Faculty of Biological Sciences Astbury Centre for Structural Molecular Biology School of Molecular and Cellular Biology
Post-doctoral Research Fellow: Structural Biology (Electron Microscopy)
Fixed term for 3 years (with possible extension to 4 or 5 years subject to funding), available from 1 November 2013, or as soon as possible thereafter
ERC Advanced Grant “Amyloid Fibril Cytotoxicity: New Insights from Novel Approaches”
Applications are invited for an ERC-funded postdoctoral research assistant to join a dynamic, interdisciplinary team focusing on the use of structural and cell biological methods to investigate the molecular mechanisms of protein aggregation into amyloid fibrils in vitro and the effects of aggregated/disaggregated species on cellular homeostasis. This large programme involves collaboration between the groups of Professor Sheena Radford and Drs David Brockwell, Neil Ranson, Eric Hewitt and Prof Alison Ashcroft within the Astbury Centre for Structural Molecular Biology. Ultimately, the programme of research will involve the appointment of four post-doctoral fellows.
We are now seeking a post-doctoral research assistant with skills in cryo-electron microscopy/tomography to join this team. You will focus on the use of cryo- electronmicroscopy/tomography, and super-resolution light microscopy to study the structure of amyloid aggregates and the interactions they make with biological membranes. You will join an integrated, well-funded team of three post-doctoral fellows who will use Protein Chemistry, Dynamic Force Spectroscopy and Cell Biology (already appointed) to achieve our project aims.
You should have (or be close to completing) a PhD in structural molecular biology, biophysics, or a related discipline. Expertise in cryo-electron microscopy is essential.
University Grade 7 (£30,424 - £36,298 p.a.)
Informal enquiries may be made to Professor Sheena E Radford, email [email protected] or Dr Neil Ranson, email [email protected]
Closing Date: 16 October 2013
Ref: FBSMB0020
Click here for further information about working at the University of Leeds www.leeds.ac.uk/info/20025/university_jobs
Responsible to: Head of School of Molecular and Cellular Biology Reports to: Professor Sheena Radford and Dr Neil Ranson
Main Duties and Responsibilities
You will use a combination of electron microscopy (EM), electron tomography (ET) and super-resolution light microscopy to examine the structure of amyloid fibrils, and how those structures interact with biological membranes, organelles and intact cells. You will:
Examine the structure of amyloid fibrils using cryo-EM Study the effect amyloid fibrils have on synthetic liposomes, including assessing the effects of bilayer composition using cryo-EM/ET Determine the structural effects of amyloid fibrils on different membranous components of the endosome/lysosome pathway using cryo-EM/ET Characterise the effect of amyloid fibrils on the trafficking of proteins and lipids in the endo-lysosomal system Characterise the effects of amyloid on mammalian cells using cryo-EM/ET and super-resolution light microscopy. Collaborate closely with all members of the research team and integrate your own results with those of others Prepare written summaries of your work and meet with the full team to discuss these reports on a monthly basis Attend group meetings and present your work to others Generate and pursue independent and original research ideas in relation to the project Design and conduct a programme of investigation together with the principal investigators Evaluate the methods and techniques used and interpret the results obtained in order to relate such evaluations appropriately to the project Communicate and present research results through publication or at meetings/conferences Understand the broader issues relating to the management of research Contribute to the supervision of less experienced researchers as appropriate Liaise with research colleagues and support staff in relation to the project Take part in knowledge transfer activities where appropriate Maintain your own continuing professional development and act as a mentor to less experienced colleagues, as appropriate Maintain a safe work environment, including ensuring compliance with legislation and the undertaking of risk assessments Undertake any other duties commensurate with the post and grade as directed by the Director of the School or nominee
Specific Duties of the post
Use and develop cryo-EM/ET and super-resolution light microscopy methods to analyse amyloid structure and interactions Combine the results of cryo-EM experiments with those from other methods obtained by the other three post-doctoral research assistants appointed to the programme Discover new insights into amyloid assembly and disassembly mechanisms Evaluate and apply new developments in electron and light microscopy assess their qualities for the project Keep informed of recent advances in the fields of amyloid structure and amyloid disease
Career Expectations
The University of Leeds is committed to developing its staff. All staff participate in the Staff Review and Development scheme and we continue to work with individuals, supporting them to maximise their potential.
