2016 Crick PhD Positions

2016 Crick PhD Student Recruitment

1

2016 Crick PhD Positions This document provides information on the PhD positions available to start the Crick PhD Programme in September 2016. Positions are listed alphabetically by supervisor’s surname.

Positions by Supervisor, in same order as this document: Dominique Bonnet | Dissecting the heterogeneity of the human Haematopoietic (blood) stem cell compartment Simon Boulton | Mechanistic analysis of pre-synaptic filament remodelling by Rad51 paralogs during homologous recombination Luiz Pedro Carvalho & Ed W. Tate | Mapping and understanding the role of protein lipidation in Mycobacterium tuberculosis Peter Cherepanov | The mechanism of retroviral DNA integration Alessandro Costa | Structural cryo-electron microscopy study of the eukaryotic DNA replication machinery Julian Downward | Activation of the immune system to eradicate KRAS oncogene driven cancers Eva Frickel | Immune-mediated cell-autonomous killing of Toxoplasma gondii by GBPs and ubiquitin in human cells Nathan Goehring | Design principles of intracellular pattern formation by cell polarity networks Alex Gould | Antioxidant roles for lipid droplets in stem cell niches Maximiliano Gutierrez | Dynamic interactions between Mycobacterium tuberculosis and autophagic organelles in macrophages Caroline Hill | The dynamics of TGF-beta signalling: mechanism and functional consequences Steve Ley | The roles of TPL-2 and ABIN-2 in lung cancer Nicholas Luscombe | Computational analysis of gene regulation on a genomic scale Neil McDonald | Structural biology of receptor tyrosine kinase signalling assemblies to define human disease mechanisms Justin Molloy | Single molecule analysis of Archael DNA processing enzymes Justin Molloy, Paula Booth & Sergi Garcia Manyes | Biological self-assembly: single molecule force methods to study the folding of membrane transport proteins Kathy Niakan | Characterising novel regulators of human pluripotency and embryogenesis Paul Nurse | Global cellular controls in eukaryotic cells Andy Oates | Control of the period of the genetic oscillations in the segmentation clock Markus Ralser & Jurg Bahler | The genetic diversity controlling metabolism Katrin Rittinger & Franca Fraternali | Dynamics and mechanisms underlying RBR E3 ligase activity


2

Peter Rosenthal | Structural Studies of Influenza Virus Entry and Assembly by Cryomicroscopy Erik Sahai | Tracking and predicting clonal competition in genetically heterogeneous tumours Guillaume Salbreux | Mechanics of cell division in a tissue Martin Singleton | Structural Biology of Chromosome Segregation Jim Smith | PAWS1 and Wnt signalling in early vertebrate development Thomas Surrey | Reverse engineering of spindle function Jesper Svejstrup | Basic mechanisms at the interface between transcription, the maintenance of genome stability, and human disease Charles Swanton | Exploiting Lung Cancer Heterogeneity by Leveraging the Host Immune Response Peter Thorpe & Attila Csikasz-Nagy | Systems level understanding of mitotic localization by microtubules Pavel Tolar & Isabel Llorente Garcia | Mechanics of receptor ligand binding in immune cell synapses Moritz Treeck | Functional analysis of kinases secreted into the host cell by the human malaria parasite Plasmodium falciparum Richard Treisman | Molecular mechanisms of signal-regulated transcription and chromatin modification Victor Tybulewicz | Novel signalling pathways controlling lymphocyte activation Victor Tybulewicz & Jeremy Green | Craniofacial development in mouse models of Down Syndrome Frank Uhlmann | The molecular mechanism of chromosome segregation Peter Van Loo | Deconvoluting normal cell and tumour cell signals from transcriptome and DNA methylome sequencing data Jean-Paul Vincent | The role of Evi/Wntless and exosomes in the trafficking and release of Wnt proteins in epithelia Andreas Wack | Factors governing epithelial damage and redifferentiation during influenza infection Michael Way | Exploring new levels of complexity within the Arp2/3 complex David Wilkinson | Spatial regulation of neurogenesis during hindbrain development Robert J Wilkinson & Robert S Heyderman | Pathogen-pathogen and host-pathogen interactome at the respiratory epithelial surface Hasan Yardimci | Understanding how the eukaryotic replication machinery deals with barriers Mariia Yuneva | Metabolic pathways as targets for anti-cancer therapy


3

Positions by Research Topic The list below tells you which positions fall under each of the Crick’s Research Topics. Biochemistry & Proteomics

Simon Boulton, Luiz Pedro Carvalho & Ed W. Tate, Peter Cherepanov, Alessandro Costa, Alex Gould, Caroline Hill, Neil McDonald, Justin Molloy, Justin Molloy, Paula Booth & Sergi Garcia Manyes, Paul Nurse, Markus Ralser & Jurg Bahler, Katrin Rittinger & Franca Fraternali, Martin Singleton, Jim Smith, Thomas Surrey, Jesper Svejstrup, Moritz Treeck, Richard Treisman, Frank Uhlmann, Michael Way, Hasan Yardimci, Mariia Yuneva

Cell Biology

Dominique Bonnet, Julian Downward, Eva Frickel, Nathan Goehring, Alex Gould, Maximiliano Gutierrez, Caroline Hill, Neil McDonald, Paul Nurse, Andy Oates, Markus Ralser & Jurg Bahler, Peter Rosenthal, Erik Sahai, Guillaume Salbreux, Jim Smith, Thomas Surrey, Jesper Svejstrup, Charles Swanton, Peter Thorpe & Attila Csikasz-Nagy, Pavel Tolar & Isabel Llorente Garcia, Moritz Treeck, Richard Treisman, Victor Tybulewicz, Frank Uhlmann, Jean-Paul Vincent, Michael Way, Robert J Wilkinson & Robert S Heyderman, Mariia Yuneva

Cell Cycle & Chromosomes

Peter Cherepanov, Alessandro Costa, Paul Nurse, Martin Singleton, Peter Thorpe & Attila Csikasz-Nagy, Richard Treisman, Frank Uhlmann, Hasan Yardimci

Chemistry & High Throughput

Luiz Pedro Carvalho & Ed W. Tate, Justin Molloy, Paula Booth & Sergi Garcia Manyes, Paul Nurse

Computational & Systems Biology

Nathan Goehring, Nicholas Luscombe, Kathy Niakan, Paul Nurse, Andy Oates, Markus Ralser & Jurg Bahler, Katrin Rittinger & Franca Fraternali, Peter Rosenthal, Guillaume Salbreux, Peter Thorpe & Attila Csikasz-Nagy, Peter Van Loo, Robert J Wilkinson & Robert S Heyderman

Developmental Biology

Nathan Goehring, Alex Gould, Kathy Niakan, Andy Oates, Guillaume Salbreux, Jim Smith, Victor Tybulewicz & Jeremy Green, Jean-Paul Vincent, Michael Way, David Wilkinson

Ecology, Evolution & Ethology

Markus Ralser & Jurg Bahler, Charles Swanton Gene Expression

Dominique Bonnet, Caroline Hill, Nicholas Luscombe, Justin Molloy, Andy Oates, Jim Smith, Jesper Svejstrup, Richard Treisman, Peter Van Loo, David Wilkinson, Robert J Wilkinson & Robert S Heyderman, Mariia Yuneva

Genetics & Genomics

Nicholas Luscombe, Paul Nurse, Andy Oates, Markus Ralser & Jurg Bahler, Jim Smith, Jesper Svejstrup, Charles Swanton, Peter Thorpe & Attila Csikasz-Nagy, Moritz Treeck, Richard Treisman, Victor Tybulewicz & Jeremy Green, Frank Uhlmann, Peter Van Loo

Genome Integrity & Repair

Simon Boulton, Alessandro Costa, Justin Molloy, Paul Nurse, Martin Singleton, Jesper Svejstrup, Charles Swanton, Frank Uhlmann, Hasan Yardimci


4

Human Biology & Physiology Dominique Bonnet, Alex Gould, Caroline Hill, Kathy Niakan, Robert J Wilkinson & Robert S Heyderman

Imaging

Dominique Bonnet, Alessandro Costa, Nathan Goehring, Maximiliano Gutierrez, Caroline Hill, Justin Molloy, Paul Nurse, Andy Oates, Peter Rosenthal, Erik Sahai, Jim Smith, Thomas Surrey, Pavel Tolar & Isabel Llorente Garcia, Victor Tybulewicz & Jeremy Green, Jean-Paul Vincent, Michael Way, Hasan Yardimci

Immunology

Steve Ley, Charles Swanton, Pavel Tolar & Isabel Llorente Garcia, Victor Tybulewicz, Andreas Wack, Robert J Wilkinson & Robert S Heyderman

Infectious disease

Luiz Pedro Carvalho & Ed W. Tate, Peter Cherepanov, Eva Frickel, Maximiliano Gutierrez, Peter Rosenthal, Pavel Tolar & Isabel Llorente Garcia, Moritz Treeck, Andreas Wack, Robert J Wilkinson & Robert S Heyderman

Metabolism

Dominique Bonnet, Alex Gould, Markus Ralser & Jurg Bahler, Moritz Treeck, Mariia Yuneva Microfabrication & Bioengineering

Justin Molloy, Paul Nurse, Andy Oates Model organisms

Nathan Goehring, Alex Gould, Paul Nurse, Andy Oates, Markus Ralser & Jurg Bahler, Jim Smith, Peter Thorpe & Attila Csikasz-Nagy, Victor Tybulewicz & Jeremy Green, Frank Uhlmann, Jean-Paul Vincent, Mariia Yuneva

Neurosciences

Alex Gould, David Wilkinson Signaling & Oncogenes

Dominique Bonnet, Julian Downward, Caroline Hill, Steve Ley, Andy Oates, Erik Sahai, Jim Smith, Charles Swanton, Moritz Treeck, Richard Treisman, Victor Tybulewicz, Jean-Paul Vincent, Michael Way, Mariia Yuneva

Stem Cells

Dominique Bonnet, Alex Gould, Kathy Niakan, Andy Oates, Jim Smith, David Wilkinson Structural Biology & Biophysics

Simon Boulton, Luiz Pedro Carvalho & Ed W. Tate, Peter Cherepanov, Alessandro Costa, Neil McDonald, Justin Molloy, Justin Molloy, Paula Booth & Sergi Garcia Manyes, Katrin Rittinger & Franca Fraternali, Peter Rosenthal, Martin Singleton, Thomas Surrey, Pavel Tolar & Isabel Llorente Garcia, Frank Uhlmann, Michael Way

Synthetic Biology

Justin Molloy, Andy Oates, Thomas Surrey Tumour Biology

Dominique Bonnet, Julian Downward, Alex Gould, Caroline Hill, Steve Ley, Erik Sahai, Charles Swanton, Richard Treisman, Peter Van Loo, Mariia Yuneva


5

Dominique Bonnet http://crick.ac.uk/research/a-z-researchers/researchers-a-c/dominique-bonnet/ Dissecting the heterogeneity of the human Haematopoietic (blood) stem cell compartment The haematopoietic system has long served as a model of choice for delineating mechanisms regulating self-renewal and differentiation. The majority of our understanding of the Haematopoietic Stem/Progenitor Cell (HSPC) compartment originated from mouse studies. However conclusions drawn from mouse data do not always translate into the human setting. Thus there is a necessity to investigate how in humans the HSPC compartment is structurally organized and regulated. So far, in human two phenotypically defined CD34+ versus CD34- HSCs have been identified. Nevertheless, our understanding of the biology of CD34- HSC and the contribution of this rare population to the maintenance of human haematopoiesis remains limited. The unique cellular/molecular features that distinguish these cells from CD34+ HSCs, as well as the signalling pathways, which regulate their properties have yet to be elucidated.

This project aims at dissecting the relationship between CD34- and CD34+ HSCs using a number of assays incluidnmg not not restricted to stem cell biology (FACS analysis, Cell sorting), molecular analysis, single cell RNA seq , lentivirus vector for gene disruption, overexpression (including Crisp-cas) and functional assays.

1. Rouault-Pierre K, et al. . HIF-2α protects human hematopoietic stem/progenitors and acute myeloid leukemic cells from apoptosis induced by endoplasmic reticulum stress. Cell Stem Cell. 2013 Nov 7;13(5):549-63.

2. Lassailly F, et al.. Multimodal imaging reveals structural and functional heterogeneity in different bone marrow compartments: functional implications on hematopoietic stem cells. Blood. 2013; 122 910): 1730-40.

3. Anjos-Afonso F, et al. CD34(-) cells at the apex of the human hematopoietic stem cell hierarchy have distinctive cellular and molecular signatures. Cell Stem Cell. 2013; 13(2):161-174.


6

Simon Boulton http://crick.ac.uk/research/a-z-researchers/researchers-a-c/simon-boulton/ Mechanistic analysis of pre-synaptic filament remodelling by Rad51 paralogs during homologous recombination Homologous recombination (HR) is an essential mechanism for the repair of DNA double strand breaks (DSBs) and stalled and collapsed replication forks across all domains of life. HR is a complex multi-step reaction, which initiates at ssDNA exposed at nucleolytically processed DSB ends or post-replicative ssDNA gaps. HR is tightly regulated at each step of the reaction by mediator proteins, including BRCA2, Rad54 and the family of Rad51 paralogs, which act as positive regulators at different steps of the HR reaction. Of the known HR mediator proteins the function of Rad51 paralog complexes has remained the most enigmatic. Ablation of Rad51 paralogs leads to severe HR defects, DNA damage sensitivity, chromosome abnormalities and defective Rad51 nuclear focus formation after DNA damage, suggestive of a major function at an early stage in the HR reaction. Like BRCA2 and PALB2, which are mutated in Fanconi anemia (FA) and breast and ovarian cancer, biallelic germline mutations in RAD51C cause a severe form of FA, while monoallelic inheritance of mutations in RAD51C and RAD51D, and RAD51B predispose individuals to ovarian and breast cancer, respectively, demonstrating an important tumour suppressor function for Rad51 paralogs. Despite extensive study, the mechanism by which Rad51 paralogs directly stimulate the recombinase activity of Rad51 had remained enigmatic for many years. Additionally, whether the Rad51 paralogs confer any intrinsic stabilization or alteration in the structural properties of the pre-synaptic Rad51 nucleoprotein filament had not been explored, nor the mechanistic importance of the conserved Walker motifs. We recently reported the characterisation of a Rad51 paralog complex, RFS-1/RIP-1, from C. elegans. RFS-1/RIP-1 is essential for HR and RAD-51 focus formation at DNA damage sites in vivo, it stimulates the recombinase activity of RAD-51, and associates directly with RAD-51 filaments in vitro. Using various biochemical and biophysical approaches (stopped flow kinetics, single molecule FRET, nuclease protection assays, electron microscopy), we demonstrated that RFS-1/RIP-1 structurally remodels the pre-synaptic RAD-51-ssDNA filament to a stabilized, “open”, flexible conformation, which facilitates strand exchange with the template duplex. Using specific mutants in the Walker boxes of RFS-1, which are compromised for stimulating strand exchange, we demonstrated that filament remodeling is critical for RFS-1/RIP-1 mediator activity. These results defined the underlying mechanism of HR stimulation by Rad51 paralogs and establish a new paradigm for HR mediator action (Taylor et al. Cell 2015). A number of outstanding questions remain to be addressed concerning the mechanism of filament remodelling induced by Rad51 paralog complexes, which will form the basis of this project: What is the role of ATP binding or hydrolysis for the reaction, is there a polarity to filament remodelling, do the Rad51 paralogs act from a specific end of the filament, is the remodelled filament more proficient to undergo the homology search, and do the vertebrate Rad51 paralog complexes also induce filament remodelling as a means to promote HR? These studies will ultimately provide a framework for comprehending the contribution of these key HR regulators and how they impact on human diseases. This is just one example of the sort of project that might be available. The precise project will be decided on in consultation with the supervisors.

1. Taylor MRG, Špírek M, Chaurasiya KR, Ward JD, Carzaniga R, Yu S, Egelman EH, Collinson LM, Rueda D, Krejci L & Boulton SJ (2015). Rad51 paralogs remodel pre-synaptic Rad51 filaments to stimulate homologous recombination. Cell, In press.

2. Adelman CA, Lolo RL, Birkbak NJ, Murina O, Matsuzaki K, Horejsi Z, Parmar K, Borel V, Skehel JM, Stamp G, D’Andrea A, Sartori AA, Swanton C & Boulton SJ (2013). HELQ promotes RAD51 paralog-dependent repair to avert germ cell attrition and tumorigenesis. Nature, 502: 381-4.

3. Chapman JR, Taylor MRG & Boulton SJ (2012). Playing the end game: DNA double-strand break repair pathway choice. Molecular Cell. 47:497-510.

4. Ward JD, Muzzini DM, Petalcorin MIR, Martinez-Perez E, Martin JS, Plevani P, Cassata G, Marini F & Boulton SJ (2010). Overlapping Mechanisms Promote Post-Synaptic RAD-51 Filament Disassembly During Meiotic Double-strand Break Repair. Molecular Cell 37:259-72.

5. Ward JD, Barber LJ, Petalcorin MIR, Yanowitz J & Boulton SJ (2007). Distinct genetic requirements for homologous recombination at impeded replication forks. EMBO J. 26:3384-96.


7

Luiz Pedro Carvalho & Ed W. Tate http://crick.ac.uk/research/a-z-researchers/researchers-a-c/luiz-pedro-carvalho/ http://www.imperial.ac.uk/people/e.tate Joint Crick/Imperial College London Position Mapping and understanding the role of protein lipidation in Mycobacterium tuberculosis Protein lipidation is an essential, covalent and usually irreversible modification that targets proteins to be inserted in membranes of a cell (Curr. Opin. Chem. Biol. 2015 24:48-57). Defects associated with lipidation contribute to a variety of health associated processes such as cancer, developmental abnormalities, infectious diseases, etc. Importantly, inhibition of protein lipidation has been recently validated as an attractive target for anti-infective drug discovery. However, in contrast to elegant and extensive work carried out on lipidation in eukaryotes using chemical proteomic tools (e.g. Nat. Chem. 2014 6(2):112-21; Nat. Comms. 2014 5:4919), the full scope of these post-translational modifications remains to be determined in bacteria.

