DFG Research Center for Experimental Biomedicine,
University of Würzburg
Annual Report
2006
Foreword
Prof. Dr. Martin Lohse
I am pleased to present the 2006 Annual Report of the Rudolf Virchow Center, the DFG Research Center for
Experimental Biomedicine of the University of Würzburg. This center is devoted to research, teaching and
public education. Its focus is on target proteins – proteins that exert key regulatory functions in a cell and
may, therefore, serve as targets for diagnostic or therapeutic purposes. These proteins are analyzed with
various sophisticated techniques and from different perspectives, ranging from their molecular structure
and mechanisms to their role in pathophysiological states, notably cardiovascular diseases and cancer.
The Rudolf Virchow Center is one of the first “Centers of Excellence” funded by the Deutsche Forschungsgemeinschaft, the German Research Foundation. Rather than enlarging existing structures, it was started in
2002 from scratch to provide a new venue for scientific excellence. Today, it houses twelve research groups
in four areas, the Junior Research Groups, the Core Center, the Research Professors and the associated
Bio-Imaging Center (funded by the State of Bavaria and the university). In addition, the new RVZ Network
funds collaborative projects with six extramural groups.
The Rudolf Virchow Center plays an active role in teaching by coordinating a research-oriented BSc/MScprogram in biomedicine as well as the Graduate School for Biomedicine together with the Faculties of
Sciences and of Medicine, and it contributes a special research focus to these programs. The biomedicine
program continues to attract highly talented students. This year saw our first MSc-graduates together
with the third year of BSc-graduates. Our graduate school was integrated into the much larger Graduate
School for Life Sciences and won funding by the “Excellence Initiative of the German Federal and State
Government”.
Last but certainly not least our Public Science Center communicates our research and raises interest in
biomedical science and research. In particular, it reaches out to elementary and high school children. The
project “Rudis Forschercamp” won the prestigious PR Report Award, while the project “ForscherReporter”
is supported by “Lernort Labor”, a BMBF-project.
The year 2006 was an important and fruitful one. It showed that the Rudolf Virchow Center has generated
a vivid research environment. Its increasing international recognition is documented by numerous scientific publications, success in obtaining extramural funding, its integration into national and international
research networks, multiple productive collaborations with companies, and in attracting young researchers
from all over the world.
Next year, the first generation of group leaders will assume new positions here or elsewhere – all of them
having gained professorial positions, either in Würzburg in the form of tenure track positions, or at other
top institutions elsewhere. The first group leader from the second generation will be Stephan Kissler, who
works on lentiviral techniques for in vivo gene silencing in order to identify proteins important for immune
tolerance and diseases. Coming from the Massachusetts Institute of Technology, he accepted a position at
the Rudolf Virchow Center in the light of a competing offer from Harvard University. We are confident that
the new generation of group leaders will continue to make the Rudolf Virchow Center a place of excellence
in biomedical research as well as teaching.
I hope you will enjoy reading our 2006 Annual Report.
Chairman Rudolf Virchow Center/
DFG Research Center for Experimental Biomedicine,
University of Würzburg
Contents
Rudolf Virchow Center
4
Research Activities
12
Junior Research Groups
14
Stefan Engelhardt
Bernhard Nieswandt
Thorsten Stiewe
Stephan Kissler
14
16
18
20
Core Center
22
Gregory Harms
Caroline Kisker
Hermann Schindelin
Albert Sickmann
22
24
26
28
Research Professors
30
Peter Friedl
Michael P. Schön
30
32
RVZ Network
34
Utz Fischer
Manfred Gessler
Thomas Hünig
Thomas Müller
Manfred Schartl
Walter Sebald
34
36
38
40
42
44
Bio-Imaging Center
46
Martin Lohse
Stephan Sigrist
46
48
Teaching & Training
50
Training Activities
52
Public Science Center
56
Appendix
58
Executive Committees and Scientific Members
Academic Members and Supporting Staff
Visiting Scientists
Teaching Committees and International
Graduate School Board
Bachelor and Master theses of the
Undergraduate Program in Biomedicine
PhD theses of the Graduate Program “Target Proteins“
Publications 2006
58
59
62
Imprint
75
63
64
67
68
3
Rudolf Virchow Center
An Overview
Framework of the Rudolf Virchow Center
The Rudolf Virchow Center is composed of different elements:
an Institute for Junior Research Groups, providing junior scientists with
funds to establish an independent research team, encompassing both
DFG-funded and third party-funded groups, with the option of extension
into research professorships or directly into tenure,
a Core Center, comprising groups that develop and utilize research
methods including structural biology, protein mass spectrometry, molecular microscopy, DNA-array and transgenic technologies, and also
provide these methods for collaborative projects,
Research Professorships, enabling successful junior or established
researchers to concentrate on a five-year, high-risk project, including
clinical research professorships,
a Bachelor and Masters Program in Biomedicine, with a focus on research training, and laboratory work to train future generations of
scientists,
a Graduate School for Biomedicine, developed from established interdisciplinary programs and in fall 2006 integrated into the new
Graduate School for Life Sciences,
a Public Science Center for information and teaching of biomedicine to
the public.
In 2006, the RVZ Network was added, a program that funds highrisk
projects of established researchers in Würzburg in collaboration with
groups at the Rudolf Virchow Center.
Closely associated with the Rudolf Virchow Center, the Bio-Imaging Center,
initiated in 2005, comprises research groups funded by the State of
Bavaria and the University of Würzburg in order to develop and apply
imaging techniques for target proteins.
4
The Rudolf Virchow Center is one of the
first three DFG Research Centers, approved
in 2001 and founded in January 2002. The
stated goal of our original application in
2001 was the creation of a new Research
Center for Experimental Biomedicine at the
interface between medicine and the natural
sciences. Today, the Rudolf Virchow Center
has become an independent entity of the
University of Würzburg that focuses on
“target proteins”. These proteins exert key
regulatory roles in a cell and their proper
function is, therefore, essential. Important
examples are receptors and other signaling
proteins at the cell surface and proteins
that bind to nucleic acids. They play an
important role in cardiovascular diseases
and in cancer, two major health problems
in our society.
