SCHOOL OF BIOCHEMISTRY

prospective postgraduates

MRC Bioimaging Studentships

Quantitative imaging of cellular dynamics

Project information
Professor George Banting: Dynamic, biophysical analysis of the organisation of lipid rafts.
Lipid rafts have been implicated in a broad range of fundamental and essential cellular functions and have been implicated in several human diseases (e.g. Alzheimer's, Parkinson's and prion diseases). This project will focus on an important lipid raft-associated protein, CD317/tetherin, and its role in the organisation of these plasma membrane domains. Extensive fixed and live cell imaging, allied with electron microscopy and single particle tracking will be used. Quantitative analysis of data from such approaches will help to elucidate the role of CD317 in human cells.

Rollason, R., Korolchuk, V., Hamilton, C. and Banting, G. (2009) A CD317(tetherin)/RICH2 complex plays a critical role in the organisation of the sub-apical actin cytoskeleton in polarised epithelial cells. J. Cell Biol. (in press)
Professor Pete Cullen: Quantitative imaging of phosphoinositide-regulated membrane trafficking in health and disease.
An increasing number of genetic diseases (e.g. various myopathies and neuropathies) are linked to perturbation in the metabolism of phosphoinositides known to regulate membrane trafficking. You will join an active research group that aims to gain new disease-related insight by defining, at the molecular level, how phosphoinositides regulate membrane trafficking. More specifically, you will use quantitative cell imaging to elucidate the spatial and temporal dynamics by which phosphoinositides regulate trafficking through the endosomal network, and how, by perturbing proteins implicated in Alzheimer’s, the dynamic state can be altered, leading to the formation of phenotypes diagnostic of this disease.

Wassmer T, Attar N, Harterink M, van Weering JRT, Traer CJ, Oakley JD, Goud B, Stephens DJ, Verkade P, Korswagen HC, Cullen PJ (2009). The retromer coat complex co-ordinates endosomal sorting and dynein-mediated transport, with carrier recognition by the trans-Golgi network. Developmental Cell 17, 110-122.
Dr Jon Hanley: Real-time confocal imaging of the AMPA receptor-associated protein PICK1.
AMPA receptor (AMPAR) trafficking is fundamental to changes in synaptic strength that underlie learning and memory. PICK1 is a BAR-domain protein that binds AMPARs and regulates their trafficking. Little is known about how PICK1 itself traffics. We will use YFP-tagged PICK1 constructs in hippocampal neurons visualised with live confocal imaging. The subcellular distribution and translocation of PICK1 will be analysed after physiological and pharmacological stimuli. PICK1 mutants will be tested to determine the molecular mechanisms of translocation. We will collaborate with experts in object tracking and positional mapping for quantitative, time-resolved positional analysis of PICK1 translocation.

Rocca D.L., Martin S., Jenkins E.L., and Hanley J.G. (2008). Inhibition of Arp2/3-mediated actin polymerisation by PICK1 regulates neuronal morphology and AMPA receptor endocytosis. Nature Cell Biology, 10: 259-271.
Professor Jeremy Henley: Application of light activated channel rhodopsin to study AMPA receptor trafficking.
Channelrhodopsin 2 is a light sensitive, nonselective cation channel that can noninvasively depolarise specific neurones to induce action potentials. It can therefore be used to control neuronal excitability and synaptic activity. This approach has tremendous potential for studying many aspects of activity dependent protein trafficking in neurones. The overall aim of this project is to do simultaneous, quantitative imaging of fluorescently tagged AMPARs and optical depolarisation of cells, axons, dendrites or individual synapses. These experiments will allow comparison of pre- and postsynaptic AMPAR trafficking and address the mechanisms underlying the transport, synaptic recruitment and retention of AMPARs.

Martin S., Nishimune A., Mellor J.R., and Henley J.M. (2007) SUMOylation regulates kainite-receptor-mediated synaptic transmission. Nature, 477: 321-325.
Dr Jon Lane: Defining the site of mammalian autophagosome assembly.
Autophagy promotes cell survival during nutrient/growth factor stress. It also delays aging and protects against diseases including cancer, neurodegeneration and infection. During autophagy cytoplasm is sequestered into double membrane autophagosomes that form de novo in the peripheral cytoplasm. The source of the autophagosome membrane in mammalian cells remains obscure. This project will use advanced live-cell imaging and in vitro motility assays to derive qualitative and quantitative information defining the site of autophagosome formation. Correlative light and electron microscopy (CLEM) with tomography will then be used to describe the ultrastructure of the autophagosome assembly site.