Progression to a higher grade is dependent on an individual taking on an increased level of responsibility. Vacancies that arise within the area or across the wider University are advertised on the HR website - http://jobs.leeds.ac.uk - to allow staff to apply for wider career development opportunities.
University Values
All staff are expected to operate in line with the university‟s values and standards, which work as an integral part of our strategy and set out the principles of how we work together. More information about the university‟s strategy and values is available at http://www.leeds.ac.uk/comms/strategy/.
Person Specification
Essential
PhD in Structural Molecular Biology, Biophysics or a related discipline (or be close to completion) Skilled experimentalist Experience in cryo-electron microscopy Experience in the processing of EM data for 3D structure determination Use of software for image analysis and data management Previous experience of combining the results of different approaches across different disciplines to develop new insights into a field of study Highly motivated and hardworking Ability to absorb new ideas and to master new techniques rapidly and efficiently Good verbal and written communication skills A high standard of English and the ability to write high quality articles for publication Ability to design, execute and write up experimental work independently Good organisational and time management skills Ability to deal with and prioritise varied tasks to meet project deadlines Ability to work independent, but able work effectively as part of a team Commitment to own professional development
Desirable
BSc in Biochemistry, Molecular Biology, Biophysics or a related subject Experience in the imaging of cells using fluorescence microscopy Experience in biochemical techniques e.g. subcellular fractionation, analysis of protein expression in mammalian cells, analysis of enzyme activity Previous experience in the analysis of lipid trafficking Experience in DNA techniques, protein expression and purification Experience in proteomic analysis of cells
Additional Information
Details of the terms and conditions of employment for all staff at the University, including information on pensions and benefits, are available on the Human Resources web pages accessible at http://hr.leeds.ac.uk/
Disclosure and Barring Service Checks
A Disclosure and Barring Service (DBS) Check is not required for this position. However, applicants who have unspent convictions must indicate this in the „other personal details‟ section of the application form and send details to the Recruitment Officer at [email protected].
Disabled Applicants
The post is located in the Faculty of Biological Sciences. Disabled applicants wishing to review access to the building are invited to contact the department direct. Additional information may be sought from the Recruitment Officer, email [email protected] or tel + 44 (0)113 343 1723.
Disabled applicants are not obliged to inform employers of their disability but will still be covered by the Equality Act once their disability becomes known.
Further information for applicants with disabilities, impairments or health conditions is available in the applicant guidance. Further Information
Background to the Programme and Composition of the Research Team This post is funded by an ERC Advanced Grant to Professor Sheena Radford, along with her collaborators Drs David Brockwell, Neil Ranson and Eric Hewitt and Professor Alison Ashcroft. These investigators are members of the Astbury Centre for Structural Molecular Biology and the School of Molecular and Cellular Biology at the University of Leeds. The programme, named AMYTOX, will ultimately employ four PDRAs and aims to use and develop a suite of novel and cutting edge biophysical, biochemical and cell biological methods to provide a step change in our understanding of amyloid fibril structure, dynamics and cytotoxicity. These findings will provide information vital for our understanding of these dynamic complexes in human disease; how these self-assembling systems associate and dissociate; and how these fascinating materials can be used in nanotechnology.
To achieve the goals of AMYTOX the principal investigator (Prof Sheena Radford) has combined her expertise in the biochemistry of amyloidosis; with collaborators who bring expertise in force spectroscopy (Dr David Brockwell), electron microscopy (Dr Neil Ranson), mass spectrometry (Prof Alison Ashcroft) and cell biology (Dr Eric Hewitt). Four post-doctoral research assistant positions will constitute the research team, alongside a technician and personal assistant.
