The School of Biological Sciences
Review and Prospectus 2002
Wellcome Trust Centre for
Cell-Matrix Research
contents
RESEARCH REVIEW
4-5
CELL-MATRIX ADHESION AND
INTRACELLULAR SIGNALLING
6-11
MATRIX ASSEMBLY AND
SUPRAMOLECULAR STRUCTURES
12-19
GENETIC CONTROL OF TISSUE
STRUCTURE AND FUNCTION
20-25
PRINCIPAL INVESTIGATOR PROFILES
26-27
FACILITIES
28
TRAINING OPPORTUNITIES
29
STAFF LIST
30
PUBLICATIONS
31-35
page 2
Wellcome Trust Centre
The Wellcome Trust Centre for Cell-Matrix
Research is an interdisciplinary research
centre embedded within the School of
Biological Sciences at the University of
Manchester. The Centre was established in
1995 under the chairmanship of Professor
Mike Grant with the long-term aims of
elucidating the structure and function of
extracellular matrices and cell-matrix
adhesions, defining the contribution of cellmatrix interactions to human diseases, and
developing approaches for preventing and
treating these diseases.
In late 2000, I assumed the position of
Director of the Centre as Professor Grant was
first made Dean of the School of Biological
Sciences, and then Pro-Vice-Chancellor for
Research of the University. I would like to
express my personal gratitude to Mike for the
momentous contributions that he has made to
cell-matrix research in general, and to the
matrix group in Manchester in particular, and
we all wish him well in his new roles.
Since the publication of our last Review &
Prospectus in 2000, we have said farewell to
Sarah Dallas who has moved to a post in the
Department of Oral Biology at the University of
Missouri at Kansas City, and in 2002 we will
see the departure of John Sheehan to the
Department of Biochemistry at the University
of North Carolina, to take up the position of
John Hooker Professor of Biophysics and
Biochemistry. In the fifteen years that John
has worked in Manchester, he has pioneered
the biochemical and biophysical analyses of
mucins, as well as being instrumental in
establishing our outstanding facilities for
purification and analysis of biomolecules. We
will greatly miss his enthusiasm and creativity.
By contrast, we welcome Dr. Keith Brennan to
a Wellcome Trust Research Career
Development Fellowship in the Centre. Keith
joins us from the Strang Cancer Research
Laboratory at Rockefeller University, New York
to work on Notch and Wnt signalling in
mammary gland development. We also
welcome Dr. Clair Baldock to a prestigious
Royal Society Olga Kennard Research
for Cell-Matrix Research
Fellowship. Clair joined the Centre a few
years ago as a postdoctoral researcher and
will now initiate an independent career in the
area of elastic microfibril structure. Further
information on Keith and Clair’s work can be
obtained from two Research in Focus articles
within this brochure.
The first twelve months of my period as
Director have turned out to be most eventful.
As detailed in this brochure, group leaders
within the Centre have continued to make
important discoveries. These advances
include contributions to the structural biology
of extracellular matrix molecules and adhesion
receptors, the biosynthesis and assembly of
extracellular matrix polymers, and the
elucidation of genotype-phenotype links in
diseases caused by mutations in extracellular
matrix genes. Advances have been made
across the entire research portfolio of the
Centre, and have been published in the
highest quality journals. In addition to
research output, which is of course of
paramount importance, I want to highlight
three other more general achievements that
augur well for the future prospects of the
Centre.
Firstly, in the summer of 2000, we learned that
an application for re-validation of our status as
a Wellcome Centre and for renewal of our
core grant had been successful. The core
grant will run for five years and provide funds
for a number of new support posts as well as
a substantial tranche of new equipment. This
improvement to our infrastructure will greatly
facilitate our research and we are extremely
grateful for the Trust’s support.
Secondly, in 2000, the School of Biological
Sciences secured a £15M grant from the
Wellcome Trust and the UK Government for
the construction of an Integrative Centre for
Molecular Cell Biology. This Joint
Infrastructure Fund award has since been
combined with further funds from the
University to create a large complex for
biological and medical research. The new
building will be constructed at a focal point in
the University’s biomedical corridor, adjacent
to physical sciences research, organismal
biology research, the pioneering Manchester
Bioscience Incubator, the Wellcome Trust
Clinical Research Facility, and the vast Central
Manchester NHS Trust. The 20,000 m2
development will be completed by the end of
2003 and will provide a new home for our
Centre in outstanding accommodation.
Thirdly, in the last few weeks, we have learned
that the School of Biological Sciences has
received two top grades of 5* in the Higher
Education Funding Council for England’s 2001
Research Assessment Exercise. These
ratings make the School the largest, top-rated
Biology department in the UK and will
determine the level of Government research
funding to the School for the next five years.
We anticipate this success enabling the
School to continue to improve its laboratory
provision and to recruit outstanding scientists.
So, as we enter the second five year period of
the Centre’s existence, the future could hardly
be brighter. Advances in basic and clinical
science make this an exciting era for
biomedical research and we have the real
prospect of translating our work into practical
benefits. I hope you enjoy reading about our
recent progress and future plans in this
Review & Prospectus.
Martin Humphries
page 3
Research Review
The scientific plans of the Centre can be viewed as three
integrated and collaborative programmes focused at three
levels of biological organisation - the cell, the tissue and the
organism. In brief, the cellular programme focuses on cellextracellular matrix adhesion and intracellular signalling,
and aims to define the molecular principles underlying the
responses of cells to their environment; the tissue programme
studies matrix assembly and supramolecular structures
with the aim of engineering tissues; and the organism
programme aims to elucidate mechanisms of genetic control
Cell-matrix adhesion and intracellular
signalling. The overarching aim of this
programme is to decipher the molecular
improved diagnosis and treatment. Whilst each programme is
mechanisms whereby adhesive cues on the
highly focused on extracellular matrices and cell-matrix
outside of cells are transduced into
interactions, the long-term promise of the work overlaps with
functional responses on the inside (and vice
versa) and to develop approaches for
some of the most important areas of biomedical research intervening
experimentally
and
signalling, tissue engineering and medical genetics.
therapeutically in these processes. The
programme combines strengths in structural
biology, biochemistry, molecular biology, bioinformatics and cell biology, with an ability to dissect signalling
pathways in vivo in genetically tractable model organisms such as the mouse, Caenorhabditis, and
Drosophila. In the long-term, an understanding of the molecular mechanisms and biological functions of
adhesion will provide insights into tissue formation and homeostasis, and will suggest approaches to
control many of the most common human diseases, including inflammatory, neoplastic, traumatic and
infectious conditions.
of tissue structure and function and uses this information for
Matrix assembly and supramolecular structure. An organised extracellular matrix is essential for the
function of all tissue and organs. This organisation depends on the cellular control of the co-ordinated
expression of a range of matrix macromolecules and extracellular assembly into higher order structures.
In this programme, multidisciplinary approaches are being employed to understand the pathway of
assembly of extracellular matrices from their initial biosynthesis to the final mature form. The functions of
different classes of matrix components are being dissected. New approaches to derive structural models
for higher order macromolecular assemblies, using electron microscopy/tomography, are being combined
with biophysical analyses, protein crystallography and mass spectrometry to define complex structures.
These strategies will provide a better understanding of extracellular matrix assembly and the molecular
basis of the changes associated with skeletal and tissue pathologies.
Genetic control of tissue structure and function. In this programme, a variety of approaches including
model vertebrate and invertebrate systems, cell culture systems and human genome scans are being
employed to gain new insights into the role of the extracellular matrix in determining tissue structure and
function.
Developmental systems under investigation include skeletogenesis, myogenesis,
vasculogenesis and tissue morphogenesis. Particular progress is being made, firstly, through elucidating
the molecular pathogenesis of major diseases affecting these organ systems, including osteoarthritis,
chondrodysplasias and cardiovascular disease, and secondly, by developing new models of diseases
involving extracellular matrices, such as muscular dystrophies. Understanding the links between the
individual molecules comprising the extracellular matrix with tissue structure and pathology, is the central
goal of this research and offers the prospect of developing novel approaches for treatment or prevention
of several of the most common chronic diseases of mankind.
page 4
Key Research Discoveries
Key Research Discoveries
C.Baldock, K.E.Kadler, C.A.Shuttleworth and
C.M.Kielty
The supramolecular organization of fibrillin-rich
microfibrils.
J. Cell Biol. 2001; 152:1045-1056.
M.Baron
Drosophila dumpy is a gigantic extracellular
protein required to maintain tension at epidermalcuticle attachment sites.
Curr. Biol. 2000; 10:559-567.
J.Bella
Staggered molecular packing in crystals of a
collagen-like peptide with a single charged pair.
J. Mol. Biol. 2001; 301:1191-1205.
P.N.Bishop M.D. Briggs and J.K.Sheehan
Identification in vitreous and molecular cloning of
opticin, a novel member of the family of leucinerich repeat proteins of the extracellular matrix.
J. Biol. Chem. 2000; 275:2123-2129.
R.P.Boot-Handford
Characterization of Hydra type IV collagen. Type
IV collagen is essential for head regeneration
and its expression is up-regulated upon exposure
to glucose.
J. Biol. Chem. 2000; 275:39589-39599.
M.D.Briggs and M.E.Grant
Mutations in the region encoding the von
Willebrand factor A domain of matrilin-3 are
associated with multiple epiphyseal dysplasia.
Nature Genet. 2001; 28:393-396.
N.J.Bulleid
Hsp47: a molecular chaperone that interacts with
and stabilizes correctly-folded procollagen.
EMBO J. 2000; 19:2204-2211.
A.E.Canfield
1 α,25-dihydroxyvitamin D(3) inhibits
angiogenesis in vitro and in vivo.
Circ. Res. 2000; 87:214-220.
A.P.Gilmore and C.H.Streuli
Integrin-mediated survival signals regulate the
apoptotic function of Bax through its
conformation and subcellular localization.
J. Cell Biol. 2000; 149:431-446.
M.J.Humphries
Generation of a minimal α5β1 integrin-Fc
fragment.
J. Biol. Chem. 2001; 276:35854-35866.
K.E.Kadler and C.Baldock
Corneal collagen fibril structure in three
dimensions: Structural insights into fibril
assembly, mechanical properties and tissue
organization.
Proc. Natl. Acad. Sci. U. S. A. 2001; 98:73077312.
R.A.Kammerer
The laminin-binding domain of agrin is
structurally related to N-TIMP-1.
Nature Struct. Biol. 2001; 8:705-709.
U.Mayer
Impaired axonal regeneration in α7 integrindeficient mice.
J. Neurosci. 2000; 20:1822-1830.
J.K.Sheehan
Identification of a non-mucin glycoprotein (gp340) from a purified respiratory mucin
preparation: evidence for an association involving
the MUC5B mucin.
Glycobiology. 2001; 11:969-977.
C.A.Shuttleworth, C.M.Kielty and C.Baldock
The role of the C1 and C2 A-domains in type VI
collagen assembly.
J. Biol. Chem. 2001; 276:7422-7430.
C.H.Streuli
Desmosomal adhesion regulates epithelial
morphogenesis and cell positioning. Nature Cell
Biol. 2001; 3:823-830.
D.S.Tuckwell and M.J.Humphries
Monoclonal antibodies identify residues 199-216
of the integrin α2 vWFA domain as a functionally
important region within α2β1.
Biochem. J. 2000; 350:85-493.
G.A.Wallis and M.E.Grant
Coordinated expression of matrix Gla protein is
required during endochondral ossification for
chondrocyte survival.
J. Cell Biol. 2001; 154:659-666.
T.E.Hardingham
The analysis of intermolecular interactions in
concentrated hyaluronan solutions suggest no
evidence for chain-chain association.
Biochem. J. 2000; 350:329-335.
page 5
Cell-matrix adhesion and
intracellular signalling
MARTIN J. HUMPHRIES
BSc PhD
Wellcome Trust Principal
Research Fellow and Professor of
Biochemistry
Key Publications:
Mould,A.P., J.A.Askari and M.J.Humphries.
2000. Molecular basis of ligand recognition by
integrin α5β1. I. Specificity of ligand binding
is determined by amino acid sequences in the
second and third NH2-terminal repeats of the
α subunit. J. Biol. Chem. 275:2032420336.
Humphries,J.D., J.A.Askari, X.P.Zhang,
Y.Takada, M.J.Humphries and A.P.Mould.
2000. Molecular basis of ligand recognition by
integrin α5β1. II. Specificity of Arg-Gly-Asp
binding is determined by Trp157 of the α
subunit. J. Biol. Chem. 275:20337-20345.
Coe,A.P., J.A.Askari,A.D.Kline, M.K.Robinson,
H.Kirby, P.E.Stephens and M.J.Humphries.
2001. Generation of a minimal α5β1
integrin-Fc fragment. J. Biol. Chem.
276:35854-35866.
page 6
Molecular basis of integrin-dependent
cell adhesion
The interactions of integrin adhesion
receptors with their extracellular matrix
ligands are important for many aspects of cell
function. Most notably, they organise
signalling complexes to modulate
differentiation and cell fate, provide physical
support for cells in order to maintain
cohesion, and permit the generation of
traction forces to enable movement. Animal
model studies have also shown integrins to
contribute to the progression of many
common diseases, and have implicated them
as potential therapeutic targets in
cardiovascular and inflammatory diseases.
This laboratory is employing a combination of
biochemical, molecular biological, biophysical
and cell biological techniques to understand
the molecular basis of integrin function. The
aims of this research are to elucidate the
mode of interaction between integrins and
their ligands and effectors, usually at the
atomic level. The work involves narrowing
down sites within integrin ligands that serve
as receptor contact sites, determining the
ligand-binding pocket within integrins, and
complementing this information through
tertiary structure determination by X-ray
crystallography. Such studies have relevance
for the design of clinically useful anti-adhesive
agents, and close links with industry are
facilitating such developments. Recently, the
regulation of integrin activity has become a
major emphasis of the research. Integrin
activation is accomplished through alterations
in receptor shape that are being probed using
divalent cations, conformation-dependent
monoclonal antibodies, and site-directed
mutagenesis. These studies are now
translating into investigations of the molecular
basis of transmembrane signal transduction
by integrins, in particular integrin-mediated
organisation of the cytoskeleton through
agonist-dependent association of cytoskeletal
proteins. It is hoped that this work will
provide insights into the mechanisms by
which cells sense and respond to their
extracellular environment.
