Joshua Z Rappoport

University of Birmingham, Birmingham, England, United Kingdom

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Publications (46)276.97 Total impact

  • [Show abstract] [Hide abstract]
    ABSTRACT: Total internal reflection fluorescence (TIRF) microscopy has gained popularity in recent years among cell biologists due to its ability to clearly visualize events that occur at the adherent plasma membrane of cells. TIRF microscopy systems are now commercially available from nearly all microscope suppliers. This review aims to give the reader an introduction to the physical basis of TIRF and considerations that need to be made when purchasing a commercial system. We explain how TIRF can be combined with other microscopy modalities and describe how to use TIRF to study processes such as endocytosis, exocytosis, and focal adhesion dynamics. Finally, we provide a step-by-step guide to imaging and analyzing focal adhesion dynamics in a migrating cell using TIRF microscopy.
    Methods in molecular biology (Clifton, N.J.) 01/2015; 1251:1-23. · 1.29 Impact Factor
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    ABSTRACT: Tight junctions (TJs) link adjacent cells and are critical for maintenance of apical-basolateral polarity in epithelial monolayers. The TJ protein occludin functions in disparate processes, including wound healing and Hepatitis C Virus infection. Little is known about steady-state occludin trafficking into and out of the plasma membrane. Therefore, we determined the mechanisms responsible for occludin turnover in confluent Madin-Darby canine kidney (MDCK) epithelial monolayers. Using various biotin-based trafficking assays we observed continuous and rapid endocytosis of plasma membrane localised occludin (the majority internalised within 30 minutes). By 120 minutes a significant reduction in internalised occludin was observed. Inhibition of lysosomal function attenuated the reduction in occludin signal post-endocytosis and promoted co-localisation with the late endocytic system. Using a similar method we demonstrated that ∼20% of internalised occludin was transported back to the cell surface. Consistent with these findings, significant co-localisation between internalised occludin and recycling endosomal compartments was observed. We then quantified the extent to which occludin synthesis and transport to the plasma membrane contributes to plasma membrane occludin homeostasis, identifying inhibition of protein synthesis led to decreased plasma membrane localised occludin. Significant co-localisation between occludin and the biosynthetic secretory pathway was demonstrated. Thus, under steady-state conditions occludin undergoes turnover via a continuous cycle of endocytosis, recycling and degradation, with degradation compensated for by biosynthetic exocytic trafficking. We developed a mathematical model to describe the endocytosis, recycling and degradation of occludin, utilising experimental data to provide quantitative estimates for the rates of these processes.
    PLoS ONE 11/2014; 9(11):e111176. · 3.53 Impact Factor
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    ABSTRACT: Previously we have shown that expression of the deubiquitinating enzyme USP17 is required for cell proliferation and motility. More recently we reported that USP17 deubiquitinates RCE1 isoform 2 and thus regulates the processing of 'CaaX' motif proteins. Here we now show that USP17 expression is induced by epidermal growth factor and that USP17 expression is required for clathrin mediated endocytosis of epidermal growth factor receptor. In addition, we show that USP17 is required for the endocytosis of transferrin, an archetypal substrate for clathrin mediated endocytosis, and that USP17 depletion impedes plasma membrane recruitment of the machinery required for clathrin mediated endocytosis. Thus, our data reveal that USP17 is necessary for epidermal growth factor receptor and transferrin endocytosis via clathrin coated pits, indicate this is mediated via the regulation of the recruitment of the components of the endocytosis machinery and suggest USP17 may play a general role in receptor endocytosis.