Rather than simply extending already
existing institutions, the Rudolf Virchow
Center intended to take a more innovative, but also more risky approach which
involved the creation of a completely new
entity: A new research and teaching center
under a common roof. Right from the beginning, our purpose was not only to attract
excellent established scientists as Research
Professors to work on high-risk projects,
but also to provide highly promising junior
researchers with the possibility to work
independently. Additionally, a Core Center
with groups developing and utilizing new
and expensive technologies in the area of
optical microscopy, mass spectrometry and
X-ray crystallography was created. These
groups not only pursue their own research
programs, but also serve as a backbone for
collaborative projects within the center and
for biomedical research at the university
in general.
In the meantime, these structures have
evolved into a successful and fruitful environment for cutting edge research in the
center itself and in national as well as international collaborations. The scientific
success of all Junior Research Groups has
propelled them into independent careers:
all of them have developed internationally
visible research projects and all of them
have been offered professorial positions
quite early during their 5-year period at the
Rudolf Virchow Center.
In all its elements, the Rudolf Virchow
Center has been guided by the successful
principle to give talented scientists the opportunity to realize their scientific dreams.
Thus, while the general topics of research
are defined by the overall theme, it is the
primary goal of this center to attract and to
support the very best scientists.
Training the next generation of scientists
Attracting the best means attracting them
early. This is why, in addition to research,
we also established programs for young
children, high school students and undergraduate as well as graduate students.
In combining these elements, the Rudolf
Virchow Center plays a role at all stages of
academic education and careers.
excellent students, allowing us to be highly
selective since there are on average ~30
applicants per slot. In 2006, the first students received their MSc diplomas (all of
them with excellent results), while already
the third group of BSc students completed
their studies. We are excited to see that most
of them continue their careers in science.
Structured graduate training also helps
to attract good graduate students and offers them what they need for a career in
science. To this end, the Rudolf Virchow
Center has initiated together with other
graduate programs the foundation of the
Graduate School for Biomedicine. The
goal is to offer training programs (laboratory courses, soft skills and seminars) that
complement their research projects. The
year 2006 witnessed a major re-structuring
in order to accommodate the increasing
number of graduate programs that participate in this effort. A new Graduate School
for Life Sciences, initiated and directed
by members of the Rudolf Virchow Center
(Dean: Markus Riederer), was formed and
won support by the national “Excellence
Initiative“. The first interviews with applicants from all over the world have just
been conducted.
Attracting people to science
In our concept of attracting the best people
to science we start with the very young. Our
Public Science Center serves to communicate
our research to the public and to enhance
the direct dialog between scientists and
the public in order to explain the needs,
problems and opportunities of biomedical
research and to maintain public support. In
this context, working with young people is
an integral part of the overall idea of the
Rudolf Virchow Center.
In our attempts to raise interest in science, we created several new programs for
children and high school students. More
than 300 children already participated in
Rudis Forschercamp, where schoolchildren
aged 8 to 12 pursue simple and yet fundamental experiments in biology, medicine,
physics and chemistry. This project won the
prestigious PR Report award. In its third
year, demand still remains high, and children who have been in the program now
ask for follow-up projects.
For high school students aged 16 to 19
the Public Science Center established the
new project ForscherReporter, a BMBFfunded project that allows students to carry out experimental work, but in addition
to observe scientists at the Rudolf Virchow
Center from the perspective of a reporter.
This project, conducted in collaboration
with the Bayerischer Rundfunk, has so far
reached about a third of the high schools
in the Würzburg area, and evaluations show
very positive responses.
The project Rudolf Virchow Paten involves
mentoring of individual projects in the
“Jugend forscht” competition. This year,
our “godchild“ Constanze Rieckmann
achieved the first place with a project on
wound healing.
Undergraduate and graduate training
A key element of the Rudolf Virchow Center is the close combination of research
with undergraduate and graduate training.
This sets it apart from similarly equipped
non-university institutes. The purpose of
our contribution to teaching is to attract
talented students and to offer early participation in cutting edge research. Therefore,
new forms of undergraduate and graduate
training were developed together with the
Faculties of Sciences and Medicine.
A new research-oriented undergraduate
BSc/MSc program “Biomedicine“ was established and is coordinated by the Rudolf
Virchow Center. This program is aimed at
future scientists. Therefore, the students
are integrated early into the research environment of the Rudolf Virchow Center as
well as other research institutes. The program continues to attract large numbers of
High school students explore science by doing experiments on their own and by producing a radio
report for publication on the “ForscherReporter” web site and for presentation at their school.
5
Rudolf Virchow Center
Research Program
Target proteins
Target proteins are at the heart of research in the Rudolf Virchow Center.
These are proteins that act as key regulators of biological functions and may,
at least potentially, serve diagnostic or
therapeutic purposes. The importance of
target proteins results from the fact that
most diseases are caused by malfunctioning proteins. Two types of target proteins
of particular biomedical importance constitute the focus of the Rudolf Virchow
Center: cell surface receptors and nucleic
acid binding proteins. These proteins are
analyzed at four levels of complexity:
molecular structure and function, biochemical mechanisms, cellular responses,
and (patho)physiological roles. To fully
understand a protein it has to be studied
at all of these levels and the results have
to be integrated into a common framework. These studies constitute the core of
our research.
Fig. 1:
Target proteins investigated
at the Rudolf Virchow Center.
Cell surface receptors
Receptors at the cell surface sense the environment and trigger cellular responses;
they mediate cellular interactions as well
as cell-cell communication. Projects in this
area deal with key functions of receptors:
ligand binding, protein-protein interactions and downstream signaling, and include heptahelical (G-protein-coupled) as
well as single pass receptors of the bone
morphogenetic protein receptor family, integrins and selectins, ligand-gated
channels, and the cell surface receptors
6
of platelets, cardiomyocytes, T cells and
neurons. These proteins often function as
part of larger assemblies that are required
for proper functioning, and the study of
the resulting supramolecular complexes
becomes increasingly important.