Co-workers:
Janet Askari BSc
Wellcome Trust Technician
Stephanie Barton BSc
Wellcome Trust Technician
Mark Bass BSc MRes PhD
Wellcome Trust Research Associate
Tanja Benkert Dipl Biol
Aventis Research Assistant
Patrick Buckley BSc PhD
Wellcome Trust Research Associate
Sue Craig BSc
Wellcome Trust Technician
Jon Humphries BSc MPhil
Wellcome Trust Research Assistant
Lynn McKeown BSc
BBSRC Student
Anthea Messent BSc PhD
Wellcome Trust Research Associate
Zohreh Mostafavi-Pour BSc MSc PhD
Wellcome Trust Research Associate
Paul Mould BSc PhD
Wellcome Trust Research Associate
Eileen Pinnington
Wellcome Trust Technician
Adam Shaw BSc
BBSRC Student
Stephen St. George Smith BSc PhD
BBSRC Research Associate
Emlyn Symonds BSc
MRC Student
Mark Travis BSc
BBSRC Student
Dimitra Valdramidou BSc MRes
Aventis Research Assistant
Mark Watson BSc
BBSRC Student
JORDI BELLA BSc PhD
Lecturer in Biochemistry
Key publications:
Kramer,R.Z., M.G.Venugopal, J.Bella, P.Mayville,
B.Brodsky, and H.M.Berman. 2000. Staggered
molecular packing in crystals of a collagen-like
peptide with a single charged pair. J. Mol. Biol.
301:1191-1205.
Kramer,R.Z., J.Bella, P.Mayville, B.Brodsky, and
H.M.Berman. 1999. Sequence dependent
conformational variations of collagen triplehelical structure. Nat. Struct. Biol. 6:454457.
Xiao,C., C.M.Bator,V.D.Bowman, E.Rieder,Y.He,
B.Hebert, J.Bella,T.S.Baker, E.Wimmer, R.J.Kuhn,
and M.G.Rossmann. 2001. Interaction of
coxsackievirus A21 with its cellular receptor,
ICAM-1. J.Virol. 75:2444-2451.
Co-workers:
Paul McEwan BSc
BBSRC Student
Structural biology of cell-matrix proteins
Our current knowledge of the three-dimensional organisation of
the extracellular matrix and its interaction with cell-surface
molecules is still rudimentary. My group is particularly interested
in the structure and function of collagens, as quintessential
components of all types of extracellular matrices. Collagens are a
family of proteins characterised by containing at least one domain
region with a very specific conformation, the collagen triple helix,
and also by forming higher order macromolecular assemblies.
Our understanding of the molecular subtleties of the collagen
triple helix has greatly improved thanks to the crystallographic
determination of model systems at high resolution. However, key
questions still remain. For example, we aim to elucidate the basis for molecular recognition of
collagen, as the triple helix is used as a binding motif for several extracellular macromolecules.
We will combine crystallographic studies on small versions of collagen with electron microscopy
reconstructions of entire collagen assemblies. We are also studying the co-assembly of
collagens with proteins that may control the dimensions or organisation of fibre structures. Major
efforts are directed at complexes found in cornea and vitreous, and at the molecules mediating
mineralisation. Finally, we are studying fragments of fibronectin, a molecule that is responsible
for cell adhesion and migration through its interaction with cell-surface integrins. Solving the
structures of extracellular matrix proteins, and determining the molecular basis of their
interaction with other molecules, will contribute to an understanding of their function both in
normal and diseased states.
ANDREW P GILMORE BA
PhD
Wellcome Trust Research
Career Development Fellow
Key publications:
Streuli,C.H. and A.P.Gilmore. 1999.The role of
adhesion in regulating mammary epithelial cell
survival. J. Mammary Gland Biol.
Neoplasia. 4:183-191.
Gilmore,A.P.,A.D.Metcalfe, L.H.Romer and
C.H.Streuli. 2000. Integrin mediated survival
signals regulate the apoptotic function of Bax
through conformation and subcellular
localization. J. Cell Biol. 149:431-445.
Hadjiloucas,I.,A.P.Gilmore, N.J.Bundred and
C.H.Streuli. 2001.Assessment of apoptosis in
human breast tissue using an antibody against the
active form of caspase 3: relation to
histopathological characteristics. Brit. J.
Cancer. 85:1522-1526.
Co-workers:
Anthony Valentijn BSc
Wellcome Trust Research Assistant.
Nadia Zouq BSc MRes
MRC Student
Regulation of cell survival
by cell-extracellular
matrix adhesion
Our primary interest lies in extracellular matrix-dependent suppression of apoptosis, which we are
using as a model for understanding how adhesion-mediated signalling events at the membrane
are transmitted to other cellular compartments. Apoptosis, or programmed cell death, is a default
pathway by which damaged or displaced cells are destroyed whilst avoiding a damaging
inflammatory response, and constant survival signals from the environment are required for cells
to suppress its activation. The plethora of survival signals are integrated through the Bcl-2 family
of apoptosis regulators, proteins which function as a gateway between a cells decision to live or
die. We have found that cell-matrix adhesion activates signalling pathways that suppress the
activity of Bax, an apoptosis-promoting member of the Bcl-2 family. Bax exerts its death-promoting
activity at the outer membrane of mitochondria, but in adherent cells adhesion-initiated signals
keep Bax in the cytosol. Upon inhibition of adhesion, Bax rapidly translocates to mitochondria
where it initiates apoptosis.
Our research is currently pursuing two complementary aims. First, we aim to identify the integrinproximal signalling pathways that suppress Bax activity in adherent cells, focussing on tyrosine
kinases activated upon cell adhesion. In addition, the use of GFP-fusions is allowing us to follow
the movement of Bax in the cytosol of live cells following manipulation of signalling pathways.
Second, we are examining post-translational modifications of Bax that promote its association with
mitochondria in cells where the normal adhesion-mediated signalling has been blocked. A variety
of approaches are being employed, including high-resolution gel filtration and mass spectrometry.
page 7
CHARLES H STREULI MA PhD
Wellcome Trust Senior Research
Fellow
Key publications:
Gilmore,A.P.,A.D.Metcalfe, L.H.Romer and
C.H.Streuli. 2000. Integrin mediated survival
signals regulate the apoptotic function of Bax
through conformation and subcellular
localization. J. Cell Biol. 149:431-445.
Klinowska,T.C., C.M.Alexander, E.GeorgesLabouesse, R.Van der Neut, J.A.Kreidberg,
C.J.Jones,A.Sonnenberg and C.H.Streuli.
2001. Epithelial development and
differentiation in the mammary gland is not
dependent on α3 or α6 integrin subunits.
Dev. Biol. 233:449-467.
Runswick,S.K., M.J.O’Hare, L.Jones,
C.H.Streuli and D.R.Garrod. 2001.
Desmosomal adhesion regulates epithelial
morphogenesis and cell positioning. Nature
Cell Biol. 3:823-830.
Co-workers:
Nasreen Akhtar BSc PhD
Wellcome Trust Research
Associate
Kirsty Green BSc
BBSRC Student
Emma Lowe BSc
HEFCE-funded Technician
Emma Marshman BSc PhD
BBSRC Resarch Associate
QingQiu Pu BS MB PhD
AstraZeneca Research Associate
Pengbo Wang BSc MSc PhD
MRC Research Associate
Harriet Watkin BSc
Wellcome Trust Prize Student
page 8
Adhesion-dependent signalling pathways that
regulate differentiation and apoptosis
The goal of our research is to understand
how the extracellular matrix regulates
epithelial cell phenotype. Matrix proteins
interact with cell surface integrin receptors to
modulate cellular architecture and intracellular
signal transduction pathways. Adhesion to
basement membranes has a central role in
controlling the differentiation, apoptosis, and
morphogenesis of mouse mammary gland
and human breast epithelium. We are
dissecting the signalling pathways that control
these aspects of epithelial cell behaviour.
Mammary epithelial cells retain their ability to
express differentiation products in culture, but
this is dependent on signal transduction
pathways that are triggered by lactogenic
hormones and extracellular matrix proteins.
We have discovered that the signalling
pathways triggered by integrins and prolactin
and insulin receptors converge at the level of
protein tyrosine kinases and protein tyrosine
phosphatases. A current aim is to identify
integrin-regulated signalling proteins that
modulate the pathways driven by lactogenic
hormones, thereby controlling mammary
differentiation.
Breast epithelia undergo rapid and dramatic
apoptosis after periods of lactation. We are
dissecting the mechanisms that regulate
apoptosis, with a particular focus on integrin
and growth factor control of Bcl-2 family
proteins. These proteins are associated with
mitochondrial membranes, and we are
investigating how two family members, Bax
and BAD, control apoptotic decisions. We
have recently found that BAD phosphorylation
is regulated at the lactation-involution switch
in vivo, that IGF-BP5 blocks IGF signalling
and is a potent inducer of apoptosis, and that
an essential adaptor protein linking IGF
receptor activation with intracellular signals,
insulin receptor substrate-1, disappears at
this time through the activation of a specific
caspase. Moreover, the ligand-binding
conformation of the β1 integrin is altered to a
non-binding state at the onset of apoptosis in
vivo.
The morphogenesis of mammary gland
involves the formation of collecting ducts and
lactational alveoli, both of which are bilayered
epithelia. We are investigating how adhesion
regulates these processes, and have shown
that mammary gland development in vivo is
dependent on β1 integrin function. In
addition, we have identified new functions for
desmosomal cell-cell adhesion molecules in
the morphoregulation of alveolus formation.
We have also discovered that these
molecules provide essential cues to regulate
spatial positioning of the epithelial cell types
in these bilayered structures.
DANNY S.TUCKWELL BSc PhD
Modular proteins and the extracellular matrix
BBSRC Advanced Research Fellow
Key publications:
Knight,C.G., L.F.Morton,A.R.Peachey,
D.S.Tuckwell, R.W.Farndale and M.J.Barnes.
2000.The collagen-binding A-domains of
integrins α1β1 and α2β1 recognize the same
specific amino acid sequence, GFOGER, in
native (triple-helical) collagens. J. Biol.
Chem. 275:35-40.
Tuckwell, D. 2002. Identification and analysis
of collagen α1(XXI), a novel member of the
FACIT collagen family. Matrix Biology. 21:
63-66.
Aquilina,A., M.Korda, J.M.Bergelson,
M.J.Humphries, R.Farndale and D.S.Tuckwell.
2002.A novel gain of function mutation in the
integrin α2 VWFA domain. European
Journal of Biochem. in press
Co-workers:
Claire Johnston BSc MSc
BBSRC Student
The protein components of extracellular
matrices are typically modular in structure, and
investigations of the properties of these
modules can provide insights into the functions
of the proteins that contain them. Much of the
research in this laboratory focuses on the 200
amino acid von Willebrand factor A domain
module, or VWFA domain, which is found in a
range of proteins, including integrins, collagens
and blood coagulation proteins. The
overarching aim of this work is to determine
relationships between structure, function and
evolution within the domain family. Three major
approaches are being taken:
1. Integrins: Many cell types interact with
collagens to regulate normal tissue
organisation and to mediate more dynamic
processes such as blood coagulation and
tumour metastasis. These interactions are
mediated by members of the integrin family of
receptors. We have showed that it is the
VWFA domain within the integrin that binds to
collagens, and recent work has characterised
the molecular basis of this interaction.
Currently, we are studying the interaction of the
α2 VWFA domain with non-collagenous ligands
and naturally occurring inhibitors, with a view to
developing novel therapeutic agents.
2. Caenorhabditis elegans: Bioinformatics
analysis shows that there are 33 VWFA
domain-containing proteins in the nematode C.
elegans. We have used an integrative
approach to study the novel VWFA domain
protein DDA-1 and shown that it is required for
the correct formation of the cuticle. We now
intend to use genetic and biochemical
approaches to identify the proteins interacting
with DDA-1, thereby linking matrix genes with
specific functions within the organism.
3. Bioinformatics and phylogeny: Much of our
work employs bioinformatics approaches to
complement and extend lab-based studies,
including structure prediction, homology
modelling and phylogenetic analysis. We
recently identified a novel human collagen
[collagen α1(XXI)] using bioinformatics tools,
and showed that it diverged early in vertebrate
collagen evolution. Over the next few years,
bioinformatics methodologies will be used to
elucidate the functional evolution of other
modules, including vertebrate collagen Cpropeptides and C. elegans cuticle ZP
domains.
In conclusion, our studies have resulted in the
detailed characterisation of known interactions
and the discovery of novel functions. VWFA
domains will continue to be a rewarding field for
research; moreover, the strategies outlined
above will also facilitate the exploration of other
domain families.
page 9
MARTIN BARON BSc PhD
Lecturer in Biochemistry
Key publications:
Fostier,M., D.Evans, S.Artavanis-Tsakonas and
M.Baron. 1998. Genetic characterisation of the
Drosophila melanogaster Suppressor of deltex:A
regulator of Notch signalling. Genetics
150:1477-1485.
Cornell,M., D.Evans, R.D.Mann, M.Fostier,
M.Flasza, M.Monthatong, S.Artavanis-Tsakonas
and M.Baron. 1999 The Suppressor of deltex gene
a regulator of the Notch receptor signalling
pathway is an E3 class ubiquitin ligase. Genetics
152:567-576.
Baron,M.,V.O’Leary, D.A.P.Evans, M.Hicks and
K.Hudson. 2000. Multiple Roles of the Dcdc42
GTPase during wing development in Drosophila
melanogaster. Molecular and General
Genetics 264:98-104.
Regulation of Notch receptor signalling
The control of cell differentiation during development requires communication between cells
via diffusible growth factors and cell-cell adhesion mediated signalling molecules. The Notch
receptor is an example of the latter class and regulates the timing and outcome of cell
differentiation decisions in many different tissues
during development. The Notch signal must be
precisely regulated to prevent inappropriate signalling.
Notch was first identified in Drosophila but has
subsequently been identified in a range of vertebrate
species, including four versions of the gene in
humans. The activity of Notch in vivo is precisely
controlled spatially and temporally. Using the
Drosophila model system, our group has identified a
negative regulator of Notch called Suppressor of
Deltex [Su(dx)], which belongs to an E3 class of
ubiquitin ligase molecules that regulate endocytosis
and proteolytic degradation of target molecules. We
are currently investigating the mechanism of Notch
pathway regulation by Su(dx) using a combination of
genetic, biochemical and cell biological approaches.
