Publications (72)505.67 Total impact
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Article: Super-resolution imaging by localization microscopy.
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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 example, 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.Methods in molecular biology (Clifton, N.J.) 01/2013; 950:81-93. -
Article: Creating adhesive and soluble gradients for imaging cell migration with fluorescence microscopy.
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ABSTRACT: Cells can sense and migrate towards higher concentrations of adhesive cues such as the glycoproteins of the extracellular matrix and soluble cues such as growth factors. Here, we outline a method to create opposing gradients of adhesive and soluble cues in a microfluidic chamber, which is compatible with live cell imaging. A copolymer of poly-L-lysine and polyethylene glycol (PLL-PEG) is employed to passivate glass coverslips and prevent non-specific adsorption of biomolecules and cells. Next, microcontact printing or dip pen lithography are used to create tracks of streptavidin on the passivated surfaces to serve as anchoring points for the biotinylated peptide arginine-glycine-aspartic acid (RGD) as the adhesive cue. A microfluidic device is placed onto the modified surface and used to create the gradient of adhesive cues (100% RGD to 0% RGD) on the streptavidin tracks. Finally, the same microfluidic device is used to create a gradient of a chemoattractant such as fetal bovine serum (FBS), as the soluble cue in the opposite direction of the gradient of adhesive cues.Journal of Visualized Experiments 01/2013; -
Article: Characterization of a new series of fluorescent probes for imaging membrane order.
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ABSTRACT: Visualization and quantification of lipid order is an important tool in membrane biophysics and cell biology, but the availability of environmentally sensitive fluorescent membrane probes is limited. Here, we present the characterization of the novel fluorescent dyes PY3304, PY3174 and PY3184, whose fluorescence properties are sensitive to membrane lipid order. In artificial bilayers, the fluorescence emission spectra are red-shifted between the liquid-ordered and liquid-disordered phases. Using ratiometric imaging we demonstrate that the degree of membrane order can be quantitatively determined in artificial liposomes as well as live cells and intact, live zebrafish embryos. Finally, we show that the fluorescence lifetime of the dyes is also dependent on bilayer order. These probes expand the current palate of lipid order-sensing fluorophores affording greater flexibility in the excitation/emission wavelengths and possibly new opportunities in membrane biology.PLoS ONE 01/2013; 8(2):e52960. · 4.09 Impact Factor -
Article: Sub-resolution lipid domains exist in the plasma membrane and regulate protein diffusion and distribution.
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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 example, 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.Nature Communications 12/2012; 3:1256. · 7.40 Impact Factor -
Article: Conformational states of the kinase Lck regulate clustering in early T cell signaling.
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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.Nature Immunology 12/2012; · 26.01 Impact Factor -
Article: Fluorescence localization microscopy: The transition from concept to biological research tool.
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ABSTRACT: Localization microscopy techniques are super-resolution fluorescence imaging methods based on the detection of individual molecules. Despite the relative simplicity of the microscope setups and the availability of commercial instruments, localization microscopy faces unique challenges. While achieving super-resolution is now routine, issues concerning data analysis and interpretation mean that revealing novel biological insights is not. Here, we outline why data analysis and the design of robust test samples may hold the key to harness the full potential of localization microscopy.Communicative & integrative biology 07/2012; 5(4):345-9. -
Article: Using an electrical potential to reversibly switch surfaces between two states for dynamically controlling cell adhesion.
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ABSTRACT: Smart surfaces presenting both antifouling molecules with a charged functional group at their distal end, and molecules that are terminated by RGD peptides for cell adhesion, were fabricated and characterized (see picture). By applying potentials of +300 or -300 mV, the surfaces could be dynamically switched to make the peptide accessible or inaccessible to cells.Angewandte Chemie International Edition 06/2012; 51(31):7706-10. · 13.45 Impact Factor -
Article: The lipid raft hypothesis revisited--new insights on raft composition and function from super-resolution fluorescence microscopy.
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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 hampered 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.BioEssays 06/2012; 34(9):739-47. · 4.95 Impact Factor -
Article: HIV taken by STORM: super-resolution fluorescence microscopy of a viral infection.