A highlight from this research area is a
project investigating the organization of
receptors and signaling proteins in a synapse. Synapses are contact sites between
neurons and their recipient cells – for example a muscle –, where chemical media-
tors are secreted from the neuron’s “active
zone” and excite (or inhibit) the recipient
cell via specific receptors on its surface. The
group of Stephan Sigrist has discovered a
protein in Drosophila, called “Bruchpilot”
(because flies with reduced levels of this
protein cannot fly properly), which appears
to organize the presynaptic “active zone”
that contacts and excites the postsynaptic
“receptor zone”. In collaboration with the
group of Stefan Hell (Max Planck Institute
for Biophysical Chemistry and DFG Research
Center “Molecular Physiology of the Brain”
in Göttingen), the new STED-microscopy
technique with a resolution well below the
classical limit of 0.2 µm was used to visualize the molecular organization of the
synapse (Fig. 2). This technique promises
to carry optical microscopy into the realm
of individual protein complexes and will be
an important method in coming years.
Fig. 2:
STED microscopy uncovers supramolecular architectures at synaptic membranes. A: Organization of the junction between a neuron and a
muscle cell, the neuromuscular synapse, in Drosophila. Left: A synaptic “bouton” with multiple synapses, composed of presynaptic “active
zones” (green: staining “Bruchpilot”) and postsynaptic glutamate receptors (red). B: Conventional confocal versus C: STED-microscopy of
Bruchpilot in synapses. Scale bar is 1 µm.
Nucleic acid binding proteins
Nucleic acid binding proteins are involved
in maintaining the structural integrity of
the genome and in regulating gene expression and the cell cycle, cell death and differentiation. Examples of proteins studied
in this area comprise the machinery performing repair of damaged DNA, the p53
tumor suppressor family of DNA-binding
proteins, and the RNA-binding proteins
that mediate the so-called TOP response of
translational regulation.
As in the case of cell surface receptors,
the analysis of protein-nucleic acid complexes is of central importance. Studies on
the mechanisms of DNA repair illustrate
how the combination of biochemical approaches and X-ray crystallography leads
to a mechanistic understanding. DNA is
constantly damaged, for example by UV-irradiation or reactive chemicals, and thus
it is in constant need of repair. Nucleotide
excision repair is the most versatile repair
system which repairs a broad range of DNA
damages and is present in eukaryotic and
bacterial cells. It involves a sequence of
steps: the recognition of DNA damage, followed by incision, removal of the damaged
DNA segment and subsequent repair. The
group of Caroline Kisker utilizes assays for
the individual biochemical steps and has
elucidated the structures of the proteins
that carry them out. Future studies will
now attempt to combine the pieces of the
puzzle and to obtain the structures of the
higher order complexes that are formed in
the repair cascade (Fig. 3).
Fig. 3:
Model for the interaction between UvrB (gray) and UvrC (ribbon presentation)
leading to the successful incision of the damaged DNA strand.
7
Rudolf Virchow Center
Research Program
Biomedical focus
The wide-spread distribution of most target
proteins in the body often leads to their
involvement in several, yet very different
physiological functions. Thus, the study of
a given protein can provide insights into
a whole variety of physiological processes
and diseases. For example, interactions of
cell surface selectins and integrins mediate interactions between diverse types of
cells and regulate both cell adhesion and
motility. Thus, similar basic processes underlie the adhesion of platelets to blood
vessels, the recruitment of immune cells in
inflammation, and the cell motility in cancer metastasis. This means that the study
of a single target protein leads into new
venues of disease-oriented research, which
have not been anticipated before.
To focus the biomedical impact of our research, we concentrate on two major areas:
cardiovascular diseases and cancer. These
topics are not only of great medical importance, but they are key research topics in
Würzburg and thus offer ample opportunities for collaborations.
In the cardiovascular field, platelets are
our most intensely studied cell type. Research groups in the Rudolf Virchow Center (Bernhard Nieswandt, Albert Sickmann)
have teamed up with groups in Clinical
Biochemistry (Ulrich Walter) and Bioinformatics (Thomas Dandekar) in the “Virtual
Platelet” project. This collaboration will result in an inventory of all platelet proteins,
in particular of its membrane proteome,
the analysis of protein modifications (e.g.
phosphorylation), and the modeling of
functional protein networks in these tiny
cells that serve to stop bleeding, but also
play a role in atherosclerosis and thus can
cause, among other diseases, myocardial
infarction and stroke.
Mechanisms of cancer development are
studied at the molecular level (DNA damage and repair, Caroline Kisker), at the
level of cellular decisions about cell death,
survival and differentiation (p53 protein
family, Thorsten Stiewe), cell-cell interactions in cancer cell defense and invasion
(Michael Schön), and finally cell migration
8
as the basis of metastasis (Peter Friedl).
The analysis of mechanisms in melanoma
development require both relevant animal
models (Manfred Schartl) as well as clinical
expertise and direct access to tissues from
patients, assured through the research
professorships of Peter Friedl and Michael
Schön, who also hold appointments in the
Department of Dermatology. The combined
efforts to understand the molecular steps
of cancer progression and resistance to anticancer drugs have led to novel therapeutic strategies, including inhibition of the
p53 antagonist protein ∆Np73, anti-tumor
small molecule immune response modifiers
and the recently patented strategy to inhibit IKKβ signaling.
Central technologies
One of the central aims of the Rudolf Virchow Center was to provide a backbone for
the biomedical community in Würzburg, in
a way that is achieved in Germany most
commonly only in non-university institutions. Key technologies established in our
research groups comprise structural biology/X-ray crystallography (Caroline Kisker,
Hermann Schindelin and Thomas Müller,
Walter Sebald), proteomics and mass spectrometry (Albert Sickmann) and molecular
microscopy (Gregory Harms, Peter Friedl).