Co-workers:
Ann-Marie Carbery BSc MSc, BBSRC Student
Maggy Fostier BSc PhD, Wellcome Trust Research Associate
Jenny Higgs BSc, BBSRC Student
Sabine Mazaleyrat BSc, BBSRC Student
Ngoc-sa Nguyen Huu BSc MSc, MRC Research Assistant
Marian Wilkin BSc PhD, MRC Research Associate
KEITH R. BRENNAN BA PhD
Wellcome Trust Research
Career Development Fellow
Key publications:
Brennan,K., M.Baylies and A.Martinez Arias.
1999.A repressive function of Notch required
prior to Wingless signalling during muscle
progenitor cell development in Drosophila.
Current Biology. 9:707-710.
Brennan,K.,T.Klein, E.Wilder and A.Martinez
Arias 1999. Wingless modulates the effects of
dominant negative Notch molecules in the
developing wing of Drosophila. Implications for
Notch/Wingless signalling. Developmental
Biology. 216:210-229.
Lawrence,N.,T.Langdon, K.Brennan and
A.Martinez Arias. 2001. Notch signalling targets
the Wingless response element of a Ubx enhancer
in Drosophila. Current Biology. 11:375-385.
page 10
Interactions between signalling pathways
during mammalian development
The interactions between signalling pathways are crucial for their regulation during embryonic
development and the maintenance of adult tissues, and disruption of these feedback mechanisms
can lead to developmental defects and disease. The Notch and Wnt pathways are two of the most
important developmental signalling pathways and are required for multiple aspects of mammalian
development. Frequently, signalling through both pathways is required for the formation of an organ
although they typically have opposing effects on cell fate decisions. Through detailed genetic
analysis in Drosophila, and recently in mammals, we have highlighted a novel role of Notch in the
repression of Wnt target genes prior to the receipt of a Wnt signal. This function does not require
known elements of the Notch signalling pathway, although
recent experiments suggest that the cytoplasmic protein Deltex
is required. Current experiments seek to elucidate the
molecular mechanism that underlies this regulatory interaction.
Continuation of the cell culture experiments, using established
Wnt and Notch signalling assays and cell lines whose
differentiation is regulated by both pathways, will identify the
point(s) of regulation and the proteins involved. In parallel,
interactions between the pathways will be examined in vivo
within the developing mammary gland where signalling through
both pathways is required and intersects. Transgenic mice and
conditional knockouts will be used to manipulate Wnt and
Notch signalling. Careful examination of the resulting
mammary gland phenotypes will reveal whether signalling
through one pathway can regulate the other.
Research in Focus
Notch signal regulation and cell proliferation
In defining a body plan and regulating cellular
responses to positional cues, organisms do
not use a different signal for each individual
cell-fate decision, but repeatedly use a
relatively small set of cellular signalling
pathways. The Notch receptor and its
membrane bound ligands, Delta and Serrate,
were first identified in Drosophila and are vital
components of one such developmentally
important signalling pathway. Four
homologues of Notch are found in humans
and aberrant Notch signalling levels have
been linked to diverse human diseases
including neural degenerative diseases,
developmental disorders and cancer, making
this pathway an important target for
therapeutic interventions.
Notch is best known for its role in lateral inhibition
signalling where a small number of cells are selected
to adopt a particular fate from a larger group of
precursor cells. This lateral inhibition signalling
depends on a ligand-dependent cleavage of Notch,
which allows its intracellular domain to activate a
transcriptional regulator, Suppressor of Hairless
[Su(H)], in the nucleus. However, recent work has
indicated that there is a second intracellular signalling
pathway, which requires the Notch interacting protein
Deltex, acting downstream of the receptor.
The regulation of Notch signalling occurs at
many levels, including cross-talk with other
signalling pathways, covalent modifications
including glycosylation, phosphorylation and
ubiquitination, and intracellular trafficking of
the receptor and ligands. Our aim is to
unravel these intersecting mechanisms of
regulation.
One mechanism of regulation under
investigation is the role played by ubiquitin
ligase proteins which covalently modify target
proteins and regulate their intracellular
trafficking and turnover. Several Notch
regulatory proteins have been identified
which have either been shown to act as
ubiquitin ligases or contain ubiquitin ligaserelated domains. In our laboratory, a
negative regulator of Notch signalling,
Suppressor of deltex [Su(dx)], has been
identified that belongs to the E3 class of
ubiquitin ligases. Like Deltex, this protein
associates with the Notch intracellular
domain. However, it is currently unclear how
Su(dx) regulates Notch signalling and
whether regulation occurs through Su(dx)dependent ubiquitination of Notch. In
addition, genetic experiments indicate that
Su(dx) and Deltex may interact to regulate
certain aspects of Notch signalling.
Understanding the mechanism of action of
these proteins and their relationship to the
two intracellular signalling pathways
downstream of Notch are important goals of
our research.
A second important avenue of research is the
investigation of the cross-talk between the
Notch signalling pathways and another
developmentally important signal, the
Wingless (Wnt-1 in vertebrates) pathway.
Our genetic experiments indicate that Notch
signalling through the Deltex-dependent
pathway suppresses Wingless target genes
prior to the receipt of a Wingless signal in
Drosophila. In addition, our results suggest
that an important step in Wingless signalling
is to attenuate Notch signalling via Deltex.
Finally, preliminary experiments indicate that
regulatory interactions occur between the
Notch and Wnt pathways in mammals.
Understanding the molecular basis for this
regulatory interaction will give insights into
how these two pathways collaborate to
control developmental patterning.
In summary, our research has highlighted two
distinct mechanisms for regulating Notch
signalling. By using a wide variety of genetic,
cell biological and biochemical methods we
are currently unravelling the molecular
mechanisms behind these regulatory
interactions. Finally we are translating our
results from the model organism, Drosophila,
to human disease.
Martin Baron and Keith Brennan
page 11
Matr ix Assembly and
Supramolecular Structures
KARL E. KADLER BSc PhD
Professor of Biochemistry
Key publications:
Graham,H.K., D.F.Holmes, R.B.Watson and
K.E.Kadler. 2000. Identification of collagen
fibril fusion during vertebrate tendon
morphogenesis.The process relies on unipolar
fibrils and is regulated by collagenproteoglycan interaction. J. Mol. Biol.
295:891-902.
Garrigue-Antar,L., C.Barker and K.E.Kadler.
2001. Identification of amino acid residues in
bone morphogenetic protein-1 important for
procollagen C-proteinase activity. J. Biol.
Chem. 276:26237-26242.
Holmes,D.F., C.J.Gilpin, C.Baldock, U.Ziese,
A.J.Koster and K.E.Kadler. 2001. Corneal
collagen fibril structure in three dimensions:
Structural insights into fibril assembly,
mechanical properties and tissue organization.
Proc. Natl.Acad. Sci. U. S.A 98:73077312.
The molecular and cellular basis of
collagen fibrillogenesis
My laboratory aims to determine the
molecular and cellular basis of collagen
fibrillogenesis during tissue assembly. Tissue
assembly in animals requires the formation of
an extracellular matrix containing millimetrelong collagen fibrils arranged in elaborate
three-dimensional architectures such as
parallel bundles (in tendons and ligaments),
basket weaves (in skin and bone) and
orthogonal lattices (in cornea). We are taking
a multidisciplinary approach to studying
collagen fibrillogenesis, by using a
combination of electron microscopy (to
determine the three-dimensional structure of
individual collagen fibrils), site-directed
mutagenesis and recombinant protein
expression (to understand how collagen is
synthesised), and immunolocalisation
techniques (to study protein trafficking leading
to collagen fibrillogenesis). Our work has
direct relevance to protein trafficking of large
and multimeric proteins, and in understanding
the aetiology of some of the most debilitating
diseases of man and animals, such as
fibrosis and arthritis, that are characterised by
ectopic and unusual synthesis of collagen
fibrils.
Analysis of the synthesis of large multimeric
polymers, that are orders of magnitude longer
than the cells that produce them, is difficult
because they are refractory to conventional
approaches. We have used transmission
electron microscopy, which has led to the
discovery that the fibrils are synthesised as
two types of ‘collagen early fibrils’ that have
the ability to fuse (end-to-end and end-toside) to generate long fibrils that, in turn,
anastomose and form branched networks in
developing tissues. Recently, we have used
page 12
automated electron tomography and 3D
reconstruction to obtain the first threedimensional structure of individual collagen
fibrils. A new focus of interest is to understand
how the trafficking of key proteins involved in
fibrillogenesis, including procollagen and
bone morphogenetic protein-1, influences
collagen fibril assembly, as well as the
structure and function of the extracellular
matrix.
Co-workers:
Adetola Adesida BSc PhD
Wellcome Trust/Catalyst Biomedica Ltd
Research Associate
Elizabeth Canty BSc PhD
Wellcome Trust Research Associate
Laure Garrigue-Antar BSc PhD
Wellcome Trust Research Associate
Hanane Gouizi BSc
Algerian Government Student
Nicola Hartigan BSc
Wellcome Trust Prize Student
David Holmes BSc PhD
Wellcome Trust Research Associate
Matthew Leighton BSc PhD
Wellcome Trust Research Associate
Roger Meadows BSc MSc
Wellcome Trust technician
Vasiliki Petropolou BSc MSc
University of Manchester Student
Susan Richardson BSc PhD
Wellcome Trust/Catalyst BioMedica Ltd
Research Associate
Tobias Starborg BSc PhD
BBSRC/MRC/EPSRC Research Associate
NEIL J. BULLEID BSc PhD
Professor of Biochemistry
Key publications:
Tasab,M., M.R.Batten and N.J.Bulleid. 2000.
Hsp47: a molecular chaperone that interacts with
and stabilizes correctly-folded procollagen.
EMBO J. 19:2204-2211.
Wilson,C.M., M.R.Farmery and N.J.Bulleid.
2000. Pivotal role of calnexin and mannose
trimming in regulating the endoplasmic
reticulum-associated degradation of major
histocompatibility complex class I heavy chain. J.
Biol. Chem. 275:21224-21232.
Bottomley,M.J., M.R.Batten, R.A.Lumb and
N.J.Bulleid. 2001. Quality control in the
endoplasmic reticulum. PDI mediates the ER
retention of unassembled procollagen Cpropeptides. Curr. Biol. 11:1114-1118.
Co-workers:
Claire Bithell BSc
BBSRC Student
Seema Chakravarthi BSc MSc
Wellcome Trust Technician
Alexandra Hillebrand BSc
Marie Curie Visiting Student
Catherine Hoare BSc MSc
Wellcome Trust Research Associate
Lynsey Jenkinson BSc
Wellcome Trust Prize Student
Richard Lumb BSc
BBSRC Student
Pooli Ragesekariah BSc PhD
MRC Research Associate
Mohammed Tasab BSc PhD
Wellcome Trust Research Associate
Mark Warren BSc PhD
Wellcome Trust Research Associate
Rachel Watkins BSc
Wellcome Trust Technician
The maturation and secretion of procollagen
A major aim of the research in this laboratory is to elucidate the fundamental molecular
interactions that control the biosynthesis and folding of extracellular matrix proteins. The lumen
of the endoplasmic reticulum contains a number of proteins that have been shown previously to
interact with polypeptide chains during their maturation and transport through the ER to the
Golgi apparatus. These proteins include enzymes that catalyse folding events and molecular
chaperones that interact with folding polypeptide chains to prevent premature aggregation or
non-specific interactions.
Procollagen is an excellent example of a molecule that interacts with a number of enzymes and
molecular chaperones during its folding and assembly. If the initial folding of the procollagen is
prevented, the unfolded chains associate with BiP, which eventually leads to proteasomemediated degradation. Once the C-propeptide domains have folded, they either assemble to
form trimers immediately or associate with protein disulfide isomerase until interacting chains are
synthesised. Folding of the triple helical domain follows this trimerisation event. Trimeric, nontriple helical molecules interact with prolyl-4-hydroxylase (P4H), an interaction that depends on
the folding status of the protein rather than hydroxylation state. Once the triple helix has formed,
the protein is transported to the Golgi apparatus where it forms higher order aggregates
resulting in characteristic distensions of the Golgi cisternae.
This is not the complete picture, however, as procollagen chains have also been shown to
interact with a heat shock protein (Hsp47). What role could this protein be playing in
procollagen biosynthesis? Clearly it is an essential protein as mice deficient in Hsp47 die before
birth. We are currently investigating a variety of different hypotheses as to the role of this novel
molecular chaperone, which is one of the few examples of a protein-specific chaperone.
page 13
TIMOTHY E HARDINGHAM
PhD DSc
Professor of Biochemistry
Key publications;
Day,J.M.,A.D.Murdoch, and T.E.Hardingham.
1999.The folded protein modules of the Cterminal G3 domain of aggrecan can each
facilitate the translocation and secretion of the
extended chondroitin sulfate attachment sequence.
J. Biol. Chem. 274:38107-38111.
Gribbon,P., B.C.Heng, and T.E.Hardingham.
2000.The analysis of intermolecular interactions
in concentrated hyaluronan solutions suggest no
evidence for chain-chain association. Biochem. J.
350 Pt 1:329-335.
Kolettas,E., H.I.Muir, J.C.Barrett, and
T.E.Hardingham. 2001. Chondrocyte phenotype
and cell survival are regulated by culture
conditions and by specific cytokines through the
expression of Sox-9 transcription factor.
Rheumatology. (Oxford) 40:1146-1156.
Co-workers:
Chris Brew BSc MBChB MRCS
ARC Clinical Training Fellow
Alan Murdoch BSc PhD
BBSRC/MRC/EPSRC Research
Fellow
Janine Prince BSc MSc
BBSRC/MRC/EPSRC Technician
Richard Rauchenberg BSc
BBSRC Student
Bertrand Raynal BSc MSc
Wellcome Trust Research Assistant
Simon Tew BSc PhD
BBSRC/MRC/EPSRC Research
Associate
page 14
Proteoglycan functions in the extracellular matrix
The formation, turnover and repair of the extracellular matrix are essential determinants of
the growth and development of tissues into organs. Proteoglycans are key components of
the extracellular matrix, and aggrecan is the major proteoglycan of cartilaginous tissue.