The project Diseases associated with the deposition of normally soluble proteins as insoluble amyloid fibrils are a major threat to human health and are predicted to dominate world health needs in the coming century. Despite major research efforts in this area (with >58,000 publications on the topics of „amyloid and amyloidosis‟ over the last 20 years), a cure for amyloid diseases remains remote. Not all amyloid fibrils are deleterious, however, and examples of functional amyloid fibrils in microorganisms and man have been discovered. The unique physical properties of the cross- structure of amyloid have also been Figure 1. The amyloid assembly cascade results in a recognised, and amyloid fibrils multitude of species with different properties which can contribute to cell death. are being actively studied as potential new nanomaterials. The potential of amyloid fibrils as novel biomaterials and our hopes of finding a cure for amyloid diseases cannot be realised until the molecular mechanisms of amyloid assembly are better understood. There is an urgent need to determine the structure, stability and dynamics of amyloid fibrils in all-atom detail. We also need to identify the species responsible for amyloid-associated cytotoxicity and to determine how these species cause cell death (Fig. 1). In this programme of work we plan to examine amyloid-associated cytotoxicity in a new way, by focusing on the nature of fibril ends and their role in fibril stability and cytotoxic potential, which was inspired by recent results in which we demonstrated that fibril-associated cytotoxicity is enhanced when the fibrils are shortened to nanoscale length (100-300nm) particles by fragmentation (Fig. 2). We have also used cell biological and biophysical studies to show that purified lipid membranes are disrupted by amyloid fibrils. Based on these observations, we Figure 2. Cytotoxicity of short (nanoscale) have proposed that amyloid fibril ends and long (micron length) (1M monomer (which are increased in number by equivalent concentration) measured by MTT fragmentation) possess unique structural reduction in RAW 264.7 and SH-SY5Y and/or dynamic properties that endow cells. their cytotoxic potential and that interaction of fibril ends with biological membranes triggers cell death. Fibril ends are also of paramount importance in that they are uniquely able to recruit protein monomers and to seed fibril elongation. Overall, the goals of AMYTOX are to determine: (i) the structure of amyloid fibril ends and how this relates to their dynamics and cytotoxic properties; (ii) the nature of protein-protein and fibril-membrane interactions in amyloid formation in vitro and in vivo at the level of single molecules; and (iii) the cellular mechanisms by which amyloid fibrils disrupt cellular function.
Methods to be Employed To achieve the objectives of AMYTOX a broad range of complementary methods will be employed including non-covalent mass spectrometry, single molecule fluorescence, Theme 1 Theme 2 force spectroscopy, cell biology, electron FIBRIL STRUCTURE FIBRIL /STABILITY INTERACTIONS tomography and high resolution imaging, using Non-covalent MS Single mol. Single mol. force spectroscopy In vitro expts purified samples in vitro, as well as fluorescence experiments with intact cells. By combining AMYTOX Interaction Electron with cells these approaches with our ability to create tomography In cell +/- membranes electron fibrils with known, highly controlled or designed, tomography proteomics physical, dynamic and biological properties, the PALM STORM Objectives of AMYTOX (Fig. 3) are to: siRNA Cell biology CYTOTOXICTY use single molecule fluorescence Theme 3 spectroscopy and non-covalent mass spectrometry to identify and quantify the Figure 3: Overall aims of AMYTOX products of fibril dissociation and to determine whether fibril ends contribute to amyloid-associated cytotoxicity by molecular shedding. (Protein Chemist- already appointed); use single molecule dynamic force spectroscopy to determine the nature of the earliest protein-protein interactions in amyloid assembly and to examine the interaction between amyloid fibrils, fibril ends, membranes and cells (Dynamic Force Spectroscopist- already appointed); use cell biological and biochemical assays to elucidate how amyloid fibrils disrupt cellular function (Cell Biologist- already appointed). use super resolution imaging techniques (including stochastic optical reconstruction microscopy (STORM) and photoactivated localisation microscopy (PALM)), cryo-electron microscopy and electron tomography to determine the conformation of amyloid fibrils and to visualise amyloid fibrils in the act of disrupting membranes and cellular functions (Electron microscopist, this advertised position). The research will focus on three examples of amyloid fibrils, chosen to represent fibrils associated with disease (2-microglobulin (2m), the culprit protein of dialysis- related amyloidosis); functional amyloid (csgA, an E. coli protein that assembles into curli and is responsible for biofilm formation); and amyloid as a nanomaterial (using a de novo designed sequence that is evolutionarily unrelated to any known protein sequence (cc-p)). Other fibril samples will also be used where necessary.