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ABSTRACT: The visualization of viral proteins has been hindered by the resolution limit of conventional fluorescent microscopes, as the dimension of any single fluorescent signal is often greater than most virion particles. Super-resolution microscopy has the potential to unveil the distribution of proteins at the resolution approaching electron microscopy without relying on morphological features of existing characteristics of the biological specimen that are needed in EM. Using direct stochastic optical reconstruction microscopy (dSTORM) to achieve a lateral resolution of 15-20 nm, we quantified the 2-D molecular distribution of the major structural proteins of the infectious human immunodeficiency virus type 1 (HIV-1) before and after infection of lymphoid cells. We determined that the HIV-1 matrix and capsid proteins undergo restructuring soon after HIV-1 infection. This study provides the proof-of-concept for the use of dSTORM to visualize the changes in the molecular distribution of viral proteins during an infection.Virology Journal 05/2012; 9:84. · 2.34 Impact Factor -
Chapter: The Actin Cytoskeleton and Membrane Organisation in T Lymphocytes
01/2012: pages 103-122; , ISBN: 978-1-61779-787-3 -
Chapter: Optical Techniques for Imaging Membrane Domains in Live Cells (Live-Cell Palm of Protein Clustering)
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ABSTRACT: It is now recognized that the plasma membrane is not homogeneous but instead contains a variety of membrane microdomains. These include lipid microdomains (lipid rafts) and protein clusters which exist on a range of size and time scales but are often small and short-lived. The small size and dynamic nature of membrane domains has made them difficult to study by conventional fluorescence microscopy approaches. Photoactivated localization microscopy (PALM) is a super-resolution technique capable of localizing the positions of individual molecules with tens of nanometers precision. Here, we describe a method for imaging membrane proteins using PALM, including live-cell PALM, to detect the molecular clustering of plasma membrane proteins using a statis-tical cluster-analysis method based on Ripley's K-function. While the method is applicable to a wide variety of proteins in various biological systems, to illus-trate the technique, we will image and analyze the clustering behavior of the adaptor protein Linker for activation of T cells (LAT) at the T cell immunological Methods in Enzymology, Volume 504 # 2012 Elsevier Inc.01/2012; -
Article: How does the kinase Lck phosphorylate the T cell receptor? Spatial organization as a regulatory mechanism.
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ABSTRACT: T cell signaling begins with the ligation of the T cell antigen receptor (TCR) by a cognate peptide and the phosphorylation of the receptor's immunoreceptor tyrosine-based activation motif domains by the kinase Lck. However, the canonical receptor model is insufficient to explain how the constitutively active kinase Lck can discriminate between non-ligated and ligated TCRs. Here, we discuss the factors that are thought to regulate the spatial distribution of the TCR and Lck, and therefore critically influence TCR signaling initiation.Frontiers in immunology. 01/2012; 3:167. -
Article: Optical techniques for imaging membrane domains in live cells (live-cell palm of protein clustering).
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ABSTRACT: It is now recognized that the plasma membrane is not homogeneous but instead contains a variety of membrane microdomains. These include lipid microdomains (lipid rafts) and protein clusters which exist on a range of size and time scales but are often small and short-lived. The small size and dynamic nature of membrane domains has made them difficult to study by conventional fluorescence microscopy approaches. Photoactivated localization microscopy (PALM) is a super-resolution technique capable of localizing the positions of individual molecules with tens of nanometers precision. Here, we describe a method for imaging membrane proteins using PALM, including live-cell PALM, to detect the molecular clustering of plasma membrane proteins using a statistical cluster-analysis method based on Ripley's K-function. While the method is applicable to a wide variety of proteins in various biological systems, to illustrate the technique, we will image and analyze the clustering behavior of the adaptor protein Linker for activation of T cells (LAT) at the T cell immunological synapse [Williamson, D. J., Owen, D. M., Rossy, J., Magenau, A., Wehrmann, M., Gooding, J. J. , and Gaus, K. (2011). Pre-existing clusters of the adaptor Lat do not participate in early T cell signaling events. Nat. Immunol.12, 655-662.].Methods in enzymology 01/2012; 504:221-35. · 1.90 Impact Factor -
Article: The integration of signaling and the spatial organization of the T cell synapse.