Currently, the identity and overall function of the lipoproteome in the human pathogen Mycobacterium tuberculosis is unknown, although there is strong evidence that protein lipidation is essential for successful infection of host cells, since key proteins in their biogenesis are required for virulence (Mol. Microbiol. 2004 52(6):1543-1552). Preliminary chemical proteomic profiling studies carried out by our groups has revealed for the first time that protein lipidation is common in M. tuberculosis membrane proteins. Identification of the enzymes involved in this modification in M. tuberculosis will allow the characterization of this pathway during infection, and perhaps the validation of a novel target for antibacterial drug discovery. In addition, it will allow for the first time a broad description of this post-translational modification in M. tuberculosis. This knowledge will have important implications for the understanding of protein function in bacteria, pathogenesis, cell biology and synthetic biology.

In this project we propose you will (1) identify the enzymes involved in protein lipidation in the Mtb genome; (2) identify their protein substrates using chemical tagging technologies; (3) identify the role and the impact of protein lipidation in M. tuberculosis, using genetic knockout and knockdown strains; and (4) evaluate the role of these processes during experimental infection (in collaboration).

The successful candidate will receive substantial training in protein chemistry, proteomics and enzymology, and in molecular biology, mycobacteriology and bacterial genetics. In addition, the successful candidate will take advantage of a highly diverse and collaborative environment with substantial expertise in synthetic chemistry, chemical biology, proteomics, bacterial metabolism, mycobacteriology, protein chemistry, enzymology and structural biology.

The ideal candidate will have a strong interest in chemical proteomics, biochemistry and microbiology, with clear evidence of dedication to scientific research, as evidenced by successful research placements. We are chiefly interested in a biochemist, chemist or chemical biologist, willing to work with virulent M. tuberculosis, a class 3 human pathogen. Prior experience in molecular biology/genetics, microbiology, protein purification, mass spectrometry, and/or synthetic chemistry would be advantageous.

1. Curr Opin Chem Biol 2015 24:48-57 2. Nat. Chem. 2014 6(2):112-21; Nat. Comms. 2014 5:4919 3. Mol. Microbiol. 2004 52(6):1543-1552

NOTE: Additional eligibility criteria apply to this position: As well as meeting the standard eligibility criteria, applicants to this position will be expected to hold either a 4-year undergraduate degree at 2.1 level or higher, or a 3-year undergraduate degree plus a Masters degree. Non-EU applicants are not eligible for the funding for this project.


8

Peter Cherepanov http://crick.ac.uk/research/a-z-researchers/researchers-a-c/peter-cherepanov/ The mechanism of retroviral DNA integration A retrovirus, such as HIV, must insert a DNA replica of its genome into a host cell chromosome to establish successful infection. This essential process is catalyzed by integrase, a specialized DNA recombinase carried by the virus (reviewed by Li et al., 2011). To accomplish this function, a multimer of integrase assembles at the ends of viral DNA forming a highly stable complex termed intasome (Hare et al., 2010). Upon nuclear entry, the intasome inserts 3’ ends of viral DNA molecule into chromosomal DNA (Maertens et al., 2010; Maskell et al., 2015). While recent research unraveled many structural and mechanistic details of this process, we are far from understanding the rules of engagement between the retroviral integration machinery and the host cell environment. How is the intasome trafficked in the cell, and how does the virus select appropriate chromosomal locations for integration? How is the thermodynamically stable post-integration intermediate disassembled, and which cellular DNA repair enzymes are hijacked by the virus to complete the integration process? These are examples of the sorts of projects that may be available in this research group. Only one studentship is available with this group and the precise project will be decided on consultation with the supervisor.

1. Hare, S., Gupta, S.S., Valkov, E., Engelman, A., and Cherepanov, P. (2010). Retroviral intasome assembly and inhibition of DNA strand transfer. Nature 464, 232-236.

2. Li, X., Krishnan, L., Cherepanov, P., and Engelman, A. (2011). Structural biology of retroviral DNA integration. Virology 411, 194-205.

3. Maertens, G.N., Hare, S., and Cherepanov, P. (2010). The mechanism of retroviral integration from X-ray structures of its key intermediates. Nature 468, 326-329.

4. Maskell, D.P., Renault, L., Serrao, E., Lesbats, P., Matadeen, R., Hare, S., Lindemann, D., Engelman, A.N., Costa, A., and Cherepanov, P. (2015). Structural basis for retroviral integration into nucleosomes. Nature, doi: 10.1038/nature14495.


9

Alessandro Costa http://crick.ac.uk/research/a-z-researchers/researchers-a-c/alessandro-costa/ Structural cryo-electron microscopy study of the eukaryotic DNA replication machinery DNA replication is essential for the propagation of life and its tight control preserves chromosome integrity, preventing the onset of cancer [1]. In eukaryotes, a core player in this process is the 24-member Replisome Progression Complex (RPC), an assembly of enzymes that couple parental duplex-DNA unwinding (by the CMG helicase) with daughter strand synthesis (by three replicative polymerases named Pol alpha, delta and epsilon) [2]. Our group combines structural cryo-electron microscopy, molecular modelling and biochemistry to study the RPC structure and function. Using these integrated methods, we have recently described the molecular architecture of the 11-member CMG nanomotor and characterized the conformational changes that promote helicase translocation along DNA [3]. We are now interested in understanding how the CMG helicase activity is coupled with DNA synthesis during replication fork elongation. To address this issue, we have developed a new method to reconstitute the link between the CMG helicase and Pol alpha [4]. Our new PhD student will build on these advances to determine the structure of a helicase/polymerase super-assembly. These efforts will help establish the basis for the coordinated DNA unwinding/synthesis by the eukaryotic RPC, a key process for genome stability maintenance.

1. Costa, A., Hood, I.V., and Berger, J.M. (2013). Annual review of biochemistry 82, 25-54. 2. Georgescu, R.E., Schauer, G.D., Yao, N.Y., Langston, L.D., Yurieva, O., Zhang, D., Finkelstein, J.,

and O'Donnell, M.E. (2015). eLife 2015 Apr 14;4. 3. Costa, A., Renault, L., Swuec, P., Petojevic, T., Pesavento, J.J., Ilves, I., MacLellan-Gibson, K.,

Fleck, R.A., Botchan, M.R., and Berger, J.M. (2014). eLife 2014 Aug 12;3:e03273. 4. Simon, A.C., Zhou, J.C., Perera, R.L., van Deursen, F., Evrin, C., Ivanova, M.E., Kilkenny, M.L.,

Renault, L., Kjaer, S., Matak-Vinkovic, D., Labib, K., Costa, A. & Pellegrini L. (2014). Nature 510, 293-297.


10

Julian Downward http://crick.ac.uk/research/a-z-researchers/researchers-d-h/julian-downward/ Activation of the immune system to eradicate KRAS oncogene driven cancers The KRAS oncogene is the most frequently mutated oncogenic driver in human cancer, being activated in some 15% of tumours, including many poor prognosis cancers such as those of the lung and the pancreas. Despite huge research efforts, at present it is not possible to target the KRAS protein directly, although drugs have been developed that target downstream signaling pathways controlled by it, such as the RAF/MEK pathway and the PI 3-kinase/AKT pathway. MEK inhibitory drugs have shown some modest benefit in KRAS mutant lung cancer, but are only able to delay progression of the disease by a couple of months before resistance develops.

In order to move beyond treatments that only delay advanced cancers for a few months or, at best, years, we need to understand how to eradicate tumour cells completely, not leaving minor populations that go on to develop drug resistance and cause disease relapse. A very interesting area of investigation in this regard is that of immunotherapy. Tumours have to find ways to avoid recognition as foreign by the immune system, and recent clinical trials have achieved remarkable response rates using immune checkpoint inhibitors as immunotherapies in certain advanced cancers. This has illustrated how efficiently immune surveillance is suppressed locally by tumours and how powerful the intrinsic anti-tumour response can be once this suppression is overcome. However, response rates to immunotherapies are highly variable and it is entirely unclear how these therapies can be combined to best effect with existing treatments. Systematic investigation of how immunotherapy can be combined optimally with targeted or chemotherapeutic agents will require good pre-clinical model system which, unfortunately, are currently lacking.

Much work on the development of therapeutic agents to target oncogene driven cancers in recent years has relied on the use of genetically engineered mouse models (GEMMs) of cancer. However, we have found that these models have limited value in the study of the interaction of the tumour with the immune system, largely because they lack immunogenicity as they have very low mutation rates and low levels of aneuploidy, in sharp contrast to real human tumours. We are working to develop improved mouse models of oncogene driven cancers which contain rates of mutation and aneuploidy elevated to the level seen in human cancers due to alterations of critical processes implicated in genetic instability in the clinic. These include compromise of the spindle assembly checkpoint, leading to aneuploidy, and increase in mutagenic processes implicated in mutational signatures relevant to the specific cancer type. We refer to these improved mouse models as immunogenic GEMMs (iGEMMs).

In this project, we plan to use iGEMMs modelling KRAS mutant lung and pancreatic cancer to investigate the interplay between the immune system and the tumour and how this is influenced by targeted and cytotoxic agents, as well as immunotherapies. We will explore the possibility that that some targeted agents, including epigenetic modifiers, might be able to increase exposure of tumour neo-antigens to the immune system in a manner that may allow effective combination with immune checkpoint blockade, with particular attention paid to scheduling and multi-component combinations.

1. J. Downward (2003) Nature Reviews Cancer 3, 11-22. Targeting Ras signaling pathways in cancer therapy.

2. M.S. Kumar, D.C. Hancock, M. Molina-Arcas, M. Steckel, P. East, M. Diefenbacher, E. Armenteros-Monterros, F. Lassailly, N. Matthews, E. Nye, G. Stamp, A. Behrens, J. Downward (2012) Cell 149, 642-655. The GATA2 transcriptional network is requisite for RAS oncogene-driven non-small cell lung cancer.

3. R. Fritsch, I. de Krijger, K. Fritsch, R. George, B. Reason, M.S. Kumar, M. Diefenbacher, G. Stamp, J. Downward (2013) Cell 153, 1050-1063. RAS and RHO families of GTPases directly regulate distinct phosphoinositide 3-kinase isoforms.”

4. E. Castellano, C. Sheridan, M.Z. Thin, E. Nye, B. Spencer-Dene, M.E. Diefenbacher, C. Moore, M.S. Kumar, M.M. Murillo, E. Gronroos, F. Lassailly, G. Stamp, J. Downward (2013) Cancer Cell 24, 617-630. Requirement for interaction of PI 3-kinase p110α with RAS in lung tumor maintenance.

5. S.L. Topalian, C.G. Drake, D.M. Pardoll (2015) Cancer Cell 27, 450-461. Immune checkpoint blockade: a common denominator approach to cancer therapy.


11

Eva Frickel http://crick.ac.uk/research/a-z-researchers/researchers-d-h/eva-frickel/ Immune-mediated cell-autonomous killing of Toxoplasma gondii by GBPs and ubiquitin in human cells Infection with the intracellular parasite Toxoplasma gondii leads to the rapid production of the proinflammatory cytokine interferon gamma (IFNγ), which stimulates cell-autonmous defence mechanisms to kill and restrict the pathogen. We have identified two effector pathways of IFNγ-dependent killing of Toxoplasma in human non-hematopoetic cells. This project will investigate the detailed mechanism of one or both of these pathways and interrogate if they are connected.

Firstly, IFNγ upregulates the p65 Guanylate Binding Proteins (GBPs). The GBPs are a family of GTPases that are conserved amongst vertebrates and 7 Gbps have been identified in humans. Mouse GBPs have been shown to restrict Toxoplasma by disrupting the parasitophorous vacuole (PV) (Yamamoto et al, 2012)., however, we have shown that human GBPs are not found directly at the PV. Weak data implies a role of human GBPs in viral restriction and no other immune-related function is known. We have found that in GBP1CRISPR knockout human epithelial cells IFNγ-mediated cell autonomous killing of Toxoplasma is impaired.

Secondly, we have determined that in IFNγ-stimulated human cells, the PV of avirulent, but not virulent Toxoplasma is decorated with the cellular protein ubiquitin. Ubiquitination of intracellular pathogens can lead to autophagic clearance of the pathogen via host adaptor proteins (Sorbara & Girardin, 2015). We indeed find p62 and NDP52, two such autophagy adaptor proteins, at the PV of avirulent Toxoplasma. After 4h the ubiquitinated PVs acidify and the parasite dies. Using mass spectrometry we have identified a set of candidate host proteins that are ubiquitinated upon Toxoplasma infection.

You will investigate 1) how hGBP1 can restrict Toxoplasma without localising directly to the PV and 2) what role the novel ubiquitinated host proteins play in killing of the parasite.

For the first part of the project we hypothesise that hGBP1 can regulate levels of p62 and authopagy via β-catenin. β-catenin is part of a signal transduction pathway and decreased β-catenin levels are linked to increased autophagy and p62 levels (Petherick et al, 2013). Overexpression of hGBP1 in epithelial cells leads to the degradation of β-catenin and enhanced cell proliferation (Capaldo et al, 2012). You will investigate the protein status of β-catenin in hGBP1 CRISPR knock out cells and determine if an increased percentage of the PVs are acidified and decorated with p62. Should this be the case, then you will study the precise mechanism of how hGBP1 can regulate β-catenin by either directly or indirectly interfering with its stabilisation machinery.

For the second part of the project you will localise the novel host ubiquitinated substrate proteins to either the PV or other cellular locations by immunofluorescence microscopy. Upon knock down of these candidate effector proteins, you will determine the cell’s killing ability of Toxoplasma and acidification status of the PVs. The host proteins that are able to interfere with Toxoplasma acidification and death will be studied. For these candidates, this will include generating CRISPR knock down cell lines, conducting ubiquitin linage analysis and determining their cellular trafficking patterns and interaction partners.

In summary, this project will lead to the enhanced understanding of IFNγ-mediated restriction of Toxoplasma in human cells.

1. Capaldo CT, Beeman N, Hilgarth RS, Nava P, Louis NA, Naschberger E, Stürzl M, Parkos CA & Nusrat A (2012) IFN-γ and TNF-α-induced GBP-1 inhibits epithelial cell proliferation through suppression of β-catenin/TCF signaling. Mucosal Immunol 5: 681–690

2. Petherick KJ, Williams AC, Lane JD, Ordóñez-Morán P, Huelsken J, Collard TJ, Smartt HJM, Batson J, Malik K, Paraskeva C & Greenhough A (2013) Autolysosomal β-catenin degradation regulates Wnt-autophagy-p62 crosstalk. EMBO J 32: 1903–1916

3. Sorbara MT & Girardin SE (2015) Emerging themes in bacterial autophagy. Curr Opin Microbiol 23: 163–170

4. Yamamoto M, Okuyama M, Ma JS, Kimura T, Kamiyama N, Saiga H, Ohshima J, Sasai M, Kayama H, Okamoto T, Huang DCS, Soldati-Favre D, Horie K, Takeda J & Takeda K (2012) A Cluster of Interferon-γ-Inducible p65 GTPases Plays a Critical Role in Host Defense against Toxoplasma gondii. Immunity 37: 302-313


12

Nathan Goehring http://crick.ac.uk/research/a-z-researchers/researchers-d-h/nathan-goehring/ Design principles of intracellular pattern formation by cell polarity networks The development of an organism from a single undifferentiated cell is enormously complex. An essential feature of this process is the formation spatial patterns by so-called morphogens, which provide landmarks for organising developmental processes. At the tissue scale, such patterns provide cues to guide the location and fate of cells within an organism. So too, intracellular patterns govern cells’ internal organisation, providing a coordinate system to enable proper orientation with respect to neighbours and the environment.

Our lab focuses on understanding intracellular pattern formation by the conserved PAR polarity machinery, which compartmentalizes the cell membrane into complementary domains that define the polarity axis. Polarization is essential for animal development from worms to humans, and is implicated in axis specification, tissue organization and asymmetric stem cell divisions. Defects in polarity are associated with cancer progression and metastasis. Most of the molecules required for PAR polarity have been identified. Our current challenge is to develop a systems-level understanding of how spatial organization at the cellular scale emerges from the individual activities of and interactions between these molecules.

The nematode worm Caenorhabditis elegans is an ideal model for this work. It has a reproducible and rapid development, its early developmental stages are accessible to manipulation and live imaging, and there is a robust set of genetic and RNAi-based tools for probing protein function in living animals. We are particularly interested in understanding the network properties and design principles of the PAR polarity pathway that enable this highly conserved set of proteins to polarize diverse cell types during animal development. Examples of questions we are currently addressing include: How are PAR distributions shaped by the physical properties of proteins and their local environment? How does this network adapt to changes in cell size / shape that occur during development? What is the wiring logic of the protein network that permits mutually exclusive protein distributions? How is the wiring of the system regulated to ensure cells polarize at the proper time and place? To address these questions, we are combining traditional cell and molecular biology with the development of new tools for manipulating the activities and physical properties of proteins in living embryos at high spatiotemporal precision as well as the development of mathematical models to quantitatively test models for pattern formation.

We are looking for a highly motivated student to tackle these fundamental questions within a multidisciplinary environment. This project potentially involves a broad range of techniques including 3-D time-lapse confocal microscopy, quantitative image analysis, photobleaching and photoactivation, RNAi, biochemistry, quantitative proteomics, chemical biology, and computational modeling, which will be tailored to the student’s interests and aptitude. A variety of backgrounds will be considered (e.g. physics, biology, biochemistry) and a general curiosity and willingness to embrace team effort will be essential. Note that this project description illustrates the types of questions that occupy us in the lab. Individual projects will be developed together with the supervisor, taking into account the student’s background and specific scientific interests. This specific project will be part of an international consortium working on various aspects of cell polarity and will involve secondments and training opportunities with other partner laboratories in Europe.

1. Goehring NW: PAR polarity: From complexity to design principles. Exp. Cell Res. 2014, doi:10.1016/j.yexcr.2014.08.009.

2. Goehring NW, Grill SW: Cell polarity: mechanochemical patterning. Trends Cell Biol 2013, 23:72–80.

3. Goehring NW, Trong PK, Bois JS, Chowdhury D, Nicola EM, Hyman AA, Grill SW: Polarization of PAR proteins by advective triggering of a pattern-forming system. Science 2011, 334:1137–1141.

4. Goehring NW, Hoege C, Grill SW, Hyman AA: PAR proteins diffuse freely across the anterior-posterior boundary in polarized C. elegans embryos. J Cell Biol 2011, 193:583–594.