Aggrecan, which comprises a multi-domain protein core attached to a large number of
chondroitin sulphate and keratan sulphate glycosaminoglycan chains, forms supramolecular
aggregates by binding to hyaluronan. We are using biochemical, molecular biology and
biophysical techniques to investigate the protein domain and glycosaminoglycan functions of
aggrecan. A new approach using fluorescence recovery after photobleaching with a confocal
microscope (confocal-FRAP) has been developed to study molecular interactions of
proteoglycans, hyaluronan and other glycoconjugates, such as mucins. With this technique,
which employs high molecular concentrations close to those found physiologically, the
networks formed by hyaluronan and aggrecan in solution and their permeability to FITClabelled probes are being investigated. This approach is also being applied to sections of
cartilage to follow permeability changes that accompany matrix damage and new matrix
assembly, and we are studying the changes in gene expression in chondrocytes that
characterise osteoarthritis.
We also have an active programme in cartilage tissue engineering. Chondrocytes are
responsible for the production of cartilage by extracellular matrix, but lose the ability to make
extracellular matrix in cell culture. We are investigating the expression of SOX genes in
human chondrocytes during the loss of phenotype in cell culture and the effects of
transfected SOX genes on the recovery of phenotype. The synthesis and assembly of
cartilage matrix by chondrocytes in culture will be assessed by composition analysis,
confocal microscopy, and by confocal-FRAP analysis to monitor matrix network formation.
This will identify the key interactions in the initial assembly of extracellular matrix by
chondrocytes and will lead to new strategies for generating cartilage matrix in culture.
CAY M. KIELTY BSc PhD
MRC Senior Research Fellow
and Professor of Medical
Biochemistry
ADRIAN SHUTTLEWORTH
BSc PhD
Reader in Medical Biochemistry
Key publications:
Baldock,C.,A.J.Koster, U.Ziese, M.J.Rock,
M.J.Sherratt, K.E.Kadler, C.A.Shuttleworth, and
C.M.Kielty. 2001.The supramolecular
organization of fibrillin-rich microfibrils. J. Cell
Biol. 152:1045-1056.
The structure and function of extracellular matrix
macromolecules in health and disease
Our aims are to understand how cells regulate the formation of supramolecular assemblies in
health and disease, and how such assemblies are organised within, and contribute functionally
to, diverse extracellular matrices. We are correlating genotype with phenotype in heritable
connective tissue disorders, and investigating the processes underlying vascular matrix
remodelling. Molecular, cellular and microscopy approaches are being applied to define the
structure and function of three major supramolecular assemblies of the extracellular matrix.
Ball,S.G., C.Baldock, C.M.Kielty, and
C.A.Shuttleworth. 2001.The role of the C1 and
C2 a-domains in type VI collagen assembly. J.
Biol. Chem. 276:7422-7430.
1. Fibrillin-rich microfibrils are complex, extensible polymers that play a key role in elastic fibre
formation. Their essential contribution to connective tissue integrity is highlighted by linkage of
mutations in fibrillin genes to Marfan syndrome and related diseases associated with
cardiovascular, skeletal and ocular defects. We are expressing fibrillin in recombinant form to
investigate its assembly and molecular interactions, and using electron microscopy and atomic
force microscopy to study microfibril organisation and elasticity.
Chaudhry,S.S., J.Gazzard, C.Baldock, J.Dixon,
M.J.Rock, G.C.Skinner, K.P.Steel, C.M.Kielty and
M.J.Dixon. 2001. Mutation of the gene encoding
fibrillin-2 results in syndactyly. Hum. Mol.
Genet. 10:835-843.
2. Type VI collagen microfibrils play a key linking role in the extracellular matrix. Mutations in
this collagen cause the heritable skeletal muscle disorder, Bethlem myopathy. We are
investigating the unique hierarchical assembly of type VI collagen microfibrils by analysing the
specific interactions between its triple helix and von Willebrand factor A-domains, and identifying
genotype-to-phenotype correlations that affect microfibril assembly.
3. Type VIII collagen is a structural component of the vasculature that is upregulated following
vascular injury and during remodelling. Mutations in this collagen cause two forms of corneal
endothelial dystrophy. Type VIII collagen forms hexagonal lattices that may provide pericellular
structural support for proliferating vascular smooth muscle cells and endothelial cells.
Recombinant expression approaches are being used to examine its chain composition,
supramolecular assembly and biological interactions.
Co-workers:
Craig Barley BSc
ARC Research Assistant
David Murray MBChB MRCS
BBSRC/MRC/EPSRC Clinical Training Fellow
Sarah Bernard BSc
BBSRC Student
Steve Ball BSc PhD
BBSRC Research Associate
Dan Bax BSc
BHF Research Associate
Claire Crouchley BSc
BBSRC/MRC/EPSRC Student
Nigel Hodson BSc PhD
MRC Co-op Research Associate
Karen Johnston BSc PhD
BBSRC/MRC/EPSRC Research Associate
David Lee BSc PhD
BBSRC/MRC/EPSRC Research Associate
Amanda Lomas BSc
BBSRC/MRC/EPSRC Technician
Kieran Mellody BSc MSc
HEFCE-funded Technician
Amanda Morgan BSc
HEFCE-funded Technician
Suzannah Phillips BSc
BBSRC Student
Gareth Pugh BSc PhD
BBSRC Research Associate
Matt Rock BSc PhD
MRC Research Associate
Mike Sherratt BSc PhD
MRC Research Associate
Simon Stephan BSc PhD
BHF Research Associate
page 15
PAUL N. BISHOP
PhD FRCS FRCOphth
Wellcome Trust Senior Research
Fellow in Clinical Science
Key Publications:
Reardon,A.J., M.Le Goff, M.D.Briggs, D.McLeod,
J.K.Sheehan, D.J.Thornton and P.N.Bishop. 2000.
Identification in vitreous and molecular cloning of
opticin, a novel member of the family of leucinerich repeat proteins of the extracellular matrix.
J. Biol. Chem. 275:2123-2129.
Bos,K.J., D.F.Holmes, K.E.Kadler, D.McLeod,
N.P.Morris and P.N.Bishop. 2001.Axial structure
of the heterotypic collagen fibrils of vitreous
humour and cartilage. J. Mol. Biol. 306:10111022.
Takanosu,M.,T.C.Boyd, M.Le Goff, S.P.Henry,
Y.Zhang, P.N.Bishop and R.Mayne. 2001.
Structure, chromosomal location and tissue-specific
expression of the mouse opticin gene.
Invest.Ophthalmol.Vis. Sci. 42:2202-2210.
Co-workers:
Jane Bradley BSc
Wellcome Trust Technician
Magali Le Goff BSc MSc
Wellcome Trust Research Associate
V. John Hindson BSc PhD
Wellcome Trust Research Associate
Tom Jowitt BSc
Iris Fund Research Associate
Lisa Macrory BSc
Wellcome Trust Prize Student
Wang Jing MD
Clinical Research Fellow
Matrix biology of the eye
The gel state of the vitreous humour is maintained by a loose network of heterotypic collagen fibrils, containing collagens types II, IX,
and V/XI. These collagen fibrils are coated with non-collagenous structural macromolecules and we hypothesise that these
molecules also play a key role in the supramolecular organisation of the vitreous gel. A major focus of our research has been to
understand how these collagenous and non-collagenous components are organised within the vitreous humour and how this
organisation is perturbed in ageing and disease. Using electron microscopy combined with computer modelling, we have elucidated
the axial distribution of collagen molecules within the fibrils. We have also determined how the collagen fibrils are distributed through
the vitreous gel and the effects of ageing upon these different levels of organisation. We isolated and characterised a pool of
collagen-associated molecules from the vitreous gel and this work led to the discovery of a novel member of the extracellular matrix
small leucine-rich repeat protein family that we have named opticin. Opticin expression is virtually restricted to the non-pigmented
ciliary epithelium of the eye and the onset of expression coincides with ciliary body differentiation, thereby providing a marker of this
developmental process. Opticin is highly expressed by both the embryonic and adult eye and we are currently elucidating its
functions using a combination of genetic, biochemical and cell biological strategies.
page 16
JOHN K SHEEHAN
BSc MSc PhD
Reader in Physiological
Biochemistry
The structure and function of mucus
DAVID J.THORNTON BSc PhD
Senior Experimental
Officer/Senior Research
Scientist
Key Publications:
Sheehan,J.K., C.Brazeau, S.Kutay, H.Pigeon,
S.Kirkham, M.Howard and D.J.Thornton. 2000.
Physical characterization of the MUC5AC mucin:
a highly oligomeric glycoprotein whether isolated
from cell culture or in vivo from respiratory
mucous secretions. Biochem. J. 347: 37-44.
Thornton,D.J.,T.Gray, P.Nettesheim, M.Howard,
J.S.Koo and J.K.Sheehan. 2000. Characterization
of mucins from cultured normal human
tracheobronchial epithelial cells. Am. J. Physiol.
Lung Cell Mol. Physiol. 278:L1118-L1128.
Thornton,D.J., J.Davies, S.Kirkham,A.Gautrey,
N.Khan, P.S.Richardson and J.K.Sheehan. 2001.
Identification of a non-mucin glycoprotein (gp340) from a purified respiratory mucin
preparation: evidence for an association involving
the MUC5B mucin. Glycobiology. 11: 969977.
Our work is centred on understanding the supramolecular assembly
and function of mucus gels, with a specific interest in the role of
mucus in the human airways in health and disease. Mucus is central
to the protection and the maintenance of homeostasis of the lung.
This highly hydrated gel, in conjunction with ciliated epithelial cells,
forms the mucociliary escalator, which along with cough, is essential
for the sterility of the airways. By contrast, overproduction of mucus
with altered physical properties is an important factor in the morbidity
and mortality of chronic airways disease such as asthma, cystic
fibrosis and chronic obstructive pulmonary disease. Our work until
recently has centred upon the identification, characterisation and
quantitation of the large oligomeric O-linked glycoproteins (mucins)
responsible for the properties of the mucus gels found in healthy and
diseased airways secretions. We have been successful in devising
probes to identify distinct mucin species secreted from different
cellular sources in the respiratory tract (i.e. from the surface epithelia
or underlying submucosal glands). It has become clear that the
mucins are the organising framework around which many protective
molecules are condensed. In the coming phase of our work we are
seeking to understand the higher-order organisation of mucins and
the role other proteins play in mucus structure. A large part of the
organisation arises from the initial mechanisms of biosynthesis and
packaging of the mucins into intracellular storage granules prior to
their secretion and thus our interests in these processes are
expanding. We are also pursuing a vigorous proteomics approach
for the identification and quantitation of other proteins and
glycoproteins involved in mucus organisation and at the same time
are devising new methods and strategies for assaying the network
properties of the mucus gel itself.
Co-workers:
Linda Eyers BSc
BBSRC Student
Sara Kirkham BSc
Wellcome Trust Research Assistant
Matthew Wakefield BSc
Wellcome Trust Prize Student
page 17
RICHARD A. KAMMERER
Dipl Biochemistry PhD
Wellcome Trust Research
Career Development Fellow
Key Publications:
Kammerer,R.A.,V.A.Jaravine, S.Frank,
T.Schulthess, R.Landwehr,A.Lustig, C.GarciaEcheverria,A.T.Alexandrescu, J.Engel, and
M.O.Steinmetz. 2001.An intrahelical salt bridge
within the trigger site stabilizes the GCN4 leucine
zipper. J. Biol. Chem. 276:13685-13688.
Stetefeld,J., M.Jenny,T.Schulthess, R.Landwehr,
J.Engel, and R.A.Kammerer. 2000. Crystal
structure of a naturally occurring parallel righthanded coiled coil tetramer. Nat. Struct. Biol.
7:772-776.
Coiled coils in extracellular matrix proteins
The goal of our research is to improve our understanding of protein-protein interactions in
extracellular matrices in order to understand the processes determining their formation and
their disruption in disease. We are using the coiled-coil element as a model system to
study these interactions. Coiled coils mediate subunit oligomerisation in a large number of
proteins and are implicated in a wide variety of biological functions. The left-handed coiledcoil motif is a type of protein structure consisting of two to five right-handed amphipathic αhelices that “coil” around each other in a slight super-twist. The sequences of left-handed
coiled coils are characterized by a heptad repeat of seven residues denoted a to g with a
3,4-hydrophobic repeat of mostly apolar amino acids at positions a and d. The helices in
coiled-coil assemblies can be arranged in a parallel or anti-parallel manner. In addition,
coiled-coil interactions can result in homo- or heterotypic oligomers. The simplicity of its
structure makes the coiled-coil structural motif an attractive system for studying both the
intra- and inter-molecular interactions that govern the folding and stability of multi-subunit
proteins. Using a combination of molecular biology, biochemistry, biophysical chemistry,
and structural biology, our efforts are aimed at understanding these interactions.
Stetefeld,J., M.Jenny,T.Schulthess, R.Landwehr,
B.Schumacher, S.Frank, M.A.Ruegg, J.Engel, and
R.A.Kammerer. 2001.The laminin-binding
domain of agrin is structurally related to NTIMP-1. Nat. Struct. Biol. 8:705-709.
Co-workers:
Philip Macdonald BSc
Wellcome Trust Research Assistant
CLAIR BALDOCK BSc PhD
Royal Society Olga Kennard
Research Fellow
Structural studies on microfibrillar
components of the
extracellular matrix
The aim of the research in this laboratory is to investigate
the structure and function of microfibrillar components of
the extracellular matrix using a combination of electron
microscopy and X-ray crystallography. Two different
microfibrillar assemblies are being studied - the fibrillin-rich
microfibrils and collagen VI microfibrils - each of which
makes an essential contribution to normal tissue elasticity.
Key Publications:
Baldock,C.,A.J.Koster, U.Ziese, M.J.Rock,
M.J.Sherratt, K.E.Kadler, C.A.Shuttleworth and
C.M.Kielty. 2001.The supramolecular
organization of fibrillin-rich microfibrils. J. Cell
Biol. 152:1045-1056.
Ball,S.G., C.Baldock, C.M.Kielty and
C.A.Shuttleworth. 2001.The role of the C1 and
C2 A-domains in type VI collagen assembly. J.
Biol. Chem. 276:7422-7430.