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ABSTRACT: Engagement of the T cell antigen receptor (TCR) triggers signaling pathways that lead to T cell selection, differentiation and clonal expansion. Superimposed onto the biochemical network is a spatial organization that describes individual receptor molecules, dimers, oligomers and higher order structures. Here we discuss recent findings and new concepts that may regulate TCR organization in naïve and memory T cells. A key question that has emerged is how antigen-TCR interactions encode spatial information to direct T cell activation and differentiation. Single molecule super-resolution microscopy may become an important tool in decoding receptor organization at the molecular level.Frontiers in immunology. 01/2012; 3:352. -
Article: Electrochemical "switching" of Si(100) modular assemblies.
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ABSTRACT: We report on a modular approach for producing well-defined and electrochemically switchable surfaces on Si(100). The switching of these surfaces is shown to change a Si(100) surface from resistant to cell adsorption to promoting cell adhesion. The electrochemical conversion of the modified electrode surface is demonstrated by X-ray photoelectron spectroscopy, X-ray reflectometry, contact angle and cell adhesion studies.Journal of the American Chemical Society 12/2011; 134(2):844-7. · 9.91 Impact Factor -
Article: Annexin A6 is an organizer of membrane microdomains to regulate receptor localization and signalling.
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ABSTRACT: Annexin A6 (AnxA6) belongs to the conserved annexin protein family--a group of Ca(2+) -dependent membrane binding proteins. It is the largest of all annexin proteins and upon activation, binds to negatively charged phospholipids in the plasma membrane and endosomes. In addition, AnxA6 associates with cholesterol-rich membrane microdomains termed lipid rafts. Membrane cholesterol triggers Ca(2+) -independent translocation of AnxA6 to membranes and AnxA6 levels determine the number of caveolae, a form of specialized rafts at the cell surface. AnxA6 also has an F-actin binding domain and interacts with cytoskeleton components. Taken together, this suggests that AnxA6 has a scaffold function to link membrane microdomains with the organization of the cytoskeleton. Such a link facilitates AnxA6 to participate in plasma membrane repair and it would also impact on receptor signalling at the cell surface, growth factor, and lipoprotein receptor trafficking, Ca(2+) -channel activity and T cell activation. Hence, the regulation of cell surface receptors by AnxA6 may be facilitated by its unique structure that allows recruitment of interaction partners and simultaneously bridging specialized membrane domains with cortical actin surrounding activated receptors.International Union of Biochemistry and Molecular Biology Life 11/2011; 63(11):1009-17. · 3.51 Impact Factor -
Article: Phagocytosis of IgG-coated polystyrene beads by macrophages induces and requires high membrane order.
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ABSTRACT: The biochemical composition and biophysical properties of cell membranes are hypothesized to affect cellular processes such as phagocytosis. Here, we examined the plasma membranes of murine macrophage cell lines during the early stages of uptake of immunoglobulin G (IgG)-coated polystyrene particles. We found that the plasma membrane undergoes rapid actin-independent condensation to form highly ordered phagosomal membranes, the biophysical hallmark of lipid rafts. Surprisingly, these membranes are depleted of cholesterol and enriched in sphingomyelin and ceramide. Inhibition of sphingomyelinase activity impairs membrane condensation, F-actin accumulation at phagocytic cups and particle uptake. Switching phagosomal membranes to a cholesterol-rich environment had no effect on membrane condensation and the rate of phagocytosis. In contrast, preventing membrane condensation with the oxysterol 7-ketocholesterol, even in the presence of ceramide, blocked F-actin dissociation from nascent phagosomes and particle uptake. In conclusion, our results suggest that ordered membranes function to co-ordinate F-actin remodelling and that the biophysical properties of phagosomal membranes are essential for phagocytosis.Traffic 08/2011; 12(12):1730-43. · 4.92 Impact Factor -
Article: Spacing of integrin ligands influences signal transduction in endothelial cells.