NOTE: Additional eligibility criteria apply to this position: As well as meeting the standard eligibility criteria, applicants to this position must not have resided in the UK for more than 12 months in the last 3 years immediately prior to commencing the role.


13

Alex Gould http://crick.ac.uk/research/a-z-researchers/researchers-d-h/alex-gould/ Antioxidant roles for lipid droplets in stem cell niches The overarching goal of our research is to harness the advanced genomics and tissue-specific genetics available in the fruit fly Drosophila (http://flybase.org/) to identify conserved mechanisms relevant to human metabolic health and disease. Much of our work focuses on lipid metabolism, which is essential for health and its disruption can lead to obesity and type 2 diabetes.

One project on offer in our laboratory involves lipid droplets. These cytoplasmic organelles are known to form inside cells in response to various environmental stresses and also during metabolic disease, cancer and neurodegeneration. In most of these pathological contexts, it is still not clear whether lipid droplets play a harmful role in disease progression or whether they are a beneficial part of the body's protective response. The starting point for this project is recent work in our laboratory on the neural stem cell niche of Drosophila (Bailey et al. 2015). This work has revealed a new and novel role for lipid droplets as antioxidant organelles that function to protect developing neural stem cells from damage by reactive oxygen species (ROS). These lipid droplets are particularly important for safeguarding neural stem cells against ROS that are generated from polyunsaturated fatty acids consumed in the diet. Hence, although omega 3 and omega 6 polyunsaturated fatty acids are important for health, they also have a dark side such that consuming high amounts can generate enough ROS to harm dividing neural stem cells. The aims of this project are two fold. First, to use tissue-specific RNAi knockdowns to identify the unknown molecular mechanism by which ROS induce the biosynthesis of lipid droplets in the Drosophila neural stem cell niche. And second, to test whether lipid droplets also play antioxidant functions in other stem cell systems, including those of mammals. This project will provide training in genetics, embryology, transgenesis, molecular biology, biochemistry, cell biology, microscopy, metabolomics and bioinformatics.

1. Bailey AP, Koster G, Guillermier C, Hirst EM, MacRae J, Lechene CP, Postle AD and Gould AP (2015). An antioxidant role for lipid droplets in a stem cell niche of Drosophila (Cell, under revision)

2. Ragan TJ, Bailey AP, Gould AP and Driscoll PC (2013). Volume determination with two standards allows absolute quantification and improved chemometric analysis of metabolites by NMR from submicroliter samples. Anal. Chem. 85, 12046-54.

3. Steinhauser ML, Bailey AP, Senyo SE, Guillermier C, Perlstein TS, Gould AP, Lee RT and Lechene CP (2012). Multi-isotope imaging mass spectrometry quantifies stem cell division and metabolism. Nature 481, 516-519.

4. Cheng LY, Bailey AP, Leevers SJ, Ragan TJ, Driscoll PC and Gould AP (2011). Anaplastic Lymphoma Kinase Spares Organ Growth during Nutrient Restriction in Drosophila. Cell 146, 435-47.

5. Sousa-Nunes R, Yee LL and Gould AP (2011). Fat cells reactivate quiescent neuroblasts via TOR and glial insulin relays in Drosophila. Nature 471, 508-512


14

Maximiliano Gutierrez http://crick.ac.uk/research/a-z-researchers/researchers-d-h/maximiliano-gutierrez/ Dynamic interactions between Mycobacterium tuberculosis and autophagic organelles in macrophages Autophagy in the immune response to M. tuberculosis The ability of M. tuberculosis to survive in host cells is central to the pathogenesis of tuberculosis. M. tuberculosis impairs phagosome maturation to avoid elimination (Russell, 2001). However, M. tuberculosis killing can be restored by induction of autophagy through diverse stimuli in a process that targets mycobacteria to autophagosomes and then degradation (Gutierrez et al., 2004). Although this process is less understood, mycobacteria are also able to induce autophagy after infection via activation of specific intracellular pathways (Bradfute et al., 2013). M. tuberculosis interactions with autophagic compartments The targeting of M. tuberculosis to autophagosomes is a complex and dynamic process that can be modulated by numerous cellular and bacterial factors. However, the molecular players that mediate the interactions of M. tuberculosis with components of the autophagic pathway are ill defined (Songane et al., 2012). Many questions remain unanswered regarding the dynamic interactions of spatio-temporal targeting of M. tuberculosis to autophagosomes , primarily due to the lack of imaging tools to visualize this process. Correlative Live-Cell and Superresolution imaging in tuberculosis Superresolution microscopy techniques such as stochastic optical reconstruction microscopy (STORM) or photoactivation localization microscopy (PALM) made possible to surpass the diffraction limit in fluorescence microscopy (Henriques et al., 2011). Conventionally, these techniques rely on determining the position of sparsely activated photoswitchable probes. Although live-cell superresolution has been hampered by imaging speed and photodamage, recent strides in the field are considerably improving these elements opening the possibility to observe fast phagophore/autophagosome dynamics on the autophagic pathway during xenophagy with the needed spatiotemporal resolution. Aim of the project To define using correlative live-cell and super resolution imaging the dynamic events during targeting of M. tuberculosis to autophagosomes for degradation in macrophages. Specific aims 1-Development of tools and workflow for correlative live-cell and super resolution (SR) imaging studies. We will focus our analysis in three components of the selective autophagic machinery, namely the expression ubiquitin probes, p62 and LC3. In addition, we will investigate the association of two proteins that are associated with the isolation membrane: Rab33B and Atg16L1. Finally, we will monitor bacterial DNA recognition by expressing cGAS. Here, fusion proteins with appropriate fluorophores for live cell imaging, PALM and STORM will be generated. 2-Characterization of the spatio-temporal dynamics of autophagosome-mycobacteria interactions at the super-resolution level. We will investigate the dynamic association of these proteins during the targeting to autophagosomes of M. tuberculosis. For that mouse and human macrophages expressing the different autophagic proteins will be infected with M. tuberculosis and analysed by live-cell and SR imaging studies. Complementary studies will be performed using endogenous proteins when validated antibodies are available. Here, we will analyze how these factors associate with mycobacteria during the infection and the response after activation with immune mediators of autophagy such as interferon-gamma. We aim here to define the steps and timing of association and quantitatively measure the different subpopulations of bacteria targeted to degradation. Moreover, we will characterize the association of the different proteins after phagosomal escape or damage. For that, we will use mutants of M. tuberculosis lacking specific virulence factors such as the ESX-1 secretion system. 3-Identification of the molecular components required for autophagic sequestering of M. tuberculosis. Once we have a global picture of the sequencial steps in the process of M. tuberculosis sequestering in resting or activated cells, we will use different knockdown/knockout (shRNA, siRNA and Crispr/Cas9


15

approaches to analyze the role of these autophagic proteins in the killing or replication of M. tuberculosis in macrophages. For that, colony forming units (CFU) and cytokine release will be monitored and correlated with data from aim 2.

1. Bradfute, S.B., Castillo, E.F., Arko-Mensah, J., Chauhan, S., Jiang, S., Mandell, M., and Deretic, V. (2013). Autophagy as an immune effector against tuberculosis. Current opinion in microbiology 16, 355-365.

2. Gutierrez, M.G., Master, S.S., Singh, S.B., Taylor, G.A., Colombo, M.I., and Deretic, V. (2004). Autophagy is a defense mechanism inhibiting BCG and Mycobacterium tuberculosis survival in infected macrophages. Cell 119, 753-766.

3. Henriques, R., Griffiths, C., Hesper Rego, E., and Mhlanga, M.M. (2011). PALM and STORM: unlocking live-cell super-resolution. Biopolymers 95, 322-331.

4. Russell, D.G. (2001). Mycobacterium tuberculosis: here today, and here tomorrow. Nature reviews. Molecular cell biology 2, 569-577.

5. Songane, M., Kleinnijenhuis, J., Netea, M.G., and van Crevel, R. (2012). The role of autophagy in host defence against Mycobacterium tuberculosis infection. Tuberculosis 92, 388-396.


16

Caroline Hill http://crick.ac.uk/research/a-z-researchers/researchers-d-h/caroline-hill/ The dynamics of TGF-beta signalling: mechanism and functional consequences Our lab focuses on the transforming growth factor beta (TGF-β) superfamily of ligands which comprises the TGF-βs, Activins, Nodal, BMPs and GDFs. These ligands control many aspects of embryonic development and adult tissue homeostasis, and deregulated signalling is associated with cancer and fibrosis (1, 2). Ligands bind to a type II receptor, which complexes with and transphosphorylates a type I receptor that subsequently phosphorylates the receptor-regulated Smads (R-Smads), the effectors of the pathway. The activated Smads then accumulate in the nucleus where they regulate gene transcription in conjunction with other DNA-binding transcription factors (3).

In the context of investigating TGF-β signalling dynamics, we discovered that acute stimulation of cells with TGF-β leads to cells becoming refractory to further acute stimulation (4). This behaviour is the result of very rapid internalisation of receptors upon ligand binding. Signalling competence is only restored once the ligand is depleted and the receptors have reaccumulated on the cell surface, which occurs approximately 48 hours after the initial stimulation. This refractory behaviour appears to be specific to TGF-β. Cells treated with Nodal and Activin show sustained signalling and do not become desensitized, and cells stimulated with BMPs show an oscillatory response, as determined by phosphorylation of R-Smads. We anticipate that the dynamics of TGF-β superfamily signalling are crucial for normal development and tissue homeostasis. Interestingly, several diseases, cancer and fibrosis, are associated with high and sustained levels of TGF-β signalling. The refractory behaviour we observed in response to TGF-β stimulation is incompatible with this, suggesting that in these pathogenic contexts, either the pathway is rewired, or the signalling is actually due to a different TGF-β family ligand.

The PhD project is focused on understanding the mechanism underlying the distinct signalling dynamics for the different ligands, and the functional consequences.

Our current work suggests that receptor trafficking, both of newly synthesized receptors and of ligand-bound receptors, is a crucial factor in determining the dynamics of signalling. The project will involve generating novel biosensors for tracking receptors and monitoring receptor activity. For tracking, the student will fuse a photo-convertible fluorescent tag to the type I and type II receptors, and for measuring receptor activity they will fuse a circularly permutated YFP that fluoresces upon activation of the type I receptor (5). CRISPR/Cas9 technology will be used to knock these tags into the endogenous genes. The student will also take advantage of several genome-wide loss-of-function screens that we have performed in the lab to identify factors involved in determining TGF-β signalling dynamics.

The second part of the project will focus on the functional consequences of the distinct signalling dynamics. This will be explored genome-wide at the level of transcriptional responses in a variety of tissue culture systems, including human ES cells. Finally, the student will determine how high levels of TGF-β signalling are sustained in the contexts of cancer and fibrosis using cells derived from relevant mouse models and patient samples, aiming to distinguish between rewiring of the TGF-β pathway versus the involvement of different ligands.

1. Wu, M. Y., Hill, C. S., (2009) TGF-β superfamily signaling in embryonic development and homeostasis. Dev Cell 16, 329-343.

2. Calon, A., Tauriello, D. V., Batlle, E., (2014) TGF-β in CAF-mediated tumor growth and metastasis. Semin Cancer Biol 25, 15-22.

3. Schmierer, B., Hill, C. S., (2007) TGFβ-SMAD signal transduction: molecular specificity and functional flexibility. Nat Rev Mol Cell Biol 8, 970-982.

4. Vizan, P., Miller, D. S., Gori, I., Das, D., Schmierer, B., Hill, C. S., (2013) Controlling long-term signaling: receptor dynamics determine attenuation and refractory behavior of the TGF-β pathway. Sci signal 6, ra106.

5. Michel, M., Raabe, I., Kupinski, A. P., Perez-Palencia, R., Bokel, C., (2011) Local BMP receptor activation at adherens junctions in the Drosophila germline stem cell niche. Nature Commun 2, 415.


17

Steve Ley http://crick.ac.uk/research/a-z-researchers/researchers-k-o/steve-ley/ The roles of TPL-2 and ABIN-2 in lung cancer TPL-2 kinase (1) regulates activation of the ERK1/2 MAP kinase pathway in innate immune responses (Figure 1). C-terminally truncated TPL-2 promotes tumourigenesis in mice, and TPL-2 is overexpressed in multiple human cancers, enhancing cell survival and proliferation. However, based on experiments using Tpl2-/- mice, a tumour suppressor function for TPL-2 has been proposed in non-small cell lung carcinoma (NSCLC), the most common form of lung cancer in humans.

Tpl2-/- cells are profoundly deficient in the expression of ABIN-2 (2), an NF-κB inhibitor with which TPL-2 interacts. Significantly, ABIN-2 has been suggested to function as a tumour suppressor in a subtype of human B cell lymphoma (3). It is therefore possible that TPL-2 deficiency enhances the susceptibility to lung carcinogenesis indirectly via a reduction in ABIN-2 expression, which may drive lung cancer progression by increasing the activation of NF-κB.

The aim of this project is to investigate whether TPL-2 and/or ABIN-2 function as tumour suppressors in the LSL-KRasG12D mouse model for NSCLC. This will involve the use two knock-in mouse strains in which the specific functions of TPL-2 and ABIN-2 have been separately blocked by point mutation. Analyses of these mutant mice crossed with LSL-KRasG12D mice will enable the independent assessment of TPL-2 and ABIN-2 signalling activity in the development and progression of NSCLC.

A clear understanding of the distinct roles of TPL-2 and ABIN-2 in lung cancer is essential to establish whether TPL-2 functions as a tumour suppressor. This is an important consideration in the safety of TPL-2 inhibitors as potential anti-inflammatory drugs to treat rheumatoid arthritis and inflammatory bowel disease.

This project will involve a number of techniques, including immunohistochemistry, qRT-PCR, ELISA, RNA sequencing, flow cytometry, tissue culture and western blotting.

1. Gantke T, Sriskantharajah S, Sadowski M, & Ley SC (2012) IκB kinase regulation of the TPL-2/ERK MAPK pathway. Immunol. Rev. 246:168 - 182.

2. Sriskantharajah S, et al. (2014) Regulation of experimental autoimmune encephalomyelitis by TPL-2. J. Immunol. 192:3515 - 3529.

3. Dong D, et al. (2011) A20, ABIN-1/2 and CARD11 mutations and their prognostic value in gastointestinal diffuse large B-cell lymphomas. Clin. Cancer Res. 17:1440 - 1451.


18

Nicholas Luscombe http://crick.ac.uk/research/a-z-researchers/researchers-k-o/nicholas-luscombe/ Computational analysis of gene regulation on a genomic scale Our research takes a genomic, integrative approach to understand gene regulation and evolution. We combine genome sequence, gene expression, ChIP-seq and iCLIP data to gain insights into: • How gene expression is controlled; • How this system regulates biologically important behaviours; • And how a breakdown in this system leads to diseases. Recent research successes include: investigations of evolutionary processes in bacterial genomes (Martincorena et al, Nature 2012); qualitative models of nucleosome-positioning and transcriptional regulation (Zaugg & Luscombe, Genome Research 2012); understanding how RNA-binding proteins control transcript stability and translation (Zarnack et al, Cell 2013; Sugimoto et al, Nature 2015); and how the three-dimensional conformation of chromosomes contribute to gene expression control (Mifsud et al, Nature Genetics 2015). Ongoing projects • How do we measure spatial organisation of chromosomes in the nucleus using HiC techniques? • How does this chromosomal arrangement affect gene activities, and do chromosomes rearrange themselves between different cellular conditions? • Which regions of the genome do regulatory proteins bind, and how do they control gene activities? • How do their binding patterns change over time? • How do these regulatory processes alter or break down during diseases such as bacterial infections and cancer progression? Though our main research focus is genomics and transcriptional regulation, the laboratory is open to people who wish to develop research on other related areas of computational biology and biostatistics. Much of our work is purely computational using publicly available genomic data, but we also encourage close collaborations with experimental laboratories.

1. Mifsud B*, Tavares-Cadete F*, Young AN*, Sugar R, Schoenfelder S, Ferreira L, Wingett S, Andrews S, Grey W, Ewels PA, Herman B, Happe S, Higgs A, LeProust E, Follows GA, Fraser P, Luscombe NM+, and Osborne CS+. (2015). Mapping long-range promoter contacts in human cells with high-resolution capture Hi-C. Nature Genet. 47:598-606.

2. Sugimoto Y, Vigilante A, Darbo E, Zirra A, Militti C, D'Ambrogio A, Luscombe NM+, and Ule J. (2015).hiCLIP reveals the in vivo atlas of mRNA secondary structures recognized by Staufen 1. Nature. 519:491-494.

3. Ilsley GR, Apweiler R, Jasmin Fisher, DePace AH+, and Luscombe NM+. (2013). Cellular resolution models of even skipped regulation in the entire Drosophila embryo. eLife. 2:e00522.

4. Zarnack K*, König J*, Tajnik M, Martincorena I, Eustermann S, Stévant I, Reyes A, Anders S, Luscombe NM+, and Ule J+. (2013). Direct competition between hnRNP C and U2AF65 protects the transcriptome from the uncontrolled exonization of Alu elements. Cell. 152:453-66.

5. Martincorena I, Seshasayee ASN, and Luscombe NM. (2012). Evidence of non-random mutation rates suggests a risk management strategy for evolution. Nature. 485:95-8.


19

Neil McDonald http://crick.ac.uk/research/a-z-researchers/researchers-k-o/neil-mcdonald/ Structural biology of receptor tyrosine kinase signalling assemblies to define human disease mechanisms Receptor tyrosine kinases (RTKs) respond to extracellular ligands received at the cell membrane and undergo ligand-dependent activation triggering intracellular signaling pathway activation. We study the RET receptor as a model RTK to understand how its ligands are recognised and how this interaction drives tyrosine kinase activation by allosteric or/and clustering mechanisms. We also investigate the basis for assembly of downstream effector complexes associated with an activated RET. These studies are important as RET signalling is crucial for both embryonic and adult development, whilst RET missense mutations underlie at least three human diseases (Hirschsprung’s disease, kidney agenesis and cancer) [1]. We have determined structures of both extracellular and intracellular portions of RET, alone and in complex with ligand [2,3,4]. Insights into human disease mechanisms have come from a concerted effort to combine structural, biochemical, mass spectrometry and cellular data to understand how RET missense mutations drive disease [5]. In this project, activated RET signalling complexes will be produced containing either a full-length RET receptor or its intracellular portion together with effector molecules using stable cell lines. Such complexes will be characterized by biochemical assay, by structural methods (crystallography and cryo-electron microscopy) and by biophysical analysis (mass spectrometry and affinity measurements). In parallel, disease-associated mutations will be prepared and analysed in both contexts (full length and intracellular domain). Differences underlying wild type and disease mutations will be explored further as well as in a cellular context using stable cell lines producing tagged forms of RET. Several related RTKs will also be investigated using similar approaches.

