A*STAR-Dundee Partnership (ADP) Research Projects
1. The role of PIP2 in cell movement
Bob Robinson (Institute of Molecular & Cell Biology, Singapore) and Nicholas
Leslie (Division of Molecular Physiology, College of Life Sciences)
2. Curing Cancer with Computers: virtual drug design from the inhibition of
growth factor signalling to translation
Chandra Verma (Bioinformatics Institute, Singapore) and Ruth Brenk (Division of
Biological Chemistry and Molecular Microbiology, College of Life Sciences)
3. Defects in the skin barrier and their role in eczema and psoriasis
E. Birgit Lane (Institute of Medical Biology, Singapore) and W.H. Irwin McLean,
(Division of Molecular Medicine, College of Life Sciences and Medicine, Dentistry
& Nursing)
4. Relationship(s) between two the tumour suppressors p53 and APC in
cancer
David Lane (Institute of Molecular & Cell Biology, Singapore) and Inke Nathke
(Division of Cell & Developmental Biology, College of Life Sciences)
5. Specificity and regulation of interactions between 14-3-3 proteins and actin
binding proteins
Ed Manser (Institute of Molecular & Cell Biology, Singapore) and Carol
MacKintosh (MRC Protein Phosphorylation Unit, College of Life Sciences)
6. Lowe syndrome protein (OCRL-1) and endosome-to-TGN trafficking
Wanjin Hong (Institute of Molecular & Cell Biology, Singapore) and John Lucocq
(Division of Cell Biology & Immunology, College of Life Sciences,
7. The role of the cell cycle during vertebrate somite segmentation
Yun-Jin JIANG (Institute of Molecular & Cell Biology, Singapore) and Miguel
Maroto (Division of Cell & Developmental Biology)
8. Novel cellular targets of Human Papilloma Virus – implications in disease
Francoise Thierry (Institute of Medical Biology, Singapore) and Sam Crouch
(Division of Molecular Medicine, College of Life Science and Medicine, Dentistry
& Nursing)
9. Characterisation of the Wnt and GSK3 signalling and their role in cancer
David Virshup (Institute of Medical Biology, Singapore) and Dario Alessi (MRC
Protein Phosphorylation Unit, College of Life Sciences)
10. Mutations in type VII collagen and their role in wound healing and cancer
E. Birgit Lane (Institute of Medical Biology, Singapore) and Andrew South,
(Division of Medical Sciences, Medicine, College of Medicine, Dentistry &
Nursing)
11. Analysis into the function and regulation of Kirrel in the paraxial mesoderm
of the chick embryo
Mike Jones (Institute of Medical Biology, Immunos, Singapore ) and Kim Dale
(Division of Cell & Developmental Biology, College of Life Sciences)
12. Function of the proto-oncogene ect2 in cell motility and cell signalling
Ed Manser, (Institute of Molecular & Cell Biology, A*STAR, Singapore) and Arno
Muller, Division of Cell & Developmental Biology, College of Life Sciences)
13. Exploring and exploiting conserved synthetic lethal interactions of DNA
double strand break repair factors.
David Lane (Institute of Molecular & Cell Biology, Singapore) and Anton Gartner,
(Wellcome Trust Centre for Regulation and Expression)
14. Mathematical/Systems biology, Computational Biology/Bioinformatics
Vladimir Kuznetsov (Bioinformatics Institute, Singapore) and Mark Chaplain
(Division of Mathematics)
15. The role of Aurora A in the cell cycle and transformation
Ed Manser (Institute of Molecular & Cell Biology, Singapore) and Ron Hay
(College of Life Sciences)
16. The role of the Cdc7 kinase in DNA replication and in the DNA damage
response
Philipp Kaldis (Institute of Molecular & Cell Biology, Singapore) and Julian Blow
(Wellcome Trust Centre for Regulation and Expression, College of Life Sciences)
DETAILS OF PROJECTS
(1) The Role of PIP2 in Cell Movement
Supervisors: Dr Bob Robinson (Institute of Molecular & Cell Biology, Singapore)
and Dr Nicholas Leslie (College of Life Sciences, University of Dundee)
Actin polymerization provides the force that drives cellular movement. Concerted
polymerization, in a particular direction, is required in order for a cell to push forward its
leading edge. There are a host of actin-binding proteins that regulate the spatial and
temporal patterning of this actin polymerization. The list of actin-interacting proteins now
exceeds 150 classes of proteins. Actin-filament uncapping and actin-filament nucleation
are the two known mechanisms through which a cell can produce the explosive actin
filament elongation that is required for cell movement. However, filament capping (to
prevent non-productive polymerization), filament crosslinking (to provide a strong base
against which to push), and actin filament recycling (to provide an inexhaustible supply
of actin monomers) are also vital components of this system.
Phosphatidylinositol 4,5-bisphosphate (PIP2), a phospholipid, is a major regulator of
many of these actin-binding proteins, generally favouring the transition to polymeric
actin. PIP2 is, however, just one of several related phosphatidylinositol signalling
molecules and the precursor for the second messengers diacylglycerol, inositol 1,4,5trisphosphate, and phosphatidylinositol 3,4,5-trisphosphate (PIP3). The first phase of
this project is a cell biology approach to investigate how a single phospholipid species
fulfils such a range of independent functions. This will involve quantitative and spatial
analysis of PIP2 in motile cells and under conditions where PIP2 levels are manipulated
by pharmacological and molecular biological means. The effects of sequestering PIP2 in
vivo, using specific lipid binding protein domains, on the distribution and functionality of
the actin network will also be analysed. A second aim of this phase is to identify the
protein partners of PIP2 that regulate cell movement, using affinity isolation techniques.
This portion of research will be carried out in the first 2 years of the PhD in Dundee
under the supervision of Pete Downes, who is a world renowned leader in the
phosphatidylinositol signalling field.
www.dundee.ac.uk/biocentre/SLSBDIV6cpd.htm
The second half of the PhD, years 3 and 4, will be carried out in Singapore. In this phase
the molecular basis of the interactions of PIP2 with actin-binding proteins and actin will
be studied in vitro. Proteins that were identified in the first phase as potential PIP2controlled actin regulators will be characterized in biochemical assays to determine
these relationships. PIP2-protein complexes will be subjected to protein crystallographic
studies in order to unravel the specificity of these proteins for PIP2 over PIP3 or inositol
1,4,5-trisphosphate. Bob Robinson is a PI at IMCB specializing in the structural basis of
cell movement.
http://www.imcb.a-star.e.sg/research/research_group/
This project is intended to enhance our understanding of the balance between cell
proliferation, cell growth and apoptosis through inositol signalling and PIP2 initiated
movement. These questions are particularly pertinent to tumour growth and metastasis.
To apply or to obtain more information on this PhD opportunity, please contact Pete or
Bob directly:
Nick Leslie
n.r.leslie@dundee.ac.uk
(2) Curing Cancer with Computers: virtual drug design from the inhibition
of growth factor signalling to translation
Supervisors: Chandra Verma (Bioinformatics Institute, Singapore) and Ruth
Brenk (College of Life Sciences, University of Dundee)
Introduction: Translation in eukaryotic systems begins with the recognition/binding of 5’
mRNA by the eukaryotic initiation factor 4E (eIF4E)1,2. eIF4G then binds eIF4E thereby
enabling the formation of a larger translational assembly which then anchors to the
mRNA. Formation of this complex is crucial for recruiting the ribosome to the initiation
codon and subsequent translation. Translation is suppressed when 4E binding protein
(4E-BP) binds to the same site as eIF4G, preventing the formation of the translation
initiation complex (see figure below). This pathway is of great medical interest as
translational control appears to be a major route of action of rapamycin the
immunosuppressive drug and now a promising anti-cancer agent. mTOR, the kinase
inhibited by rapamycin, phosphorylates the BP1 proteins and blocks their binding to
eIF4E, thus allowing eIF4E to bind eIF4G and initiate translation of capped mRNA's. The
BP1 and eIF4G proteins share a common peptide motif that binds to the peptide binding
pocket on eIF4e.