    Oncotarget 07/2014; · 6.63 Impact Factor
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    ABSTRACT: RhoJ is a RhoGTPase expressed in endothelial cells and tumour cells which regulates cell motility, invasion, endothelial tube formation and focal adhesion numbers. This study aimed to further delineate the molecular function of RhoJ. Using timelapse microscopy RhoJ was found to regulate focal adhesion disassembly; siRNA-mediated knockdown of RhoJ increased focal adhesion disassembly time, while expression of an active mutant (daRhoJ) decreased it. Further, daRhoJ co-precipitated with the GIT-PIX complex, a regulator of focal adhesion disassembly. An interaction between daRhoJ and GIT1 was confirmed using yeast-2-hybrid, which depended on the Spa homology domain of GIT1. GIT1, GIT2, β-PIX and RhoJ all co-localised in focal adhesions and depended on each other for their recruitment to focal adhesions. Functionally, the GIT-PIX complex regulated endothelial tube formation, with knockdown of GIT1/2 or β-PIX phenocopying RhoJ knockdown. RhoJ knockout mice showed reduced tumour growth and diminished tumour vessel density, identifying a role for RhoJ in mediating tumour angiogenesis. These studies give novel insight into the molecular function of RhoJ in regulating cell motility and tumour vessel formation.
    Journal of cell science. 06/2014;
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    ABSTRACT: Directed cell migration is critical for numerous physiological processes including development and wound healing. However chemotaxis is also exploited during cancer progression. Recent reports have suggested links between vesicle trafficking pathways and directed cell migration. Very little is known about the potential roles of endocytosis pathways during metastasis. Therefore we performed a series of studies employing a previously characterised model for chemotactic invasion of cancer cells to assess specific hypotheses potentially linking endocytosis to directed cell migration. Our results demonstrate that clathrin‐mediated endocytosis is indispensable for epidermal growth factor (EGF) directed chemotactic invasion of MDA‐MB‐231 cells. Conversely, caveolar endocytosis is not required in this mode of migration. We further found that chemoattractant receptor (EGFR) trafficking occurs by clathrin‐mediated endocytosis and is polarised towards the front of migrating cells. However, we found no role for clathrin‐mediated endocytosis in focal adhesion disassembly in this migration model. Thus, this study has characterised the role of endocytosis during chemotactic invasion and has identified functions mechanistically linking clathrin‐mediated endocytosis to directed cell motility.
    Traffic 06/2014; 15(6). · 4.71 Impact Factor
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    ABSTRACT: Directed cell migration is critical for numerous physiological processes including development and wound healing. However chemotaxis is also exploited during cancer progression. Recent reports have suggested links between vesicle trafficking pathways and directed cell migration. Very little is known about the potential roles of endocytosis pathways during metastasis. Therefore we performed a series of studies employing a previously characterised model for chemotactic invasion of cancer cells to assess specific hypotheses potentially linking endocytosis to directed cell migration. Our results demonstrate that clathrin-mediated endocytosis is indispensable for epidermal growth factor (EGF) directed chemotactic invasion of MDA-MB-231 cells. Conversely, caveolar endocytosis is not required in this mode of migration. We further found that chemoattractant receptor (EGFR) trafficking occurs by clathrin-mediated endocytosis and is polarised towards the front of migrating cells. However, we found no role for clathrin-mediated endocytosis in focal adhesion disassembly in this migration model. Thus, this study has characterised the role of endocytosis during chemotactic invasion and has identified functions mechanistically linking clathrin-mediated endocytosis to directed cell motility.
    Traffic (Copenhagen, Denmark). 06/2014; 15(6):648-64.
  • Sylwia Jones, Joshua Z Rappoport
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    ABSTRACT: Epidermal growth factor (EGF) receptor (EGFR) signalling regulates diverse cellular functions, promoting cell proliferation, differentiation, migration, cell growth and survival. EGFR signalling is critical during embryogenesis, in particular in epithelial development, and disruption of the EGFR gene results in epithelial immaturity and perinatal death. EGFR signalling also functions during wound healing responses through accelerating wound re-epithelialisation, inducing cell migration, proliferation and angiogenesis. Upregulation of EGFR signalling is often observed in carcinomas and has been shown to promote uncontrolled cell proliferation and metastasis. Therefore aberrant EGFR signalling is a common target for anticancer therapies. Various reports indicate that EGFR signalling primarily occurs at the plasma membrane and EGFR degradation following endocytosis greatly attenuates signalling. Other studies argue that EGFR internalisation is essential for complete activation of downstream signalling cascades and that endosomes can serve as signalling platforms. The aim of this review is to discuss current understanding of intersection between EGFR signalling and trafficking.