1. RET revisited: expanding the oncogenic portfolio. Mulligan LM. Nat Rev Cancer. 2014 Mar;14(3):173-86.

2. RET recognition of GDNF-GFRα1 ligand by a composite binding site promotes membrane-proximal self-association. Goodman KM, Kjær S, Beuron F, Knowles PP, Nawrotek A, Burns EM, Purkiss AG, George R, Santoro M, Morris EP, McDonald NQ. Cell Rep. 2014 Sep 25;8(6):1894-904.

3. Mammal-restricted elements predispose human RET to folding impairment by HSCR mutations. Kjaer S, Hanrahan S, Totty N, McDonald NQ. Nat Struct Mol Biol. 2010 Jun;17(6):726-31.

4. Structure and chemical inhibition of the RET tyrosine kinase domain. Knowles PP, Murray-Rust J, Kjaer S, Scott RP, Hanrahan S, Santoro M, Ibáñez CF, McDonald NQ. J Biol Chem. 2006 Nov 3;281(44):33577-87.

5. Oncogenic RET kinase domain mutations perturb the autophosphorylation trajectory by enhancing substrate presentation in trans. Plaza-Menacho I, Barnouin K, Goodman K, Martínez-Torres RJ, Borg A, Murray-Rust J, Mouilleron S, Knowles P, McDonald NQ. Mol Cell. 2014 Mar 6;53(5):738-51.


20

Justin Molloy http://crick.ac.uk/research/a-z-researchers/researchers-k-o/justin-molloy/ Single molecule analysis of Archael DNA processing enzymes This PhD project is a collaboration between two laboratories with complementary expertise in Archaeal DNA processing enzymes, in particular RNA polymerase (Werner Lab, UCL) and in single molecule enzymology (Molloy, the Crick Institute). The aim of the PhD is to further our understanding of the way in which Archaeal organisms process DNA and manipulate the topology of their genomes. Archaea are evolutionarily ‘ancient’ single-celled organisms that often inhabit extreme environmental niches such as hydrothermal vents, hot springs and salt lakes. They are of great interest to biotechnologists because their heat-stable enzymes are (for example) key to PCR and other DNA technologies and their novel metabolic enzymes are of interest for use in bioremediation. Archaeal proteins are also helpful in furthering our understanding of basic molecular mechanisms because they exhibit unusual properties that can shed light on how proteins work. The research project is to study a protein called “DNA Gyrase”: The Archaeal form of this protein has unusual and fascinating properties.

Eukaryotic genomes are negatively supercoiled – underwound – and DNA gyrase is responsible for unwinding DNA in other words opening up the DNA helix. Hyperthermophilic (heat-loving) members of the archaea have positively supercoiled – overwound - genomes. They encode the enzyme reverse DNA gyrase that works ‘backwards’ and tends to wind-up DNA and tighten the classical Watson-Crick helix. It is not clear why Archaea have evolved a reverse gyrase (although one might speculate that the conditions of their habitat tends to melt the DNA helix); it is also not fully understood how the gyrase works. The aim of the project is to apply a battery of single molecule approaches, including magnetic and optical tweezers and single fluorophore imaging, in order study gyrase and reverse gyrase activity on isolated, individual DNA molecules in real-time.

As the project progresses and the single molecule techniques have been mastered by the student the work will be extended to explore the effect of negative and positive supercoiling on DNA binding of histones, the proteins that compact genomes and regulate gene expression. Ultimately, we would like to study RNA polymerases transcription and how it is affected by supercoiling. The project is exciting and ambitious but also has many “fall-back” options if aspects of the work do not succeed at an early stage.

1. Peeters, E., Driessen, R.P.C., Werner, F. et al. (2015) The interplay between nucleoid organization and transcription in archaeal genomes. Nature Reviews Microbiology 13:333-341

2. Grohmann, D., et al. & Werner, F. (2011) The Initiation Factor TFE and the Elongation Factor Spt4/5 Compete for the RNAP Clamp during Transcription Initiation and Elongation. Molecular Cell 43:263-274

3. Werner F. & Grohmann D. (2011) Evolution of the RNA polymerases in the three domains of life. Nature Reviews Microbiology 9:86-98

4. Takagi, Y., et al. & Molloy, J.E. (2014) Myosin-10 produces its power-stroke in two phases and moves processively along a single actin filament under low load. Proc. Natl. Acad. Sci. 111:E1833-1842

5. Fili, N., et al. & Molloy, J.E. (2010) Visualizing helicases unwinding DNA at the single molecule level. Nucleic Acids Res. 38:4448-4457

6. Skinner, G.M., Molloy, J.E. et al. (2004) Promoter binding, initiation, and elongation by bacteriophage T7 RNA polymerase - A single-molecule view of the transcription cycle. J. Biol. Chem. 279:3239-3244


21

Justin Molloy, Paula Booth & Sergi Garcia Manyes http://crick.ac.uk/research/a-z-researchers/researchers-k-o/justin-molloy/ https://www.kcl.ac.uk/nms/depts/chemistry/people/core/boothpaula.aspx https://www.kcl.ac.uk/nms/depts/physics/people/academicstaff/garcia-manyes.aspx Joint Crick/King’s College London position

Biological self-assembly: single molecule force methods to study the folding of membrane transport proteins Natural systems are reliant on the efficient and accurate self-assembly of their constituent components. Understanding of how newly synthesised proteins fold to their correct structure will give insight into how cells assemble one of their key components. Current knowledge of the folding phenomenon – often referred to as cracking the second half of the genetic code – is largely limited to experimentally amenable water-soluble proteins. This project addresses the more challenging and highly important class of proteins that are integral to cell membranes. State-of-the-art, single molecule force microscopy methods will be combined in a new approach to gain unprecedented insight into the forces underlying correct assembly of membrane transport proteins.

Membranes surround cells forming a barrier to the outside world. The basic fabric of the membrane is a lipid bilayer which contains proteins that regulate the transfer of matter and information across the membrane. These proteins form the vast majority of drug targets. This work addresses the dominant class of alpha helical membrane proteins that are ubiquitous across prokaryotes and eukaryotes. There are very few methods available to probe the details of membrane protein folding. This project exploits the potential of single molecule force spectroscopy to quantify molecular interactions both within the protein itself, as well as with the neighbouring lipids. Helical membrane proteins require unfolding forces that typically lie in the range spanning 20-150 pN, which is amenable to force spectroscopy AFM. However, the reverse mechanism, encompassing the folding phenomena, occurs at seemingly low forces of 5-25 pN, which can be readily measured using force spectroscopy magnetic tweezers under force-clamp conditions. By combining both state-of-the art single molecule mechanical techniques, our measurements will elucidate key mechanistic detail on unfolding/refolding of membrane proteins. Emphasis will be placed on their mechanical interaction with the distinct neighbouring lipid moietiesthat are known to influence protein insertion, folding and stability.

It has previously been shown that helical membrane proteins unfolded by pulling out of the membrane at a high stretching force, spontaneously re-insert into the membrane when the mechanical force is withdrawn. This was demonstrated for proteins with simple helical topological structures, namely a single domain bundle of transmembrane helices linked by short loops. This project will involve the study of more complex structures involving more than one domain and greater complexity in structure and topology. The focus will be exemplar members of key helical transport protein families. This study will also provide a new quantitative method for investigating the insertion of proteins into membranes, and breaching of the lipid bilayer during pathological processes such as mammalian host-cell invasion and egress by viral particles, bacteria and parasitic organisms and normal physiological processes such as endo- and exocytosis. Our work plan is to first establish a model system and then collaborate with Crick Biologists who have immediate interest in this area of science: Pavel Tolar (immunologist), Mike Blackman and Tony Holder (parasitologists).

1. Natkanski, E., Lee, W. Y., Mistry, B., Casal, A., Molloy, J. E. & Tolar, P. (2013). B cells use mechanical energy to discriminate antigen affinities. Science 340, 1587-90.

2. Findlay, H. E., Rutherford, N. G., Henderson, P. J. F. & Booth, P. J. (2010). The unfolding free energy of a two-domain transmembrane sugar transport protein. Proc Natl Acad Sci U S A 107, 18451-18456.

3. Popa, I., Kosuri, P., Alegre-Cebollada, J., Garcia-Manyes, S. & Fernandez, J. M. (2013). Force dependency of biochemical reactions measured by single-molecule force-clamp spectroscopy. Nat Protoc 8, 1261-76.

4. Oesterhelt, F., Oesterhelt, D., Pfeiffer, M., Engel, A., Gaub, H. E. & Müller, D. J. (2000). Unfolding pathways of individual bacteriorhodopsins. Science 288, 143-146.

5. Harris, N. J., Findlay, H. E., Simms, J., Liu, X. & Booth, P. J. (2014). Relative domain folding and stability of a membrane transport protein. J Mol Biol 426, 1812-25.

NOTE: Additional eligibility criteria apply to this position: Non-EU applicants are not eligible for the funding of this position.


22

Kathy Niakan http://crick.ac.uk/research/a-z-researchers/researchers-k-o/kathy-niakan/ Characterising novel regulators of human pluripotency and embryogenesis This project will characterise key regulators of human embryogenesis. We have identified multiple transcription factors that are highly expressed in pluripotent epiblast cells of the developing human embryo1. The pluripotent epiblast has the unique potential to give rise to the entire fetus in vivo and can self-renew indefinitely as embryonic stem cells (hESCs) in vitro. Understanding the molecular basis of pluripotency in human cells is of fundamental biological importance and has significant clinical implications for the use of hESCs to treat diseases. Importantly, the transcription factors we identified as enriched in human embryos are not expressed in mouse embryos at the equivalent developmental stage, further suggesting differences in pluripotency mechanisms between these species2. The aim of the project is to functionally test these putative regulators of human pluripotency and embryogenesis. The specific objectives of the project are:

1. The student will evaluate protein expression of putative pluripotency factors in human embryos by immunofluorescence and confocal microscopy. Proteins highly expressed specifically in epiblast cells will be good candidates for future investigation. We have validated some of the human epiblast-enriched factors, including KLF17 (Fig. 1). However, other candidates have yet to be tested, such as ARGFX and VENTX. This first objective is essential for subsequent objectives and only candidates that have been validated will be further investigated.

2. The student will test the functional requirement of the putative pluripotency factors in recently established ‘naïve’ hESCs3. Some of the factors we identified (i.e. KLF17 and ARGFX) are also expressed in ‘naïve’ hESCs that more closely resemble the in vivo epiblast3, compared to conventional hESCs. To test their requirement for the establishment and maintenance of pluripotency in naïve hESCs, CRISPR/Cas9 mutagenesis will be used to disrupt the gene. Established stem cell self-renewal and pluripotency assays will be used to comprehensively characterize the mutant cells4. Transcriptome analysis will be performed to investigate gene expression changes resulting from CRISPR-induced loss-of-function. These experiments will provide functional proof of the role of these factors in pluripotency.

3. Depending on the outcome above, the student will test the activity of putative pluripotency factors to enhance reprogramming of somatic cells to induced pluripotent stem cells (iPS) cells. Derivation of iPS cells is currently inefficient and only a small fraction of somatic cells become fully reprogrammed. Therefore, the student will induce expression of novel factors together with, or independent of, the Yamanaka factors (OCT4, SOX2, MYC and KLF4). A number of established techniques in the lab will be used to evaluate the efficiency of reprogramming including transcriptome and immunofluorescence analyses. Depending on the outcome of the second or third objective, ChIP-sequencing analysis5 will be used to investigate how the factor(s) fits into the well-defined human pluripotency gene regulatory network. A number of publically available ChIP-sequencing and transcriptome databases will be integrated together with any data generated from these studies. Importantly, we have bioinformatics expertise in the lab to provide training in analyzing these data.

Through these experiments the student will provide fundamental insights into human biology with direct relevance to stem cell biology.

1. Blakeley P., Fogarty N.M.E., del Valle I., Hu T.X. Elder K., Snell P., Christie L., Robson P. and Niakan K.K. Single-cell RNA-seq defines the three cell lineages of the human blastocyst, manuscript under review at Development.

2. Niakan K.K. and Eggan K. (2013) Lineage-specifying transcription factor expression dynamics in human preimplantation embryos reveals precocious OCT4 expression relative to mouse. Developmental Biology 375: 54-64.

3. Takashima Y., Guo G., Loos R., Nichols J., Ficz G., Krueger F., Oxley D., Santos F., Clarke J., Mansfield W., Reik W., Bertone P., Smith A. (2014) Resetting transcription factor control circuitry toward ground-state pluripotency in human. Cell 158(6): 1254-1269.

4. Dimos J.T., Rodolfa K.T., Niakan K.K., Weisenthal L.M., Mitsumoto H., Chung W., Croft G.F., Saphier G., Leibel R., Goland R., Wichterle H., Henderson C.E. and Eggan K. (2008). Induced


23

pluripotent stem cells generated from patients with ALS can be differentiated into motor neurons. Science 321(5893): 1218-21.

5. Wamaitha S.E., del Valle I., Cho L.T., Wei Y., Fogarty N.M.E., Blakeley P., Sherwood R.I., Ji H. and Niakan K.K. (2015) Gata6 potently initiates reprogramming of pluripotent and differentiated cells to extraembryonic endoderm stem cells. Genes and Development, 29(12): 1239-1255.


24

Paul Nurse http://crick.ac.uk/research/a-z-researchers/researchers-k-o/paul-nurse/ Global cellular controls in eukaryotic cells The laboratory is interested in the global networks that regulate and couple the eukaryotic cell cycle, cell form and cell growth. These processes are central to the development of living organisms and can become deregulated in disease states. We take a multidisciplinary approach to the study of these problems, and explore a variety of methodologies when trying to tackle them.

A potential PhD project would investigate the regulation of cell size and its integration with cell cycle control. The regular cylindrical shape and well characterized cell cycle control network of fission yeast Schizosaccharomyces pombe make it ideal to study cell size control; extensive genetic, genomic, chemical and cell biological tools and resources offer a unique opportunity to gain insight into this problem.

The advent of new single cell methodologies has reinvigorated the field of cell size research. In the lab, we use microfluidics devices, advanced fluorescence microscopy and automated image analysis to extract phenotypic information from populations of growing cells. We are developing synthetic and chemical biology approaches to combine with imaging to provide direct readouts of cellular physiology in real time at the single cell level.

Analysis of the large-scale data sets generated from these approaches benefits from a computational approach. Experimentally driven mathematical modelling could enhance our understanding of potential cell size control mechanisms.

Investigation of the molecular mechanisms of cell size control will benefit from the wide range of genetic tools available in S. pombe, for example genome-wide gene deletion collections and a fluorescently tagged cDNA library. An extensive array of phenotypic information provided by recent screens and studies in the lab (Kim et al, 2010), (Navarro & Nurse, 2012) provides a rich resource of candidate regulators. A previously developed minimal cell cycle control network (Coudreuse & Nurse, 2010) provides a simplified system in which to study the integration of size control with cell cycle control.

This project is just one example of the sort of question you could ask in this research group. The precise project will be developed with the supervisor and driven by the individual student's interests and curiosities. The range of methodologies used will depend on the nature of the research question. This provides a unique PhD experience, allowing independence and the creative freedom to investigate your interests with support, training and guidance from other lab members.

1. Coudreuse D, Nurse P (2010) Driving the cell cycle with a minimal CDK control network. Nature 468: 1074-1079

2. Kim DU et at. (2010) Analysis of a genome-wide set of gene deletions in the fission yeast Schizosaccharomyces pombe. Nature biotechnology 28: 617-623

3. Navarro FJ, Nurse P (2012) A systematic screen reveals new elements acting at the G2/M cell cycle control. Genome biology 13: R36

4. Kaykov, A. Nurse, P. (2015) The spatial and temporal organization of origin firing during the S-phase of fission yeast. Genome Res. 25: 391-401


25

Andy Oates http://crick.ac.uk/research/a-z-researchers/researchers-k-o/andrew-oates/ Control of the period of the genetic oscillations in the segmentation clock The generation of a segmented body axis is one of the fundamental early patterning processes in developmental biology. The period of somite formation in the embryo is influenced by the period of genetic oscillations in the segmentation clock, a population of progenitor cells. Cells in this rhythmic tissue maintain a near-constant period while in a progenitor state then appear to slow down as they differentiate. Current understanding of these processes focuses on a transcription-translation negative feedback oscillator in which the period of this feedback loop depends on the half-life of the transcription factor mRNA and proteins, the delays in producing them, and their production rates. However, these hypotheses have not been satisfactorily tested in any system. We have generated transgenic zebrafish that allow us to visualize the genetic oscillations in real time; tissue level dynamics and the behavior of individual cells both in vivo and in vitro have been observed. We have generated a series of transcription factors with altered half-lives, and made maps of the regulatory regions of their genes. In this project, we will make transgenic zebrafish with these and other variants and search for those lines with an altered somitogenesis period. In parallel, we will use quantitative fluorescence imaging in vivo and in vitro to measure the number of proteins produced in each cycle. Combined, these experiments will allow us to test our understanding and explore new avenues in the molecular control of the oscillations. They will also provide key data to inform our quantitative and theoretical models of the process.

This is just one example of the sort of project that might be available in this research group. The precise project will be decided on in consultation with the supervisor.

1. A Doppler effect in embryonic pattern formation. Soroldoni D, Jörg DJ, Morelli LG, Richmond DL, Schindelin J, Jülicher F, Oates AC. Science. 2014 Jul 11;345(6193):222-5.

2. Topology and dynamics of the zebrafish segmentation clock core circuit. Schröter C, Ares S, Morelli LG, Isakova A, Hens K, Soroldoni D, Gajewski M, Jülicher F, Maerkl SJ, Deplancke B, Oates AC. PLoS Biol. 2012;10(7):e1001364.