Project: The project will characterize the interactions of the mRNA cap and the 4E-BP in
detail using simulation and bioinformatics tools. This will then be used to design
ligands/inhibitory-peptides for both pockets. High resolution crystal structures3 and
assays for measuring the interactions are available in the laboratory of Prof Sir David
Lane. The project will complement efforts in the Lane lab at optimizing protein aptamers
for eIF4E inhibition4 that can be used as proof of concept in genetic animal model
systems for the anti-tumour affect of eIF4E inhibition.
4EBP/eIF4E
eIF4E
mRNA cap
1. Richter JD, Sonenberg N (2005) Regulation of cap-dependent translation by
eIF4E inhibitory proteins. Nature. 433:477-80.
a. Curing Cancer with Computers: virtual drug design from the
inhibition of growth factor signalling to translation
(continued from previous page)
2. Gingras AC, Raught B, Sonenberg N. (1999) eIF4 initiation factors: effectors of
mRNA recruitment to ribosomes and regulators of translation. Annu Rev
Biochem.68:913-63.
3. Quiocho FA, Hu G, Gershon PD. (2000) Structural basis of mRNA cap
recognition by proteins. Curr Opin Struct Biol. 10:78-86
4. Herbert TP, Fahraeus R, Prescott A, Lane DP, Proud CG (2000) Rapid induction
of apoptosis mediated by peptides that bind initiation factor eIF4E. Curr Biol.
10:793-6.
(3) Defects in the skin barrier and their role in eczema and psoriasis
Supervisors: E. Birgitte Lane (Institute of Medical Biology, Singapore) and W.H.
Irwin McLean (Division of Molecular Medicine, University of Dundee)
This project is aimed at identifying the cause of one of the most common groups of
diseases in the developed world. Atopic disease (including atopic dermatitis (eczema),
allergy and asthma) is becoming increasingly common (Holgate 1999) and now affects
~20% of the population in the developed world. Predisposition to atopic disease is
highly heritable (Van Eerdewegh et al. 2002). Similarly, psoriasis affects about 4% of
population and has as strong genetic component. Attempts to identify the genes
involved have mostly focused on immunological mechanisms, although a defective
epithelial barrier has been suggested (Cookson and Moffatt 2002). Both atopic dermatitis
and psoriasis show genetic linkage to the “epidermal differentiation locus” on
chromosome 1q21, where many genes encoding epidermal proteins are found. One of
these is filaggrin (FLG), a key protein that facilitates terminal differentiation of the
epidermis and also formation of the skin barrier to water loss – an extremely important
function of the epidermis.
Prof Birgit Lane (Singapore) and Prof Irwin McLean (Dundee) have collaborated for
many years in studies of genetic skin disorders. In a recent important breakthrough, Prof
McLean’s group in Dundee showed that two independent loss-of-function genetic
variants in the gene for filaggrin (FLG) are the cause of ichthyosis vulgaris, the most
common inherited disorder of keratinisation (Smith et al., Nature Genetics 2006).
Because many ichthyosis patients also have eczema, the group also looked at these
filaggrin variants in atopic disease (eczema) and showed that filaggrin mutations are
also very strong predisposing factors for eczema (Palmer et al. 2006). There was also a
highly significant association with the subtype of asthma that occurs in association with
atopic dermatitis (eczema). This work has established a key role for impaired skin
barrier function in the development of atopic disease. This project will extend these
studies into other clinically important areas, building on synergistic resources between
Singapore and Dundee.
The two FLG variants, R510X and 2282del4, are carried by ~9% of people of European
origin, but it is not yet known whether these same variants are important in Asian
populations. Singaporean patients with ichthyosis vulgaris will therefore also be
screened initially for filaggrin mutations. Other closely-related genes may also be
involved in these and related disorders. Filaggrin is one of a cluster of 7 large “fused
S100” genes that lie within the 1q21 locus (http://genome.ucsc.edu). The other genes
are filaggrin-2 (FLG2), trichohyalin (THH), trichohyalin-like 1 (THHL1), cornulin (CRNN),
repetin (no gene symbol yet assigned) and hornerin (HRNR). All these proteins are
involved in terminal differentiation of the epidermis and skin barrier function. About 40%
of the UK paediatric atopic dermatitis patients do not carry the prevalent filaggrin
mutations and may have defects in the related fused S100 genes. Pilot studies on UK
patients suggest that the filaggrin variants linked to atopic dermatitis may be in inverse
phase linkage with psoriasis, as individuals generally develop either atopic dermatitis or
psoriasis and seldom, if ever, develop both. One interpretation is that filaggrin variants
lead to atopic dermatitis and variants in a neighbouring gene cause psoriasis. Thus, the
remaining fused S100 proteins are prime candidates for psoriasis.
Skin and DNA samples from patients with these disorders will be collected for this study
in collaboration with Dr Jean Ho of the National Skin Centre. Biopsies from UK and
Singapore patients with atopic dermatitits and psoriasis showing no filaggrin mutations
will be carefully analysed for any changes in expression of other fused S100 proteins by
immunohistochemistry, immunoblotting and quantitative RT-PCR. Prof McLean’s lab
have developed long-range PCR methods for analysis of the filaggrin gene (Smith et al.
2006) which are being adapted for mutation detection strategies for the remaining 6
genes. Also, no antibodies currently exist against the THHL1 protein and limited
reagents are available to the others; there is a particular need for reagents against
additional epitopes on filaggin-2, cornulin, repetin and hornerin. Using the expertise for
antibody generation in Prof Lane’s lab antibodies will be raised against the six proteins
and used for immunohistochemistry and protein biochemistry analysis of biopsies. This
will lead to identification of target genes for analysis, and to identification of further
predisposing genes for atopic dermatitis, psoriasis and other diseases precipitated by
skin barrier defects. Depending on the nature of any fused S100 variants identified,
expression studies in organotypic keratinocyte cultures will be performed to assess the
functional consequences of these genetic defects, which may lead to development of
therapeutic strategies for these widespread disorders.
References:
Cookson WO, Moffatt MF (2002) The genetics of atopic dermatitis. Curr Opin Allergy
Clin Immunol 2:383-387
Holgate ST (1999) The epidemic of allergy and asthma. Nature 402:B2-4
Palmer CNA, Irvine AD, Terron-Kwiatkowski A, Zhao Y, Liao H, Lee SP, Goudie DR,
Sandilands A, Campbell LE, Smith FJD, O'Regan GM, Watson RM, Cecil JE,
Bale SJ, Compton JG, DiGiovanna JJ, Fleckman P, Lewis-Jones S,
Arseculeratne G, Sergeant A, Munro CS, El Houate B, McElreavey K, Halkjaer
LB, H. B, Mukhopadhyay S, McLean WHI (2006) Common loss-of-function
variants of the epithelial barrier protein filaggrin are a major predisposing factor
for atopic dermatitis. Nature Genetics, in press.