Holmes,D.F., C.J.Gilpin, C.Baldock, U.Ziese,
A.J.Koster and K.E.Kadler. 2001. Corneal
collagen fibril structure in three dimensions:
Structural insights into fibril assembly,
mechanical properties and tissue organization.
Proc. Natl.Acad. Sci. U. S.A 98:73077312.
page 18
Resolving extracellular matrix polymers using conventional structural biology techniques has
previously proved difficult due to their size and complexity, but now these problems can be
circumvented with the new technique of electron tomography. By marrying cryo-electron
microscopy data with high-resolution structures of key domains, high resolution information can be
extrapolated to complex extracellular assemblies.
The goal of this research is to provide new insights into the molecular architecture of these large
and ultrastructurally complex microfibrils. This structural information will provide a better
understanding of their biological properties in health and disease.
Co-workers:
Andrew Marson BSc
BBSRC Student
Research in Focus
The supramolecular organisation of fibrillin-rich microfibrils
The elastic fibre system makes a key
contribution to the structure and function of
organs that require elasticity, such as large
arteries, lung and skin. The principal
components of elastic fibres that endow
them with their special physical properties
are polymerised elastin and fibrillin-rich
microfibrils. The major aims of our research
are to determine the mechanisms of
assembly and organisation of fibrillin-rich
microfibrils, and to elucidate their role in
elastic fibre formation.
Fibrillin-rich microfibrils are a unique class of
multicomponent extracellular matrix microfibril that
endow connective tissues with long-range elasticity.
In the untensioned state, they have a repeating
periodicity of 56nm with a ‘beads-on-a-string’
appearance and a diameter of 15nm. Their principal
structural molecule is fibrillin, a large cysteine-rich
glycoprotein (~350 kDa) that forms the molecular
scaffold of this class of microfibril. The importance
of fibrillin-rich microfibrils is emphasised by the
linkage of fibrillin mutations to Marfan syndrome and
related connective tissue disorders that are
associated with severe cardiovascular, ocular and
skeletal defects. Until now, however, the
arrangement of fibrillin molecules within microfibrils
has remained poorly defined.
Automated electron tomography was used
to generate 3D microfibril reconstructions,
which revealed many new organisational
details of untensioned microfibrils, including
heart-shaped beads from which two arms
emerge, and inter-bead diameter variation.
The reconstructions revealed that
microfibrils comprise two in-register
filaments with a longitudinal symmetry axis,
with up to eight fibrillin molecules in crosssection.
Antibody epitope mapping of untensioned
microfibrils revealed the overlap of epitopes
at the N- and C-terminus and the
juxtaposition of two internal epitopes that
would be 42 nm apart in unfolded
molecules, which infers intramolecular
folding. Comparison of colloidal gold and
antibody binding sites in untensioned
microfibrils and those extended in vitro
highlight conformational changes and
intramolecular folding.
which involves folding at predicted “hinge”
regions and that microfibril elasticity is
dependent on a conformation-dependent
maturation to the ~one-third staggered
arrangement.
Armed with this new structural information,
we are now able to test our model by
analysing the predicted hinge regions
biochemically and through high-resolution
structure determination. We are also
defining higher-order microfibril packing
arrangements by examining the biophysical
properties of microfibril bundles and by
studying microfibril interactions and crosslinks within bundles. Insights into the
crucial role of microfibrils in elastic fibre
formation are emerging from analysis of
interactions with tropoelastin and elastinmicrofibril interface proteins.
Clair Baldock and Cay Kielty
Together our data indicate that an ~onethird stagger is adopted in untensioned
microfibrils, but a molecular head-to-tail
arrangement could occur in highly extended
microfibrils. Our model suggests a
molecular basis for microfibril extensibility
page 19
Genetic Control of Tissue
Structure and Function
MICHAEL D BRIGGS BSc PhD
ARC Research Fellow
MICHAEL E GRANT BScTech
D Phil
Pro-Vice-Chancellor and
Professor of Medical Biochemistry
Key Publications:
Chapman,K.L., G.R.Mortier, K.Chapman,
J.Loughlin, M.E.Grant, and M.D.Briggs. 2001.
Mutations in the region encoding the von
Willebrand factor A domain of matrilin-3 are
associated with multiple epiphyseal dysplasia.
Nat. Genet. 28:393-396.
Holden,P., R.S.Meadows, K.L.Chapman,
M.E.Grant, K.E.Kadler, and M.D.Briggs. 2001.
Cartilage oligomeric matrix protein interacts with
type IX collagen, and disruptions to these
interactions identify a pathogenetic mechanism in
a bone dysplasia family. J. Biol. Chem.
276:6046-6055.
Briggs,M.D. and Chapman, K.L. 2002.
Pseudoachondroplasia and multiple epiphyseal
dysplasia: mutation review, molecular interactions
and genotype-phenotype correlation. Human
Mutation. in press.
Co-workers:
Helen Attisha BSc
Nuffield Foundation Student
Kathryn Chapman BSc PhD
ARC Research Associate
Gail Skinner BSc
European Commission Research
Associate
Molecular genetics and
cell-matrix pathology of human
monogenetic bone diseases
The skeletal dysplasias
are an extremely
diverse and complex
group of genetic
disorders, which primarily affect
the development of the osseous
skeleton. There are over 200 unique
and well-characterised phenotypes, which range in severity from
relatively mild to severe and lethal forms. Many of these
individual phenotypes have been grouped into ‘bone dysplasia
families’, on the basis of a similar clinical or radiographic
presentation. One such family is the multiple epiphyseal
dysplasia (MED) and pseudoachondroplasia (PSACH) group of
diseases.
PSACH appears to result almost exclusively from mutations in
the gene encoding cartilage oligomeric matrix protein (COMP).
Some forms of MED are allelic with PSACH, but MED shows
considerable genetic heterogeneity and can also result from
mutations in the genes encoding the α1, α2 and α3 chains of
type IX collagen, COL9A1 (EDM6), COL9A2 (EDM2) and
COL9A3 (EDM3), respectively. Furthermore, we have recently
identified mutations in the gene encoding matrilin-3 (MATN3), a
member of the matrilin family of extracellular oligomeric matrix
proteins, which cause a distinctive mild form of MED (EDM5).
The non-allelic genetic heterogeneity of the MED disease
spectrum can best be explained by the observation that the
protein products encoded by the COMP, type IX collagen and
matrilin-3 genes interact to form large macromolecular
assemblies in the cartilage ECM. Disruptions to these
interactions are likely to have a fundamental effect on the
development and homeostasis of the cartilage ECM and
ultimately result in phenotypes within the PSACH-MED disease
spectrum.
We are using a multidisciplinary approach to study the molecular genetics and cell-matrix pathology
of the PSACH and MED disease spectrum. Particular emphasis is being placed on understanding
the non-allelic genetic heterogeneity of MED resulting from the disruption of supramolecular
assemblies and the effect of specific disease causing mutations on the structure and function of the
relevant gene products. Furthermore the cell, matrix and tissue pathology of matrilin-3 defects will
be studied through the generation and analysis of an engineered mouse model.
page 20
RAYMOND BOOTHANDFORD BSc PhD
Reader in Biochemistry and
Molecular Biology
Key Publications:
Marks,D.S., C.A.Gregory, G.A.Wallis,A.Brass,
K.E.Kadler, and R.P.Boot-Handford. 1999.
Metaphyseal chondrodysplasia type Schmid
mutations are predicted to occur in two distinct
three-dimensional clusters within type X collagen
NC1 domains that retain the ability to trimerize.
J. Biol. Chem. 274:3632-3641.
Fowler,S.J., S.Jose, X.Zhang, R.Deutzmann,
M.P.Sarras, Jr., and R.P.Boot-Handford. 2000.
Characterization of hydra type IV collagen.Type
IV collagen is essential for head regeneration and
its expression is up-regulated upon exposure to
glucose. J. Biol. Chem. 275:39589-39599.
Green,H.,A.E.Canfield, M.C.Hillarby,
M.E.Grant, R.P.Boot-Handford,A.J.Freemont, and
G.A.Wallis. 2000.The ribosomal protein QM is
expressed differentially during vertebrate
endochondral bone development. J. Bone Miner.
Res. 15:1066-1075.
Co-workers:
Marianne Ellin BSc
ARC Technician
Darren Hitchen BSc
Wellcome Trust Technician
Majid Shahbazi BSc PhD
ARC Research Associate
Biology, pathology and evolution
of extracellular matrices
The research in this laboratory covers two major areas: bone development and the
pathogenesis of osteoarthritis, and invertebrate models for studying the pathology and evolution
of extracellular matrix.
Bone development and osteoarthritis. Much of the human skeleton is originally laid down during
development as a cartilaginous template. The chondrocytes manufacturing this template are
destined to progress through a highly coordinated differentiation pathway in the growth plate
(see figure below) involving proliferation, maturation, hypertrophy (enlargement or swelling) and
apoptosis. Cells brought to the location by vascular invasion erode the base of the growth plate
and the matrix is converted from cartilage (type II collagen-rich) to bone (type I collagen-rich).
The only chondrocytes not consumed in this process, known as endochondral ossification, are
those that form the articular cartilage. These chondrocytes maintain the articular cartilage
throughout life. However, in osteoarthritis, a disease characterised by a destruction of the
articular cartilage matrix, the phenotype of articular chondrocytes is altered and they appear to
de-differentiate and attempt to reinitiate the pattern of differentiation seen in the growth plate.
We are using a number of different approaches, including gene targeting in vivo and cell culture
models, to investigate the fundamental differences between growth plate and articular
chondrocytes and determine whether the altered phenotype of these cells is a key factor in the
pathogenesis of osteoarthritis.
Invertebrate models. Hydra vulgaris is an evolutionarily ancient, simple, fresh water invertebrate
composed of two epithelial cell sheets (ectoderm and endoderm) separated by a basement
membrane-like matrix known as the mesoglea. Our interest in this organism stems from the fact
that exposure to glucose leads to a thickening of the mesoglea in a fashion similar to that seen
in basement membranes during diabetic microangiopathy – the secondary long term
complication of diabetes which causes blindness and kidney failure. The glucose-induced
mesoglea thickening in Hydra takes a few days whereas in diabetic patients, basement
membrane thickening takes months to years. We have cloned the basement membrane (type
IV) collagen gene from Hydra, as well as several other collagens that appear to contribute to the
function of mesoglea, and shown it to be highly conserved in comparison to mammals. In
addition, type IV collagen gene expression is upregulated within 48 hours of exposure of the
Hydra to glucose. One aim is now to examine the mechanism by which glucose directly induces
increases in type IV collagen synthesis in this relatively simple in vivo system since this may
provide strong indications as to the aetiology of basement membrane thickening in diabetes.
page 21
ULRIKE MAYER
Dipl Chemistry PhD
Lecturer in Dental Genetics
Key Publications:
Cohn,R.D., U.Mayer, G.Saher, R.Herrmann,
.A.van der Flier,A.Sonnenberg, L.Sorokin, and
T.Voit. 1999. Secondary reduction of alpha7B
integrin in laminin alpha2 deficient congenital
muscular dystrophy supports an additional
transmembrane link in skeletal muscle. J. Neurol.
Sci. 163:140-152.
Miosge,N., C.Klenczar, R.Herken, M.Willem, and
U.Mayer. 1999. Organization of the myotendinous
junction is dependent on the presence of
alpha7beta1 integrin. Lab Invest 79:15911599.
Werner,A., M.Willem, L.L.Jones, G.W.Kreutzberg,
U.Mayer, and G.Raivich. 2000. Impaired axonal
regeneration in alpha7 integrin-deficient mice. J.
Neurosci. 20:1822-1830.
Co-workers:
Melanie Klein MTA
Wellcome Trust Technician
Peter Latham BSc PhD
Wellcome Trust Research Associate
Synnva Ullensvang BSc
Wellcome Trust Technician
page 22
Function of the laminin-nidogen-1
interaction and the laminin-binding
integrin α7β1
The work of our group mainly focuses on the analysis of the basement membrane proteins
laminin and nidogen in vivo and the receptor-mediated interaction of basement membranes
with cells by gene targeting approaches. The combination of the analysis of the
supramolecular organisation of basement membranes and the function of their cellular
interactions will lead to a better understanding of a variety of biological processes.
To study the potential involvement of the laminin-binding integrin α7β1 during myogenesis, and
its role in muscle integrity and function, we have generated a null allele of the α7 gene in the
germline of mice. Mice homozygous for the mutation are viable and fertile but develop a
muscular dystrophy. We observed histopathological changes that strongly indicate an
impairment of function of the myotendinous junctions. These findings demonstrate that α7β1
integrin represents an indispensable linkage between muscle fibers and the extracellular matrix
which is independent of the well characterised interaction of the cytoskeleton with the muscle
basement membrane mediated by the dystrophin-dystroglycan complex. Ongoing studies
include the elucidation of compensatory mechanisms by other integrin α subunits and the
structural and functional relationship of the dystrophin/sarcoglycan complex with α7β1 integrin
during myogenesis and muscle maturation.
We are also studying the role of the laminin-nidogen-1 interaction in basement membrane
assembly and function during embryogenesis and organ development. We have constructed a
targeting vector for homologous recombination in embryonic stem cells in which the nidogen-1binding module γ1III4 was deleted. This approach allows the study of a specialised interaction
between two proteins without interfering with other biological functions known to be mediated
by laminins. Mice homozygous for the deletion live to birth, but die soon after due to renal
agenesis and impaired lung development. Future experiments include the generation of mice
carrying subtle point mutations through which the laminin-nidogen-1 interaction is only
weakened but not abolished.
GILLIAN A.WALLIS BSc PhD
Senior Lecturer in Medicine
Key Publications:
Roby,P., S.Eyre, J.Worthington, R.Ramesar,
H.Cilliers, P.Beighton, M.Grant, G.Wallis. 1999.
Autosomal dominant (Beukes) premature
degenerative osteoarthropathy of the hip joint
maps to an 11cM region on chromosome 4q35.
Am J Hum Genet 64:904-908.
White,A. and G.Wallis. 2001. Endochondral
ossification:A delicate balance between growth
and mineralisation. Current Biol. 11:R589-91.
Newman,B., L.I.Gigout, L.Sudre, M.E.Grant and
G.A.Wallis. 2001. Coordinated expression of
matrix Gla protein is required during
endochondral ossification for chondrocyte survival.