3. Patterning embryos with oscillations: structure, function and dynamics of the vertebrate segmentation clock. Oates AC, Morelli LG, Ares S. Development. 2012 Feb;139(4):625-39.

4. Segment number and axial identity in a segmentation clock period mutant. Schröter C, Oates AC. Curr Biol. 2010 Jul 27;20(14):1254-8.


26

Markus Ralser & Jurg Bahler http://crick.ac.uk/research/a-z-researchers/researchers-p-s/markus-ralser/ https://www.ucl.ac.uk/gee/gee-staff/academic-staff/jurg-bahler Joint Crick/University College London position The genetic diversity controlling metabolism With ~1500-2000 participating enzymes, the metabolic network is the largest, interconnected biochemical system within the cell. With the recent advent of advanced analytical methods it becomes clear that the metabolic system is highly dynamic and an integral component of the cell regulatory system. Changes in metabolism have been associated with several complex human disorders such as cancer, diabetes and neurodegeneration. The genetic mechanisms that control cellular metabolic networks are however far from understood.

To learn about genetic traits underlying metabolism in eukaryotes, we will take advantage of a genotypically and phenotypically diverse collection of ~100 natural isolates of fission yeast (Schizosaccharomyces pombe). Our recent analyses have uncovered substantial heterogeneity among these wild strains in free amino acid concentrations (Jeffares et al. 2015), which reflect flux endpoints for key energy and biosynthetic pathways. We have also sequenced the genomes of all strains to sample genetic variations, and a genome-wide association study has highlighted several candidate loci that affect metabolic patterns (Jeffares et al. 2015). We will analyze genotype-phenotype relationships underpinning intra-species differences in metabolism. We will use mass-spectrometry-based metabolic profiling and protein quantification to depict differences in metabolism. The student will apply an innovative proteomics pipeline, SWATH-MS, which allows to quantify ~70% of yeast metabolic enzymes in every strain, and to assay the range of enzyme abundance in the yeast collection. Having genotype information available, this approach will allow mapping of protein quantitative trait loci (pQTLs, Melzer et al. 2008) and combine those with the existing metabolite quantitative trait loci (mQTLs, Jeffares et al. 2015). Then we will cross strains with distinct amino-acid signatures and analyse the resulting signatures of progeny strains. The genomes of progeny with distinct metabolic signatures will be sequenced to check what genetic variants of the parental strains are present (linkage analysis) (Liti & Louis 2012).

Integrating these unique sets of information will guide systematic analyses into regulatory mechanisms underlying the cellular metabolism. The high density of genetic markers revealed by re-sequencing of haploid yeast strains will facilitate the pinpointing of causal variations that affect metabolite concentrations. These analyses will inform about key genetic factors in coding and non-coding regions, and associated changes in genome regulation, that determine cellular metabolism. These findings can be exploited to also analyse gene-environment and gene-gene interactions underlying metabolic pathways. The student will then validate the phenotypic effects of key metabolic factors by reconstructing selected variants in a suitable reference strain using the CRISPR-Cas9 system for precise genome editing (Jacobs et al. 2014). Finally, computional analysis will reveal the evolutionary conservation of the identified metabolite regulators and map them on the human genome, for which such information is largely absent.

In conclusion, this exciting multi-disciplinary project will yield unique insights into the genetic basis and genome-scale regulation underlying metabolic networks. The project will allow the student to advance in state of the art genetics and biological analytics. This research will help us to understand the systems-level relationships between genotype and metabolism, which in turn will guide research into analogous processes that underpin complex phenomena such as cancer, metabolic diseases and ageing for which altered metabolism is a key characteristic.

1. Jeffares DC, Rallis C, Rieux A, Speed D, Převorovský M, Mourier T, Marsellach FX, Iqbal Z, Lau W, Cheng TM, Pracana R, Mülleder M, Lawson JL, Chessel A, BalaS, Hellenthal G, O'Fallon B, Keane T, Simpson JT, Bischof L, Tomiczek B, BittonDA, Sideri T, Codlin S, Hellberg JE, van Trigt L, Jeffery L, Li JJ, Atkinson S, Thodberg M, Febrer M, McLay K, Drou N, Brown W, Hayles J, Carazo Salas RE, Ralser M, Maniatis N, Balding DJ, Balloux F, Durbin R, Bähler J. The genomic and phenotypic diversity of Schizosaccharomyces pombe. Nature Genetics. 2015 Mar;47(3):235-41. doi: 10.1038/ng.3215

2. Mülleder M, Capuano F, Pir P, Christen S, Sauer U, Oliver SG, Ralser M. Aprototrophic deletion mutant collection for yeast metabolomics and systems biology. Nature Biotechnology. 2012 Dec;30(12):1176-8. doi: 10.1038/nbt.2442.


27

3. Liti G, Louis EJ. Advances in quantitative trait analysis in yeast. PLoS Genetics. 2012;8(8):e1002912. doi: 10.1371/journal.pgen.1002912. Epub 2012 Aug 16.

4. Melzer D, Perry JR, Hernandez D, Corsi AM, Stevens K, Rafferty I, Lauretani F, Murray A, Gibbs JR, Paolisso G, Rafiq S, Simon-Sanchez J, Lango H, Scholz S, Weedon MN, Arepalli S, Rice N, Washecka N, Hurst A, Britton A, Henley W, van de Leemput J, Li R, Newman AB, Tranah G, Harris T, Panicker V, Dayan C, Bennett A, McCarthy MI, Ruokonen A, Jarvelin MR, Guralnik J, Bandinelli S, Frayling TM, Singleton A, Ferrucci L. A genome-wide association study identifies protein quantitative trait loci (pQTLs). PLoS Genetics. 2008 May 9;4(5):e1000072. doi: 10.1371/journal.pgen.1000072.

5. Jacobs JZ, Ciccaglione KM, Tournier V, Zaratiegui M. Implementation of the CRISPR-Cas9 system in fission yeast. Nat Commun. 2014 Oct 29;5:5344. doi: 10.1038/ncomms6344


28

Katrin Rittinger & Franca Fraternali http://crick.ac.uk/research/a-z-researchers/researchers-p-s/katrin-rittinger/ http://www.kcl.ac.uk/lsm/research/divisions/randall/research/sections/structural/fraternali/fraternalifranca.aspx Joint Crick/King’s College London position Dynamics and mechanisms underlying RBR E3 ligase activity Ubiquitination is a post-translational modification that regulates a vast array of cellular processes ranging from protein degradation to immune signalling. The modification of proteins with ubiquitin is catalysed by the sequential activity of three enzymes. The last enzyme in this cascade, the E3 ubiquitin ligase, selects the substrate to be modified and in some cases also the topology of the ubiquitin chain to be synthesised and is hence the key determinant of the ubiquitination reaction. RBR family ligases are a subfamily of E3 ubiquitin ligases that are associated with multiple cellular functions; misregulation of their activity is linked to a number of diseases including immune disorders and early-onset Parkinson’s disease. Members of this family contain a RBR (RING-between-RING) domain that habors the catalytic activity and consists of 3 subdomains (RING1, IBR and RING2) separated by flexible linkers. Ubiquitination of substrate proteins by RBR ligases is a multi-step process that starts with the recognition of the ubiquitin-loaded E2 by the RING1 domain. Next, the ubiquitin is transferred onto a conserved cysteine in RING2 to form a thioester intermediate, which in the final step is attached to the protein substrate or to another ubiquitin molecule to form a poly-ubiquitin chain. Our recent work (Rittinger group) has shed light on how the final ubiquitin transfer step occurs during the synthesis of linear ubiquitin chains by HOIP, a subunit of the multimeric LUBAC E3 ligase. We now want to characterise the conformational changes that occur in the preceding steps that promote formation of the thioester intermediate. In fact, we believe the flexibility of the linkers connecting RING1, IBR and RING2 to be key for the activity of this ligase family. These linkers orchestrate the orientation and therefore the presentation of the RING1 and RING2 domains for trans-thiolation to occur. This is a highly dynamic process and therefore not directly accessible to structural analysis by X-ray crystallography alone. To study such an intrinsic dynamic phenomenon, we propose an integrative approach combining bioinformatics, molecular modelling and enhanced sampling molecular dynamics techniques of the interdomain linkers with structural and biophysical characterisation of partial and intact RBR domains by solution methods such as NMR spectroscopy and SAXS (small angle X-ray scattering). The ultimate goal is to produce realistic models of the structures formed during the individual transfer steps. We will use the RBR domain of HOIP as our primary target system, as this protein is well behaved and we can track activity via thioester-intermediate formation and synthesis of unanchored ubiquitin chains using established biochemical assays.

1. Berndsen, C.E. and Wolberger C. (2014) New insights into ubiquitin E3 ligase mechanism. Nature Structural&Molecular Biology, 21 , 301-307. (Review)

2. Stieglitz, B., Morris-Davies, A.C., Koliopoulos, M.G., Christodoulou, E., and Rittinger, K. (2012). LUBAC synthesizes linear ubiquitin chains via a thioester intermediate. EMBO Rep 13, 840-846.

3. Stieglitz, B. Rana, R.R. Koliopoulos, M.G., Morris-Davies, A.C., Schaeffer, V., Christodoulou, E., Howell, S., Brown, N.R., Dikic, I. and Rittinger, K. (2013) Structural basis for ligase-specific conjugation of linear ubiquitin chains by HOIP. Nature, 503, 422-426.

4. Fornili A, Pandini A, Lu HC, Fraternali F. (2013) Specialized Dynamical Properties of Promiscuous Residues Revealed by Simulated Conformational Ensembles. J Chem Theory Comput., 9, 5127-5147.

5. De Simone A, Corrie JE, Dale RE, Irving M, Fraternali F. (2008) Conformation and dynamics of a rhodamine probe attached at two sites on a protein: implications for molecular structure determination in situ. J Am Chem Soc., 130, 17120-17128.


29

Peter Rosenthal http://crick.ac.uk/research/a-z-researchers/researchers-p-s/peter-rosenthal/ Structural Studies of Influenza Virus Entry and Assembly by Cryomicroscopy Our group studies the architecture of large protein assemblies in order to understand basic molecular mechanisms that control protein and membrane traffic in the cell and in virus infection. Recently, the technique of cryomicroscopy (cryoEM) has made revolutionary advances in its ability to image biological specimens at high resolution. We apply cryoEM to directly image protein structure in vitro and in the cell, in association with other physical techniques. The lipid-enveloped influenza virus is a major human pathogen. We are interested in understanding how influenza virus enters the cell by binding to cell surface receptors, how the virus membrane fuses with host membranes, and how the virus assembles and releases from the cell. We have previously performed high-resolution studies of influenza virus ultrastructure by cryotomography that have shown us the internal architecture of the virus as well as the structure of envelope glycoproteins in situ. The main goal of this project is to apply cryoEM to image steps in virus entry and assembly. Approaches will include direct imaging of influenza with model membranes and/or the imaging of influenza virus infection in cells. We are interested in structural transformations in the influenza hemagglutinin that mediate membrane fusion as well as other ultrastructural changes in the virus and host cell important to entry and assembly. The laboratory is interested in a range of problems from the molecular to the cellular scale using single particle analysis and tomography. We also work to improve experimental imaging and develop new computational methods for image analysis. While a single studentship is available, other topics for study will be considered in consultation with the advisor. The student will receive training in experimental cryomicroscopy, image analysis, protein and virus methods, and imaging of cells by light microscopy.

1. Rosenthal, P.B. 2015 From high symmetry to high resolution in biological electron microscopy. Philos. Trans. R. Soc. Lond. B Biol. Sci. 370, 2014035

2. Sader, K., Stopps, M., Calder, L.J., and Rosenthal, P.B. 2013 Cryomicroscopy of Radiation Sensitive Specimens on Unmodified Graphene Sheets: Reduction of Electron Optical Effects of Charging. Journal of Structural Biology 183, 531-6.

3. Wasilewski, S., Calder, L.J., Grant, T., and Rosenthal, P.B. 2012 Distribution of Surface Glycoproteins on Influenza A Virus Determined by Electron Cryotomography. Vaccine 30, 7368-73.

4. Calder, L.J., Wasilewski, S., Berriman, J.A., and Rosenthal, P.B. 2010 Structural Organization of Filamentous Influenza A Virus. Proc. Natl. Acad. Sci. U.S.A. 107, 10685-10690.

5. Berriman, J., Li, S., Hewlett, Li, S., Wasilewski, S., Kiskin, F., Carter, T., Hannah, M., and Rosenthal, P.B. 2009 Structural Organization of Weibel-Palade Bodies Revealed by Cryo-EM of Vitrified Endothelial Cells. Proc. Natl. Acad. Sci. U.S.A. 107, 10685-10690.


30

Erik Sahai http://crick.ac.uk/research/a-z-researchers/researchers-p-s/erik-sahai/ Tracking and predicting clonal competition in genetically heterogeneous tumours A likely project that exemplifies research goals currently being pursued in the Sahai group is described. The goal of this work is to develop predictive models of the response of genetically heterogeneous tumours to varying therapies. To understand how intra-tumour heterogeneity affects the efficacy of targeted therapy we will generate experimental models in which genetic heterogeneity is a controlled experimental variable. These will begin in vitro and progress through organotypic models to in vivo multiphoton tumour imaging. In addition to our empirical approach, we will collaborate with computational scientists. Detailed experimental measurements will be used to parameterise theoretical models. Through repeated iterations of computational modelling and experiments, we will be able to determine the role of paracrine/bystander interactions between clones in response to therapy. Following validation, our mathematical model will be able to computationally probe a huge range of treatment variations that could not be explored experimentally. Those predicted to be most effective will be experimentally tested. This work should provide new paradigms for determining the optimum chronology and phasing of targeted chemotherapy.

1. Gerlinger et al. Intratumor heterogeneity and branched evolution revealed by multiregion sequencing. New England Journal of Medicine (2012).

2. Marusyk, A. et al. Non-cell-autonomous driving of tumour growth supports sub-clonal heterogeneity. Nature 514, 54-58 (2014).

3. MB Meads, RA Gatenby, WS Dalton. Environment-mediated drug resistance: a major contributor to minimal residual disease. Nature Reviews Cancer (2009). Intravital Imaging Reveals How BRAF Inhibition Generates Drug-Tolerant Microenvironments with High Integrin β1/FAK Signaling

4. Hirata et al.Intravital Imaging Reveals How BRAF Inhibition Generates Drug-Tolerant Microenvironments with High Integrin β1/FAK Signaling. Cancer Cell 27 (4), 574-588 (2015)


31

Guillaume Salbreux http://crick.ac.uk/research/a-z-researchers/researchers-p-s/guillaume-salbreux/ Mechanics of cell division in a tissue In our research group, we use approaches from theoretical physics of soft matter and statistical physics to address biological questions. The general strategy is to identify key physical parameters playing a role in cellular and developmental process and ask how molecules in the cell regulate them. To understand this, we collaborate with experimentalists working in the fields of cell and developmental biology.

During tissue growth, cells must round up, orient their mitotic spindle, elongate, and establish their plane of division. These events must be coordinated and involved the generation of mechanical forces. Cells are for instance able to sense their elongation and establish a division plane perpendicular to it. The aim of the PhD project is to develop a theoretical model to understand the mechanics of cell division in the context of an epithelium. The model will describe the mechanics of key elements of the cytoskeleton in the dividing cell, as well as take into account overall forces acting in the tissue. The role of forces exerted by external cells, fluctuations in cell shape and cellular forces, and mechanisms leading to orientation of the mitotic spindle will be investigated. The project involves collaborative work with experimentalists working on this question.

Candidates with a background in theoretical physics or applied maths and interested in answering biological questions are encouraged to apply.

This is just one example of the sort of project that might be available in this research group. The precise project will be decided on in consultation with the supervisor.


32

Martin Singleton http://crick.ac.uk/research/a-z-researchers/researchers-p-s/martin-singleton/ Structural Biology of Chromosome Segregation Our lab is interested in understanding the molecular mechanisms of chromosome segregation in eukaryotic cells. This precisely controlled process requires the coordinated action of many multi-protein complexes associated with chromosomes. We are particularly interested in the kinetochore and the cohesin complex. Kinetochores are the large proteinaceous structures that comprise the connection between chromosomes and the mitotic spindle. In addition to providing a physical link between these huge cellular components, the kinetochore is responsible for generating the spindle assembly checkpoint (SAC), which ensures that all chromatids are correctly attached to the spindle before anaphase onset. The duplicated sister chromatids themselves are held together by the cohesin complex, which ensures that each sister is physically linked from the time of DNA replication to anaphase. This large, ring-shaped complex is loaded onto, and removed from chromosomes in a multi-stage process. Some of the factors required for this process have been identified, but their exact activities are still not yet fully understood.

We are studying protein and protein-nucleic acid complexes involved both of these systems, primarily by X-ray crystallography and electron microscopy. By determining their three-dimensional structures we can generate hypotheses about the underlying mechanisms that can then be tested both in vitro and in vivo, and inform further structural approaches. We are seeking a motivated Ph.D. student who wishes to answer fundamental questions about the mechanisms of chromosome segregation using multiple biophysical, biochemical, and genetic techniques.

These are examples of the sorts of project that might be available in this research group. Only one studentship is available with this group and the precise project will be decided on consultation with the supervisor.

1. Chatterjee, A., Zakian, S., Hu, X.-W., and Singleton, M.R. (2013). Structural insights into the regulation of cohesion establishment by Wpl1. EMBO J, Volume 32. pp. 677-687.

2. Santaguida, S., and Musacchio, A. (2009). The life and miracles of kinetochores. EMBO J, Volume 28. pp. 2511-2531.

3. Uhlmann, F. (2009). A matter of choice: the establishment of sister chromatid cohesion. EMBO Rep, Volume 10. pp. 1095-1102.


33

Jim Smith http://crick.ac.uk/research/a-z-researchers/researchers-p-s/jim-smith/ PAWS1 and Wnt signalling in early vertebrate development During early vertebrate development, the embryo integrates the activities of several signalling pathways to specify its dorso-ventral axis and to generate and pattern its three germ layers: endoderm, mesoderm and ectoderm1,2. Two of the most important families of signalling molecules are the TGF-ß superfamily (including the nodals, activin and the bone morphogenetic proteins, or BMPs), and the Wnt family. We are interested in understanding how these signalling pathways are regulated, and to do so we are studying new molecules that either modulate signalling or regulate intracellular trafficking or turnover of key signalling intermediates.