Smith FJD, Irvine AD, Terron-Kwiatkowski A, Sandilands A, Campbell LE, Zhao Y, Liao
H, Evans AT, Goudie DR, Lewis-Jones S, Arseculeratne G, Munro CS, Sergeant
A, O'Regan G, Bale SJ, Compton JG, Digiovanna JJ, Presland RB, Fleckman P,
McLean WHI (2006) Loss-of-function mutations in the gene encoding filaggrin
cause ichthyosis vulgaris. Nature Genetics (e-pub ahead of print).
Van Eerdewegh P, Little RD, Dupuis J, Del Mastro RG, Falls K, Simon J, Torrey D, et al.
(2002) Association of the ADAM33 gene with asthma and bronchial
hyperresponsiveness. Nature 418:426-430
(4) Relationship between the tumour suppressor proteins APC and p53
Supervisors: Professor David Lane (Institute of Molecular & Cell Biology,
Singapore) and Dr Inke Nathke (College of Life Sciences, University of Dundee)
Introduction: Mutations in APC are common to most colorectal cancers and occur
extremely early in the disease. Inactivating APC in other tissue also leads to cancer
particularly breast, kidney and liver. Similarly, mutations in p53 or proteins involved in
the regulation of its activity are also extremely common to many different types of
cancer. A relationship between these two important tumour suppressors has been
established for a number of specialised cases. However, it is not at all clear how the
activity of these two proteins is linked to the initiation and progression of cancer. The
aim of this proposal is to establish how the activity of APC affects the activity of p53 and
vice versa in a number of cell and animal systems.
Background: APC acts as a scaffold for the formation of a protein complex that
regulates the amount of β-catenin available for transcriptional repression of TCF/LEF
type transcription factors. Mutations in APC that compromise this function correlate with
an increase in the available β-catenin and lead to the activation of a host of genes
implicated in differentiation and proliferation. One of the genes that is induced by βcatenin/Lef is p19-Arf which in turn induces p53. As a consequence, overexpression of
β-catenin can lead to senescence in a p53-dependent manner. On the other hand,
increasing p53 itself can target β-catenin for degradation by activating the Siah1 protein
which links β-catenin to the degradation machinery. The relationship between these
different pathways and their relative contribution to the progression and/or initiation of
cancer is not at all clear. It is highly like that different tissues rely on these pathways to
different degrees to protect themselves. For instance, in the kidney, APC mutant cells
appear to be selectively depleted unless p53 is inactive, suggesting that in this tissue the
accumulation of β-catenin activates p53 to cause cells to arrest and possibly die. In the
colon on the other hand, APC-deficient cells propagate well and cause tissue
abnormalities almost immediately after APC is lost. When p53 is deleted in addition to
APC in this tissue, tumours become more aggressive but their early progression does
not appear to be altered dramatically.
Furthermore, we recently discovered that loss of APC induces tetra- and aneupoloidy in
cultured cells and tissue. The concomitant decrease in apoptosis leads to a significant
increase in aneuploid cells almost immediately after APC inactivation. Usually p53dependent pathways control such situations and the lack of an appropriate response in
cells lacking APC suggests that p53-mediated response may be compromised.
To investigate the relationship between APC and p53 the project intends to
(1) measure the activity of p53 in APC-deficient cells and tissues.
The student will use a variety of cultured cell systems where APC can be inactivated
conditionally (Cre-flox fibroblasts and RNAi). The student will also have access to a
mouse that carries floxed APC so that APC can be inactivated specifically in gut tissue.
(2) measure the activity of APC in p53-deficient cells. Assays include: Top/Fop reporter
assay to determine β-catenin activity, determine MT stability (simple depolymerisation
assays)
(3) using Zebra fish to inactivate either or both proteins in different tissues to look at
their interdependence.
(5) The specificity and regulation of interactions between 14-3-3 and actin
binding proteins
Supervisors: Ed Manser (Institute of Molecular & Cell Biology, Singapore) and
Carol MacKintosh (School of Life Sciences, University of Dundee)
Introduction
14-3-3 proteins have central regulatory roles that they exert by binding to specific
phosphorylated sites on target proteins. The binding of 14-3-3s can force conformational
changes in a target and/or affect its interactions with other proteins. Recently, the
MacKintosh lab & others have described hundreds of proteins via 14-3-3-affinity
purification methods (1, 2), confirming that 14-3-3s likely mediate the regulation of a wide
range of cellular processes. Specificity is suggested by the fact that proteins were
selectively eluted from immobilised 14-3-3 by competition with 14-3-3-binding
(phospho)peptides; far-Westerns assays indicate eluted fractions are highly enriched in
phospho-proteins which yet need to be individually tested.
Prominent among the 14-3-3 affinity purified proteins are actin-binding proteins (ABPs):
these included proteins that are involved in the dynamic remodelling of the actin
cytoskeleton by nucleating F-actin assembly or elongating, capping, severing and
crosslinking actin filaments including cortactin (HIP-55), VASP and Lim kinase. Since 143-3 often provides critical regulatory input following phosphorylation, uncovering the
mechanism and function of 14-3-3 binding allows access to cellular regulation
mechanisms.
Specific aims of the project:
1. VASP, one of the Ena family of proteins, enhances actin filament elongation by
recruiting profilin:actin complexes to sites of active actin remodelling such as the tips of
spreading lamellipodia (3). The name VASP, for Ena/ vasodilator-stimulated
phosphoprotein, comes from the original discovery of VASP as a protein that was rapidly
phosphorylated by PKA and PKG in platelets (4, 5). Three sites of phosphorylation are
well established and which can be acted upon by multiple kinases, however these do not
represent consensus 14-3-3 binding sites (nor are responsible for 14-3-3 binding,
preliminary data). In order to identify the 14-3-3 binding sites various deletion constructs
of VASP will be analysed for 14-3-3 binding with or without calyculin treatment. The
tagged phospho-protein recovered in these expts will also be analysed by LC-MS in order
to establish in vivo sites of phosphorylation. Once important sites are identified, phosphospecific antibodies will be generated in order to study the temporal and spatial pattern of
modification in cells. Of interest to the work of the Manser lab is the fact that the
important adaptor protein Nck may be co-regulated with respect to complex formation
with VASP and/or the kinase PAK1.
2. Cofilin is a critical element in the organization of actin, and is negatively regulated by
LIM kinase phosphorylation of serine 3 and subsequent binding of 14-3-3 (6).
Interestingly there is one report of LIMK1 interaction with both Lim and PDZ domains of
this kinase but the function of binding has not been establish (7). Spatial and temporal
regulation of cofilin activity by LIM kinase (LIMK) and Slingshot is critical for directional
cell migration (8, 9). We propose to investigate both binding partners for these two
domains of LIM kinase 1 and establish how 14-3-3 binding might regulate these binding
activities. Since LIM kinase 1 is found in focal adhesions (a focus of Manser lab with
respect to upstream kinase PAK1) the co-ordinated regulation of these kinases at cell
adhesions and the potential range of substrates at this site will be investigated. We hope
to uncover mechanisms by which two kinases are connected as well as the coordinated
input from other Rho regulated protein kinases such as ROK and MRCK.
References.
1. Jin J, Smith FD, Stark C, Wells CD, Fawcett JP, Kulkarni S, Metalnikov P, O'Donnell
P, Taylor P, Taylor L, Zougman A, Woodgett JR, Langeberg LK, Scott JD, Pawson T.
Proteomic, functional, and domain-based analysis of in vivo 14-3-3 binding proteins
involved in cytoskeletal regulation and cellular organization. Curr Biol. 2004 14:1436-50.