Detailed Objectives of this post The objective of the work to be carried out is to determine the structure of amyloid fibril ends and how amyloid fibrils interact with cellular membranes and alter cellular function
The integrated programme of cell biological experiments described above will generate an array of information about the cellular machineries that interact with fibrils to cause cell dysfunction. In recent work we have used cryo-EM to show that short fibrils interact with cellular membranes predominantly via their ends and that binding vesicle distorts and disrupts the membrane bilayer (Fig. 4), leading to liposome leakage. The PDRA appointed to this position will extend these studies by using cryoEM/ET to examine: fibril
the structure of amyloid fibrils and amyloid fibril ends liposome the effect of amyloid fibrils on synthetic liposomes of
different lipid composition as well as natural Figure 4. Surface membranes rendered view the effect of amyloid fibrils on the trafficking of superposed on a tomogram section of proteins and lipids in the endo-lysosomal system short fibrils binding to the effects of amyloid on mammalian cells using liposomes (Radford & cryo-EM/ET and super-resolution light microscopy. Saibil (PNAS 2012). .. Recent Relevant Publications
Recent publications relevant to the post advertised include: Organelle proteomics: Identification of the exocytic machinery associated with the natural killer cell secretory lysosome. Casey, T.M., Meade, J.L. and Hewitt, E.W. (2007). Mol Cell Proteomics. 6, 767-80.
Investigation into the role of macrophages in the formation and degradation of 2- microglobulin amyloid fibrils. Morten, IJ, Gosal, WS, Radford, SE and Hewitt, EW. (2007). J Biol Chem. 282, 29691-700.
Globular tetramers of 2-microglobulin assemble into elaborate amyloid fibrils. White, H.E., Hodgkinson, J.L., Jahn, T.R., Cohen-Krausz, S., Gosal, W.S., Müller, S., Orlova, E.V., Radford, S.E. & Saibil, H.R. (2009) J. Mol. Biol. 389, 48-57
Amyloid fibril length distribution quantified by atomic force microscopy single particle image analysis. Xue, W.-F., Homans, S.W & Radford, S.E. (2009) PEDS, 22, 489- 496
Fibril fragmentation enhances amyloid cytotoxicity. Xue, W.F., Hellewell, A.L., Gosal, W.S., Homans, S.W., Hewitt, E.W. & Radford, S.E. (2009) J. Biol. Chem. 284, 34272- 34282
Stacked sets of parallel, in register -strands in 2-microglobulin amyloid fibrils revealed by site-directed spin labelling and chemical labeling. Ladner, C.L., Chen, M., Smith, D.P., Platt, G.W., Radford, S.E. & Langen, R. (2010) J. Biol. Chem., 285, 17137-7147
Fibril fragmentation in amyloid assembly and cytotoxicity – When size matters. Xue, W.F., Hellewell, A.L., Hewitt, E.W. & Radford, S.E. (2010) Prion, 4, 20-25
Conformational conversion during amyloid formation at atomic resolution. Eichner, T., Kalverda, A.P., Thompson, G.S., Homans, S.W & Radford, S.E. (2011) Molecular Cell 41, 161-172
Structure and dynamics of oligomeric intermediates in 2-microglobulin self- assembly. Smith, D.P., Woods. L.A., Radford, S.E. and Ashcroft, A.E. (2011) Biophys. J. 101, 1238-1247
Characterization of the response of primary cells relevant to dialysis-related amyloidosis to β2-microglobulin monomer and fibrils. Porter MY, Routledge KE, Radford SE, Hewitt EW. (2011). PLoS One. 6:e27353
Direct three-dimensional visualisation of membrane disruption by amyloid fibrils. Milanesi, L., Xue, W.F., Sheynis, T., Orlova, E.V., Hellewell, A.L., Jelinek, R., Hewitt, E.W., Radford, S.E. & Saibil, H.R. (2012) Proc. Natl. Acad. Sci. USA, 109, 20455- 20460
A full list of recent publications from the Principal Investigators can be found at http://www.astbury.leeds.ac.uk.