In collaboration with Gopal Sapkota (Dundee) we are studying PAWS1/FAM83G, a novel component of the BMP pathway3. PAWS1 interacts with Smad1, and plays a role in regulating BMP-dependent/Smad4-independent gene transcription. Surprisingly, however, it also regulates the expression of non-BMP target genes, and our preliminary evidence suggests that PAWS1 stimulate Wnt signalling in the early Xenopus embryo.

Preliminary experiments place PAWS1 upstream of ß-catenin in the Wnt pathway, and the project will go on to explore in detail how PAWS1 influences Wnt signalling. For example, biochemical and proteomic techniques will attempt to identify Wnt-dependent PAWS1 interactors using extracts from cell lines and Xenopus embryos. Other approaches will complement Xenopus loss-of-function experiments by use of CRISPR-Cas9 to generate PAWS1 knockouts in Wnt-reporter zebrafish lines4 (Moro et al., 2013) or in mouse intestinal organoids5. Analysis of the phenotypes of such embryos has already revealed a role for PAWS1 in ciliogenesis,

1. Wu MY and Hill CS (2009). TGF-ß superfamily signalling in embryonic development and homeostasis. Dev. Cell 16, 329.

2. De Robertis EM, Larrain J, Oelgeschlager M. and Wesely O (2000). The establishment of Spemann’s organizer and patterning of the vertebrate embryo. Nat. Rev. Genet. 1, 171.

3. Vogt J, Dingwell KS, Herhaus L, Gourlay R, Macarney T, Campbell D, Smith JC and Sapkota GP. (2014). Protein associated with SMAD1 (PAWS1/FAM83G) is a substrate for type I bone morphogenetic protein receptors and modulates bone morphogenetic protein signalling. Open Biol. 4, 130210.

4. Moro E, Vettori A, Porazzi P, Schiavone M, Rampazzo E, Casari A, Ek O, Facchinello N, Astone M, Zancan I, Milanetto M, Tiso N, and Argenton R (2013). Generation and application of signaling pathway reporter lines in zebrafish. Mol. Genet. Genomics 288, 231.

5. Baker N (2014). Adult intestinal stem cells: critical drivers of epithelial homeostasis and regeneration. Nat Rev Mol. Cell Biol. 15, 19.


34

Thomas Surrey http://crick.ac.uk/research/a-z-researchers/researchers-p-s/thomas-surrey/ Reverse engineering of spindle function A major challenge in cell biology is to understand the behaviour of the cell as a system. During cell division, the microtubule cytoskeleton rearranges to build the mitotic spindle that segregates the genetic material. A multitude of proteins are involved in this intracellular reorganisation process. Today the most important players that are involved are known. However, how in their combination they define a specific form of the many dynamic arrangements that the cytoskeleton is able to build is not understood. Specifically, it is unclear what is the minimal interaction network of motor proteins, microtubule crosslinkers, chromosomal and membrane anchors that can build a spindle-like structure and that can position it correctly within the boundaries of the cell. In this project, we will us a reverse engineering approach to recapitulate the organisation of the mitotic microtubule cytoskeleton within a membrane boundary. Motor proteins like kinesins, dynein and their interaction partners will be key is this synthetic approach. We will extend existing in vitro reconstitutions to systematically explore the phase space of accessible dynamic microtubule architectures. Our goal is to better understand the design principles of intracellular order. Specifically, we will aim at understanding the control of the shape, the size and the position of asters and spindle-like structures structures within cell-sized volumes enclosed by lipid membranes. Advanced fluorescence microscopy, microfabrication, microfluidics, in vitro reconstitutions, modern protein biochemistry and mathematical modelling will be important methods used in this project.

1. Dogterom M, Surrey T. (2013) Microtubule organization in vitro. Curr Opin Cell Biol. 25, 23-9. (Review)

2. Duellberg C, Trokter M, Jha R, Sen I, Steinmetz MO, Surrey T. (2014) Reconstitution of a hierarchical +TIP interaction network controlling microtubule end tracking of dynein. Nat Cell Biol. 804-11.

3. Baumann H, Surrey T. (2014) Motor-mediated cortical versus astral microtubule organization in lipid-monolayered droplets. J Biol Chem. 289, 22524-35

NOTE: Additional eligibility criteria apply to this position: As well as meeting the standard eligibility criteria, applicants to this position must not have resided in the UK for more than 12 months in the last 3 years immediately prior to commencing the role.


35

Jesper Svejstrup http://crick.ac.uk/research/a-z-researchers/researchers-p-s/jesper-svejstrup/ Basic mechanisms at the interface between transcription, the maintenance of genome stability, and human disease The mechanism of transcription-coupled nucleotide excision repair (TC-NER) remains poorly understood. Cockayne syndrome B protein (CSB, also named ERCC6) plays a key role in both TC-NER and the global transcription response to DNA damage. It is recruited to damage-stalled RNAPII, allowing assembly of the core NER machinery around it. CSB contains a functionally important ubiquitin-binding domain and is itself ubiquitylated, but their precise function, and possible inter-connections, remain unknown. Importantly, some CSB ubiquitylation is carried out by a ubiquitin ligase complex containing CSA, another key TC-NER factor of poorly understood function. However, the relevant targets of the CSA ubiquitin ligase complex, also in other proteins than CSB, still need to be uncovered. We have now been able to map a number of ubiquitylation sites in different proteins, across the human proteome, which are both DNA damage- and CSA-dependent. We now need to understand the functional importance of these sites. This project will involve an unusually wide range of molecular biology-, biochemical, and cell biological approaches, including proteomics and genomics. This is just one example of the sort of project that will be available in this research group. The precise project with be decided in consultation with the supervisor.

1. Saponaro et al (2014). RECQL5 controls transcript elongation and suppresses genome instability associated with transcription stress. Cell 157, 1037-1049.

2. Close et al (2012). DBIRD integrates alternative mRNA splicing with RNA polymerase II transcript elongation. Nature 484, 386-389.

3. Anindya et al (2010). A Ubiquitin-Binding Domain in Cockayne Syndrome B Required for Transcription-Coupled Nucleotide Excision Repair. Molecular Cell 38, 637–648.

4. Anindya, R., Aygun, O., and Svejstrup, J.Q. (2007) Damage-Induced Ubiquitylation of Human RNA Polymerase II by the Ubiquitin Ligase Nedd4, but not Cockayne Syndrome Proteins or BRCA1. Molecular Cell 28, 386-397.


36

Charles Swanton http://crick.ac.uk/research/a-z-researchers/researchers-p-s/charles-swanton/ Exploiting Lung Cancer Heterogeneity by Leveraging the Host Immune Response Tumour immunotherapy is demonstrating impressive overall survival improvements in non-small cell lung cancer (NSCLC) and melanoma. However most patients with non-small lung cancer derive minimal or no benefit from immunotherapy, and acquisition of resistance to tumour immunotherapy remains a common phenomenon, the mechanisms of which are poorly understood. The lung cancer TRACERx study is a national tumour phylogenetics study, analysing 6000 deep exome datasets acquired from multiple regions of primary and metastatic tumour sites to decipher cancer evolution in 842 patients with NSCLC. Patients with no actionable mutations will be offered anti-PDL1 therapy if their disease recurs following surgery. Through serial sampling of tumours before and following drug resistance together with deep immunophenotypic analysis of tumour neo-antigen specific lymphocyte fractions during anti-PDL1 therapy, the candidate will examine mechanisms of immunotherapy response and tumour escape through immuno-editing. Through analysis of phylogenetic and RNAseq data, the candidate will examine the impact of distinct mutational and genome instability processes in lung cancer, including chromosomal instability and APOBEC induced mutagenesis upon the T cell repertoire and immune checkpoint control.

1. McGranahan N, et al Sci Transl Med. Apr 15;7(283):283ra54 (2015) 2. Murugaesu N et al. Cancer Discovery (2015) May 23 3. Mcgranahan N and Swanton C Cancer Cell Jan 12;27(1):15-26. (2015) 4. De Bruin EC Science 10;346(6206):251-6. (2014) 5. Jamal-Hanjani et al (2014) PLoS Biol. 2014 Jul 8;12(7)


37

Peter Thorpe & Attila Csikasz-Nagy http://crick.ac.uk/research/a-z-researchers/researchers-t-u/peter-thorpe/ http://www.kcl.ac.uk/lsm/research/divisions/randall/research/sections/motility/csikásznagy/home.aspx Joint Crick/King’s College London position Systems level understanding of mitotic localization by microtubules The microtubule organising centre (MTOC) is a key cellular structure that orients the plane of cell division and forms the structural scaffold for the segregation of chromosomes. Misregulation of MTOCs leads to chromosomal instability and is associated with a number of cancers (Godhino & Pellman 2014). This project aims to create a dynamic spatial model of cell cycle specific regulators of the MTOC based upon systematic studies of the MTOC in model systems. We have available a systematic dataset of the levels and location of the MTOC in yeast mutants, furthermore we have fused every protein in the proteome to the yeast MTOC and screened for growth phenotypes. These data, together with the extensive existing protein localization, genetic and physical interaction datasets combine to provide a powerful informatics dataset on the structure and function of the MTOC. This project aims to add to these data using synthetic physical interaction (SPI) screens of the yeast MTOC to produce functional models of how the MTOC changes during the cell cycle. Several cell cycle regulators, especially those that control cell division timing, are also localized at MTOCs. The obtained data will be also used to investigate the effects of perturbations in this regulatory module.

We will merge our screen data with literature data to construct a MTOC regulatory network. The SPI screen results will also inform us about the effect of constitutively localizing specific proteins to the MTOC. These results could help us to expand existing models of other biological processes and we will construct an online database to allow the SPI data to be mined. We will combine these systems-level data to initially generate models of the dynamical outcome of molecular interactions, this will lead to more complex spatial models where we take into account diffusion and microtubule based convection of key factors. Predictions from the model can be readily tested in budding yeast, where simple assays can be used to determine the effects of specific changes on MTOC assembly, MTOC orientation during mitosis, efficiency of chromosome segregation, mitotitc checkpoint and mitotic exit reulation. Such models have proved powerful to explain the asymmetry establishment between MTOCs in dividing fission yeast cells (Bajpai 2013) and spatial organization of cell polarity regulating protein clusters (Dodgson 2013).

The applicant must show evidence of excellent coding and mathematical ability. Ideally, the candidate will have an undergraduate degree in computer science, mathematics, image analysis, bioinformatics or bioengineering. The project will involve creation of new computational models and the modification of existing ones (Bajpai 2013, Dodgson 2013). It will also involve compiling, merging and analysing diverse high-throughput datasets (Vaggi 2012). The student will need to be capable of handling large data sets and extracting quantitative information.

1. Godhino SA, Pellman D. (2014) Causes and consequences of centrosome abnormalities in cancer. Philosophical Transactions of the Royal Society of London B: Biological Sciences 369(1650):20130466.

2. Bajpai A, Feoktistova A, Chen JS, McCollum D, Sato M, Carazo-Salas RE, Gould KL, Csikász-Nagy A. (2013) Dynamics of SIN Asymmetry Establishment. PLOS Computational Biology 9(7):e1003147

3. Dodgson J, Chessel A, Yamamoto M, Vaggi F, Cox S, Rosten E, Albrecht D, Geymonat M, Csikász-Nagy A, Sato M, Carazo-Salas RE. (2013) Spatial segregation of polarity factors into distinct cortical clusters is required for cell polarity control. Nature Communications 4:1834

4. Vaggi F, Dodgson J, Bajpai A, Chessel A, Jordan F, Sato M, Carazo-Salas RE, Csikász-Nagy A. (2012) Linkers of cell polarity and cell cycle regulation in the fission yeast protein interaction network. PLoS Computational Biology 8(10): e1002732


38

Pavel Tolar & Isabel Llorente Garcia http://crick.ac.uk/research/a-z-researchers/researchers-t-u/pavel-tolar/ https://www.ucl.ac.uk/phys/amopp/people/isabel_llorente_garcia Joint Crick/University College London position Mechanics of receptor ligand binding in immune cell synapses Cell surface receptors and ligands of the immune system bind each other during dynamic cell-cell contacts called immune synapses. Such interactions are important for immune recognition of foreing antigens, communication and transfer of material between cells, and spreading of pathogens. In contrast to soluble molecules, molecular interactions at cellular interfaces are influenced by the effects of the membrane environment, including passive and active mechanical forces, which can lead to modified receptor-ligand binding and unbinding rates and bond strengths, affecting the efficiency of molecular signalling events and specific cellular functions. In addition, many receptors are either organised in multivalent clusters or cluster upon ligand binding, which might lead to preferential receptor orientations or cooperative effects which could in turn influence the molecular binding mechanics. It is important to understand the specificity and potency of receptor-ligand systems, for instance, to elucidate the mechanisms of discrimination between self and non-self during cellular immune function, or to uncover the mechanisms of receptor-mediated virus entry.

Probing receptor-ligand bonds in situ has been a challenging task, but new force-sensing and force-pulling experiments at the single molecule level allow measurement of the relevant bond-rupture forces and kinetic binding rates under different force-loading conditions for various receptor-ligand systems. This project will use single molecule mechanical assays to investigate synaptic binding of B cell antigen receptors to antigens and of HIV particles to their cellular receptors. Binding will be measured using optical and magnetic tweezers with particular focus on the effects of mechanical forces and valency. These experiments will be followed by functional characterisation of the binding properties in immune cell activation and viral infection.

1. Natkanski, E. et al. B cells use mechanical energy to discriminate antigen affinities. Science 340, 1587–1590 (2013).

2. Tolar, P. & Spillane, K. M. Force generation in B-cell synapses: mechanisms coupling B-cell receptor binding to antigen internalization and affinity discrimination. Adv. Immunol. 123, 69–100 (2014).

3. Neuman KC, Nagy A. Single-molecule force spectroscopy: optical tweezers, magnetic tweezers and atomic force microscopy. Nat Methods. 2008 Jun;5(6):491–505.


39

Moritz Treeck http://crick.ac.uk/research/a-z-researchers/researchers-t-u/moritz-treeck/ Functional analysis of kinases secreted into the host cell by the human malaria parasite Plasmodium falciparum This PhD proposal aims to understand how the human malaria parasite Plasmodium falciparum remodels the red blood cell in which it resides. P. falciparum secretes ~ 20 kinases (called FIKK kinases) into the host cell, many of which are currently uncharacterized. Recent data from our lab shows specific phosphorylation of host cell proteins during parasite infection and we predict that the FIKK kinases mediate changes in the host cell by phosphorylation red blood cell proteins. In addition to regulate host cell proteins, the parasite also secretes a number of its own proteins into the host cell, many of which are also found phosphorylated. This harbours the exiting possibility that the parasite also regulates proteins after they have been secreted. We have recently developed a novel genetic system to conditionally manipulate genes in P. falciparum rapidly and currently generate conditional kinase-domain deletion mutants of the secreted kinases. The aim of this project will be to functionally characterize a subset of these conditional mutants and their role in the infection of the human red blood cell. To do that the PhD student will use state-of-the-art quantitative mass-spectrometry, cell-biology and imaging techniques.

1. A novel protein kinase family in Plasmodium falciparum is differentially transcribed and secreted to various cellular compartments of the host cell. Nunes MC, Goldring JP, Doerig C, Scherf A. Mol Microbiol. 2007 Jan;63(2):391-403. Epub 2007 Dec 20.

2. The phosphoproteomes of Plasmodium falciparum and Toxoplasma gondii reveal unusual adaptations within and beyond the parasites' boundaries. Treeck M, Sanders JL, Elias JE, Boothroyd JC. Cell Host Microbe. 2011 Oct 20;10(4):410-9. doi: 10.1016/j.chom.2011.09.004.


40

Richard Treisman http://crick.ac.uk/research/a-z-researchers/researchers-t-u/richard-treisman/ Molecular mechanisms of signal-regulated transcription and chromatin modification Work in the Signalling and Transcription group focusses on the transcriptional targets of Rho and Ras signalling, two major signalling pathways involved in oncogenic transformation, invasion and metastasis. Our main interest is in the transcription factor SRF and its two cofactor families, the TCFs and the MRTFs, which are regulated by Ras-ERK and Rho-actin signalling respectively (see Posern and Treisman 2006). We use a multidisciplinary approach, involving the biochemistry, structural biology, cell biology and genomics, applied to both tissue culture and mouse cancer and immune models. For more details see http://www.crick.ac.uk/research/a-z-researchers/researchers-t-u/richard-treisman/

Our previous studies have defined the direct genomic targets for the SRF and characterised the roles of the MRTFs and TCFs in their transcriptional activation by growth factor signals (Esnault et al, 2014, Esnault, Gualdrini et al, in preparation; Costello et al, 2015). Our current interests cover two areas: the role played by G-actin in regulating the MRTFs, and the relationship between TCF and MRTF activation and signal-induced chromatin modifications. Our recent work has used both genomic techniques and model genes to establish that ERK signalling to the TCFs is required for both transcriptional activation and induction of chromatin modifications. Correlation-based cluster analysis of five chromatin marks suggests a model in which chromatin modification proceeds stepwise, to generate a specific chromatin modification signature that correlates with active transcription. We have also used siRNA screening to identify specific factors contributing to different steps within the TCF-mediated chromatin cascade, and are working to understand their relationship to TCF activation (Gualdrini, Esnault, et al, in preparation).

We are particularly interested in the role played by G-actin in the control of the MRTFs, and the relationship between MRTF phosphorylation and transcriptional activation, and are pursuing two broad lines of investigation. Control of MRTF subcellular localisation is a major mechanism by which MRTF activity is regulated by G-actin (Miralles et al, 2003), but our previous studies have shown that nuclear G-actin suppresses MRTF target gene transcription (Vartiainen et al 2007). We are using biochemical and genomic approaches to analyse the effects of G-actin on MRTF-SRF interaction and gene targeting, and recruitment and initiation by RNA polymerase II. A second area of interest concerns the relationship between signalling to the MRTFs and chromatin modifications at their target genes. Here we will establish which the modifiers involved, the role played by the modifications in facilitating transcription, and their relation to chromatin modifications induced by the TCF proteins.

1. Posern G. and Treisman R. (2006) Actin' together: serum response factor, its cofactors and the link to signal transduction. Trends Cell Biol. 16:588-96.