In addition, transgenic mouse technologies are offered in collaboration with the
Institutes of Neurobiology (Bettina Holtmann, knockout mice) and Pharmacology
and Toxicology (Eva Schmitteckert, transgenic mice). A DNA array unit was established in collaboration with the Interdisciplinary Center for Clinical Research and
provides access to custom-made as well as
commercial array analyses (Andreas Rosenwald).
In addition, the newly established BioImaging Center groups, currently Stephan
Sigrist and Martin Lohse and two new
groups to be established in 2007 and 2008,
will complement existing optical imaging
technologies. Optical microscopy technologies available today range from single
molecule studies to fluorescence recovery
(FRAP), fluorescence resonance energy
transfer (FRET), selective plane illumination (SPIM), and fluorescence lifetime imaging (FLIM). All of these technologies are
used intensively by research groups within
the center, but also by other groups in
Würzburg and elsewhere.
Partnering with industry
Collaborations with companies of various
sizes and product portfolios are an important part of research at the Rudolf Virchow
Center. They are organized into multiple
forms. One research group on cardiac target proteins is fully funded by the biotech
company Procorde/Trigen, Martinsried,
Sanofi-Aventis, Frankfurt, and the Bavarian Ministry of Economics. Other groups
receive specific funding from companies,
mostly in the context of projects funded
by the Federal Ministry of Education and
Research (BMBF) and similar sources, but
also through direct collaborations.
Several new microscopic instruments are
being co-developed with optical companies. For example, a multi-photon platform
for optical imaging in vivo was constructed
with LaVision BioTec, Bielefeld, and new
detectors are currently being developed
as a RVZ Network project. A new type of
microscopy platform, called iMIC, is engineered with Till Photonics, Gräfelfing. A
new total internal reflection (TIRF) microscope for FRET analyses at the cell surface
is developed with Leica, Wetzlar. In 2007,
we plan to implement the new high-resolution STED microscopy, again together with
Leica. Similarly, the mass spectrometry
group enjoys close contacts with multiple
companies in order to stay at the forefront
of new developments; an example is the
development of new HPLC column materials
with Dinoex, Sunnyvale, CA. Some of our
research projects have led to common patents with biotech and pharmaceutical companies, most notably with Procorde/Trigen,
Sanofi-Aventis and Bayer.
Rudolf Virchow Center
Events
Events
Many events were organized for the general public, for students and for scientists.
These include the “Long Nights of Science“
and tours of the Rudolf Virchow Center for
various groups. The Graduate Day 2006
featured Parliamentary State Secretary
Andreas Storm of the Federal Ministry of
Education and Research. At the eve of
the Graduate Day , a screening of the movie
„Sleeper“ was arranged followed by a discussion with its director Benjamin Heisenberg about competition and ethics of conduct in science.
The new building for the Rudolf Virchow
Center and the Center for Infectious Disease Research is now well under way,
costs amounting of 62.5 million €. Almost
10,000 m2 space will be generated by a total conversion of the old surgical hospital.
A celebration on July 28th, 2006, marked
the end of the deconstruction phase and
the beginning of new constructions.
The new building for the Rudolf Virchow Center and the Center for Infectious Disease Research is under
construction.
A number of symposia and courses were
organized by the Rudolf Virchow Center
in 2006. Among recent conferences, our
series of “Dynamic Microscopy Symposia“
was continued with a three-day symposium
(October 9th-11th, 2006) combining a day
of lectures by renowned speakers from all
over the world with practical workshop
and demonstrator days, where all leading microscopy companies presented new
products and developments. For the third
time, our annual “Proteomics Workshop“
(September 17th-20th, 2006) provided an
introduction into the theory and practical
aspects of these techniques, with external
experts and tutors from the Rudolf Virchow
Center. The Rudolf Virchow Center also tutored the graduate students of the Graduate School to organize their student symposium “From Bench to Bedside - Molecular
Approaches for Novel Therapies” (October
23rd, 2006).
For the third year the Rudolf Virchow Center invited scientists for two outstanding symposia and workshops: The “Proteomics Workshop” and the “Dynamic Microscopy Symposia”
9
Rudolf Virchow Center
Output and Evaluation
Although individual projects and their results are always the key element in
evaluating our work, it is helpful to look at some commonly used indicators
in order to gauge our overall performance. Apart from the fact that we have
attracted scientists from more than 20 countries, the key figures in such analyses are grants, publications with their bibliometric analysis, awards and the
careers of our scientists.
Funding
Apart from support by the State of Bavaria
and the University of Würzburg, our main
source of support is the core funding by
the DFG with 5 million € per year. This
is complemented by grants from various
sources (Fig. 1) totaling more than 3.5
million € in 2006 (i.e. about 70% of core
funding, up from 50% in 2005).
Fig. 1:
Sources of extramural funding at the Rudolf Virchow Center with a total amount of 3.5 million € in
2006. (Abbreviations: DFG: German Research Foundation, BMBF: Federal Ministry of Education and
Research, BayStMWIVT: Bavarian Ministry of Economic Affairs, Infrastructure, Transport and Technology,
EU: European Union, MPI: Max Planck Institute, Göttingen, IZKF: Interdisziplinary Center for Clinical
Research, NIH: National Institute of Health, USA).
Publications
Most publications from the Rudolf Virchow
Center appear in high profile journals.
Thus, more than 75% of our 211 publications were in journals with an impact factor above 4, including publications in the
most prestigious journals (world average
below 8%). Benchmarking of citations with
institutes of comparable orientation in
Germany and in the US shows similar results as the Beckman Center, Stanford University, and the Max Planck Institutes for
Experimental Medicine, Göttingen, and for
Medical Research, Heidelberg (Fig. 2).