    The international journal of biochemistry & cell biology 03/2014; · 4.89 Impact Factor
  • Sarah J Fletcher, Joshua Z Rappoport
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    ABSTRACT: Epithelial layers are integral for many physiological processes and are maintained by intercellular adhesive structures. During disease, these structures can disassemble, leading to breakdown of epithelia. TJs (tight junctions) are one type of intercellular adhesion. Loss of TJs has been linked to the pathogenesis of many diseases. The present review focuses on the role of vesicle trafficking in regulation of TJs, in particular trafficking of the TJ protein occludin. We examine how endocytosis and endosomal recycling modulate occludin localization under steady-state conditions and during stimulated TJ disassembly.
    Biochemical Society Transactions 02/2014; 42(1):195-200. · 2.59 Impact Factor
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    ABSTRACT: Growth factor signalling regulates multiple cellular functions and its misregulation has been linked to cancer development and progression. Ack1 (Activated Cdc42-associated kinase 1, TNK2), a non-receptor tyrosine kinase, has been implicated in trafficking and degradation of epidermal growth factor receptor (EGFR), yet the precise functions remain elusive. In this report we investigate the role of Ack1 in EGFR trafficking and show that Ack1 partially colocalises to Atg16L-positive structures upon EGF stimulation. These are proposed to be the isolation membranes during autophagosome formation. In addition we find that Ack1 colocalises and interacts with sequestosome 1 (p62/SQSTM1), a receptor for selective autophagy, via a ubiquitin associated domain and this interaction decreases upon EGF treatment, thus suggesting that Ack1 moves away from p62/SQSTM1 compartments. Furthermore, Ack1 interacts and colocalises with NBR1, another autophagic receptor, and this colocalisation is enhanced in the presence of ectopically expressed p62/SQSTM1. Finally, Ack1 knock-down results in accelerated lysosomal localisation of EGFR upon EGF treatment. Structure-function analyses of a panel of Ack1 deletion mutants have revealed key mechanistic aspects of these relationships. The Mig6-homology domain and clathrin binding domain both contribute to the colocalisation with EGFR, whereas the UBA domain is critical for the colocalisation with p62/SQSTM1, but not NBR1. Taken together, our studies demonstrate a novel role for Ack1 in diverting activated EGFR into a non-canonical degradative pathway, marked by association with p62/SQSTM1, NBR1 and Atg16L.
    Journal of Cell Science 01/2014; · 5.33 Impact Factor
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    ABSTRACT: Calcium phosphates are used widely as orthopaedic implants and in nanocrystalline form to enable the transfer of genetic material into cells. Despite widespread use, little is known about their fate after they have crossed the cell membrane. Here we present a method of surface modification of silicon-substituted hydroxyapatite (SiHA) through a silane group, which enables the engraftment of a fluorescent dye to facilitate real-time biological tracking. Surface modification of the nanocrystal surface was undertaken using (3-mercaptopropyl)trimethoxysilane (MPTS), which presented a thiol for the further attachment of a fluorophore. Successful modification of the surface was demonstrated using zeta potential measurements and fluorescence microscopy and the number of thiol groups at the surface was quantified using Ellman's reagent. In vitro experiments using the fluorescently modified particles enabled the discrimination of the calcium phosphate particulate from other biological debris following internalisation by a population of MC3T3 (pre-osteoblast) cells and the particles were shown to maintain fluorescence for 24 hours without quenching. The successful modification of the surface of SiHA with thiol groups offers the tantalising possibility of the intracellular growth factor delivery.