J. Cell Biol. 154:659-666.
Co-workers:
Richard Aspinwall BSc MSc
ARC Research Assistant
Marianne Ellin BSc
ARC Technician
Emma Gillaspy BSc
University of Manchester Student
Carolyn Greig BSc
Wellcome Trust Research Assistant
Majid Shahbazi BSc PhD
ARC Research Associate
Kristian Spreckley BSc
Wellcome Trust Research Assistant
Laure Sudre BSc
University Of Manchester Student
The aetiology and pathogenesis of osteoarthritis
Osteoarthritis (OA) is the most common form of human joint disease and a leading cause of
pain and disability, particularly of the elderly. It is a heterogeneous condition in terms of
cause, clinical course and severity. Furthermore, the pathogenesis is complex, involving
both degradative and reparative processes of the articular cartilage and subchondral bone.
We are approaching the study of OA in two ways. Firstly, we are investigating genetic
susceptibility to the condition, and secondly, we are examining the processes whereby new
bone is laid down in the OA joint.
Epidemiological studies of OA have demonstrated that generalised nodal osteoarthritis
(GNOA) has a strong genetic component. For our studies of genetic susceptibility to
GNOA, we have obtained DNA from members of over 200 families that contain at least one
affected sibling pair. We are genotyping these DNA samples using microsatellite markers
that span the genome and analysing the data using non-parametric linkage analysis
methods. We are using a candidate gene approach to investigate promising loci.
During normal development, the endochondral ossification process is responsible for the
conversion of the cartilaginous embryonic skeleton to bone. Recent studies have
demonstrated that aspects of this process are reactivated in OA. We have identified
several genes that are upregulated in cartilage both from persons with OA and in a naturally
occurring form of OA in guinea pigs. We are investigating the function of these genes using
a cell culture system that mimics endochondral ossification in vitro. We are developing
methods for the transduction of genes that regulate the endochondral ossification process
into chondrocytes in cartilage for future use in gene therapy for OA.
The identification of genes important in the aetiology and progression of OA should allow us
to identify persons most at risk for the condition and to develop strategies for its treatment.
page 23
ANN E CANFIELD BSc PhD
Senior Lecturer in Medicine
Key Publications:
Mantell,D.J., P.E.Owens, N.J.Bundred, E.B.Mawer
and A.E.Canfield. 2000. 1 alpha,25dihydroxyvitamin D(3) inhibits angiogenesis in
vitro and in vivo. Circ. Res. 87:214-220.
Canfield,A.E., M.J.Doherty,V.Kelly, B.Newman,
C.Farrington, M.E.Grant and R.P.Boot-Handford.
2000. Matrix Gla protein is differentially
expressed during the deposition of a calcified
matrix by vascular pericytes. FEBS Lett.
487:267-271.
Canfield,A.E., C.Farrington, M.D.Dziobon,
R.P.Boot-Handford,A.M.Heagerty, S.N.Kumar
and I.S.D.Roberts. 2002.The involvement of
matrix glycoproteins in vascular calcification and
fibrosis: an immunohistochemical study. J. Path.
196: 228-234
Co-workers:
Yvonne Alexander BSc PhD
Visiting Lecturer
Georgina Collett BSc PhD
BHF Research Associate
Catherine Griffin-Jones HND
EU-Technician
Karen Howson BSc
MRC Student
Kirsty Ratcliffe BSc PhD
BBSRC/MRC/EPSRC Research
Associate
Claire Rock BSc PhD
EU-Research Associate
Neill Turner BSc
BBSRC/MRC/EPSRC Research
Assistant
page 24
Molecular and cellular mechanisms underpinning
angiogenesis and vascular calcification
Angiogenesis is the formation of new blood vessels from an existing vascular bed. It is
of fundamental importance in many physiological and pathological conditions, including
embryonic development, wound healing, atherosclerosis, diabetic retinopathy, psoriasis
and tumour growth and metastasis. Angiogenesis is a complex process involving
changes in endothelial cell phenotype, extracellular matrix remodelling and stabilisation
of the newly formed blood vessels. We are currently employing a multidisciplinary
approach: (i) to determine the contributions of specific proteins in mediating the
response of endothelial cells to angiogenic factors during the early stages of
angiogenesis, and (ii) to define the roles of specific angiogenic factors and matrix
proteins in regulating endothelial cell-pericyte interactions that are crucial for vessel
stabilisation. We aim to translate this work into the development of novel antiangiogenic and pro-angiogenic strategies for the treatment of diseases characterised by
abnormal vascularisation.
Calcification is associated with advanced complicated atherosclerosis in large arteries,
but may also occur in smaller vessels where it results in ischemic tissue necrosis. We
have shown that vascular pericytes can differentiate into osteoblast-like cells in vitro and
in vivo, and that they can deposit a calcified matrix resembling that found in calcified
atherosclerotic plaques. These results strongly suggest that pericytes may mediate, at
least in part, vascular calcification. We are currently using molecular, cellular and
biochemical approaches to elucidate the mechanisms of pericyte differentiation. This
work is likely to provide important insights into the pathogenesis of vascular calcification.
Localisation of MGP in calcified
arteries.MGP is not detected
in non-calcified vessels.
Research in Focus
Vascular Calcification
Vascular calcification, a common
complication of atherosclerosis and
diabetes, leads to an increased risk of
plaque rupture, myocardial infarction, limb
amputation and morbidity. Interestingly,
detailed studies of vascular calcification
have shown it to be a highly complex
process with many similarities to bone
formation (osteogenesis). For example,
proteins known to be involved in the
controlled calcification that occurs during
osteogenesis have also been identified in
calcified atherosclerotic lesions. These
proteins include bone morphogenetic
protein-2 (BMP-2), BMP-6, osteopontin,
osteocalcin, bone sialoprotein and matrix
Gla protein. We have now shown that
osteopontin and matrix Gla protein are also
localised to sites of calcification in
subcutaneous and dermal arteries, arterioles
and microvessels in patients with calcific
uraemic arteriolopathy (calciphylaxis),
suggesting that calcification in these different
sized vessels may actually occur by a
common mechanism.
The overarching aim of the research in my
laboratory is to develop ways to control
vascular calcification, and in recent studies
we have aimed to elucidate how vascular
calcification is regulated at the cellular and
molecular level. Our findings have
demonstrated that the deposition of mineral
in arteries may be mediated by a subpopulation of smooth muscle cells and/or
pericytes present in the vessel wall. These
cells can differentiate into ‘osteoblast-like’
cells in vivo and in vitro, and deposit a
calcified matrix resembling that found in
calcified atherosclerotic plaques. To gain a
better understanding of how this process is
regulated at the molecular level, we have
used subtractive hybridisation to identify
genes that are either up-regulated or downregulated as pericytes undergo osteogenic
differentiation and deposit a calcified matrix.
Using this approach we have shown that
genes that are implicated in the pathological
calcification of arteries, namely matrix Gla
protein, Axl receptor tyrosine kinase and
HtrA1 serine protease are all differentially
expressed during pericyte differentiation.
Gla protein and HtrA1 play crucial, but
distinct, roles in vascular calcification. The
activation of Axl by its ligand (Gas6)
appears to be required for maintaining
these cells in their vascular phenotype.
Down-regulation of Axl, or perturbation of
Axl-Gas6 interactions, enhances the
deposition of a calcified matrix by these
cells. In contrast, matrix Gla protein appears
to play a dual role in vascular cells, initially
modulating cell differentiation and
subsequently controlling matrix calcification.
Interestingly, recent studies suggest that
HtrA1 may regulate calcification by
controlling the degradation of specific matrix
proteins. We are currently conducting a
detailed investigation of the mechanism(s)
by which each of these proteins regulates
calcification.
Functional analyses of these genes using in
vitro models developed in my laboratory
have recently demonstrated that Axl, matrix
Ann Canfield
In time, this integrated programme of
research will provide a better understanding
of the molecular events regulating
physiological and pathological calcification,
and may identify potential targets for the
therapeutic manipulation of this event.
page 25
Facilities in the Centre
The work in the Centre covers a broad span of activities
encompassing approaches from pure population genetics,
through to structural and functional studies of individual
proteins and their assemblies. This breadth enables a rich
interplay of interdisciplinary contacts but also requires an
extensive infrastructure. The formation of the Centre was
coupled to the establishment of a common core of
equipment facilities. Our philosophy has been that wet
laboratory space for people is precious and that where it is
practical, equipment and other facilities are shared. This
approach has greatly benefited new scientists coming to
the Centre in rapidly establishing their laboratories and
giving them access to state-of-the-art equipment.
Biomolecular analysis
The Centre has an extensive range of shared equipment
for the purification, characterisation and study of the
interactions of biomolecules. These multi-user facilities are
housed in two fully integrated laboratories.
The first of these laboratories is focused primarily on the
isolation, identification and quantitation of proteins and
glycoproteins. This laboratory is equipped with a range of
chromatography workstations (standard, micro and
capillary LC) used either for protein purification or as inlet
devices for three of our four LC-mass spectrometers (see
below). A liquid handling workstation enables automated
multi-dimensional chromatographic separations. The mass
spectrometers (a Tof-Spec E MALDI-TOF mass
spectrometer, an LC-TOF mass spectrometer, a Quattro
electrospray quadrapole mass spectrometer and a hybrid
quadrapole-TOF mass spectrometer, all supplied by
Micromass) are used for protein/glycoprotein identification,
analysis of post-translational modifications, quantitation
and accurate mass measurements. An automated Nterminal protein sequencer (Applied Biosystems) and a
fluorescence spectrometer and CD spectrapolarimeter
(Jasco) are also present in this laboratory.
The second laboratory is focused on the physical analysis
of biomolecules in solution and the investigation of their
interactions. An extensive array of modern facilities is
available for physical analysis of proteins, glycoproteins
and polysaccharides, including a Beckman Optima XL-A
analytical ultracentrifuge, a Dawn multi-angle light
scattering photometer, a Malvern 4700 photon correlation
spectrometer and a Viscotek protein analyser.
Furthermore, this laboratory houses ultrasensitive
calorimetry equipment (Microcal ITC and DSC) for the
analysis of molecular transitions and the determination of
binding constants, stoichiometries and reaction energies
for interactions between biomolecules and their ligands.
page 26
Electron microscopy
The research of several groups in the Centre is dependent
on our electron microscopy facility which contains a new
Wellcome Trust-funded cryo-transmission electron
microscope (Tecnai12 with STEM facility). Ancillary
equipment is also available for freeze-fracture
(Cressington), high pressure freezing (EM-Pact), freeze
substitution as well as cryo-plunge equipment for use in
cryo-EM. The Centre also has a dedicated image analysis
suite that is used in automated electron tomography (AET)
studies of extracellular matrix assemblies.
Light Microscopy
A major research focus within the Centre is live-cellimaging with a particular requirement for multicolour
fluorescence. The Centre has an established
microinjection facility that is also equipped with an
environmentally controlled chamber, epi-fluorescence and
software for time-lapse imaging.
A “next generation” confocal microscopy facility is being
established. These microscopes have the ability to
separate spectral signatures and can optically section
living cells with minimal damage using a variety of
fluorescent probes including pairs of GFP variants. The x,
y, z motorised stage enables accurate point visiting during
time-lapse experiments. The system will have the
capability for FRET, FRAP and FLIM studies.
X-ray crystallography
A new, state-of-the-art X-ray diffraction facility has been
funded by the Wellcome Trust as part of the Centre
renewal. This facility will consist of controlled-temperature
crystallisation rooms, an X-ray diffraction generator
equipped with high-brilliance optics, an area detector
system, and a cryo-cooling system for diffracting frozen
crystals. The facility will also include computational
resources for data processing and analysis,
crystallographic model building and refinement, and
structural analysis.
A fermentation suite with facilities for the bulk production of
proteins from 5 – 20 L of media is also available. Five
separate fermenters can accommodate bacterial, yeast
and mammalian cultures.
Research and Training
Opportunities
The Wellcome Trust Centre offers excellent training in all aspects of the biology of the
extracellular matrix at all career levels. Staff in the Centre have an impressive track
record in helping young researchers to develop independent careers, and they also
provide opportunities for science graduates to conduct postgraduate and postdoctoral
research. In addition, opportunities are available to help young clinicians gain
appropriate support and undertake PhD or MD training. Each year opportunities exist
to apply for:
Senior Research Fellowships
Career Development Fellowships
Post-doctoral research positions
Clinical Research and Research Training Fellowships (medical & veterinary)
Postgraduate studentships
Undergraduate Bursaries
Pre-University School Bursaries
Visiting (sabbatical) Fellowships
Training for higher degrees in under the aegis of the School of Biological Sciences.
The PhD and MPhil research training offered by the School is based on supportive
supervision in an excellent research environment, a personal development
programme for each student, and a structured Graduate Training Programme. The
School also has a career development programme for Postdoctoral Research
Associates and Fellows. The excellence and innovation of the postgraduate
programme is recognised nationally and reflected in the annual award, by the
Wellcome Trust, of five 4-year PhD studentships. Each year in the School, all research
students participate in a two-day Graduate Symposium. In addition, students and
postdoctoral researchers contribute to a weekly seminar programme, and are actively
encouraged to present their results at national and international meetings.
page 25
page 27
Staff Profiles - Principal Investigators
CLAIR BALDOCK
MARTIN BARON
JORDI BELLA
1994
1984
BSc Molecular Biophysics,
1986
BA University of Oxford
University of Leeds.
1990
DPhil University of Oxford
1997
PhD University of Sheffield.
1990
1998
Royal Society Study visit,
University of Auckland,
2001
Post-doctoral Research Associate,
1991
University of Oxford
1991
New Zealand.
1998
Post-doctoral Fellow,
1994
Catalunya, Spain
1991
Lecturer, School of
University of Manchester.
Biological Sciences,
Royal Society Olga Kennard
University of Manchester
PhD Chemical Sciences,
Universitat Politècnica de
Post-doctoral Fellow,
Yale University
BSc Chemistry, Universitat de
Barcelona, Spain
Post-doctoral Fellow, Rutgers
University, Piscataway, NJ, USA
1995
Post-doctoral Research Associate,
Purdue University, West Lafayette,
Research Fellow,
IN, USA
University of Manchester.