2. Pozuelo Rubio M, Geraghty KM, Wong BH, Wood NT, Campbell DG, Morrice N,
MacKintosh C.
14-3-3-affinity purification of over 200 human phosphoproteins reveals new links to
regulation of cellular metabolism, proliferation and trafficking. Biochem J. 2004 379:395408.
3. Barzik M, Kotova TI, Higgs HN, Hazelwood L, Hanein D, Gertler FB, Schafer DA.
Ena/VASP proteins enhance actin polymerization in the presence of barbed end capping
proteins. J Biol Chem. 2005 280:28653-62.
4. Wentworth JK, Pula G, Poole AW. Vasodilator-stimulated phosphoprotein (VASP) is
phosphorylated on Ser157 by protein kinase C-dependent and -independent mechanisms
in thrombin-stimulated human platelets. Biochem J. 2006 393:555-64.
5. Lambrechts A, Kwiatkowski AV, Lanier LM, Bear JE, Vandekerckhove J, Ampe C,
Gertler FB.
cAMP-dependent protein kinase phosphorylation of EVL, a Mena/VASP relative,
regulates its interaction with actin and SH3 domains. J Biol Chem. 2000 275:36143-51.
6. Gohla A, Bokoch GM. 14-3-3 regulates actin dynamics by stabilizing phosphorylated
cofilin.
Curr Biol. 2002 12:1704-10.
7. Birkenfeld J, Betz H, Roth D. Identification of cofilin and LIM-domain-containing protein
kinase 1 as novel interaction partners of 14-3-3 zeta. Biochem J. 2003 369:45-54.
8. Nishita M, Tomizawa C, Yamamoto M, Horita Y, Ohashi K, Mizuno K. Spatial and
temporal regulation of cofilin activity by LIM kinase and Slingshot is critical for directional
cell migration.
J Cell Biol. 2005 171:349-59.
9. Soosairajah J, Maiti S, Wiggan O, Sarmiere P, Moussi N, Sarcevic B, Sampath R,
Bamburg JR, Bernard O. Interplay between components of a novel LIM kinase-slingshot
phosphatase complex regulates cofilin. EMBO J. 2005 24:473-86.
(6) Lowe syndrome protein (OCRL-1) and endosome-to-TGN trafficking
Supervisors: Wanjin Hong (Institute of Molecular & Cell Biology, Singapore) and
John Lucocq (College of Life Sciences, University of Dundee)
Background
Oculocerebrorenal syndrome of Lowe protein 1 (OCRL1) is a PI(4,5)P2 5-phosphatase
(Suchy et al., 1995 ; Zhang et al., 1995 ) originally assigned as a peripheral membrane
protein to the Golgi (Dressman et al., 2000 ) and lysosomes (Zhang et al., 1998 ).
Deficiency of OCRL1 is responsible for Oculocerebrorenal Syndrome of Lowe, a rare Xlinked disorder characterized by mental retardation, congenital cataracts, and renal
Fanconi Syndrome. The pathobiology of Lowe syndrome is obscure but cells derived
from Lowe Syndrome patients have elevated PI(4,5)P2 levels, (Zhang et al., 1998) which
could explain the altered the actin cytoskeleton, because patients' cells seem to have
fewer long actin stress fibers, abnormal punctate F-actin staining, and enhanced
sensitivity to actin depolymerizing agents (Suchy and Nussbaum, 2002 ).
Another intriguing mechanism for OCRL1 function is by altering membrane
trafficking. Recent immunofluorescence and immunoEM microscopy in the JML lab and
others (Ungewickell 2004; Konstantacopoulos et al., unpublished) has shown that while
minor fractions of OCRL1 are present in the TGN (Dressman et al., 2000) and plasma
membrane, by far the largest pool of OCRL1 is found on endosomes. Here OCRL1 is
likely involved in recycling from endosomes to the TGN because the endosomes were
found to be coated with clathrin and the clathrin adaptor AP-1 (Mallard et al., 1998). In
collaboration with Martyn Lowe (Manchester) we found that OCRL1 interacts directly
with clathrin heavy chain and can promote clathrin assembly in vitro. We found no effect
on anterograde secretory pathway or on endocytosis. JML lab has done RNAi
experiments combined with immunoEM localization of PIP2 and the OCRL1 product
PI4P, and showed that OCRL1 regulates PIP2 and PI4P levels at the trans-Golgi,
endosomes and ER (Konstantacopoulos et al., unpublished). This indicates OCRL1 is a
principal endomembrane PI(4,5)P2 5-phosphatase restraining PIP2 and maintaining
PI4P. Thus regulation of PI4P could explain OCRL1 effects on endosomal traffic. PI4P
has been implicated in traffic from endosome to the lysosome and has recently been
shown to be an important lipid for coincidence detection of trafficking proteins at the
endosome and TGN (for example clathrin adaptors AP-1 and Epsin R and ARF/PI4P
dual adaptor FAPP1 (our work with Antonella de Matteis (Godi et al., 2004)). We have
found overexpression or depletion of OCRL1 perturbs protein trafficking at the
TGN/endosome interface, suggesting a role in regulating transport between these
compartments (Choudhury et al., 2005). Thus overexpression of OCRL1 restricts M6PR
localisation and RNAi knockdown inhibits delivery of Shiga toxin to the TGN. The exact
pathway, site and mechanisms of OCRL1 action are unknown. It could act at clathrin
coat assembly on the endosome or later on this pathway, between early or late
endosomes. OCRL1 might function to regulate coat assembly by direct interaction with
coat components or more generally regulate PI4P levels in the endosome-TGN axis.
Importantly the OCRL1 knockout mouse lacks a phenotype (likely because of
compensatory phosphatase InPP5B) and studies on OCRL1 function should therefore
be limited to human cells.
There are two known pathways from endosomes to the TGN and key proteins on
these pathways have been investigated in the WJH lab. at the IMCB. The first pathway
originates from late endosomes and carries the protease furin and the lysosomal
enzyme receptor M6PR to the TGN (Mallet and Maxfield, 1999; Diaz and Pfeffer, 1998).
The other pathway leads from early endosomes, mediating traffic of Shiga toxin,
glut 4 and TGN38/46 and possibly also M6PRs. (Mallard et al., 1998; Ghosh et al.,
1998). Interestingly early/recycling endosomes depends on clathrin and epsinR, but not
AP-1 indicating redundancy or possibly parallel pathways (Saint-Pol, et al., 2004). WJH’s
lab. has successfully employed both in vivo and in vitro transport assays using
recombinant Shiga toxin B fragments (STxB) as protein cargos in order to facilitate
analysis of this transport pathway (Tai et al., 2005). We have found the Arf-like small
GTP-ase Arl1 and its effector, the GRIP domain containing Golgin 97, function along an
endosome to TGN trafficking route and suggest that Arl1 may recruit Golgins as tethers
acting late on the endosome TGN route and work prior to fusion steps that use SNARE
complex (Lu et al., 2003 and 2004). These works have established the in vivo function of
Arl1 and Golgin-97 on recycling pathways using RNAi based knockdown but also with
microinjection of anti-human Golgin-97 antibodies in human HeLa cells.
Aims
The project student will investigate the role of OCRL1 in endosome to TGN trafficking
using in vivo and in vitro assays combined with light and electron microscopy and
answer the following questions.
(1) In which endosome pathway OCRL1 acts and where with respect to coat assembly,
tethering and fusion? Does OCRL1 act on Arl/Golgin, Epsin R dependent routes, early
or late endosomes. Does OCRL1 function at budding (clathrin assembly), tethering
(Arl1) or fusion (SNARE) or some combination of these? Is the in vivo role of OCRL1 in
regulation of clathrin/adaptor dynamics at the endosome?