    J. Mater. Chem. B. 08/2013; 1(35).
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    ABSTRACT: Eps8 is involved in both cell signalling and receptor trafficking. It is a known phosphorylation substrate for two proteins involved in the fibroblast growth factor receptor (FGFR) signalling pathway: the receptor itself and Src. Here we report a differential proteomic analysis of Eps8 aimed to identify specific FGFR and Src family kinase dependent phosphosites and co-associated phosphodependent binding partners. This study reveals a total of 22 Eps8 pTyr and pSer/Thr phosphorylation sites, including those that are dependent on Src family and FGFR kinase activity. Peptide affinity purification of proteins that bind to a selection of the pTyr phosphosites has identified a range of novel Eps8 binding partners including members of the intracellular vesicle trafficking machinery (clathrin and AP-2), proteins which have been shown to regulate activated receptor trafficking (NBR1 and Vav2), and proteins involved in receptor signalling (IRS4 and Shp2). Collectively this study significantly extends the understanding of Eps8 post-translational modification by regulated phosphorylation, identifies novel Eps8 binding partners implicated in receptor trafficking and signalling, and confirms the functions of Eps8 at the nexus of receptor signalling and vesicular trafficking.
    PLoS ONE 01/2013; 8(4):e61513. · 3.53 Impact Factor
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    ABSTRACT: Fibroblast Growth Factor Receptors (FGFRs) mediate a wide spectrum of cellular responses critical for development and wound healing. However, aberrant FGFR activity leads to cancer. Activated growth factor receptors undergo stimulated endocytosis, but can continue to signal along the endocytic pathway. Endocytic trafficking controls the duration and intensity of signalling, and growth factor receptor signalling can lead to modifications of trafficking pathways. We have developed live-cell imaging methods for studying FGFR dynamics to investigate mechanisms that coordinate the interplay between receptor trafficking and signal transduction. Activated FGFR enters the cell following recruitment to pre-formed clathrin-coated pits (CCPs). However, FGFR activation stimulates clathrin-mediated endocytosis; FGF treatment increases the number of CCPs, including those undergoing endocytosis, and this effect is mediated by Src and its phosphorylation target Eps8. Eps8 interacts with the clathrin-mediated endocytosis machinery and depletion of Eps8 inhibits FGFR trafficking and immediate Erk signalling. Once internalized, FGFR passes through peripheral early endosomes en route to recycling and degredative compartments, via a Src and Eps8 dependent mechanism. Thus Eps8 functions as a key coordinator in the interplay between FGFR signalling and trafficking. This work provides the first detailed mechanistic analysis from growth factor receptor clustering at the cell surface to signal transduction and endocytic trafficking. As we have characterised the Src target Eps8 as a key regulator of FGFR signalling and trafficking, and identified the early endocytic system as the site of Eps8 mediated effects, this work provides novel mechanistic insight into the reciprocal regulation of growth factor receptor signalling and trafficking.
    Journal of Cell Science 11/2012; · 5.33 Impact Factor
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    ABSTRACT: Multiple intracellular transport pathways drive the formation, maintenance, and function of cilia, a compartmentalized organelle associated with motility, chemo-/mechano-/photosensation, and developmental signaling. These pathways include cilium-based intraflagellar transport (IFT) and poorly understood membrane trafficking events. Defects in ciliary transport contribute to the etiology of human ciliary disease such as Bardet-Biedl syndrome (BBS). In this study, we employ the genetically tractable nematode Caenorhabditis elegans to investigate whether endocytosis genes function in cilium formation and/or the transport of ciliary membrane or ciliary proteins. Here we show that localization of the clathrin light chain, AP-2 clathrin adaptor, dynamin, and RAB-5 endocytic proteins overlaps with a morphologically discrete periciliary membrane compartment associated with sensory cilia. In addition, ciliary transmembrane proteins such as G protein-coupled receptors concentrate at periciliary membranes. Disruption of endocytic gene function causes expansion of ciliary and/or periciliary membranes as well as defects in the ciliary targeting and/or transport dynamics of ciliary transmembrane and IFT proteins. Finally, genetic analyses reveal that the ciliary membrane expansions in dynamin and AP-2 mutants require bbs-8 and rab-8 function and that sensory signaling and endocytic genes may function in a common pathway to regulate ciliary membrane volume. These data implicate C. elegans endocytosis proteins localized at the ciliary base in regulating ciliary and periciliary membrane volume and suggest that membrane retrieval from these compartments is counterbalanced by BBS-8 and RAB-8-mediated membrane delivery.