1999
Lecturer, School of Biological
Sciences, University of Manchester
PAUL BISHOP
MIKE BRIGGS
RAY BOOT-HANDFORD
1980
B Med Sci University of
1989
BSc Liverpool Polytechnic
1976
BSc University College of Wales, Cardiff
Nottingham
1993
PhD MRC Clinical Research
1980
PhD University of London
Centre, Harrow
1981
1983
BM BS University of Nottingham
1988
FRCS London
1991
Post-doctoral Research Fellow,
Wellcome Trust Vision Research
Cedars Sinai Medical Center,
Training Fellow
Los Angeles
1993
PhD University of Manchester
1994
Wellcome Trust Clinician
Scientist Fellow
1998
1992
1996
ARC Research Fellow,
2001
Reader in Biochemistry and
Post-doctoral Research Fellow, UMDNJRutgers Medical School, Piscataway, NJ
1987
RNIB Research Fellow,
University of Manchester
1989
Molecular Biology
Ophthalmologist
1999
1985
University of Manchester
Honorary Consultant
Post-doctoral Fellow, University of
Manchester
School of Biological Sciences,
University of Manchester
1998
Wellcome Trust Senior Research
Wellcome Trust Research Leave
Fellowship
Fellow in Clinical Science
2001
Reader in Biochemistry and
Molecular Biology
KEITH BRENNAN
NEIL BULLEID
ANN CANFIELD
1994
BA University of Cambridge
1982
BSc University of Liverpool
1980
BSc University of Manchester
1998
PhD University of Cambridge
1985
PhD Glasgow College
1984
PhD University of Manchester
1997
Wellcome Trust Prize Fellow at the
1986
Post-doctoral Research Fellow,
1983
University of Cambridge
1999
Post-doctoral Research Fellow at
University of Kent
1989
Howard Hughes Medical Institute,
Laboratories, New York, USA and
Dallas, Texas
Weill Medical College of Cornell
1990
University, New York, USA
2001
School of Biological Sciences,
2000
Manchester
1993
Manchester
1996
Professor of Biochemistry,
Univesity of Manchester
Wellcome Trust Post-doctoral
Research Fellow, University of
Royal Society University Research
Fellow,University of Manchester
University of Manchester
2002
Visiting Post-doctoral Fellow,
the Strang Cancer Research
Post-doctoral Research Fellow,
Christie Hospital, NHS Trust,
Lecturer, Dept of Medicine,
University of Manchester
2000
Wellcome Trust Research Career
Senior Lecturer, Dept of Medicine,
University of Manchester
Development Fellow,
University of Manchester
ANDREW GILMORE
MIKE GRANT
TIM HARDINGHAM
1989
BA, Oxford University
1962
BSc Tech UMIST
1965
BSc University of Bristol
1993
PhD, University of Leicester
1966
DPhil Oxford University
1968
PhD University of Bristol
1994
EMBO Post Doctoral Fellow,
1966
Dept Medical Biochemistry
1984
DSc University of Bristol
University of Manchester
1968
Kennedy Institute of Rheumatology
Post-doctoral Fellow
1976
University of North Carolina
1997
Postdoctoral Research Associate,
1970
University of Manchester
2000
University of Pennsylvania
Wellcome Trust Research Career
1972
University of Manchester
Development Fellow,
1995
Chairman, Wellcome Trust
of Biological Sciences, University of
Centre
Manchester, (Wellcome Trust
University of Manchester
1995
for Cell-Matrix Research,
University of Manchester
2000
2001
Professor of Biochemistry, School
University Award)
2001
Director, UK Centre for
Fellow of Academy of Medical
Tissue Engineering,
Sciences
University of Manchester
Pro-Vice-Chancellor, and
Professor of Medical
page 28
MRC Travelling Fellow, NIDR
Bethesda, USA
Biochemistry,
University of Manchester.
MARTIN HUMPHRIES
KARL KADLER
RICHARD KAMMERER
1980
BSc University of Manchester
1980
BSc University of Salford
1992
1983
PhD University of Manchester
1984
PhD University of Manchester
National Cancer Institute, NIH, USA
1984
1983
and Howard University Cancer
1995
Wellcome Trust Senior Research
NJ USA
1986
Jefferson University, Philadelphia,
Wellcome Trust Principal Research
PA USA
1989
University of Manchester
2000
Fellow of Academy of
Medical Sciences
2000
1996
Post-doctoral Research Fellow,
Biozentrum, University of Basel,
Switzerland
2000
Wellcome Trust Senior Research
Fellow University of Manchester
2000
PhD Biochemistry, Biozentrum,
University of Basel, Switzerland
Assistant Professor, Thomas
Fellow University of Manchester
Fellow and Professor of Biochemistry
1996
UMDNJ-Rutgers Medical School,
Center, Washington, D.C. USA
1988
Post-doctoral Research Fellow
Dipl Phil Biochemistry, Biozentrum,
University of Basel, Switzerland
Habilitation, University of Basel,
Switzerland
2000
Wellcome Trust Career
Professor of Biochemistry,
Development Fellow,
University of Manchester
University of Manchester
Director, Wellcome Trust Centre for
Cell-Matrix Research,
University of Manchester
CAY KIELTY
ULRIKE MAYER
JOHN SHEEHAN
1978
BSc Kings College, London
1984
Dipl. Chemistry, Albert-Ludwig
1968
1981
PhD University College, London
University Freiburg, Germany
1970
1981
Post-doctoral Research Fellow,
1990
1988
PhD Biochemistry,
Max-Planck-Institute for Biochemistry,
1973
Wellcome Trust Post-doctoral
Martinsried, Germany
1973
1988
Post-doctoral Research Associate,
1993
MRC Senior Research Fellow
Max-Planck-Institute for Biochemistry,
2000
Professor of Medical Biochemistry,
Martinsried, Germany
University of Manchester
2001
1993
Fellow of Academy of
Post-doctoral Research Associate,
and Immunology, Erlangen, Germany
1994
1976
1979
Lecturer in Dental Genetics,
Post-doctoral Research Fellow,
Research Fellow,
University of Lund, Sweden
1981
Post-doctoral Research Fellow,
University of Lancaster
1988
Germany
2000
Post-doctoral Research Fellow,
University of Lancaster
Research Fellow, Max-Planck-Institute
for Biochemistry, Martinsried,
PhD Biophysics, University of Bristol
University of Bristol
Max-Planck-Group for Rheumatology
Medical Sciences
MSc Materials Science,
University of Bristol
University of Manchester
Research Fellow
BSc Physics, University of Leicester
Wellcome Trust Senior Lecturer,
University of Manchester
1998
University of Manchester
Reader in Physiological Biochemistry,
School of Biological Sciences,
University of Manchester
ADRIAN SHUTTLEWORTH
CHARLES STREULI
DANNY TUCKWELL
1964
BSc University of Liverpool
1979
BA University of Cambridge
1986
BSc University of Bristol
1967
PhD University of Liverpool
1982
MA University of Cambridge
1990
PhD University of Nottingham
1967
Post-doctoral Research Fellow,
1983
PhD University of Leicester
1990
Harvard Medical School
1982
Post-doctoral Research Fellow, ICRF,
1968
London
Post-doctoral Research Fellow,
Northwestern University Medical
1985
Lecturer, Dept of Medical
1987
University of Manchester
Senior post-doctoral Scientist,
Berkeley, USA
Manchester
Reader in Medical Biochemistry,
BBSRC Advanced Research Fellow
Lawrence Berkeley Laboratory,
Biochemistry, University of
1996
1997
Post-doctoral Research Fellow,
RPMS, London
School, Chicago
1969
Post-doctoral Research Fellow,
University of Manchester
1992
Wellcome Trust Senior Research
Fellow, University of Manchester
GILLIAN WALLIS
1982
BSc University of Cape Town,
South Africa
1985
PhD University of Cape Town,
South Africa
1985
Senior Research Associate, Dept
of Human Genetics, UCT
1988
Post-doctoral Research Fellow,
University of Washington,
Seattle, USA
1991
Post-doctoral Research Fellow,
University of Manchester
1996
Lecturer Dept of Medicine,
University of Manchester
2000
Senior Lecturer, Dept of Medicine,
University of Manchester
page 29
Staff List January 2002
DEVELOPMENT OFFICER
Linda Green
COMPUTER MANAGER
Adam Huffman
SECRETARY
Carol McMurdo
SENIOR EXPERIMENTAL
OFFICER
Dave Thornton
TECHNICAL STAFF
Susan Allan
Janet Askari
Stephanie Barton
Jane Bradley
Seema Chakravarthi
Sue Craig
Marianne Ellin
Catherine Griffin Jones
Darren Hitchen
Marj Howard
Emma Keevill
Tracy Kilbride
Melanie Klein
Jane Kott
Amanda Lomas
Emma Lowe
Roger Meadows
Kieran Mellody
Amanda Morgan
Eileen Pinnington
Janine Prince
Synnva Ullensvang
Rachel Watkins
CLINICAL FELLOWS AND
LECTURERS
Yvonne Alexander
Chris Brew
Dave Murray
Wang Jing
page 30
POSTDOCTORAL RESEARCH
ASSOCIATES
Adetola Adesida
Nasreen Akhtar
Steve Ball
Mark Bass
Dan Bax
Patrick Buckley
Elizabeth Canty
Kathryn Chapman
Georgina Collett
Maggy Fostier
Laure Garrique-Antar
John Hindson
Catherine Hoare
Nigel Hodson
David Holmes
Karen Johnston
Tom Jowitt
Saiqa Khan
Peter Latham
Magali Le Goff
David Lee
Matthew Leighton
Emma Marshman
Anthea Messent
Zohreh Mostafavi-Pour
Paul Mould
Alan Murdoch
QingQiu Pu
Gareth Pugh
Pooli Ragesekariah
Kirsty Ratcliffe
Susan Richardson
Claire Rock
Matt Rock
Majid Shahbazi
Mike Sherratt
Gail Skinner
Stephen St. George Smith
Toby Starborg
Simon Stephan
Mohammed Tasab
Simon Tew
Pengbo Wang
Mark Warren
Marian Wilkin
RESEARCH ASSISTANTS
Richard Aspinwall
Craig Barley
Tanja Benkert
Carolyn Greig
Jon Humphries
Sara Kirkham
Philip Macdonald
Ngoc-sa Nguyen Huu
Bertrand Raynal
Kristian Spreckley
Neill Turner
Dimitra Valdramidou
Anthony Valentijn
POSTGRADUATE STUDENTS
Helen Attisha
Sarah Bernard
Claire Bithell
Ann-Marie Carbery
Claire Crouchley
Linda Eyers
Emma Gillaspy
Hanane Gouizi
Kirsty Green
Nichola Hartigan
Jenny Higgs
Alexandra Hillebrand
Karen Howson
Lynsey Jenkinson
Claire Johnston
Richard Lumb
Lisa Macrory
Andrew Marson
Sabine Mazaleyrat
Paul McEwan
Lynn McKeown
Vasiliki Petropoulou
Suzannah Phillips
Richard Rauchenberg
Adam Shaw
Laure Sudre
Emlyn Symonds
Mark Travis
Matthew Wakefield
Harriet Watkin
Nadia Zouq
PUBLICATIONS 2000 - 2001
Addinall,S.G., P.S.Mayr, S.Doyle,
J.K.Sheehan, P.G.Woodman and V.J.Allan.
2001. Phosphorylation by cdc2-CyclinB1
kinase releases cytoplasmic dynein from
membranes. J. Biol. Chem.
276:15939-15944.
Alexandrescu,A.T., M.W.Maciejewski,
M.A.Ruegg, J.Engel and R.A.Kammerer.
2001. 1H, 13C and 15N backbone
assignments for the C-terminal globular
domain of agrin. J. Biomol. NMR
20:295-296.
Almond,A., A.Brass and J.K.Sheehan. 2000.
Oligosaccharides as model systems for
understanding water-biopolymer interaction:
hydrated dynamics of a hyaluronan
decamer. J. Phys. Chem. B.
104:5634-5640.
Almond,A. and J.K.Sheehan. 2000.
Glycosaminoglycan conformation: do
aqueous molecular dynamics simulations
agree with x-ray fibre diffraction?
Glycobiology 10:329-338.
Anderton,L. and P.Bishop. 2001. Acute
visual loss following thioridazine overdose.
Am. J. Psychiatry. 158: 818.
Aquilina,A., M.Korda, J.M.Bergelson,
M.J.Humphries, R.Farndale and
D.S.Tuckwell. 2002. A novel gain of function
mutation in the integrin α2 VWFA domain;
European Journal of Biochem. in press
Ashworth,J.L., C.M.Kielty and D.McLeod.
2000. Fibrillin and the eye. Br. J.
Ophthalmol. 84:1312-1317.
Aszódi,A., J.F. Bateman, E.Gustafsson,
R.P.Boot-Handford and R.Fässler. 2000
Mammalian skeletogenesis and extracellular
matrix: What can we learn from knockout
mice? Cell Structure and Function
25:71-82.
Baldock,C., A.J.Koster, U.Ziese, M.J.Rock,
M.J.Sherratt, K.E.Kadler, C.A.Shuttleworth
and C.M.Kielty. 2001. The supramolecular
organization of fibrillin-rich microfibrils. J.
Cell Biol. 152:1045-1056.
Ball,S.G., C.Baldock, C.M.Kielty and
C.A.Shuttleworth. 2001. The role of the C1
and C2 A-domains in type VI collagen
assembly. J. Biol. Chem. 276:7422-7430.
Baron,M., V.O’Leary, D.A.Evans, M.Hicks
and K.Hudson. 2000. Multiple roles of the
Dcdc42 GTPase during wing development in
Drosophila melanogaster. Mol. Gen. Genet.
264:98-104.
Bella,J. and H.M.Berman. 2000. Integrincollagen complex: a metal-glutamate
handshake. Structure Fold. Des.
8:R121-R126.
Bella,J. and M.G.Rossmann. 2000. The
dynamics of receptor recognition by human
rhinoviruses: response. Trends Microbiol.
8:254.
Bella,J. and M.G.Rossmann. 2000. ICAM-1
receptors and cold viruses.
Pharm. Acta Helv. 74:291-297.
Bishop,P.N. 2000. Structural
macromolecules and supramolecular
organisation of the vitreous gel. Prog. Retin.
Eye Res. 19:323-344.
Bishop,PN. 2000. Laser treatments in
age-related macular degeneration.