Our “field-normalized impact” RI – a sizeindependent comparator of impact relative to the world average in the same field
(Center for Science and Technology Studies
CWTS, Leiden) – is 2.78 in experimental
and 3.27 in clinical journals. These values
represent the top 1% of biomedical research institutes, well above the range of
large institutions such as the NIH (2.46),
but below higher values for top individual
research groups.
10
Fig. 2:
Benchmarking of citations with institutes of comparable orientation in Germany and in the
US shows similar results. (Abbrevations: RVZ: Rudolf Virchow Center, MPI Göttingen: Max
Planck Institute for Experimental Medicine, Göttingen, MPI Heidelberg: Max Planck Institute
for Medical Research, Heidelberg, Beckmann: Beckman Center, Stanford University, USA)
Collaborations
A large number of collaborations show that
the Rudolf Virchow Center plays an increasing role in the biomedical research community, locally, nationally and in international
networks. Local collaborations indicate that
it fullfils its intended role as a backbone
for biomedical research in Würzburg. Most
major collaborative research projects in the
life sciences in Würzburg involve the active
participation of the Rudolf Virchow Center
(Fig. 3).
To foster collaborations with outside
groups, we have initiated the RVZ Network
in 2006. Following review by the Scientific
Advisory Board high-risk projects are funded, which are carried out by research groups
in Würzburg in collaboration with groups at
the Rudolf Virchow Center. Groups of the
Rudolf Virchow Center itself can also apply
for these funds, thus allowing a flexible allocation of resources. Seven such projects
are currently funded.
Fig. 3:
Collaborations of the Rudolf Virchow Center at the University of Würzburg. Shown is the active participation (i.e. common
projects with publications and funding) in collaborative projects (DFG funded projects: blue, BMBF funded projects: yellow).
Awards
Science Careers
Members of the Rudolf Virchow Center received numerous prestigious national and
international awards. During the past year
this included the European Young Investigator Award and the Arthur Weber Prize
(Stefan Engelhardt), the Paul Langerhans
Award and the Award of the German Skin
Cancer Foundation (Michael Schön) and the
Bavarian Order of Merit (Martin Lohse).
Many of our members, including all junior
group leaders, have received offers for professorial positions in Germany and abroad.
While some members have left Würzburg,
successful competing offers were made to
Stefan Engelhardt, Martin Lohse, Thomas
Müller, Bernhard Nieswandt and Manfred
Schartl. In doing so, the junior group leaders
Stefan Engelhardt and Bernhard Nieswandt
became associate professors with tenure.
It is too early for a formal evaluation of
the success of our teaching programs by
looking where former students go and how
they fare. However, it is a good sign that
most students and graduates have stayed in
science and have continued their research
in top institutions either here in Würzburg
or in Germany and abroad.
11
Sc ience
Junior Research Groups
Stefan Engelhardt
Bernhard Nieswandt
Thorsten Stiewe
Stephan Kissler
Cardiac Target Proteins
Vascular Biology
Molecular Tumor Biology
Immune Tolerance
Core Center
Gregory Harms
Caroline Kisker
Herrmann Schindelin
Albert Sickmann
Molecular Microscopy
Structural Biology: DNA Repair and Structure-Based Drug Design
Structural Biology: Protein Folding, Function and Degradation
Functional Proteomics
Research Professors
Peter Friedl
Michael P. Schön
Molecular Cell Dynamics
Inflammation and Tumor Biology
RVZ Network Projects
Utz Fischer
Manfred Gessler
Thomas Hünig
Thomas Müller
Manfred Schartl
Walter Sebald
Translational Regulation: TOP-response Proteins
Hey Factors in Cardiac Development
T Cell Surface Proteins
Ligand-Receptor Recognition
Posttranslational Gene Regulation
BMP Receptors Struture and Function
Bio-Imaging Center
Martin Lohse
Stephan Sigrist
12
Receptor-Cyclic Nucleotide Signaling
Synapse Architecture
13
Cardiac Target Proteins-Stefan Engelhardt
E-mail: [email protected]
Phone: +49(0)931 201 487 10
Fax.:
+49(0)931 201 481 23
http://www.rudolf-virchow-zentrum.de/forschung/engelhardt.html
Heart failure is one of the leading causes of death in industrialized countries. Despite some therapeutic
advances in recent years, the incidence of heart failure continues to rise and the five-year survival rate
remains poor. Our work aims to identify intracellular signaling mechanisms mediating the development
of this disease. We focus on mechanisms leading to pathological growth of cardiac myocytes (hypertrophy) and cardiac fibrosis, two key steps in the pathogenesis of heart failure.
MicroRNAs in cardiac disease
A role for caspase-1 in heart failure
These recently discovered modulators of
gene expression putatively regulate translation and stability of a large portion of
eukaryotic mRNAs. The human genome
contains more than 500 genes encoding
microRNAs. The function of microRNAs in
the adult heart is unclear.
Using microRNA Arrays, we identified a
set of microRNAs deregulated in the left
ventricular myocardium during the development of heart failure. After validation
by Northern blot analysis in mouse models
of the disease and in human heart failure,
our current aim is to assign a function to
several of these deregulated microRNAs. We
have successfully generated transgenic mice
with cardiomyocyte-specific overexpression
of a set of cardiac microRNAs which are currently undergoing phenotypic analysis.
Apoptosis of cardiomyocytes increases in
heart failure and has been implicated in
disease progression. The activation of proapoptotic caspases represents a key step in
cardiomyocyte apoptosis. In contrast, the
role of pro-inflammatory caspases (caspases 1, 4, 5, 11, 12) is unclear. Within
this project, we are studying the cardiac
function of caspase-1.
Gene array analysis in a murine heart failure model showed upregulation of myocardial caspase-1. In addition, we found increased expression of caspase-1 protein in
murine and human heart failure. Mice with
cardiomyocyte-specific overexpression of
caspase-1 developed heart failure in the
absence of detectable formation of IL-1β
or IL-18 and inflammation. Transgenic
caspase-1 induced primary cardiomyocyte
Fig. 1:
Deregulation of miR
expression in cardiac failure.