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ABSTRACT: The physical attributes of the extracellular matrix play a key role in endothelium function by modulating the morphology and phenotype of endothelial cells. Despite the recognized importance of matrix-cell interactions, it is currently not known how the arrangement of adhesive ligands affects the morphology, signal transduction processes, and migration of endothelial cells. We aimed to study how endothelial cells respond to the average spatial arrangement of integrin ligands. We designed functionalized silicon surfaces with average spacing ranging from nanometers to micrometers of the peptide arginine-glycine-aspartic acid (RGD). We found that endothelial cells adhered to and spread on surfaces independently of RGD-to-RGD spacing. In contrast, organization within focal adhesions (FAs) was extremely sensitive to ligand spacing, requiring a nanoscaled average RGD spacing of 44 nm to form lipid raft domains at FAs. The localized membrane organization strongly correlated with the signaling efficiencies of integrin activation and regulated vascular endothelial growth factor (VEGF)-induced signaling events. Importantly, this modulation in signal transduction directly affected the migratory ability of endothelial cells. We conclude that endothelial cells sense nanoscaled variations in the spacing of integrin ligands, which in turn influences signal transduction processes. Average RGD spacing similar to that found in fibronectin leads to lipid raft accumulation at FAs, enhances sensitivity to VEGF stimulation, and controls migration in endothelial cells.Biophysical Journal 08/2011; 101(4):764-73. · 3.65 Impact Factor -
Article: Different functionalization of the internal and external surfaces in mesoporous materials for biosensing applications using "click" chemistry.
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ABSTRACT: We report the use of copper(I)-catalyzed alkyne-azide cycloaddition reaction (CuAAC) to selectively functionalize the internal and external surfaces of mesoporous materials. Porous silicon rugate filters with narrow line width reflectivity peaks were employed to demonstrate this selective surface functionalization approach. Hydrosilylation of a dialkyne species, 1,8-nonadiyne, was performed to stabilize the freshly fabricated porous silicon rugate filters against oxidation and to allow for further chemical derivatization via "click" CuAAC reactions. The external surface was modified through CuAAC reactions performed in the absence of nitrogen-based Cu(I)-stabilizing species (i.e., ligand-free reactions). To subsequently modify the interior pore surface, stabilization of the Cu(I) catalyst was required. Optical reflectivity measurements, water contact angle measurements, Fourier transform infrared spectroscopy (FTIR), and X-ray photoelectron spectroscopy (XPS) were used to demonstrate the ability of the derivatization approach to selectively modify mesoporous materials with different surface chemistry on the exterior and interior surfaces. Furthermore, porous silicon rugate filters modified externally with the cell-adhesive peptide Gly-Arg-Gly-Asp-Ser (GRGDS) allowed for cell adhesion via formation of focal adhesion points. Results presented here demonstrate a general approach to selectively modify mesoporous silicon samples with potential applications for cell-based biosensing.Langmuir 01/2011; 27(1):328-34. · 4.19 Impact Factor -
Article: Mesoporous silicon photonic crystal microparticles: towards single-cell optical biosensors.
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ABSTRACT: In this paper we demonstrate the possibility of modifying porous silicon (PSi) particles with surface chemistry and recognition molecules (antibodies) such that these devices could potentially be used for single-cell identification or sensing. This is achieved by modifying PSi Rugate filters via hydrosilylation with surface chemistry that serves firstly, to protect the silicon surfaces from oxidation; secondly, renders the surfaces resistant to nonspecific adsorption of proteins and cells and thirdly, allows further functionality to be added such as the coupling of antibodies. The surface chemistry remained unchanged after sonication of the PSi to form PSi microparticles. The ability to monitor the spectroscopic properties of microparticles, and shifts in the optical signature due to changes in the refractive index of the material within the pore space, is demonstrated. The particles are shown to remain stable in physiological buffers and human blood for longer than one week. Finally, the modification of the PSi particles with functional antibodies is achieved.Faraday Discussions 01/2011; 149:301-17; discussion 333-56. · 5.00 Impact Factor
Top co-authors
Top Journals
Institutions
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2013
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University of South Wales
Pontypridd, WLS, United Kingdom
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2003–2013
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University of New South Wales
- • Lowy Cancer Research Centre
- • School of Chemistry
- • Centre for Vascular Research (CVR)
- • School of Medical Sciences
Kensington, New South Wales, Australia -
Heart Research Institute
Newtown, New South Wales, Australia
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2011
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INSERM, GIP CYCERON
Caen, Basse-Normandie, France
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2009
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Université Pierre et Marie Curie Paris 6
- Centre de Recherche des Cordeliers
Paris, Ile-de-France, France
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2007–2009
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University of Oxford
- Sir William Dunn School of Pathology
Oxford, ENG, United Kingdom
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