2. Esnault, C., Stewart, A., East, P., Horswell, S., Mathews, N., Gualdrini, F. and Treisman, R. (2014) Rho-actin signalling to the MRTF coactivators dominates the immediate transcriptional response to serum in fibroblasts. Genes and Development 28: 943-58.

3. *Esnault, C., *Gualdrini, F., Horswell, S., Mathews, N., Stewart, A., and Treisman, R. The TCF-SRF transcription factor partnership plays a central role in the immediate transcriptional response to ERK activation (in preparation)

4. Costello, P., Sargent, M., Maurice, D., Esnault, C., Foster, K., Afonso, F.A., Treisman, R*. (2015) MRTF-SRF signaling is required for seeding of HSC/Ps in bone marrow during development. Blood. 125(8):1244-55.

5. *Gualdrini, F., *Esnault, C., Horswell, S., East, P., Mathews, N., Stewart, A., and Treisman, R. Global analysis of ERK-induced chromatin modifications identifies a central role for the Ternary Complex Factors in establishment of chromatin modification and transcriptional activation (in preparation)

6. Miralles, F., Posern, G., Zaromytidou, A-I., and Treisman, R. (2003) Actin dynamics control SRF activity by regulation of its coactivator MAL. Cell 113, 329-342.

7. Vartiainen, M.K., Guettler, S., Larijani, B. and Treisman, R (2007). Nuclear actin regulates dynamic subcellular localization and activity of the SRF cofactor MAL. Science 316 (5832) 1749-52 (Abstract)


41

Victor Tybulewicz http://crick.ac.uk/research/a-z-researchers/researchers-t-u/victor-tybulewicz/ Novel signalling pathways controlling lymphocyte activation Our group is interested in understanding signalling pathways in lymphocytes that control development, survival, migration and activation of both B and T lymphocytes. We approach these using a combination of mouse genetics, biochemistry and cell biology. In a recent RNA interference screen we identified a novel pathway controlling T cell adhesion and migration. We showed that the serine-threonine kinase WNK1 is a negative regulator of integrin-mediated adhesion and a positive regulator of T cell migration. Furthermore we found that WNK1 regulates migration through the related OXSR1 and STK39 kinases and the SLC12A2 Na+/K+/Cl- co-transporter. This discovery was unexpected, since the WNK1 pathway had been previously studied in kidney epithelial cells where it controls reuptake of salt back into the bloodstream, but nothing was known about this pathway in lymphocytes.

One possible PhD project stemming from this work is to investigate the roles of this WNK1 pathway in immune responses. T cell adhesion and migration are critical for the participation of T cells in immune responses. T cells are activated following binding of their T cell antigen receptors to peptide-MHC complexes on antigen presenting cells (APCs), which in turn results in firm adhesion between T cells and APCs, which is mediated by integrins. We have already shown that WNK1 is a negative regulator of conjugation between T cells and APCs, and thus it may influence the activation of T cells during immune responses. Following activation, T cells migrate towards the border of the T cell:B cell areas in lymphoid organs where they form conjugates with B cells, and then migrate together with the B cells into B cell follicles to establish germinal centres. In doing so, they differentiate into T follicular helper (Tfh) cells, and support somatic hypermutation and affinity selection of B cells and hence the production of high-affinity antibodies.

In this project we will investigate the role of this kinase and its downstream OXSR1/STK39/SLC12A2 pathway in T-dependent antibody responses, investigating T cell activation, migration, differentiation into Tfh cells, establishment of germinal centres, and the generation of high affinity antibody responses. Key tools for these studies include mouse strains with mutations in WNK1, OXSR1, STK39 and SLC12A2, all of which have already been established, as well as various TCR transgenic strains, which will allow monitoring of antigen-specific T cell responses both in vitro and in vivo. The immune responses of T cells with mutations in WNK1 or in downstream components of the pathway will be followed using flow cytometry, histology and imaging. In particular we will use intra-vital imaging to follow the movement of T cells within lymphoid organs and evaluate their ability to interact with APCs. This is just one example of the sort of project that might be available in this research group. The precise project will be decided on in consultation with the supervisor.

1. Schweighoffer, E., Vanes, L., Nys, J., Cantrell, D., McCleary, S., Smithers, N., and Tybulewicz, V. L. J. (2013). The BAFF receptor transduces survival signals by co-opting the B cell antigen receptor signaling pathway. Immunity, 38, 475-488.

2. Hartweger, H., Schweighoffer, E., Davidson, S., Peirce, M. J., Wack, A., Tybulewicz, V. L. J. (2014). Themis2 is not required for B cell development, activation and antibody responses. J Immunol,193, 700-707.

3. Ackermann, J. A., Nys, J., Schweighoffer, E., McCleary, S., Smithers, N., and Tybulewicz, V. L. J. (2015). Syk tyrosine kinase is critical for B cell antibody responses and memory B cell survival. J Immunol, 194, 4650-4656.

4. Köchl, R., Thelen, F., Vanes, L., Brazao, T. F., Fountain, K., Xie, J., Huang, C.-L., Lyck, R., Stein, J. V., Tybulewicz, V. L. J. (2015). WNK1 kinase balances T cell adhesion and migration in vivo. Submitted.


42

Victor Tybulewicz & Jeremy Green http://crick.ac.uk/research/a-z-researchers/researchers-t-u/victor-tybulewicz/ https://kclpure.kcl.ac.uk/portal/jeremy.green.html Joint Crick/King’s College London position Craniofacial development in mouse models of Down Syndrome Down Syndrome (DS) is caused by trisomy of human chromosome 21 (Hsa21) and is the most common human aneuploidy (1 in 700 live births). DS results in multiple phenotypes including learning deficits, cardiac defects, early-onset Alzheimer’s and facial dysmorphology. The mechanisms resulting in these phenotypes are largely unknown, but presumably result from an extra copy of one or more of the ~300 genes on Hsa21. The Tybulewicz lab (Crick) and Elizabeth Fisher (UCL) have constructed mouse strains with duplications of regions of mouse chromosome 16 (Mmu16) orthologous to Hsa21. One strain, Dp1Tyb, carries a duplication spanning the entire orthologous region. Additional strains carry a nested series of duplications functions as a “mapping panel” that can be used to identify the location of genes causing specific DS phenotypes. Craniofacial dysmorphology in people with DS is highly characteristic and instantly recognisable. The face is flatter (mid-facial retrusion), the jaw shorter (micrognathia), and eye shape is altered. These changes are reproduced in a mouse model of DS, the Dp(16)1Yey strain (Starbuck et al., 2014), essentially identical to the Dp1Tyb strain to be used in this project and thus readily accessible for analysis. These changes were shown using micro-CT (computer tomographic) X-ray 3D-images of adult Dp(16)1Yey skulls to record locations of defined landmarks in normal and Dp(16)1Yey adults and a multivariate analysis of shape differences using established methods and software. A similar analysis on the Dp1Tyb mouse is the first step in the proposed project.

Applying this analysis to the mapping panel will then enable the causal chromosomal region to be mapped. If necessary, the student will be involved in making new strains carrying novel duplications to achieve finer mapping. Once we have narrowed the region down to a small number of genes, candidate genes will be picked based on known function or patterns of expression and duplication strains will be crossed with knockouts of these candidates to identify causative genes.

While landmark morphometry provides a useful assay, to understand the cellular and molecular causes of dysmorphology, it is necessary to analyse its embryonic origins. The Green lab has developed analytical approaches and software tools that break down directional tissue growth into its component cellular processes: localised cell proliferation, cell rearrangement, cell shape change and oriented cell division (Economou et al., 2013). These essential elements of morphogenesis will be mapped anatomically onto the developing embryonic face. Access to a new state-of-the-art multiphoton microscope and the extensive instrumentation of the KCL Nikon imaging centre will provide the ability to image rapidly thick tissue at subcellular resolution. Imaging facilities available at the Crick will also allow optimal allocation of instrument time. By applying this analysis to successive stages of development, the student will be able to link genotype to cellular phenotype in the DS mouse strains. This will establish a critical understanding of the biology of this syndrome and complement and more convincingly resolve the assignment of gene function to phenotypic outcome. It therefore represents an exciting fine-scale strategy for genetic mapping and molecular understanding of DS.

1. O’Doherty, A., Ruf, S., Mulligan, C., Hildreth, V., Errington, M. L., Cooke, S., Sesay, A., Modino, S., Vanes, L., Hernandez, D., Linehan, J. M., Sharpe, P. T., Brandner, S., Bliss, T. V. P., Henderson, D. J., Nizetic, D., Tybulewicz, V. L. J., Fisher, E. M. C. (2005). An Aneuploid Mouse Strain Carrying Human Chromosome 21 with Down syndrome phenotypes. Science 309, 2033-2037.

2. Starbuck, J.M., Dutka, T., Ratliff, T. S. Reeves, R. H., Richtsmeier, J. T. (2014). Overlapping trisomies for hiuman chromosome 21 orthologs produce similar effects on skull and brain morphology of Dp(16)1Yey and Ts65Dn mice. Am J Med Gen 164A, 1981-1990.

3. Economou, AD, Brock, LJ, Cobourne, MT & Green JBA (2013) Whole population cell analysis of a landmark-rich mammalian epithelium reveals multiple elongation mechanisms. Development 140:4740-50.


43

Frank Uhlmann http://crick.ac.uk/research/a-z-researchers/researchers-t-u/frank-uhlmann/ The molecular mechanism of chromosome segregation Sister chromatid cohesion is the basis for the recognition of chromosomal DNA replication products for their bipolar segregation in mitosis. Fundamental to sister chromatid cohesion is the ring-shaped cohesin complex, which is loaded onto chromosomes well before the initiation of DNA replication and is thought to hold replicated sister chromatids together by topological embrace. What happens to cohesin when the replication fork approaches, and how cohesin recognises newly synthesised sister chromatids, are poorly understood. The characterization of a number of ‘cohesion establishment factors’ has started to provide hints as to the reactions involved. Cohesin is a member of the evolutionarily conserved family of Smc subunit-based protein complexes that contribute to many aspects of chromosome biology by mediating long-range DNA interactions. The establishment of sister chromatid cohesion may entail selective stabilisation of those cohesin-mediated DNA interactions that link sister chromatids in the wake of replication forks. How these are singled out and how cohesion establishment factors help the cohesin complex to hold together two strands of DNA, will be investigated. We use a combination of biochemical and molecular genetic approaches to approach these questions.

1. F. Uhlmann (2009) A matter of choice: the establishment of sister chromatid cohesion. EMBO Rep. 10, 1095-1102

2. L. Lopez-Serra et al. (2013) Budding yeast Wapl controls sister chromatid cohesion maintenance and chromosome condensation. Curr. Biol. 23, 64-69

3. V. Borges et al. (2013) An Eco1-independent sister chromatid cohesion establishment pathway in S. cerevisiae. Chromosoma 122, 121-134

4. Y. Murayama & F. Uhlmann (2014) Biochemical reconstitution of topological DNA binding by the cohesin ring. Nature 505, 367-371


44

Peter Van Loo http://crick.ac.uk/research/a-z-researchers/researchers-v-y/peter-van-loo/ Deconvoluting normal cell and tumour cell signals from transcriptome and DNA methylome sequencing data Genomic changes play a key role in the development and evolution of cancer. Thanks to advances in sequencing technology, we are now beginning to understand what the key changes are that drive carcinogenesis. As the cancer genome gradually reveals its secrets, the next challenges come into focus: can we dissect the transcriptome and DNA methylome in similar depth? How do (epi)genomic alterations lead to transcriptomic (and proteomic, interactomic, …) changes driving cancer development and evolution? Considering that tumour samples contain both cancerous and admixed normal cells, any measurement of the genome, transcriptome and DNA methylome represents a mix of signals, confounding the interpretation of the state of the tumour (and normal) cells.

We have previously developed methods to deconvolute tumour cell and normal cell genomes from genome sequencing data. We will now take the next step and separate tumour and normal transcriptomes and DNA methylomes from RNA- and bisulfite sequencing data. Our efforts will be guided by single-cell sequencing data and will allow for unique views into cancer transcriptomes and DNA methylomes, as well their interaction during tumour development.

In this project, the successful candidate will: (i) develop approaches to deconvolute tumour-cell-specific expression signals from normal-cell-specific expression signals, using RNA sequencing data; (ii) develop similar approaches to separate tumour-cell-specific and normal-cell-specific DNA methylation signals from whole-genome bisulfite sequencing data; (iii) extend these approaches to model multiple populations of tumour cells and (iv) build upon the methods to study the influence of genomic and epigenomic changes in cancer on transcription at the gene or transcript level and at the transcriptome level.

This position is suitable for a computational biologist, or a statistician, mathematician or physicist with a strong interest in biology, or a biologist or geneticist with a passion for computational cancer biology.

The projects above is one example of a research project available in the Cancer Genomics group – the exact project will be decided in consultation with the supervisor.

1. Nik-Zainal S#, Van Loo P#, Wedge DC#, et al. The life history of 21 breast cancers. Cell. 2012;149:994-1007.

2. Gundem G, Van Loo P, Kremeyer B, Alexandrov LB, Tubio JM, Papaemmanuil E, Brewer DS, Kallio HM, Högnäs G, Annala M, Kivinummi K, Goody V, Latimer C, O'Meara S, Dawson KJ, Isaacs W, Emmert-Buck MR, Nykter M, Foster C, Kote-Jarai Z, Easton D, Whitaker HC; ICGC Prostate UK Group, Neal DE, Cooper CS, Eeles RA, Visakorpi T, Campbell PJ, McDermott U, Wedge DC, Bova GS. The evolutionary history of lethal metastatic prostate cancer. Nature. 2015;520:353-357.

3. Van Loo P#, Nordgard SH#, Lingjærde OC, Russnes HG, Rye IH, Sun W, Weigman VJ, Marynen P, Zetterberg A, Naume B, Perou CM, Børresen-Dale AL#, Kristensen VN#. Allele-specific copy number analysis of tumors. Proc Natl Acad Sci U S A. 2010;107(39):16910-5.

4. Van Loo P, Voet T. Single cell analysis of cancer genomes. Curr Opin Genet Dev. 2014;24:82-91.


45

Jean-Paul Vincent http://crick.ac.uk/research/a-z-researchers/researchers-v-y/jean-paul-vincent/ The role of Evi/Wntless and exosomes in the trafficking and release of Wnt proteins in epithelia Wnts are lipid-modified secreted proteins that activate a conserved signal transduction pathway that has been linked to development, stem cell maintenance and tumorigenesis. Therefore, understanding the mechanisms that regulate the production of active Wnt is of great interest. In this respect, the lipid moiety on Wnt, which is essential for signalling activity, raises interesting challenges, as it is likely to greatly limit solubility and ability to spread in the extracellular space. We focus much of our work on Wnt trafficking in epithelial tissues, using mainly wing imaginal discs of Drosophila but also extending our studies to mammalian epithelia grown in culture. In wing imaginal discs, Wingless (the main Drosophila Wnt) is produced by a stripe of cells. We have shown that, in these Wingless-secreting cells, Wingless traffics to the apical surface and then undergoes transepithelial transcytosis before being released to activate signalling in surrounding cells. There has been much debate about the mechanism of release from producing cells. One possibility is that it is mediated by a lipid-binding extracellular protein of the lipocalin family. Another model is that Wingless is released on exosomes as a complex with Evi, a mutlipass transmembrane protein. Evi has extensively been shown to be required for progression of Wingless from the Golgi to the plasma membrane but its role in Wingless release remains controversial. Evi’s role in transepithelial transcytosis is also unknown. In a joint effort between the Vincent lab (Crick Institute) and Henriques lab (UCL LMCB) we are proposing to apply high-resolution and super-resolution microscopy techniques to study the association of Evi and Wingless in Wingless secreting cells and beyond. The primary aim will be to determine where Wingless and Evi separate, at the apical surface, during transcytosis or after release from secreting cells. For this aim we will conventional livecell microscopy as well as state of the art super-resolution imaging approaches to track in real-time protein associations down to nanoscale resolution. Wingless and Evi association kinetics will be assessed both in subcellular comparmtent and the extracellual space through a robust statistical framework bridging information from single-molecule tracking, FRAP and/or FRET, as needed. We will also devise tests of the role of exosomes in Wingless release and other processes. If time permits experimental approaches will be extended to mammalian cells in culture.

1. *Beckett, K., Monier, S., Palmer, L., Alexandre, C., Green, H., Bonneil, E., Raposo, G., Thibault, P., Le Borgne, R., and Vincent, J.P. (2013). Drosophila S2 cells secrete wingless on exosome-like vesicles but the wingless gradient forms independently of exosomes. Traffic 14, 82-96.

2. *Gross, J.C., Chaudhary, V., Bartscherer, K., and Boutros, M. (2012). Active Wnt proteins are secreted on exosomes. Nat Cell Biol 14, 1036-1045.

3. *Kakugawa, S., Langton, P.F., Zebisch, M., Howell, S.A., Chang, T.-H., Liu, Y., Feizi, T., Bineva, G., O'Reilly, N., Snijders, A.P., et al. (2015). Notum deacylates Wnt proteins to suppress signalling activity. Nature 519, 187-192.

4. *Mulligan, K.A., Fuerer, C., Ching, W., Fish, M., Willert, K., and Nusse, R. (2012). Secreted Wingless-interacting molecule (Swim) promotes long-range signaling by maintaining Wingless solubility. Proceedings of the National Academy of Sciences of the United States of America 109, 370-377.

5. *Herbert, S., Soares, H., Zimmer, C., Henriques, R. (2012). Single-molecule localization super-resolution microscopy: deeper and faster. Microscopy and Microanalysis 18 (06), 1419-1429.


46

Andreas Wack http://crick.ac.uk/research/a-z-researchers/researchers-v-y/andreas-wack/ Factors governing epithelial damage and redifferentiation during influenza infection Infection by the influenza virus remains an important public health burden, and symptoms vary from mild to fatal disease. Predictors of disease severity are the virulence and pathogenicity of the infecting virus strain versus the host's ability to control and eliminate the virus, which is facilitated by pre-existing adaptive immunity gained through previous exposure to virus or vaccines that are antigenically similar. Another important determinant of severity is the degree of tissue damage caused by the anti-viral immune response, and avoidance of excessive immune-mediated tissue damage during infection is crucial for survival. We have previously compared influenza-resistant and -susceptible mouse strains and found that, in contrast to resistant C57BL/6 mice, susceptible mice such as the 129 strain showed dramatically increased lung damage, caused by increased levels of type I interferon (IFNαβ). We also identified the cellular source of IFNαβ and the IFNαβ-dependent mechanisms leading to lung damage [1]. This disease-promoting effect of IFNαβ in influenza infection was unexpected, as IFNαβ is known for its antiviral function. We therefore hypothesize that in mice and humans, host-determined excessive IFNαβ can be deleterious during influenza infection [2].