Fig. 2:
Cardiomyocyte apoptosis after ischemia reperfusion depends on Caspase-1. (Merkle et al., Circ Res, 2007)
14
apoptosis before structural and molecular
signs of myocardial remodeling occurred.
In contrast, deletion of endogenous caspase-1 was beneficial in the context of
myocardial infarction-induced heart failure. Furthermore, caspase-1-deficient mice
were protected from ischemia-reperfusioninduced cardiomyocyte apoptosis. Studies
in primary rat cardiomyocytes indicated
that caspase-1 induces cardiomyocyte
apoptosis primarily through activation of
caspases-3 and -9.
In contrast to previous findings, which
imply a pro-inflammatory role of caspase-1,
these data suggest a primary proapoptotic
role for caspase-1 in cardiomyocytes. Our
findings indicate a functional role for caspase-1-mediated myocardial apoptosis contributing to the progression of heart failure.
β-adrenergic signaling in heart failure
Heart failure is typically accompanied by
chronic activation of the sympathetic nervous system and enhanced release of catecholamines. This leads to chronic stimulation of β1- and β2-adrenergic receptors on
cardiac myocytes. β1- and β2-adrenergic receptors (βARs) are known to differentially
regulate cardiomyocyte contraction and
growth. We tested the hypothesis that
these differences are due to spatial compartmentation of the second messenger cAMP.
Using a fluorescent resonance energy transfer (FRET)-based approach, we directly
monitored the spatial and temporal distribution of cAMP in adult cardiomyocytes.
We developed a new cAMP-FRET sensor
(called HCN2-camps) based on a single
cAMP binding domain of the hyperpolarization activated cyclic nucleotide-gated
potassium channel 2 (HCN2). Its cytosolic
distribution, high dynamic range and sensitivity make HCN2-camps particularly
well suited to monitor subcellular localization of cardiomyocyte cAMP. We generated
HCN2-camps transgenic mice and performed
single cell FRET imaging on freshly isolated
cardiomyocytes. Localized β1AR-stimulation generated a cAMP-gradient propagating throughout the cell, whereas local
β2AR-stimulation did not elicit marked
cAMP diffusion.
Our data reveal that in adult cardiac myocytes, β1ARs induce far-reaching cAMP-signals, whereas β2AR-induced cAMP remains
locally confined.
Extramural Funding
Fig. 3:
Temporal and spatial resolution of cardiomyocyte cAMP (Nikolaev et al., Circ Res, 2006).
AG Kardiale Targetproteine:
Wirtschaftsministerium, Aventis, Trigen
IZKF E 25-1
Fondation Leducq (Paris), Transatlantic Network
BMBF: Kompetenznetz Herzinsuffizienz
Deutsche Gesellschaft für Kardiologie
Selected Publications
In addition, we aim to directly assess
activation of the two β-adrenergic receptor
subtypes. We have successfully generated
β1- and β2-adrenergic receptor mutants
that allow real-time determination of receptor activation by FRET. We use fluorescent resonance energy transfer (FRET)based approaches to directly monitor activation of the β1AR and downstream
signaling. While the commonly used βARantagonists metoprolol, bisoprolol and
carvedilol displayed varying degrees of inverse agonism on the Gly389-variant of the
receptor (i.e. actively switch off the β1AR),
surprisingly only carvedilol showed very
specific and marked inverse agonist effects
at the more frequent Arg389-variant. These
specific effects of carvedilol on the β1AR
Arg389-variant were also seen for frequency
control in cardiac myocytes expressing the
two receptor variants. This FRET-sensor permits the direct observation of activation of
the β1-adrenergic receptor in living cells in
real-time. It reveals that β1-adrenergic receptor variants differ dramatically in their
response to diverse β-blockers, with possible consequences for their clinical use.
Cell-cell communication in the mammalian heart
Myocardial hypertrophy and fibrosis are key
findings in the majority of heart failure
patients and likely contribute to progression of the disease. Conditioned medium
from isolated cardiomyocytes exerts strong
growth promoting effects on cardiac fibroblast cell cultures. We hypothesize that yet
unknown secreted factors contribute to
cardiac fibrosis.
We performed a systematic search for
secreted proteins using a genetic yeast
secretion trap to screen a murine cardiac
cDNA-library. Out of 1.7 x 107 screened
yeast transformants we identified 54 cardiac genes comprising a secretion signal.
Among them are well-known genes such
as the atrial-natriuretic peptide but also
genes with so far unknown cardiac expression and/or function. Among those we are
currently focusing on protease inhibitor 16
(PI16). PI16 is a putative 489 amino acid
protein with so far unknown function. It is
strongly upregulated during early stages of
the disease. PI16 is secreted into the culture medium after transfection of neonatal
rat cardiomyocytes and secreted PI16 accumulates extracellularly in the heart.
Fig. 4:
Development of FRET-sensors for the human β1-adrenergic receptor. A naturally occurring polymorphism at
position 389 determines the conformational change upon ligand binding (Rochais et al., J Clin Invest, 2007).
Rochais, F., Vilardaga, J.-P., Nikolaev,
V.O., Bunemann, M., Lohse, M.J., and
Engelhardt, S. (2007) Real-time optical
recording of β1-adrenergic receptor
activation and signaling reveals supersensitivity of the Arg-389 variant to
carvedilol. J Clin Invest, 117, 229-35.
Nikolaev, V.O., Bunemann, M., Schmitteckert, E., Lohse, M.J., and Engelhardt,
S. (2006) Cyclic AMP imaging in adult
cardiac myocytes reveals far-reaching
β1-adrenergic but locally confined
β2-adrenergic receptor-mediated signaling.
Circ Res, 99, 1084-91.
Merkle, S., Frantz, S., Schön, M.P., Bauersachs, J., Buitrago, M., Frost, R.J.A.,
Schmitteckert, E., Lohse, M.J., and Engelhardt, S. (2007) A role for caspase-1
in heart failure. Circ Res, in press.