(2) What is the role of PI4P in endosome-TGN traffic. Does PI4P regulation (by
OCRL1/PI4kinases), and PI4P effectors such as FAPP1, regulate the retrograde
trafficking route?
(3) Is there an endosome-TGN traffic phenotype of Lowe syndrome cells? Can this traffic
phenotype be reverted by OCRL1 expression?
Plan
The student will set up the assays in the WJH lab in Singapore and test the
sites/pathways of OCRL1 regulation. Clathrin and adaptor dynamics studies, PI4P,
OCRL1/FAPP1 and immunoEM components will be done in JML Lab in Dundee.
References.
Choudhury R, Diao A, Zhang F, Eisenberg E, Saint-Pol A, Williams C,
Konstantakopoulos A, Lucocq J, Johannes L, Rabouille C, Greene LE, Lowe M. (2005)
Mol Biol Cell. 16(8):3467-79.
Diaz, E., and Pfeffer, S.R. (1998). Cell 93: 433-443.
Downes CP, Gray A, Watt SA, Lucocq JM.(2003) Methods Enzymol. 366:64-84.
Downes CP, Gray A, Lucocq JM. (2005) Trends Cell Biol. 15:259-68.
Dressman MA, Olivos-Glander IM, Nussbaum RL, Suchy SF. (2000) J Histochem
Cytochem. 48:179-90.
Godi A, Di Campli A, Konstantakopoulos A, Di Tullio G, Alessi DR, Kular GS, Daniele T,
Marra P, Lucocq JM, De Matteis MA. (2004) Nat Cell Biol. 6:393-404.
Ghosh, R.N., Mallet, W.G., Soe, T.T., McGraw, T.E., and Maxfield, F.R. (1998). J. Cell
Biol. 142: 923-936.
Janne PA, Suchy SF, Bernard D, MacDonald M, Crawley J, Grinberg A, Wynshaw-Boris
A, Westphal H, Nussbaum RL. (1998) J Clin Invest. 101:2042-53.
Lu L, Tai G, Hong W. (2004) Mol Biol Cell. 15:4426-43.
Lu L, Hong W. (2003). Mol Biol Cell. 14:3767-81.
Mallet, W.G., and Maxfield, F.R. (1999) J. Cell Biol. 146, 345-359
Suchy SF, Nussbaum RL. (2002) Am J Hum Genet. 71:1420-7.
Suchy SF, Olivos-Glander IM, Nussabaum RL. (1995) Hum Mol Genet. 4:2245-50.
Tai G, Lu L, Johannes L, Hong W. (2005) Methods Enzymol. 404:442-53.
Ungewickell A, Ward ME, Ungewickell E, Majerus PW. (2004) Proc Natl Acad Sci U S
A. 101:13501-6.
Watt SA, Kimber WA, Fleming IN, Leslie NR, Downes CP, Lucocq JM. (2004) Biochem
J. 377:653-63.
Watt SA, Kular G, Fleming IN, Downes CP, Lucocq JM. (2002) Biochem J. 363:657-66.
Zhang X, Hartz PA, Philip E, Racusen LC, Majerus PW. (1998) J Biol Chem. 273:157482.
(7) The role of the cell cycle during vertebrate somite segmentation
Supervisors: Yun-Jin JIANG (Laboratory of Developmental Signalling and Patterning,
Institute of Molecular & Cell Biology, Singapore) and Miguel Maroto (Division of Cell &
Developmental Biology, University of Dundee)
Segmentation is a key feature of the body plan of vertebrates which initiates very early in
embryonic development. The first sign of segmentation is seen when vertebrate
embryos develop somites, which are the embryonic precursors of muscles, dermis and
skeletal bones, and are produced from the presomitic mesoderm in a highly regulated
process called somitogenesis. As development proceeds, the embryo makes new
somites according to a strict periodic schedule. This aspect of tight temporal control led
scientists to develop a number of theoretical models all of which shared in common the
prediction that the process would be driven by an oscillatory mechanism. Interference
with the proper function of this molecular clock produces, as a result, the formation of
abnormal embryonic structures, which will later give rise to animals with severe
morphological segmentation defects. Many studies have substantiated one such model,
namely the ‘clock and wavefront’ model, implicating both the Notch and Wnt signalling
pathways as key components of the machinery of this molecular clock. There are,
however, findings that cannot be explained by this widely accepted model. Thus,
experiments on amphibian, avian and fish embryos provided evidence that a single heat
shock causes a disruption to segmentation visible several hours after treatment,
producing several discrete periodic segmental anomalies. In addition, treating the
embryos with a variety of inhibitors of the cell cycle causes similar periodic segmental
defects, which suggests the existence of some degree of cell cycle synchrony between
cells destined to segment together. Based on findings such as these a ‘cell cycle’ model
was proposed as an alternative model for the control of the timing of segmentation. The
hypotheses underlying the ‘cell cycle’ model are now widely disputed; however, it is still
unknown what the real function of the cell cycle is during the process of vertebrate
segmentation. Experiments outlined in this proposal aim to enhance our knowledge of
this subject using chick, mouse and zebrafish embryos as animal models.
Using chick and mouse embryos as animal models the project student will aim to
analyse cell division along the presomitic mesoderm and the forming somites, by BrdU
incorporation and immunohistochemistry using specific antibodies against components
of the cell cycle. As a complementary approach, by electroporation using specific GFP
constructs, the distribution of dividing cells along the presomitic mesoderm in chick will
be investigated. The project student will determine the real time period of the cell cycle in
these tissues and the possible existence of cell division synchrony. The student will also
analyse the effect on segmentation, both molecularly and morphologically, after
treatment with specific inhibitors known to affect the cell cycle in chick and mouse. The
project will investigate cell division in chick after interfering with the Notch pathway
implicated in the molecular mechanism of the segmentation clock. If from these results
any relationship with the Notch pathway is found then cell division distribution in different
Notch mutant mouse embryos will be characterised. This part of the project will be
performed in Dundee (UK). Using H2A transgenic zebrafish line as an animal model, it is
proposed that cell division along the presomitic mesoderm and the forming somites will
be analysed. The project student will investigate the effect on segmentation, both
molecularly and morphologically, after treatment with cell cycle inhibitors. The student
will also analyse cell division in the presomitic mesoderm of different Notch mutant
zebrafish lines. Finally, the effect on segmentation in wild-type embryos and Notch
mutants after treatment with cell cycle inhibitors will be determined. This part of the
project will be performed in Singapore.
(8) Novel cellular targets of Human Papilloma Virus - implications to disease
Supervisors
Dr D (Sam) Crouch
Dr Francoise Thierry
University of Dundee
Division of Molecular
Medicine
Scotland
Institute of Medical
Biology
A*STAR
Singapore
d.h.crouch@dundee.ac.u
k
francoise.thierry@imb.
a-star.edu.sg
Background
High risk forms of the human papilloma viruses (HPV) are major causative agents in the
development of cervical cancer, which is the second largest cause of cancer deaths in
women worldwide. A major challenge is to understand the mechanism of how these
viruses cause or contribute to disease, at both the physiological and molecular level with
a view to developing effective therapies in the future (1).