CE Optometry. 3:100-103.
Bishop,PN., 2000. Matrix metalloproteinases
and their natural inhibitors in fibrovascular
membranes of proliferative diabetic
retinopathy (editorial). Br. J. Ophthalmol.
84:1087-1088.
Biswas,S., F.L.Munier, J.Yardley, N.HartHolden, R.Perveen, P.Cousin, J.E.Sutphin,
B.Noble, M.Batterbury, C.M.Kielty,
A.Hackett, R.Bonshek, A.Ridgway,
D.McLeod, V.C.Sheffield, E.M.Stone,
D.F.Schorderet and G.C.M.Black. 2001.
Missense mutations in COL8A2, the gene
encoding the α 2 chain of type VIII collagen,
cause two forms of corneal endothelial
dystrophy. Hum. Mol. Genet.
10: 2415-2423.
Bos,K.J., D.F.Holmes, K.E.Kadler,
D.McLeod, N.P.Morris and P.N.Bishop.
2001. Axial structure of the heterotypic
collagen fibrils of vitreous humour and
cartilage. J. Mol. Biol. 306:1011-1022.
Bos,K.J., D.F.Holmes, R.S.Meadows,
K.E.Kadler, D.McLeod and P.N.Bishop.
2001. Collagen fibril organisation in
mammalian vitreous by freeze etch/rotary
shadowing electron microscopy. Micron
32:301-306.
Bottomley,M.J., M.R.Batten, R.A.Lumb and
N.J.Bulleid. 2001. Quality control in the
endoplasmic reticulum. PDI mediates the
ER retention of unassembled procollagen Cpropeptides. Curr. Biol. 11:1114-1118.
Boudko,S., S.Frank, R.A.Kammerer,
J.Stetefeld, T.Schulthess, R.Landwehr,
A.Lustig, H.P.Bächinger and J.Engel. 2002
Nucleation and propagation of the collagen
triple helix in single-chain and trimerized
peptides: transition from 3rd to 1st order
kinetics. J. Mol. Biol., in press.
Briggs,M.D. 2000. Screening for mutations
in cartilage ECM genes. Methods Mol. Biol.
139:133-145.
Briggs,M.D. and Chapman,K.L. 2002.
Pseudoachondroplasia and multiple
epiphyseal dysplasia: mutation review,
molecular interactions and genotypephenotype correlation. Human Mutation.
in press.
Brookman,J.L., G.Mennim, A.P.Trinci,
M.K.Theodorou and D.S.Tuckwell. 2000.
Identification and characterization of
anaerobic gut fungi using molecular
methodologies based on ribosomal ITS1
and 18S rRNA. Microbiology 146: 393-403.
Bulleid,N.J., D.C.John and K.E.Kadler. 2000.
Recombinant expression systems for the
production of collagen. Biochem. Soc.
Trans. 28:350-353.
Burkhard,P., R.A.Kammerer, M.O.Steinmetz,
G.P.Bourenkov and U.Aebi. 2000. The
coiled-coil trigger site of the rod domain of
cortexillin I unveils a distinct network of
interhelical and intrahelical salt bridges.
Structure Fold. Des. 8:223-230.
Cabibbo,A., M.Pagani, M.Fabbri, M.Rocchi,
M.R.Farmery, N.J.Bulleid and R.Sitia. 2000.
ERO1-L, a human protein that favors
disulfide bond formation in the endoplasmic
reticulum. J. Biol. Chem. 275:4827-4833.
Canfield,A.E., M.J.Doherty, V.Kelly,
B.Newman, C.Farrington, M.E.Grant and
R.P.Boot-Handford. 2000. Matrix Gla protein
is differentially expressed during the
deposition of a calcified matrix by vascular
pericytes. FEBS Lett. 487:267-271.
Canfield,A.E., M.J.Doherty and B.A.Ashton.
2000. Osteogenic potential of vascular
pericytes. In:Bone Engineering Eds. J.E.
Davies. em squared incorporated, Toronto,
Canada, pp143-151.
Canfield,A.E., M.J.Doherty, A.C.Wood,
C.Farrington, B.Ashton, N.Begum, B.Harvey,
A.Poole, M.E.Grant and R.P.Boot-Handford.
2000. Role of pericytes in vascular
calcification: a review. Z. Kardiol.
89 Suppl 2:20-27.
Canfield,A.E., C.Farrington, M.D.Dziobon,
R.P.Boot-Handford, A.M.Heagerty,
S.N.Kumar and I.S.D.Roberts. 2002. The
involvement of matrix glycoproteins in
vascular calcification and fibrosis: an
immunohistochemical study. J. Path.
196: 228-234
Chapman,K.L., G.R.Mortier, K.Chapman,
J.Loughlin, M.E.Grant and M.D.Briggs.
2001. Mutations in the region encoding the
von Willebrand factor A domain of matrilin-3
are associated with multiple epiphyseal
dysplasia. Nature Genet. 28:393-396.
Chaudhry,S.S., J.Gazzard, C.Baldock,
J.Dixon, M.J.Rock, G.C.Skinner, K.P.Steel,
C.M.Kielty and M.J.Dixon. 2001. Mutation of
the gene encoding fibrillin-2 results in
syndactyly in mice. Hum. Mol. Genet.
10:835-843.
Cheung,J.O., M.C.Hillarby, S.Ayad,
J.A.Hoyland, C.J.Jones, J.Denton,
J.T.Thomas, G.A.Wallis and M.E.Grant.
2001. A novel cell culture model of
chondrocyte differentiation during
mammalian endochondral ossification. J.
Bone Miner. Res. 16:309-318.
page 31
Clark,K., P.Newham, L.Burrows, J.A.Askari
and M.J.Humphries. 2000. Production of
recombinant soluble human integrin α4β1.
FEBS Lett. 471:182-186.
Coe,A.P., J.A.Askari, A.D.Kline,
M.K.Robinson, H.Kirby, P.E.Stephens and
M.J.Humphries. 2001. Generation of a
minimal α5β1 integrin-Fc fragment. J. Biol.
Chem. 276:35854-35866.
Costes,S., C.H.Streuli and M.H.BarcellosHoff. 2000. Quantitative image analysis of
laminin immunoreactivity in skin basement
membrane irradiated with 1 GeV/nucleon
iron particles. Radiat. Res. 154:389-397.
Day,A.J. and Sheehan,J.K. 2001.
Hyaluronan: polysaccharide chaos to protein
organisation. Current Opinion in Structural
Biology. 11:617-622.
Deutzmann,R., S.Fowler, X.Zhang, K.Boone,
S.Dexter, R.P.Boot-Handford, R.Rachel and
M.P.Sarras, Jr. 2000. Molecular, biochemical
and functional analysis of a novel and
developmentally important fibrillar collagen
(Hcol-I) in hydra. Development
127:4669-4680.
Engel,J. and R.A.Kammerer. 2000. What are
oligomerization domains good for? Matrix
Biol. 19:283-288.
Farmery,M.R., S.Allen, A.J.Allen and
N.J.Bulleid. 2000. The role of ERp57 in
disulfide bond formation during the
assembly of major histocompatibility
complex class I in a synchronized
semipermeabilized cell translation system. J.
Biol. Chem. 275:14933-14938.
Farmery,M.R. and N.J.Bulleid. 2001. Major
histocompatibility class I folding, assembly
and degradation: a paradigm for two-stage
quality control in the endoplasmic reticulum.
Prog. Nucleic Acid Res. Mol. Biol.
67:235-268.
Farrington,C., A.M.Heagerty and
A.E.Canfield. 2000. Thrombospondin
expression by calcifying and non-calcifying
vascular cells. In: Chemistry and Biology
of Mineralised Tissues. Eds.Goldberg, M.
Boskey, A & Robinson, C. American
Academy of Orthopaedic Surgeons.
pp 377-381.
Firth,L. J.Manchester, J.A.Lorenzen,
M.Baron and L.A.Perkins. 2000.
Identification of genomic regions that
interact with a viable allele of the Drosophila
protein tyrosine phosphatase corkscrew.
Genetics 156:733-748.
Fosang,A.J., K.Last, H.Stanton, D.B.Weeks,
I.K.Campbell, T.E.Hardingham and
R.M.Hembry. 2000. Generation and novel
distribution of matrix metalloproteinasederived aggrecan fragments in porcine
cartilage explants. J. Biol. Chem.
275:33027-33037.
page 32
Fowler,S.J., S.Jose, X.Zhang, R.Deutzmann,
M.P.Sarras, Jr. and R.P.Boot-Handford.
2000. Characterization of hydra type IV
collagen. Type IV collagen is essential for
head regeneration and its expression is upregulated upon exposure to glucose. J. Biol.
Chem. 275:39589-39599.
Frank,S., R.A.Kammerer, S.Hellstern,
S.Pegoraro, J.Stetefeld, A.Lustig, L.Moroder
and J.Engel. 2000. Toward a high-resolution
structure of phospholamban: design of
soluble transmembrane domain mutants.
Biochemistry 39:6825-6831.
Frank,S., A.Lustig, T.Schulthess, J.Engel
and R.A.Kammerer. 2000. A distinct sevenresidue trigger sequence is indispensable
for proper coiled-coil formation of the human
macrophage scavenger receptor
oligomerization domain. J. Biol. Chem.
275:11672-11677.
Frank,S., R.A.Kammerer, D.Mechling,
T.Schulthess, R.Landwehr, J.Bann, Y.Guo,
A.Lustig, H.P.Bachinger and J.Engel. 2001.
Stabilization of short collagen-like triple
helices by protein engineering. J. Mol. Biol.
308:1081-1089.
Garrigue-Antar,L., C.Barker and K.E.Kadler.
2001. Identification of amino acid residues in
bone morphogenetic protein-1 important for
procollagen C-proteinase activity. J. Biol.
Chem. 276:26237-26242.
Gilmore,A.P., A.D.Metcalfe, L.H.Romer and
C.H.Streuli. 2000. Integrin-mediated survival
signals regulate the apoptotic function of
Bax through its conformation and subcellular
localization. J. Cell Biol. 149:431-446.
Gilmore, A.P. and C.H.Streuli. 2002.
Analysing apoptosis in cultured epithelial
cells. Methods Mol. Biol. in press.
Graham,H.K., D.F.Holmes, R.B.Watson and
K.E.Kadler. 2000. Identification of collagen
fibril fusion during vertebrate tendon
morphogenesis. The process relies on
unipolar fibrils and is regulated by collagenproteoglycan interaction. J. Mol. Biol.
295:891-902.
Grant,M.E. 2001. Basement Membranes:
more matrix than membrane. Biological
Sciences Review 13:36-39.
Green,H., A.E.Canfield, M.C.Hillarby,
M.E.Grant, R.P.Boot-Handford,
A.J.Freemont and G.A.Wallis. 2000. The
ribosomal protein QM is expressed
differentially during vertebrate endochondral
bone development. J. Bone Miner. Res.
15:1066-1075.
Gregory,C.A., B.Zabel, M.E.Grant, R.P.BootHandford and G.A.Wallis. 2000. Equal
expression of type X collagen mRNA from
mutant and wild type COL10A1 alleles in
growth plate cartilage from a patient with
metaphyseal chondrodysplasia type Schmid.
J. Med. Genet. 37:627-629.
Gribbon,P., B.C.Heng and T.E.Hardingham.
2000. The analysis of intermolecular
interactions in concentrated hyaluronan
solutions suggest no evidence for chainchain association. Biochem. J.
350: 329-335.
Gribbon,P., B.C.Heng and T.E.Hardingham.
2001. Novel confocal-FRAP analysis of
carbohydrate-protein interactions within the
extracellular matrix. Methods Mol. Biol.
171:487-494.
Guo,Y., R.A.Kammerer and J.Engel. 2000.
The unusually stable coiled-coil domain of
COMP exhibits cold and heat denaturation
in 4-6 M guanidinium chloride. Biophys.
Chem. 85:179-186.
Hadjiloucas,I., A.P.Gilmore, N.J.Bundred and
C.H.Streuli. 2001. Assessment of apoptosis
in human breast tissue using an antibody
against the active form of caspase 3:
relation to histopathological characteristics.
Brit. J. Cancer 85: 1522-1526.
Hardingham,T.E. and P.Gribbon. 2000.
Confocal-FRAP analysis of ECM molecular
interactions. In: Extracellular Matrix
Protocols. Eds. Streuli C., Grant M.E.
Humana Press pp. 83-93.
He,Y., V.D.Bowman, S.Mueller, C.M.Bator,
J.Bella, X.Peng, T.S.Baker, E.Wimmer,
R.J.Kuhn and M.G.Rossmann. 2000.
Interaction of the poliovirus receptor with
poliovirus. Proc. Natl. Acad. Sci. U. S. A
97:79-84.
Hendershot,L.M. and N.J.Bulleid. 2000.
Protein-specific chaperones: the role of
hsp47 begins to gel. Curr. Biol.
10:R912-R915.
Hicks,M.S., V.O’Leary, M.Wilkin, S.E.Bee,
M.J.Humphries and M.Baron. 2001.
DrhoGEF3 encodes a new Drosophila DH
domain protein that exhibits a highly
dynamic embryonic expression pattern. Dev.
Genes Evol. 211:263-267.
Hill,J., M.Lewis, P.Mills and C.M.Kielty. 2002.
Effects of pulsed short-wave diathermy on
human fibroblast proliferation. Arch. Phys.
Med. Rehabilitation in press.
Holden,P., R.S.Meadows, K.L.Chapman,
M.E.Grant, K.E.Kadler and M.D.Briggs.
2001. Cartilage oligomeric matrix protein
interacts with type IX collagen and
disruptions to these interactions identify a
pathogenetic mechanism in a bone
dysplasia family. J. Biol. Chem.
276:6046-6055.
Holmes,D.F., C.J.Gilpin, C.Baldock, U.Ziese,
A.J.Koster and K.E.Kadler. 2001. Corneal
collagen fibril structure in three dimensions:
Structural insights into fibril assembly,
mechanical properties and tissue
organization. Proc. Natl. Acad. Sci. U. S. A
98:7307-7312.
Holmes,D.F., H.K.Graham, J.A.Trotter and
K.E.Kadler. 2001. STEM/TEM studies of
collagen fibril assembly. Micron.
32:273-285.
Humphries,J.D., J.A.Askari, X.P.Zhang,
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