Hein, P., Rochais, F., Hoffmann, C.,
Dorsch, S., Nikolaev, V.O., Engelhardt, S.,
Berlot, C.H., Lohse, M.J., and Bunemann,
M. (2006) Gs activation is time-limiting
in initiating receptor-mediated signaling. J Biol Chem, 281, 33345-51.
Hallhuber, M., Burkard, N., Wu, R., Buch,
M.H., Engelhardt, S., Hein, L., Neyses, L.,
Schuh, K., and Ritter, O. (2006) Inhibition of nuclear import of calcineurin
prevents myocardial hypertrophy.
Circ Res, 99, 626-35.
15
Vascular Biology-Bernhard Nieswandt
E-mail: [email protected]
Phone: +49(0)931 201 489 96
Fax:
+49(0)931 201 481 23
http://www.rudolf-virchow-zentrum.de/forschung/nieswandt.html
Platelets circulating in the blood survey the integrity of the vascular system. As a response to vascular
injury, platelets rapidly adhere to tissue and to one another to form a platelet plug, which, in combination with the coagulation system, allows re-establishment of normal blood flow in the disrupted
vasculature. However, neither platelets nor other components of the hemostatic process can distinguish between traumatic wounds and lesions that occur in diseased vessels, e.g. upon rupture of an
atherosclerotic plaque. Therefore, uncontrolled platelet activation in diseased vessels may lead to arterial occlusion and infarction of vital organs. Such acute ischemic cardiovascular and cerebrovascular
syndromes are still a major cause of death or serious pathological complications in Western societies.
Our group uses genetically modified mouse lines in combination with disease models to identify new
strategies to inhibit the thrombotic and/or pro-inflammatory activity of the cells while preserving their
hemostatic function.
Reduced thrombus stability in mice lacking the α2A-adrenergic receptor
The Gz-coupled α2Α-adrenergic receptor
(α2A) is known to mediate platelet responses to adrenaline but its role in thrombosis
and hemostasis has been unclear. Therefore, we analyzed α2A-deficient mice using different in vitro and in vivo models.
Adrenaline per se is only a weak platelet
agonist, but it enhances responses of other
platelet activators. Studies of platelet aggregation and activation showed that α2Adeficient platelets in contrast to wild type
platelets, did not respond to adrenaline.
This confirmed that α2A is the essential
receptor for adrenaline on the platelet surface. The importance of α2A in hemostasis
was demonstrated by prolonged bleeding
times in α2A-deficient mice and significant
protection from lethal collagen/adrenaline
induced pulmonary thromboembolism. In
a model of FeCl3-induced injury in mesenteric arterioles, α2A-deficient mice showed
a two-fold increase in embolus formation,
suggesting thrombus instability (Fig. 1).
This was confirmed in a model where the
aorta was mechanically injured and blood
flow was measured with an ultrasonic flow
probe. These results demonstrate that α2A
plays a significant role in thrombus stabilization (Pozgajóva et al., Blood, 2006).
Fig. 1:
Enhanced embolus formation in α2A-deficient mice. A: Reduced arterial occlusion of the α2A-deficient
mice. B: Mice deficient in α2A show increased embolus formation. C: Representative pictures of growing thrombi after FeCl3-induced injury on mesenteric arterioles (Pozgajóva et al., Blood, 2006).
16
Dual role of PKC in thrombus formation
Platelet activation leads to integrin activation and granule secretion (pro-aggregatory activity). Strong rises in intracellular
Ca2+ levels also lead to surface exposure
of negatively charged phosphatidylserine (PS), which represents a surface for
the pro-thrombinase complex and thereby
enhances thrombin generation (pro-coagulant activity). In platelets, the serine
threonine kinase, protein kinase C (PKC),
is involved in integrin activation, but a
negative effect of PKC on calcium mobilization has also been reported. To clarify
the role of PKC in platelet pro-aggregatory and pro-coagulant activity a study
was conducted in collaboration with Johan
Heemskerk, Maastricht University, NL. For
this, Amrei Strehl from our group received
a Marie Curie fellowship to spend 3 months
in Maastricht. PKC was blocked by different
inhibitors and integrin activation, platelet aggregation, PS exposure and calcium
mobilization were monitored after platelet
stimulation with various agonists under
static and flow conditions. Very surprisingly, we found enhanced calcium release
upon PKC inhibition under both conditions,
and similarly PS exposure was increased as
measured with fluorescently labeled annexinV. In contrast, fewer platelets adhered to
the collagen coated surface and no thrombi
were formed. This points to a dual role of
PKC in platelets (Strehl et al., J Biol Chem,
in press.).
An EF hand mutation in Stim1 causes premature platelet activation and bleeding in mice
Stromal interaction molecule 1 (STIM1)
has recently been identified as the Ca2+
sensor in the endoplasmatic reticulum that
activates Ca2+ release activated channels
(CRAC) in T cells, but its function in mammalian physiology has remained elusive.
In collaboration with J. Grosse (Ingenium
AG), we analyzed a mouse line expressing
an activating EF hand mutant of Stim1
(Sax). Very unexpectedly, heterozygous
Stim1Sax/+ animals display a profound macrothrombocytopenia that is associated
with a bleeding disorder. Basal intracellular Ca2+ levels are increased ~3-fold in
circulating platelets, resulting in their
pre-activation and rapid consumption. Furthermore, a selective unresponsiveness to
immunoreceptor tyrosine activation motif
(ITAM)-coupled, but not G protein-coupled
agonists was detected, which contributed
to the bleeding phenotype and protection
from arterial thrombosis. In contrast, only
basal Ca2+ levels, but not receptor-mediated responses were affected in mutant
T cells. These findings identify Stim1 as a
central regulator of platelet function and
suggest a cell type-specific activation or
composition of the CRAC complex (Grosse
et al., submitted). The homozygous expression of the Stim1 mutation is lethal in the
embryo (Fig. 2). Currently, we are investigating the underlying mechanisms and
preliminary results indicate a defect in
vessel formation.