HPV, a small double stranded DNA virus, encodes proteins required for its survival and
propagation. The oncogenic activity of the virus is primarily attributed to the E6 and E7
proteins, which establish and maintain malignant transformation. Generally, E6
functions as a ubiquitin ligase which down regulates the tumour suppressor, p53, by
targeting it for proteasomal degradation. In contrast, E7 controls the activity of E2F
transcription factor by degrading its regulator, the tumour suppressor pRb. The
molecular bases of how these viral oncoproteins function is still actively being
researched.
We have discovered that E6 and E7 specifically interfere with the levels of a
cytoskeleton-associated protein, EBP50, suggesting this is a significant target in disease
progression. EBP50 mediates the linkage between the ERM (ezrin, radixin, moesin)
family of proteins and the actin cytoskeleton, thereby contributing to cell:cell adhesion
and cellular polarity. It acts as both an oncogene and a tumour suppressor, depending
on its subcellular localisation (2).
The Project
The aims of this PhD proposal is to find out how this works, as this may lead to new
ideas for cervical cancer management and treatment. You would aim to characterise the
molecular mechanism and sub-cellular regulation of EBP50 and associated proteins
during HPV oncogenesis, both in vitro and in tumours in vivo. EBP50 regulation will be
characterised in the presence of both high and low risk HPV isoforms. This will be done
in a number of ways:
(1) Viral-mediated regulation of EBP50 in vitro: a comprehensive study of molecular
mechanisms underlying the transcriptional and proteolytic regulation of EBP50 (and
associated proteins) in cell lines expressing HPV-encoded proteins will be assessed.
siRNA and overexpression studies will be performed to determine the role of EBP50 in
cell growth, cell:cell adhesion and cell cycle.
(2) Expression of EBP50 in cervical carcinoma in vivo: analysis of EBP50 expression in
cervical tumours will be performed using immunohistochemistry. The subcellular
localisation and expression levels of EBP50 will be scored, and correlated with the stage
and severity of the disease, and presence of specific HPV isoforms.
References
(1) Thomas et al (2008) Human papilloma viruses, cervical cancer and cell polarity.
Oncogene 127: 7018-7030.
(2)
Georgescu MM et al (2008) Roles of NHERF1/EBP50 in cancer. Curr Mol Med.
8:459-68.
(9) Characterisation of the Wnt and GSK3 signalling and their role in cancer
David Virshup (Institute of Medical Biology, Singapore) and Dario Alessi (College
of Life Sciences, University of Dundee)
Description of science
The Wnt signalling pathway is at the nexus of a signal transduction network that
regulates stem cell maintenance, cell proliferation, cancer and development. The
Virshup and Alessi laboratories study the Wnt signaling pathways with an emphasis
on characterization of the protein kinases and phosphatases that are regulated by
Wnt, and how these control cell proliferation and development. Recent work has
revealed that a significant number of all cancers possess mutations that disrupt the
Wnt signalling pathway and these are major factors driving the inappropriate growth
and proliferation of these cells. At the heart of the Wnt signalling pathway lies a
protein kinase termed GSK3 that is inactivated by Wnt. Although much research has
been undertaken on the role of GSK3 in Wnt signalling, we still know little about the
molecular mechanism by which GSK3 is switched off by Wnt and what are the
enzymes and other regulators controlling GSK3 activity within this complex. A
central aim of this project will be to deploy state of the art research technologies to
address this key question.
Specific aims of project
1. We would isolate Wnt-regulated complexes of GSK3 bound to axin and other
regulators and undertake state of the art proteomic mass spectroscopy analysis of
these complexes. We would characterise the composition of the GSK3:axin complex
and determine the activity state of GSK3 within the complex. To determine how
GSK3 is regulated, we would also investigate the phosphorylation and ubiquitination
status of each of the components within the complex. This work will reveal how Wnt
regulates the composition and phosphorylation/ubiquitination state of the GSK3:axin
complex.
2. Appropriate mutagenesis and functional analysis will be undertaken to
characterise Wnt regulated changes in the GSK3:axin complex that are
observed. For example, if Wnt regulated the phosphorylation state of
component(s) within the GSK3 complex, we would mutate the phosphorylation
sites and analyse how this affected the integrity and function of the GSK3
complex. Phosphospecific antibodies to the characterised phosphorylation sites
would be raised in order to study these Wnt regulated processes in cells.
3. If Wnt regulated the phosphorylation or ubiquitination state of the GSK3:axin
complex, we will identify the kinases and phosphatases and/or
ligases/deubiquitinating enzymes that controlled the modification of the complex.
This could lead to the identification of novel Wnt regulated enzymes that might
represent drug targets to modulate the activity of the Wnt signalling pathway.
4. If novel kinases and/or phosphatases and/or ligases that were regulated by Wnt
were identified, we would explore the mechanism by which Wnt controlled the
activity of these enzymes. We would also search for other substrates of these
enzymes that might also comprise novel targets of the Wnt signalling pathway.
5. The laboratory of David Virshup has recently discovered that the Wnt signalling
pathway activates a protein phosphatase that dephosphorylates and activates
casein kinase-1 another enzyme known to participate in the Wnt-signalling
pathway. Functional assays will be set up assays to determine how
phosphorylation of CK1 regulates its activity. Based on these results we would
generate knock-in mice in which the key regulatory phosphorylation sites on CK1
are mutated to Ala in order to prevent phosphorylation, or to acidic residues in
order to mimic phosphorylation. The generation and analysis of knock-in mice is
routinely performed in the laboratory of Dario Alessi. We would then analyse how
these mutations affected Wnt signalling pathway in cell lines derived from these
mice. We would investigate the impact that the knock-in mutations had in mouse
development and whether the knock-in mice were predisposed to developing
cancer
Techniques to be employed
The DUKE-NUS and MRC Protein Phosphorylation Unit (MRC-PPU) are worldleading centres in analysing signal transduction pathways. Within the MRC-PPU
Unit and the other laboratories of Dundee University there are currently over 150
researchers working on signal transduction pathways. This project will involve use of
state of the art technologies and methodologies of signal transduction, mass
spectrometry, biochemistry, enzymology and protein expression. It will also involve
generation and characterisation of knock-in mice. The student undertaking this
project will have access to all available equipment, expertise and resources that are
necessitated for the project. All equipments, resource and expertise are in place in
DUKE-NUS and MRC-PPU to undertake this work.
Originality
Understanding how the GSK3 kinase is regulated and functions in the Wnt signalling
pathway is one of the most important questions in the field of signal transduction.
Although worldwide many groups are seeking answers to this problem, with the
combined DUKE-NUS and MRC-PPU resource, expertise and previous
accomplishments in this area of research, we believe that we have the potential to
make a significant impact in this area of research. The discovery of new
components of the Wnt signalling pathway and how Wnt controls the activity and
function of these will open up new opportunities to target these enzymes in drug
discovery efforts for modulating this pathway for treatment of disease such as
cancer.
Selected Publications:
Tsai, I.C., Woolf, M., Neklason, D.W., Branford, W.W., Yost, H.J., Burt, R.W., and
Virshup, D.M. 2007. Disease-associated casein kinase I delta mutation may
promote adenomatous polyps formation via a Wnt/beta-catenin independent
mechanism. International journal of cancer 120: 1005.
Tsai, I., Amack, J., Gao, Z., Band, V., Yost, H., and Virshup, D. 2007. A WntCKIvarepsilon-Rap1 pathway regulates gastrulation by modulating SIPA1L1, a Rap
GTPase activating protein. Developmental cell 12: 335.
Luo, W., Peterson, A., Garcia, B., Coombs, G., Kofahl, B., Heinrich, R.,
Shabanowitz, J., Hunt, D., Yost, H., and Virshup, D. 2007. Protein phosphatase 1
regulates assembly and function of the beta-catenin degradation complex. The
EMBO journal 26: 1511.