Location of the work You will be based in large, well-resourced laboratories within the Astbury Centre for Structural Molecular Biology (ACSMB) within the School of Molecular and Cellular Biology at Leeds. Professor Radford and Dr Ranson have large, recently refurbished laboratories that are closely located to each other and together their integrated research groups comprise of large team of ~20 post-doctoral workers and PhD students with a broad range of background and skills. These laboratories are located close to the MS facility under the directorship of Prof Ashcroft, the AFM suite used by Dr. Brockwell, and Cell Biology (Dr. Hewitt) who also form part of the ERC AMYTOX research team. The Principal Investigators also have close links with colleagues in the School of Physics and Astronomy, as well as with colleagues also working in the field of protein-protein interactions in the School of Chemistry. Our vibrant and dynamic group meets regularly both formally and informally to exchange ideas, problem solve and for social occasions. For more information about our laboratories please visit http://www.astbury.leeds.ac.uk/bmbsgi10/index.php and www.astbury.leeds.ac.uk.
Astbury Centre for Structural Molecular Biology The Astbury Centre for Structural Molecular Biology is a dynamic, multidisciplinary research centre involving 55 academic staff spanning physics, chemistry and the biological sciences at the University of Leeds. The Centre houses excellent research infrastructure for the purification and the biophysical and biochemical analysis of biomolecules. For electron microscopy (EM), the centre operates an FEI TecnaiF-20 field emission EM equipped with a Gatan US4000 CCD camera, and a GIF2002. We also run new, ccd-equipped Tecna-G2 Spirit and JEOL 1400 EMs, and a wide range of modern ancillary equipment for cryo-EM studies. For super-resolution light microscopy (LM), the Centre operates a custom built PALM/STORM setup and is funded to build a state of the art structured illumination microscope. For LM, we also operate inverted Zeiss LSM 510, LSM 700 and Delta Vision widefield deconvolution microscopes for live cell imaging and an upright Zeiss LSM 510 microscope for routine confocal microscopy. These microscopes, together with a Becton Dickenson BD-LSRFortessa for routine flow cytometry applications and a FACSAria II for cell sorting, will shortly be installed in newly refurbished space co-located with the EM unit. The Astbury Centre equipment base includes three high-field NMR instruments (500-750Mz plus cryo-probes) and a recently upgraded, in-house X-ray source, and a range of facilities for the analysis of binding using DSC, ITC, CD, FTIR, fluorescence spectroscopy, AUC and SPR, and kinetic analysis using stopped flow methods.
The facilities are run by experienced core-funded staff, who are on hand to train new users and to offer advice on experimental design.
For more information please look at http://www.fbs.leeds.ac.uk/facilities/em/ and http://www.fbs.leeds.ac.uk/facilities/cytometry/
School of Molecular and Cellular Biology The School (previously Institute), comprises some 40 principal investigators, and exists in parallel with our sister Schools of Biology and Biomedical Sciences, was formed in September 2005. The aim of the School is to provide a stimulating environment for the prosecution of world-class research. We have a strong emphasis on inter-disciplinary activity, with the aim of developing the boundaries between traditional disciplines. To this end, collaborations between members of SMCB and our sister Schools within FBS are strongly encouraged. Moreover, the Astbury Centre for Structural Molecular Biology is a cross-faculty centre that includes staff from FBS as well as the Faculty of Mathematics and Physical Sciences and the Faculty of Medicine and Health.
Faculty of Biological Sciences The Faculty of Biological Sciences is one of the leading groups of life-science researchers within the UK, offering superb facilities, providing a high quality research training environment and delivering an exceptional student education.
Our position amongst the UK elite for bioscience research was confirmed in the last government Research Assessment Exercise (RAE) 2008 when we were ranked 4th for biological sciences in the UK by the leading scientific journal Nature based on the number of staff producing research that was “world leading" or "internationally excellent”.
In addition to 110 academic staff, the Faculty has over 400 postdoctoral fellows and postgraduate students supported by a current active research grant portfolio of some £53m derived from a range of sources including charities, research councils, the European Union and industry.
With around 2000 undergraduate students and 150 taught postgraduate students, we are one of the largest life sciences faculties in the UK. Our programmes cover the breadth of the biological sciences with undergraduate programmes in the areas of biology, biochemistry, microbiology, sport and exercise sciences and medical sciences including physiology and neuroscience.
Significant investments in our infrastructure contribute to our dynamic and vibrant research environment, offering excellent opportunities for leading edge research focused around key areas, including neuroscience, sports and exercise science, membrane biology, and structural molecular biology.
The Faculty has three schools:
School of Biology School of Biomedical Sciences School of Molecular and Cellular Biology Find out more about the Faculty here.