2016 Crick PHD Positions

2016 Crick PhD Student Recruitment
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 supervisors 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
1
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
Positions by Research Topic

The list below tells you which positions fall under each of the Cricks 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
3
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

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-2 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.

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 RFS1, 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, prek 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, DAndrea A, Sartori AA, Swanton C & Boulton SJ (2013). HELQ promotes RAD51 paralogdependent 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.

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
Mycobacterium tuberculosis
the
role
of
protein
lipidation
in
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. NonEU applicants are not eligible for the funding for this project.

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.

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.

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 antitumour 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 neoantigens 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. ArmenterosMonterros, 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, 617630. Requirement for interaction of PI 3-kinase p110 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.
10

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 (IFN), which stimulates cell-autonmous defence mechanisms
to kill and restrict the pathogen. We have identified two effector pathways of IFN-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, IFN 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 IFN-mediated cell autonomous killing of Toxoplasma
is impaired.
Secondly, we have determined that in IFN-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 -catenin. -catenin is part of a signal transduction pathway and decreased -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 -catenin and enhanced cell proliferation (Capaldo et al,
2012). You will investigate the protein status of -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 -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 cells 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 IFN-mediated restriction of
Toxoplasma in human cells.
1. Capaldo CT, Beeman N, Hilgarth RS, Nava P, Louis NA, Naschberger E, Strzl M, Parkos CA &
Nusrat A (2012) IFN- and TNF--induced GBP-1 inhibits epithelial cell proliferation through
suppression of -catenin/TCF signaling. Mucosal Immunol 5: 681690
2. Petherick KJ, Williams AC, Lane JD, Ordez-Morn P, Huelsken J, Collard TJ, Smartt HJM, Batson
J, Malik K, Paraskeva C & Greenhough A (2013) Autolysosomal -catenin degradation regulates
Wnt-autophagy-p62 crosstalk. EMBO J 32: 19031916
3. Sorbara MT & Girardin SE (2015) Emerging themes in bacterial autophagy. Curr Opin Microbiol 23:
163170
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--Inducible p65 GTPases Plays a Critical Role in Host Defense against Toxoplasma
gondii. Immunity 37: 302-313
11

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 students 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 students 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, 334:11371141.
4. Goehring NW, Hoege C, Grill SW, Hyman AA: PAR proteins diffuse freely across the anteriorposterior boundary in polarized C. elegans embryos. J Cell Biol 2011, 193:583594.
NOTE:
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.
12

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
13

Maximiliano Gutierrez
http://crick.ac.uk/research/a-z-researchers/researchers-d-h/maximiliano-gutierrez/
Dynamic interactions between Mycobacterium

autophagic organelles in macrophages
tuberculosis
and
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
14

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.
15

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-) superfamily of ligands which comprises
the TGF-s, 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- signalling dynamics, we discovered that acute stimulation of
cells with TGF- 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-. 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- 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- signalling. The refractory behaviour we observed in
response to TGF- 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- 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- 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-
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- pathway versus the
involvement of different ligands.
1. Wu, M. Y., Hill, C. S., (2009) TGF- superfamily signaling in embryonic development and
homeostasis. Dev Cell 16, 329-343.
2. Calon, A., Tauriello, D. V., Batlle, E., (2014) TGF- in CAF-mediated tumor growth and
metastasis. Semin Cancer Biol 25, 15-22.
3. Schmierer, B., Hill, C. S., (2007) TGF-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-
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.
16
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-B 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-B.
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) IB 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 TPL2. 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.
17

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 highresolution 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*, Knig J*, Tajnik M, Martincorena I, Eustermann S, Stvant 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.
18

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 (Hirschsprungs 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-GFR1 ligand by a composite binding site promotes membrane-proximal
self-association. Goodman KM, Kjr 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, Ibez 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, Martnez-Torres RJ,
Borg A, Murray-Rust J, Mouilleron S, Knowles P, McDonald NQ. Mol Cell. 2014 Mar 6;53(5):738-51.
19

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:E18331842
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
20

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/Kings 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. & Mller, 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.
21

Kathy Niakan
http://crick.ac.uk/research/a-z-researchers/researchers-k-o/kathy-niakan/
Characterising
embryogenesis
novel
regulators
of
human
pluripotency
and
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 nave hESCs3. Some of the factors we identified (i.e. KLF17 and ARGFX) are also
expressed in nave 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 nave 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
22
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.
23