Swiatek, W., Kang, H., Garcia, B.A., Shabanowitz, J., Coombs, G.S., Hunt, D.F.,
and Virshup, D.M. 2006. Negative regulation of LRP6 function by casein kinase I
epsilon phosphorylation. J Biol Chem 281: 12233.
Margolis, S.S., Perry, J.A., Forester, C.M., Nutt, L.K., Guo, Y., Jardim, M.J.,
Thomenius, M.J., Freel, C.D., Darbandi, R., Ahn, J.H., Arroyo, J.D., Wang, X.F.,
Shenolikar, S., Nairn, A.C., Dunphy, W.G., Hahn, W.C., Virshup, D.M., and
Kornbluth, S. 2006. Role for the PP2A/B56delta phosphatase in regulating 14-3-3
release from Cdc25 to control mitosis. Cell 127: 759.
Eide, E., Woolf, M., Kang, H., Woolf, P., Camacho, F., Vielhaber, E., Giovanni, A.,
and Virshup, D. 2005. Control of mammalian circadian rhythm by CKIe-regulated
proteasome-mediated PER2 degradation. Molecular and Cellular Biology 25: 2795.
McManus, E.J., Sakamoto, K., Armit, L.J., Ronaldson, L., Shpiro, N., Marquez, R.
and Alessi, D.R. (2005) Role that phosphorylation of GSK3 plays in insulin and Wntsignalling defined by knockin analysis. EMBO J. 24, 1571-1583.
(10)
Mutations in type VII collagen and their role in wound healing and cancer
E. Birgitte Lane (Institute of Medical Biology), Singapore) and Andrew South,
(Division of Medical Sciences, Medicine, College of Medicine, Dentistry &
Nursing, University of Dundee)
This project will investigate the contribution of dermal fibroblasts to the defective
wound healing and the greatly increased incidence of invasive squamous cell
carcinomas (SCC) seen in patients with mutations in type VII collagen.
Defective or absent type VII collagen, caused by mutations in the COL7A1 gene, is
responsible for the inherited skin disease dystrophic epidermolysis bullosa (DEB).
DEB can be genetically dominant or recessive depending on the nature of the
COL7A1 mutations and is characterised by varying degrees of epithelial blistering in
skin and internal epithelia (oral mucosa, gastrointestinal tract). People with the
severe, mutilating Hallopeau–Siemens form of recessive dystrophic epidermolysis
bullosa (RDEB) suffer trauma-induced blisters and slow to heal skin erosions. The
chronic wounds cause considerable morbidity and lead to skin cancer, and most
RDEB patients die in their twenties of SCC.
Andrew South’s group (Dundee) have compared skin cells from RDEB patient’s
“normal” skin and SCC with those from non-RDEB site matched samples. Most
studies looking at effects of COL7A1 mutations on cell behaviour have concentrated
on the epidermal cell, the keratinocyte (Vessagowit et al, 2004; Ortiz-Urda et al,
2005). Increasing evidence now points to the dermal component as being a major
factor in wound healing and SCC invasion in non-RDEB individuals (Gaggioli et al,
2007; Wong et al, 2007). Expression profiling of RDEB keratinocytes in culture
supported by their own matrix has shown no clear expression pattern that
distinguishes RDEB from non-RDEB samples. This suggests that there is no intrinsic
difference in monocultures of COL7A1-impaired keratinocytes that predispose to
reduced wound healing and SCC development. However, the ability of keratinocytes
to invade 3-dimensional organotypic skin equivalents is increased when RDEB (but
not wild-type) fibroblasts are embedded in the matrix. These unpublished data
suggests that the dermal component is influencing the pathology of DEB, and this
hypothesis will be the target of this project.
The ability of a cell to migrate is critically dependent on the composition of its
adjacent extracellular matrix and migration is critical to both wound healing and
cancer progression. The project will use time-lapse microscopy to compare the
migration of RDEB fibroblasts with site-matched non-RDEB fibroblasts plated onto
plastic alone or plastic coated with various extracellular matrices. The migration of
both fibroblasts and keratinocytes will then be assessed on cell-derived matrix,
generated by culturing fibroblasts for 8 days before removing the fibroblasts and reseeding the used plates with cells to be assessed. This will examine whether the
matrix generated by RDEB fibroblasts significantly alters fibroblast or keratinocyte
migration. The effect of type VII collagen will be investigated either by expressing
recombinant collagen VII through viral vectors or by using purified protein.
Data from these assays will then be incorporated into models of SCC invasion
established in Andy South’s group and wound healing models established in Birgit
Lane’s group. Using these model systems known pathways involved in migration,
invasion and wound healing will be interrogated using chemical inhibitors, RNAi or
recombinant protein expression. It is envisaged that data generated from this project
will not only increase our understanding of DEB pathology, but will also be of wider
clinical significance in informing on cellular mechanisms underlying wound healing
and epithelial cancer metastasis.
References: Gaggioli C et al. 2007 Nature Cell Biology Epub ahead of print;
Ortiz-Urda et al. 2005 Science 307:1773-6 ; Wong et al. Br J Dermatol. 2007
156: 1149-55 ; Wessagowit V et al. Clin Exp Dermatol. 2004 29: 664-8.
(11) Analysis into the function and regulation of Kirrel in the paraxial mesoderm of
the chick embryo
Collaboration between Dr Kim Dale (University of Dundee, UK) and Dr Mike Jones
(Institute of Medical Biology, Immunos, Singapore )
Segmentation is a key feature of the body plan of all vertebrates, including humans, that
initiates very early in embryonic development. The first sign of segmentation is seen
when vertebrate embryos develop somites; which are the precursors of several
segmented organs such as the axial skeleton. Somites are formed from the
unsegmented presomitic mesoderm (PSM). During the formation of somites the most
mature PSM cells bud off as an epithelial sphere of cells to form the somite, with a strict
temporal periodicity.
Very exciting and critical molecular and embryological experimental data
obtained in the last ten years has shown that segmentation is governed by a molecular
oscillator called the segmentation clock that drives cyclic expression of genes in the
PSM with a periodicity that matches that of somite formation. All cyclic genes identified
to date encode either (a) components or modulators of the Notch pathway, (b)
components of the Wnt pathway or (c) components of the FGF pathway. In our lab we
study the molecular mechanism underlying this segmentation clock using the chick and
mouse animal models.
We have identified that Kirrel, a homolog of receptor proteins that organize
myoblast fusion in Drosophila melanogaster and which is necessary for muscle
precursor fusion in zebrafish is expressed at high levels in the chick PSM. We would like
to investigate the potential function of this gene in muscle development since this latter
role has clearly been conserved from fly to fish embryos. We would also like to
investigate the regulation of this gene in the PSM by addressing whether Notch, Wnt or
FGF signalling or a combination of these signalling pathways regulates Kirrel expression
and if this expression is in any way linked to the segmentation clock mechanism.
