their study has hitherto been h ered by the resolution limit of optical microscopy, making the definition of their properties problematic and contentious. New microscopy techniques circumvent the resolution limit and, for the first time, allow the fluorescence imaging of structures on length scales below 200 nm. This review describes such techniques, particularly as applied to the study of membrane organisation, synthesising newly emerging facets of lipid raft biology into a state-of-the art model.
Publisher: Wiley
Date: 02-07-2009
DOI: 10.1111/J.1600-0854.2009.00908.X
Abstract: The mobility of membrane proteins is a critical determinant of their interaction capabilities and protein functions. The heterogeneity of cell membranes imparts different types of motion onto proteins immobility, random Brownian motion, anomalous sub-diffusion, 'hop' or confined diffusion, or directed flow. Quantifying the motion of proteins therefore enables insights into the lateral organisation of cell membranes, particularly membrane microdomains with high viscosity such as lipid rafts. In this review, we examine the hypotheses and findings of three main techniques for analysing protein dynamics: fluorescence recovery after photobleaching, single particle tracking and fluorescence correlation spectroscopy. These techniques, and the physical models employed in data analysis, have become increasingly sophisticated and provide unprecedented details of the biophysical properties of protein dynamics and membrane domains in cell membranes. Yet despite these advances, there remain significant unknowns in the relationships between cholesterol-dependent lipid microdomains, protein-protein interactions, and the effect of the underlying cytoskeleton. New multi-dimensional microscopy approaches may afford greater temporal and spatial resolution resulting in more accurate quantification of protein and membrane dynamics in live cells.
Publisher: Humana Press
Date: 18-09-2012
DOI: 10.1007/978-1-62703-137-0_6
Abstract: Photoactivated localization microscopy (PALM) and the related technique of Stochastic optical reconstruction microscopy (STORM) are super-resolution imaging methods based on the precise localization of single molecules. Instruments based on these techniques are now commercially available and are capable of generating images with lateral resolutions in the tens of nanometers range. Here, we give an overview of the current state of this technology including live-cell and 3D PALM and provide an in-depth protocol for performing PALM experiments in a fixed cell monolayer. This includes both the instrumentation/acquisition aspects and the data analysis required for generating quantitative, super-resolution data of molecular distributions. In this ex le, the system under investigation will be fixed HeLa cells transfected with the photo-switchable fluorescent protein PS-CFP2 targeted to the plasma membrane by fusion to the N-terminus of the protein kinase Lck.
Publisher: IEEE
Date: 07-2015
Location: United Kingdom of Great Britain and Northern Ireland
No related grants have been discovered for David Williamson.