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
24

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, Jrg DJ, Morelli LG, Richmond DL, Schindelin J, Jlicher F, Oates AC. Science. 2014
Jul 11;345(6193):222-5.
2. Topology and dynamics of the zebrafish segmentation clock core circuit.
Schrter C, Ares S, Morelli LG, Isakova A, Hens K, Soroldoni D, Gajewski M, Jlicher 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.
Schrter C, Oates AC. Curr Biol. 2010 Jul 27;20(14):1254-8.
25

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 resequencing 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, Pevorovsk M, Mourier T, Marsellach FX, Iqbal Z, Lau W,
Cheng TM, Pracana R, Mlleder 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, Bhler J. The genomic and phenotypic
diversity of Schizosaccharomyces pombe. Nature Genetics. 2015 Mar;47(3):235-41. doi:
10.1038/ng.3215
2. Mlleder 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.
26
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
27

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/f
raternalifranca.aspx
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 Parkinsons 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, 51275147.
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.
28

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.
29

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 1/FAK Signaling
4. Hirata et al.Intravital Imaging Reveals How BRAF Inhibition Generates Drug-Tolerant
Microenvironments with High Integrin 1/FAK Signaling. Cancer Cell 27 (4), 574-588 (2015)
30

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.
31

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.
32

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- 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/Smad4independent 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 -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- 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 Spemanns
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.
33

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:
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.
34

Jesper Svejstrup
http://crick.ac.uk/research/a-z-researchers/researchers-p-s/jesper-svejstrup/
Basic mechanisms at the interface between transcription,

maintenance of genome stability, and human disease
the
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, 637648.
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.
35

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 immunoediting. 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.
2.
3.
4.
5.
McGranahan N, et al Sci Transl Med. Apr 15;7(283):283ra54 (2015)

Murugaesu N et al. Cancer Discovery (2015) May 23
Mcgranahan N and Swanton C Cancer Cell Jan 12;27(1):15-26. (2015)
De Bruin EC Science 10;346(6206):251-6. (2014)
Jamal-Hanjani et al (2014) PLoS Biol. 2014 Jul 8;12(7)
36

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/csiksznagy/h
ome.aspx
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, Csiksz-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, CsikszNagy 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, Csiksz-Nagy A.
(2012) Linkers of cell polarity and cell cycle regulation in the fission yeast protein interaction
network. PLoS Computational Biology 8(10): e1002732
37

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

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,
15871590 (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, 69100
(2014).
3. Neuman KC, Nagy A. Single-molecule force spectroscopy: optical tweezers, magnetic tweezers and
atomic force microscopy. Nat Methods. 2008 Jun;5(6):491505.
38
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.
39
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. Correlationbased 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 Gactin 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)
40

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 highaffinity 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. Kchl, 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.
41

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

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 Alzheimers 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. ODoherty, 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.
42
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
43

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-cellspecific 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, Hgns 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#, Lingjrde OC, Russnes HG, Rye IH, Sun W, Weigman VJ, Marynen P,
Zetterberg A, Naume B, Perou CM, Brresen-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.
44

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.
Evis 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 superresolution microscopy: deeper and faster. Microscopy and Microanalysis 18 (06), 1419-1429.
45

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 (IFN). We also identified the
cellular source of IFN and the IFN-dependent mechanisms leading to lung damage [1]. This diseasepromoting effect of IFN in influenza infection was unexpected, as IFN is known for its antiviral
function. We therefore hypothesize that in mice and humans, host-determined excessive IFN 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 IFN 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,
IFN and IFN (type III IFN), for the induction of antiviral effects [3], and this is possible because airway
epithelia express the receptors for both IFN and IFN [4]. We are therefore studying whether IFN and
IFN 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 redifferentiation 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
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., Terczyska-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
46

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). Arp2/3dependent 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., Kjr, S., Carlier, M-F., and Way, M. Isoform
diversity in the Arp2/3 complex determines actin filament dynamics. Submitted 2015
47

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., Kster, 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.
48

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

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 pathogenpathogen/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 antipneumococcal 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
49

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 singlemolecule 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.
50

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.
51

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2016 Crick PhD Student Recruitment