    Current biology: CB 02/2012; 22(6):451-60. · 10.99 Impact Factor
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    ABSTRACT: Hepatitis C virus (HCV) leads to progressive liver disease and hepatocellular carcinoma. Current treatments are only partially effective, and new therapies targeting viral and host pathways are required. Virus entry into a host cell provides a conserved target for therapeutic intervention. Tetraspanin CD81, scavenger receptor class B member I, and the tight-junction proteins claudin-1 and occludin have been identified as essential entry receptors. Limited information is available on the role of receptor trafficking in HCV entry. We demonstrate here that anti-CD81 antibodies inhibit HCV infection at late times after virus internalization, suggesting a role for intracellular CD81 in HCV infection. Several tetraspanins have been reported to internalize via motifs in their C-terminal cytoplasmic domains; however, CD81 lacks such motifs, leading several laboratories to suggest a limited role for CD81 endocytosis in HCV entry. We demonstrate CD81 internalization via a clathrin- and dynamin-dependent process, independent of its cytoplasmic domain, suggesting a role for associated partner proteins in regulating CD81 trafficking. Live cell imaging demonstrates CD81 and claudin-1 coendocytosis and fusion with Rab5 expressing endosomes, supporting a role for this receptor complex in HCV internalization. Receptor-specific antibodies and HCV particles increase CD81 and claudin-1 endocytosis, supporting a model wherein HCV stimulates receptor trafficking to promote particle internalization.
    Journal of Virology 02/2012; 86(8):4305-16. · 4.65 Impact Factor
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    ABSTRACT: CTLA-4 is one of the most important negative regulators of the T cell immune response. However, the subcellular distribution of CTLA-4 is unusual for a receptor that interacts with cell surface transmembrane ligands in that CTLA-4 is rapidly internalized from the plasma membrane. It has been proposed that T cell activation can lead to stabilization of CTLA-4 expression at the cell surface. Here we have analyzed in detail the internalization, recycling, and degradation of CTLA-4. We demonstrate that CTLA-4 is rapidly internalized from the plasma membrane in a clathrin- and dynamin-dependent manner driven by the well characterized YVKM trafficking motif. Furthermore, we show that once internalized, CTLA-4 co-localizes with markers of recycling endosomes and is recycled to the plasma membrane. Although we observed limited co-localization of CTLA-4 with lysosomal markers, CTLA-4 was nonetheless degraded in a manner inhibited by lysosomal blockade. T cell activation stimulated mobilization of CTLA-4, as judged by an increase in cell surface expression; however, this pool of CTLA-4 continued to endocytose and was not stably retained at the cell surface. These data support a model of trafficking whereby CTLA-4 is constitutively internalized in a ligand-independent manner undergoing both recycling and degradation. Stimulation of T cells increases CTLA-4 turnover at the plasma membrane; however, CTLA-4 endocytosis continues and is not stabilized during activation of human T cells. These findings emphasize the importance of clathrin-mediated endocytosis in regulating CTLA-4 trafficking throughout T cell activation.
    Journal of Biological Chemistry 01/2012; 287(12):9429-40. · 4.60 Impact Factor
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    ABSTRACT: Increasing production and application of nanomaterials raises significant questions regarding the potential for cellular entry and toxicity of nanoparticles. It was observed that the presence of serum reduces the cellular association of 20 nm carboxylate-modified fluorescent polystyrene beads up to 20-fold, relative to cells incubated in serum-free media. Analysis by confocal microscopy demonstrated that the presence of serum greatly reduces the cell surface association of nanoparticles, as well as the potential for internalization. However, both in the presence and absence of serum, nanoparticle entry depends upon clathrin-mediated endocytosis. Finally, experiments performed with cells cooled to 4°C suggest that a proportion of the accumulation of nanoparticles in cells was likely due to direct permeabilization of the plasma membrane.