Betin VMS & Lane JD (2009) Caspase cleavage of Atg4D stimulates GABARAP-L1 processing and triggers mitochondrial targeting and apoptosis. Journal of Cell Science 122: 2554-2566.
Professor Paul Martin: A zebrafish model for WASp function during leukocyte migration.
Zebrafish are excellent model systems to study cell migration in vivo due to their translucency. We have identified two zebrafish mutants in WASp1 (the gene disrupted in Wiskott Aldrich Syndrome) that are linked to deficiencies in extravascular leukocyte migration. In this project, morphant zebrafish will be used to investigate the capacity of WASp deficient cells to diapedese through vessels walls - a key rate limiting step in the inflammatory response. We will devise ways to track and quantify diapesesis in fluorescently tagged leukocytes, and will investigate the roles of WASp in the assembly and function of podosomes - structures thought to contribute to leukocytic probing of the endothelial cell prior to transcellular migration

Cvejic A., Hall C., Bak-Maier M., Vega Fores M., Crosier P., Redd M.J. and Martin P (2008) Analysis of WASp function during the wound inflammatory response - live-cell imaging studies in zebrafish larvae. Journal of Cell Science 121: 3196-206.
Dr Harry Mellor: Signalling pathways in angiogenesis.
Angiogenesis is essential for formation of the vasculature during development, but is subverted in cancer to allow the growth of solid tumours. We are interested in the signalling pathways that control angiogenesis. The project goal is to develop high-content screens based on an assay that recapitulates capillary formation in vitro (see image). This assay allows us to target individual signalling proteins using RNAi. We will use high-resolution imaging of capillary formation to discover new targets for anti-angiogenic therapies. An important component will be the development of software (in MATLAB) for the automated analysis of angiogenesis, allowing large-scale screening of the drugable genome.

Gampel A., Moss, L., Jones, J.C., Norman J.C., Brunton, V. and Mellor H. (2006) VEGF regulates the mobilisation of VEGFR-2/KDR from an intracellular endothelial storage compartment. Blood 108, 2624-2631.
Dr Kate Nobes: Regulation of cell migration by Eph-ephrin signalling.
Cell-cell interactions play fundamental roles during embryonic development, and are critically involved in tumour progression and cancer metastasis. We work on Eph receptors and their ligands (the ephrins) - key mediators of social interactions between cells that play important roles in cancer. Ephrin/Eph receptor expression is often deregulated in cancer, and high expression of ephrins correlates with aggressive, metastatic tumour phenotypes. This project will develop and utilise quantitative live cell imaging probes (including FRET sensors) to measure Rho GTPase activity and cell migration in models of prostate cancer cell invasion.

Marston DJ, Dickinson S, Nobes CD. (2003) Rac-dependent trans-endocytosis of ephrinBs regulates Eph-ephrin contact repulsion. Nat Cell Biol. 5, 879-88.
Dr David Stephens: Mitotic regulation of ER-to-Golgi membrane traffic..
The first membrane trafficking step in mammalian cells is the export of secretory cargo from the ER. ER export is stopped during mitosis 1 but little is known of the mechanism underlying this mitotic inhibition. Understanding the mechanisms by which ER export is inhibited on entry into mitosis and restarted on exit from mitosis are therefore central to our understanding of cellular organization and function. This project seeks to define the mechanisms used by cells to ensure ordered inheritance of organelles during mitosis. The student will integrate skills in advanced microscopy, notably live cell imaging, with a systems analysis of membrane trafficking.

Stephens, D.J. De novo formation, fusion and fission of mammalian COPII-coated endoplasmic reticulum exit sites. EMBO rep. 4, 210-217 (2003).
Professor Jeremy Tavaré: Dynamics of GLUT4 trafficking and membrane fusion.
GLUT4 is an insulin sensitive glucose transporter that is expressed in fat and muscle cells. Insulin stimulates the trafficking of GLUT4-containing vesicles to the plasma membrane where they dock, dwell, occasionally translocate, and then either fuse or return back into the bulk cytoplasmic compartment - events that can be visualised by total internal reflection (TIRF) microscopy. We will develop computational methods to measure the kinetics of GLUT4 vesicle trafficking through automated inspection of TIRF images. Using this methodology, the impact of over-expression or depletion of key molecular components will be addressed in work that will help to define potential new therapeutic routes for the treatment of Types I and II diabetes.

Welsh, G.I., Leney, S.E., Lloyd-Lewis, B., Wherlock, M., Lindsay, A.J., McCaffrey, M. and Tavaré, J.M. (2007) Rip11 is a Rab11 and AS160-RabGAP binding protein required for insulin-stimulated glucose uptake in adipocytes. Journal of Cell Science 120, 4197-208.
Dr Paul Verkade: Studying intracellular sorting events.
Sorting inside the cell is essential to provide specificity and give organelles their unique identity. We try to understand how cells perform these tasks. The combination of live cell imaging of fluorescently labelled molecules with high resolution Electron Microscopy (so-called Correlative Light Electron Microscopy, CLEM) and even 3-Dimensional electron microscopy is currently the most powerful method to perform such transport studies. We feel it is essential to use the most advanced imaging tools to fully understand all the intracellular sorting routes. In order to have access to those advanced techniques we spent considerable time in developing new tools and techniques to tackle our scientific questions.

- Brown, E., J. Mantell, D.A. Carter, G. Tilly, and P. Verkade (2009). Studying intracellular transport using High-Pressure Freezing and Correlative Light Electron Microscopy. Seminars in Cell and developmental Biology. doi:10.1016/j.semcdb.2009.07.006.