(12) Function of the proto-oncogene ect2 in cell motility and cell signalling
Supervisors: Dr. Arno Muller, College of Life Sciences, University of Dundee
Dr. Ed Manser, IMCB, A*STAR, Singapore
Background: Cancer is one of the most devastating diseases of our present time; for
example worldwide over 200,000 people die annually of pancreatic cancer. The
Epithelial Cell Transformation Sequence 2 (ECT2) oncogene is known as diagnostic
marker for cancers like pancreatic and lung cancer, esophageal carcinomas and
malignant gliomas (1, 2). Moreover Ect2 function has been correlated with invasive
behavior in metastatic glioblastomas (3). The molecular mechanisms how Ect2 promotes
invasiveness of tumors are not clear, but involve its activity as a guanine nucleotide
exchange factor for the Rho family of small GTPases. Research in vertebrates and in
Drosophila has demonstrated highly conserved mechanisms of Ect2 activity in the
process of cytokinesis. In addition, our research on fly embryos showed that the
homolog of Ect2 (Pebble (Pbl)) plays a role in epithelial-mesenchymal transition (EMT)
and FGF-triggered cell migration, processes that are relevant to transformation and
tumor progression (4).
Project: A structure-function analysis carried out in vivo has revealed that the conserved
C-terminal tail (CTT) of Pbl plays a role in determining the localization and substrate
preference of the protein (5). This function of the CTT can be brought about either by
conformational changes in response to posttranslational modification, and/or by binding
other regulatory proteins. One part of the project (to be performed in Dundee) will utilize
Drosophila genetics and biochemical methods to investigate potential binding partners of
the CTT in order to understand the function of this domain in regulating Pbl’s activity in
EMT and cell migration in gastrulation. A mutational analysis of conserved
phosphorylation sites within the CTT will be performed to test their functions in the
embryo.
To translate the findings in Drosophila to vertebrates, complementing genetic
approaches will be applied in zebrafish, Dani rerio (in Singapore). Using zebrafish one
can follow cell movements in the embryos during epiboly and gastrulation (0-8h post
fertilization). Injection of antisense morpholino oligonucleotide (MO) at the one-cell stage
allows highly effective knockdown of protein expression.
Human Ect2 mRNA
(unaffected by the zebrafish-targeted MO) will be used to rescue the phenotype, and
therefore address vertebrate Ect2 contributions to development and migration. One
would anticipate that mutants can be derived that segregate cell cycle from cell
migratory functions of Ect2. The mapping of phosphorylation sites in human Ect2 will
complement work done in Drosophila. The notion that Ect2 supports both RhoA and
Cdc42 activation is supported by the recent identification of a role for Ect2 in polar body
emission (6).
Conclusion: The genetic and biochemical dissection of ECT2 function in two different
model organisms will provide insights into the role of this oncogene in embryonic
development and advance our understanding of its transforming activity in cancer cells.
These studies might also help to define Ect2 as a potential target for anti-invasive
treatment of certain cancers.
References:
1.
2.
3.
4.
5.
6.
Hirata, D., Yamabuki, T., Miki, D., Ito, T., Tsuchiya, E., Fujita, M., Hosokawa, M.,
Chayama, K., Nakamura, Y. & Daigo, Y. (2009) Clin Cancer Res 15, 256-66.
Sano, M., Genkai, N., Yajima, N., Tsuchiya, N., Homma, J., Tanaka, R., Miki, T.
& Yamanaka, R. (2006) Oncol Rep 16, 1093-8.
Salhia, B., Tran, N. L., Chan, A., Wolf, A., Nakada, M., Rutka, F., Ennis, M.,
McDonough, W. S., Berens, M. E., Symons, M. & Rutka, J. T. (2008) Am J
Pathol 173, 1828-38.
Schumacher, S., Gryzik, T., Tannebaum, S. & Muller, H. A. (2004) Development
131, 2631-40.
van Impel, A., Schumacher, S., Draga, M., Herz, H. M., Grosshans, J. & Muller,
H. A. (2009) Development 136, 813-822.
Zhang, X., Ma, C., Miller, A. L., Katbi, H. A., Bement, W. M. & Liu, X. J. (2008)
Dev Cell 15, 386-400.
(13) Exploring and Exploiting conserved synthetic lethal interactions of DNA
double strand break repair factors.
Dr Anton Gartner, Wellcome Trust Centre for Regulation and Expression, University of
Dundee
Professor David Lane, A*STAR Singapore
DNA double strand breaks (DSBs) are amongst the most deleterious lesions a cell has
to deal with. Such breaks arise from genotoxic agents, but also happen during normal
DNA replication. Homologous Recombination (HR) provides an error free DNA repair
pathway to deal with DSBs. Late steps of recombinational repair are mediated by
redundant mechanisms.
Paradoxically cancer therapy largely depends on genotoxic agents, many of which kill
cancer cells by generating excessive levels of DSBs. However, these approaches also
affect normal cells, resulting into unwanted side effects and a rather small therapeutic
window. Redundancy within DNA repair pathways can be exploited to more selectively
kill cancer cells. The genome of cancer cells is inherently unstable, often resulting into
the loss of DNA repair genes. Due to the redundancy of repair pathways these cells are
viable. However, if a second redundant repair pathway is pharmacologically targeted
cancer cells can be selectively killed.
Key intermediates of recombinational repair are 4-way structures called Holliday
Junctions (HJs). HJ have been first proposed to occur in 1964. However, only very
recently two HJ resolving enzymes, GEN1 and the SLX4/SLX1 complex, have been
described in animals. We know next to nothing about how cells chose between HJ
resolving enzymes and how these enzymes are regulated. A biological function for
GEN1 has not been reported. Besides HJ resolution, HJ can also be untangled by a
process referred to as HJ dissolution, which requires the combined action of a
toposiomerase and the Blooms helicase.
We use the nematode worm C. elegans as a genetically tractable model system to better
understand DNA damage response pathways. Our recent analysis of C. elegans GEN-1
points towards the intriguing and unexpected possibility that the GEN-1 HJ resolvase
may be a dual function protein that co-ordinates HJ resolution with DNA damage
signaling leading to cell cycle arrest and apoptosis. We want to understand how GEN-1
works; to further define the GEN-1 DNA damage checkpoint pathway and to screen for
genes that act redundantly with gen-1 to mediate HJ resolution, and already found
several such candidates. Given that GEN-1 is deleted in some cancer cells we hope, in
the long-term, to contribute to cancer therapies based on synthetic lethal interactions we
expect to uncover.
As part of this studentship we, in the first two years, plan on undertaking C. elegans,
large scale, genome wide RNAi based synthetic lethal screens to uncover further
synthetic lethal interactions with gen-1 and other genes involved in late stage
recombinational repair. These synthetic lethal interactions will be validated by double
mutant interactions, and further characterized by C. elegans genetic and cytological
approaches. As part of the second part of the studentship, we wish to rapidly test
whether the same synthetic lethal interactions also occur in vertebrate systems. We
therefore aim at exploiting the Zebrafish model and inactivate fish GEN1 or GEN1 in
conjunction with candidate genes that cause synthetic lethality in the worm by
morpholino-based approaches. DNA damage response phenotypes, as well as the
sensitivity to DNA damaging agents, associated with single and double knockouts will be
studied in more detail. Strong synthetic lethal interactions will also be confirmed in
mammalian tissue culture models.
(14) Mathematical/Systems biology, Computational Biology/Bioinformatics
Vladimir Kuznetsov (Bioinformatics Institute, Singapore) and Mark Chaplain (Division
of Mathematics, University of Dundee)
(15) The role of Aurora A in the cell cycle and transformation
Ed Manser (Institute of Molecular & Cell Biology, Singapore) and Ron Hay (College of
Life Sciences, University of Dundee)
(16) Role of Cdc7 kinase in DNA replication and in the DNA damage response
Philipp Kaldis (Institute of Molecular & Cell Biology, Singapore) and Julian Blow
(Wellcome Trust Centre for Regulation and Expression, College of Life Sciences)