    International Journal of Nanomedicine 01/2012; 7:2045-55. · 4.20 Impact Factor
  • W.T.E. Pitkeathly, J.Z. Rappoport, E. Claridge
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    ABSTRACT: The dynamics of vesicle trafficking via the cell membrane and the tracking of their subsequent movements within the cell are of great interest in the biomedical sciences. One challenge of image analysis is following the vesicle's fate continuously from its formation at the membrane to its final destination, as it requires different microscopy techniques to image the complete journey. Total internal reflection fluorescence (TIRF) microscopy is used for imaging events at the cell membrane and laser scanning confocal microscopy (LSCM) is used for imaging the interior of the cell. We present a simple and robust method for co-registration of data sets from the two microscopy techniques. This method is validated on images generated by computer simulation of the image formation process in TIRF and in LSCM. The registration parameters are recovered with error less than 1% in presence of Gaussian noise up to SNR of 3.8dB. Registration of real microscopy data is shown and the accuracy of the retrieved parameters is compared and agrees well with values obtained manually and a difference in squares measure.
    Biomedical Imaging (ISBI), 2012 9th IEEE International Symposium on; 01/2012
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    ABSTRACT: Vesicle trafficking has long been suggested to play mechanistic roles in regulating directed cell migration. Recent evidence demonstrates that specific cell types and modes of migration involve transport of particular cargo through particular pathways. Epithelial wound healing is essential in tissue repair. However, investigations into the mechanisms regulating cell migration have mainly focused upon other models such as fibroblast-derived cells. Roles for vesicle trafficking pathways in regulating directed cell migration have been identified in recent studies, but mechanisms through which endocytosis might be involved in epithelial wound healing have not been as well studied. Therefore, we analysed potential regulatory roles for endocytosis pathways during epithelial cell motility, with a particular focus on cell adhesion. Specifically, and in contrast to studies in fibroblasts, we find no evidence for a link between endocytosis and the distribution of focal adhesions. However, the localisation of occludin, an essential component of tight junctions, is regulated through endocytosis. We identified epithelial monolayer wounding as a stimulus for endocytosis of occludin and have shown that internalisation of occludin from the wound edge occurs through clathrin-mediated endocytosis (CME) into a rab5-positive compartment. Thus, these studies have evaluated mechanistic roles for dynamin-dependant, CME and caveolar endocytosis during epithelial wound healing and have provided contrasting observations between analyses of cell motility in fibroblast models and epithelial cells. In conclusion, these studies have identified a novel mechanism for regulation of occludin during wound healing.
    Biology of the Cell 12/2011; 104(4):238-56. · 3.87 Impact Factor
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    ABSTRACT: Multi-modality microscopes incorporate multiple microscopy techniques into one module, imaging through a common objective lens. Simultaneous or consecutive image acquisition of a single specimen, using multiple techniques, increases the amount of measurable information available. In order to benefit from each modality, it is necessary to accurately co-register data sets. Intrinsic differences in the image formation process employed by each modality result in images which possess different characteristics. In addition, as a result of using different measurement devices, images often differ in size and can suffer relative geometrical deformations including rotation, scale and translation, making registration a complex problem. Current methods generally rely on manual input and are therefore subject to human error. Here, we present an automated image registration tool for fluorescence microscopy. We show that it successfully registers images obtained via total internal reflection fluorescence (TIRF), or epi-fluorescence, and confocal microscopy. Furthermore, we provide several other applications including channel merging following image acquisition through an emission beam splitter, and lateral stage drift correction. We also discuss areas of membrane trafficking which could benefit from application of Auto-Align. Auto-Align is an essential item in the advanced microscopist's toolbox which can create a synergy of single or multi-modality image data.
    Traffic 11/2011; 13(2):204-17. · 4.71 Impact Factor
  • Gut 09/2011; 54. · 13.32 Impact Factor

Publication Stats

805 Citations
276.97 Total Impact Points


  • 2008–2014
    • University of Birmingham
      • School of Biosciences
      Birmingham, England, United Kingdom
  • 2003–2010
    • The Rockefeller University
      • Laboratory of Cellular Biophysics
      New York City, NY, United States
  • 2007
    • Université René Descartes - Paris 5
      Lutetia Parisorum, Île-de-France, France