In this PhD programme, we plan to focus on a damage-related aspect that is of great importance but comparably less well studied: the efficiency of tissue repair during the recovery phase of influenza infection. The lung is a vital organ as it allows oxygen supply to the organism, and the ability of lung epithelia to perform gas exchange has to be maintained throughout respiratory infections. Therefore, damage inflicted on lung epithelia during influenza infection must be efficiently repaired to avoid lung organ failure or allow bacterial superinfections, a common complication of influenza [5]. Our preliminary data show that IFNαβ not only has the antiviral and proinflammatory effects mentioned above, but it also regulates lung epithelial repair and re-differentiation. The molecular mechanisms involved are not understood. In addition, we have previously shown that lung epithelia rely on two redundant IFN systems, IFNαβ and IFNλ (type III IFN), for the induction of antiviral effects [3], and this is possible because airway epithelia express the receptors for both IFNαβ and IFNλ [4]. We are therefore studying whether IFNαβ and IFNλ have similar or divergent effects on epithelial re-differentiation and how they interact. We have also discovered that a transcription factor with known function in the immune system is important for airway epithelial differentiation, and we are trying to understand the molecular mechanisms involved in this process.

The aim of this PhD programme is therefore to build on our accumulating knowledge of lung epithelial repair and identify mechanisms underlying the IFN-dependent regulation of lung epithelial re-differentiation that takes place during recovery from influenza infection. Another aim is to understand how the transcription factor we identified interacts with IFNs in the regulation of airway repair. The proposed PhD project will be within this area of research and will be delineated in detail closer to the commencement date of the student in consultation with the supervisor. The PhD student will learn and use state-of-the-art technology to assess in vitro and in vivo the mechanisms underlying epithelial differentiation, to understand better the impact of epithelial repair in recovery from influenza infection.

1. Davidson, S., Crotta, S., McCabe, T., and Wack, A. (2014). Pathogenic potential of interferon αβ in acute influenza infection. Nat. Comm. 5, 3864. PMID: 24844667

2. Davidson, S., Maini, M.K. and Wack, A. (2015). Disease promoting effects of type I interferons in viral, bacterial and co-infections. J. Interferon Cytokine Res. 35, 252-64.

3. Crotta, S., Davidson, S., Desmet, C., Buckwalter, M., Albert, M., Staeheli, P., and Wack, A. (2013). Type I and type III interferons drive redundant amplification loops to induce a transcriptional signature in influenza-infected airway epithelia. PLoS Pathog. 9, e1003773.

4. Wack, A., Terczyńska-Dyla, E. and Hartmann, R. (2015). Guarding the frontiers: The biology of type III interferons. Nature Immunology, in press.

5. Ellis, G.T., Davidson, S., Crotta, S., Branzk, N., Papayannopoulos, V. and Wack, A. (2015). TRAIL+ monocytes and monocyte-related cells cause lung damage and thereby increase susceptibility to influenza-Streptococcus pneumoniae coinfection. EMBO Rep. 2015 Aug 11. pii: e201540473, PMID: 26265006


47

Michael Way http://crick.ac.uk/research/a-z-researchers/researchers-v-y/michael-way/ Exploring new levels of complexity within the Arp2/3 complex The Arp2/3 complex consists of seven proteins (actin related proteins; Arp2 and Arp3, and Arp2/3 complex subunits; ARPC1-5) that are conserved in all eukaryotes, with the exception of some algae, microsporidia and protists. The complex plays an essential role in a wide variety of cellular processes, including lamellipodia-mediated cell migration, endocytosis, autophagosome biogenesis and phagocytosis, by virtue of its ability to generate branched actin filament networks (Campellone and Welch 2010). Arp2/3-dependent actin polymerization is also used by a number of intracellular pathogens such Listeria and Vaccinia virus to enhance their cell-to-cell spread (Weisswange et al., 2009; Welch and Way, 2013; Donnelly et al., 2013).

In isolation, the Arp2/3 complex has little or no actin filament nucleating activity. Consequently, the complex needs to be activated by so called nucleation promoting factors (NPFs) (Campellone and Welch 2010). NPFs include WASP, N-WASP, WAVE1-3, WASH, WHAMM and JMY all of which have a conserved C-terminal VCA domain that interacts with the Arp2/3 complex and actin. NPFs are highly divergent outside their VCA domains and capable of interacting with a variety of regulatory or scaffolding proteins. Numerous protein and lipid interactions also regulate the localisation of NPFs and their ability to stimulate the activity of the Arp2/3 complex downstream of a variety of signalling pathways.

Interestingly, in many higher eukaryotes, several Arp2/3 complex subunits are encoded by more than one gene. For example, in humans, Arp3, ARPC1 and ARPC5 are each represented by two isoforms that are 91, 67 and 67% identical respectively. The sequence differences between the two human ARPC1 and ARPC5 isoforms are spread throughout their length. Moreover many of these sequence differences are surface exposed, thus offering the opportunity for different interactions with Arp2/3 complex binding partners and NPFs. This raises the possibility that Arp2/3 complexes with different properties may exist. We have recently demonstrated that the Arp2/3 complex in higher eukaryotes is actually a family of complexes with different properties (Abella et al., 2015). Using actin based motility of Vaccinia virus as a model system we have uncovered a previously unappreciated complexity in the regulation of Arp2/3 generated actin networks. The project will use a combination of biochemistry and cell biology to further examine roles of the different Arp2/3 complexes during a variety of cellular and developmental processes. The project will involve a variety of methods including single molecule analysis, in vitro assays, advanced imaging and generation of stable cell lines using lentivirus and CRISPR/Cas approaches. The precise project will be decided on consultation with the supervisor during the interview.

1. Campellone, K.G. & Welch, M.D. A nucleator arms race: cellular control of actin assembly. Nat Rev Mol Cell Biol 11, 237-251 (2010).

2. Weisswange, I., Newsome, T.P., Schleich, S. & Way, M. The rate of N-WASP exchange limits the extent of ARP2/3-complex-dependent actin-based motility. Nature 458, 87-91 (2009).

3. Welch, M.D. & Way, M. Arp2/3-mediated actin-based motility: a tail of pathogen abuse. Cell Host Microbe 14, 242-255 (2013).

4. Donnelly, S.K., Weisswange, I., Zettl, M. & Way, M. WIP Provides an Essential Link between Nck and N-WASP during Arp2/3-Dependent Actin Polymerization. Curr Biol 23, 999-1006 (2013).

5. Abella, J.V.G., Galloni, G., Pernier, J., Barry, D.J., Kjær, S., Carlier, M-F., and Way, M. Isoform diversity in the Arp2/3 complex determines actin filament dynamics. Submitted 2015


48

David Wilkinson http://crick.ac.uk/research/a-z-researchers/researchers-v-y/david-wilkinson/ Spatial regulation of neurogenesis during hindbrain development Development of the nervous system requires precise spatial and temporal control of the differentiation of neural stem cells to form a diversity of neuronal and glial cell types. Central to this are inhibitory mechanisms which restrain differentiation and underlie the correct balance between maintenance of progenitors and generation of neurons and glia. In specific regions of the developing nervous system, there is a spatially-restricted inhibition of neurogenesis such that discrete neurogenic zones are formed. A striking example occurs in the zebrafish hindbrain, in which neurogenic zones form adjacent to segment borders. We have shown that this patterning of neurogenesis involves fgf20 signals emanating from specific neurons positioned at segment centres. However, there is a limited understanding of how fgf20 expression is regulated, how it inhibits neurogenesis, and how it acts with other signals to organise the neurogenic zones. This project seeks to identify and study the function of further components of the signaling and transcriptional network that regulates neurogenesis in the developing hindbrain. It will use the advantages of zebrafish for genome manipulation and imaging, in combination with high throughput sequencing and bioinformatic analysis.

1. Cheng, Y.-C., Amoyel, M., Qiu, X., Jiang, Y.-J., Xu, Q. and Wilkinson, D.G. (2004) Notch activation regulates the segregation and differentiation of rhombomere boundary cells in the zebrafish hindbrain. Developmental Cell 6, 539-550.

2. Nikolaou, N., Watanabe-Asaka, T., Gerety, S., Distel, M., Köster, R.W. and Wilkinson, D.G. (2009) Lunatic fringe promotes the lateral inhibition of neurogenesis. Development 136, 2523-2533.

3. Sobieszczuk, D.F., Poliakov, A., Xu, Q. and Wilkinson, D.G. (2010) A feedback loop mediated by degradation of an inhibitor is required to initiate neuronal differentiation. Genes and Development 24, 206-218.

4. Gonzalez-Quevedo, R., Lee, Y., Poss, K.D. and Wilkinson, D.G. (2010) Neuronal regulation of the spatial patterning of neurogenesis. Developmental Cell 18, 136-147.

5. Terriente, J., Gerety, S.S., Watanabe-Asaka, T., Gonzalez-Quevedo, R. and Wilkinson, D.G. (2012) Signaling from hindbrain boundaries regulates neuronal clustering that patterns neurogenesis. Development 139, 2978-2987.


49

Robert J Wilkinson & Robert S Heyderman http://crick.ac.uk/research/a-z-researchers/researchers-v-y/robert-j-wilkinson/ https://www.uclh.nhs.uk/OurServices/Consultants/Pages/ProfRobertHeyderman.aspx Joint Crick/University College London position Pathogen-pathogen and host-pathogen interactome at the respiratory epithelial surface Severe acute respiratory tract infections (SARI) are associated with a high burden of death and disability, particularly in immunocompromised hosts. Worldwide, tuberculosis and pneumonia caused by Streptococcus pneumoniae are prominent contributors to this disease burden. It is increasing recognised that patients with SARI are frequently co-colonised at the epithelial surface with multiple pathogens but how these interact with each other and with the host to cause disease is uncertain. We hypothesise that co-infection with Mycobacterium tuberculosis and S. pneumoniae and the consequent pathogen-pathogen/host-pathogen interactions undermine mucosal defences, facilitating colonisation, invasion and onward transmission. We further propose that the nature of this complex respiratory epithelial surface interactome is further imprinted by the overall microbiome at the mucosa. Ultimately, these studies may lead to clinical trials of adjunctive antibiotic therapy aimed at commensal or collaborating bacteria that may reduce the infectivity and transmission of M. tuberculosis.

Exploiting internationally recognised expertise in TB immunology and pathogenesis (Wilkinson, Crick Institute), and mucosal immunology and bacterial pathogenesis (Heyderman, UCL), this studentship will first explore this complex relationship using human TB patient samples and then gain a mechanistic understanding of these in vivo observations in laboratory epithelial models. Exploration of the interactome with the wider microbiome will be supported by Professor Young (Crick Institute).The student will establish the relationship between pneumococcal and mycobacterial load, and the microbiome; and then determine the complex impact of co-colonisation on both bacterial and host gene expression at the mucosal surface in both immunocompetent and immunocompromised hosts. Epithelial models will then be used to further explore the signalling pathways and metabolic adaptations to co-infections identified. Were necessary mutant bacterial strains, host cell reporter and gene knockdown experiments will be performed to gain a more precise insight into these mechanisms. The student will gain expertise and training in molecular microbiology, immunology, cell biology and bioinformatics.

1. Glennie SJ, Banda D, Gould K, Hinds J, Kamngona A, Everett DB, Williams NA, Heyderman RS. Defective pneumococcal-specific Th1 responses in HIV-infected adults precedes a loss of control of pneumococcal colonization. Clin Infect Dis. 2012;56:291-9

2. Pido-Lopez J, Kwok W, Mitchell TJ, Heyderman RS, Williams NA. Maturation of mucosal anti-pneumococcal effector and regulatory CD4+ T cell immunity sequestered within upper human respiratory tract lymphoid tissue. PloS Pathog. 2011;7:e1002396

3. Wilkinson, K.A., Walker, N.F., Meintjes, G., Deffur, A., Nicol, M.P., Skolimowska, K.H., Matthews, K., Tadokera, R., Seldon, R.R., Maartens, G., Rangaka, M.X., Besra, G., Wilkinson, R.J. Cytotoxic mediators in paradoxical HIV-tuberculosis immune reconstitution inflammatory syndrome Journal of Immunology (2015) 194(4):1748-54

4. Coussens, A.K., Wilkinson, R.J., Martineau, A.R. Phenylbutyrate is Bacteriostatic against Mycobacterium tuberculosis and Regulates the Macrophage Response to Infection, Synergistically with 25-hydroxy-vitamin D� PLOS Pathogens in press


50

Hasan Yardimci http://crick.ac.uk/research/a-z-researchers/researchers-v-y/hasan-yardimci/ Understanding how the eukaryotic replication machinery deals with barriers Before a cell divides it has duplicate its genome so that two identical copies of the DNA content can be partitioned into daughter cells. In eukaryotic cells, DNA replication is initiated at thousands of origins on the DNA, each resulting in the assembly of two replisomes that travel away from the initiation site in opposite directions. Complete and high-fidelity duplication of the genome is essential for faithful transmission of genetic information. When DNA replication goes awry, the result could be cells with mutations, missing or extra genetic material a hallmark of the genomic instability seen in most cancers.

We investigate processes involved in eukaryotic replication using conventional biochemistry and single-molecule imaging tools. A significant advantage of single-molecule methods is that one can directly observe individual proteins that provide new insight into their dynamics and reaction mechanisms. To study eukaryotic replication at the single molecule level we use a number of model systems including Xenopus egg extracts [1-3], SV40 replication system [4], and extracts derived from budding yeast. Using novel methodologies that combine single-molecule imaging and DNA nanomanipulation in extract-based systems and conventional bulk assays, we previously differentiated between different models of unwinding by the replicative helicases MCM2-7 [2-3] and SV40 large T-antigen [4], and discovered how large T-antigen deals with replication barriers [4]. In the future, we aim to gain a comprehensive understanding of the overall architecture and dynamics of the eukaryotic replisome by fluorescently labeling individual components of the replisome and visualizing labeled proteins in real-time during replication. The goal of this project is to understand how the eukaryotic replisome deals with replication barriers such as DNA-protein crosslinks [5] and DNA interstand-crosslinks.

Consideration will be given to talented and motivated students from various backgrounds including biochemistry, biophysics, physics, and biology. During the course of graduate studies, the student will develop skills in molecular biology such as cloning, expression and purification of proteins and single-molecule techniques including total internal fluorescence microscopy and optical tweezers.

1. Yardimci, H; Loveland, AB; van Oijen, AM; Walter, JC. (2012) Single-molecule analysis of DNA replication in Xenopus egg extracts. Methods 57, 179

2. Fu, YV; Yardimci, H; Long, DT; Ho, TV; Guainazzi, A; Bermudez, VP; Hurwitz, J; van Oijen, AM; Scharer, O; Walter, JC. (2011) Selective bypass of a lagging strand roadblock by the eukaryotic replicative DNA helicase. Cell 146, 931

3. Yardimci, H; Loveland, AB; Habuchi, S; van Oijen, AM; Walter, JC. (2010) Uncoupling of eukaryotic sister replisomes. Mol. Cell 40, 834.

4. Yardimci, H; Wang, X; Loveland, AB; Tappin, I; Rudner, DZ; Hurwitz, J; van Oijen, AM; Walter, JC. (2012) Bypass of a protein barrier by a replicative DNA helicase. Nature 492, 205.

5. Duxin, JP; Dewar, JM; Yardimci, H and Walter, JC. (2014) Repair of a DNA-protein crosslink by replication-coupled proteolysis. Cell 159, 346.


51

Mariia Yuneva http://crick.ac.uk/research/a-z-researchers/researchers-v-y/mariia-yuneva/ Metabolic pathways as targets for anti-cancer therapy One of the hallmarks and driving forces of cancer is altered metabolism (1). Although metabolic changes can provide cancer cells with the advantage in proliferation and survival, they can also make cancer cells addicted to certain nutrients or metabolic pathways (2). The metabolic patterns of tumors are often associated with the expression of enzyme isoforms different from the ones expressed in the original normal tissues (3). Targeting these tumor-specific enzyme isoforms represents a brilliant opportunity for uncovering the metabolic vulnerabilities of various tumors and developing new anti-cancer therapies.

Cancer is extremely complex and heterogenous disease and metabolic changes and nutrient requirements of tumors depend on the genetic lesions, tissue of origin as well as on a tumor environment (4). The projects in the lab are evaluating the role of metabolic enzyme isoforms regulating major pathways of glucose and glutamine metaboilsm in tumorigeneisis induced by specific oncogenes in specific tissues in vivo. Mouse genetics as well as molecular biological approaches are being used to manipulate the expression of tumour-specific enzyme isoforms in a tissue-specific manner before tumor intiation and during tumor progression. The effect of these manipulations on tumour metabolism is assessed by metabolomics approaches including the employment of labled substrates, nuclear magnetic resonance (NMR) and mass spectrometry. Finally, the molecular mechanisms of tumour dependence/independence on identified metabolic pathways are addressed.

1. Hanahan D., and Weinberg R.A. 2011. Hallmarks of cancer: the next generation. 2. Hanahan D, Weinberg RA. Cell, 144, 646-674. 3. Yuneva M., Zamboni N., Oefner P., Sachidanandam R. and Lazebnik Y. 2007. Deficiency in

glutamine but not glucose induces MYC-dependent apoptosis in human cells. JCB, 178, 93-105. 4. Hamanaka R.B. and Chandel N.S. 2012. Targeting glucose metabolism for cancer therapy. JEM,

209, 211-215. 5. Yuneva M., Fan T.M., Allen T.D., Higashi R.M., Ferraris D.V., Tsukamoto T., Mates J.M., Alonso

F.J., Wang C., Seo Y., Chen X. and Bishop J.M. 2012. The metabolic profile of tumors depends on both the responsible genetic lesion and tissue type. Cell Metabolism, 15, 157-170.

2016 Crick PhD Positions

Documents

Transcript of 2016 Crick PhD Positions