Fig. 2:
Severe haemorrhage in different regions
of the body of Stim1Sax/Sax embryos.
Diverging signaling events control the
pathway of GPVI down-regulation in vivo
The activating platelet collagen receptor
glycoprotein (GP)VI associates non-covalently with the Fc receptor γ-chain (FcRγ),
which signals through its immunoreceptortyrosine-based-activation-motif (ITAM) via
the adaptor LAT, leading to activation of
phospholipase Cγ2 (PLCγ2) (Fig. 3). GPVI
is a promising anti-thrombotic target since
anti-GPVI antibodies induce the irreversible loss of the receptor from circulating
platelets by yet undefined mechanisms in
humans and mice and long-term antithrombotic protection in the latter. However, we
have demonstrated that this treatment is
associated with transient but severe thrombocytopenia and reduced platelet reactivity to thrombin, thus questioning its clinical usefulness (Schulte et al., Aterioscler
Thromb Vasc Biol, 2006). Therefore, the
mechanisms underlying ´therapeutic´ GPVI
downregulation and the undesired side effects were studied in vivo. Our results show
that GPVI downregulation occurs through
two distinct pathways, namely ectodomain
shedding or internalization/degradation and
that both processes are abrogated in mice
carrying a point mutation in the FcRγ-associated ITAM. In mice lacking LAT or PLCγ2
GPVI shedding is abolished but the receptor is irreversibly down-regulated through
internalization/degradation. This route of
GPVI loss is not associated with thrombocytopenia or altered thrombin responses. These results reveal the existence of
two distinct signaling pathways downstream
of FcRγ-ITAM and show that it is possible to
uncouple GPVI downregulation from undesired side effects with obvious therapeutic
implications (Rabie et al., Blood, in press.).
Extramural Funding
IZKF Würzburg (E 30)
DFG (SFB 688 TP A1; TP A3; TP B1),
(Ni 556/4-3)
Selected Publications
Kleinschnitz, C., Stoll, G., Bendszus, M.,
Schuh, K., Pauer, U., Burfeind, P., Renne,
C., Gailani, D., Nieswandt, B.,
and Renne, T. (2006) Targeting coagulation factor XII provides protection from
pathological thrombosis in cerebral
ischemia without interfering with hemostasis. J Exp Med, 203, 513-518.
Pozgajova, M., Sachs, U., Hein, L., and
Nieswandt, B. (2006) Reduced thrombus
stability in mice lacking the α2A adrenergic receptor. Blood, 108, 510-514.
Sachs, U. and Nieswandt, B. (2006)
In vivo thrombus formation: what
can we learn from murine models?
Circ Res, in press.
Schulte, V., Reusch, P., Pozgajova,
M., Varga-Szabó, D., Gachet, C., and
Nieswandt, B. (2006) Two-phase antithrombotic protection after anti-GPVI
treatment in mice. Arterioscler Thromb
Vasc Biol, 26, 1640-1647.
Sayeh, E., Crow, M., Webster, M.L.,
Nieswandt, B., Freedman, J., and Ni, H.
(2006) Distinctive efficacy of IVIG in
ameliorating thrombocytopenia induced
by anti-platelet GPIIbIIIa versus antiGPIbα antibodies. Blood, 108, 943-946.
Fig. 3:
The signaling cascade downstream of GPVI. The LAT signalosome appears to regulate the
activity of a metalloproteinase that cleaves the receptor (Rabie et al., Blood, in press).
17
Molecular Tumor Biology-Thorsten Stiewe
E-mail: [email protected]
Phone: +49(0)931 201 487 22
Fax:
+49(0)931 201 481 23
http://www.rudolf-virchow-zentrum.de/forschung/stiewe.html
Cancer is a genetic disorder caused by mutations in genes critically involved in the control of cell
proliferation. Long-lived organisms, such as humans, have evolved strategies to restrict the development of potentially malignant cells. The most important defense against cancer is provided by the p53
family of tumor suppressor genes. Activated in response to DNA damage and to oncogenic signaling
the three proteins of this family - p53, p63 and p73 - cooperate to induce apoptosis and thus restrict
tumor formation by eliminating potentially malignant cells. However, p53 family proteins are ancient
proteins also expressed in short-lived organisms such as Drosophila or C. elegans, which typically do not
suffer from cancer. Using ∆Np73 as a potent antagonist of the p53 family, we investigated whether p53
family members have functions apart from apoptosis control that contribute to their tumor suppressor
activity in humans.
∆Np73 interferes with normal embryonic
development
Many cancer-related genes are essential
for control of embryonic development
and understanding their normal biological function has frequently revealed the
mechanistic basis for their role during
tumorigenesis. To test for p53 family
functions in development we generated
transgenic mice expressing the p53 family antagonist ∆Np73 in a regulated fashion. Surprisingly, activation of the ∆Np73
transgene in the embryo interfered with essential steps of development and resulted
in embryonic lethality (Fig. 1).
Fig. 1:
Transgenic expression of ∆Np73α
induces embryonic lethality.
18
This severe phenotype caused by inhibition of the total p53 family by overexpression of ∆Np73 was not predicted from the
knockout phenotypes of the individual p53
family members. p53-knockout mice have
a high rate of spontaneous tumorigenesis
but are developmentally normal. In striking
contrast, the phenotype of p53 depletion
in Xenopus embryos leads to gastrulation
failure and defects in mesoderm formation.
Whereas p53 is the only p53 family member
expressed during early Xenopus embryogenesis, mouse embryos express the other two
family members p63 and p73 in addition to
p53. It has therefore been suspected that
the lack of a developmental phenotype in
p53 knockout mice is due to redundancy
within the p53 family. Since ∆Np73 is a
potent inhibitor of all p53 family members,
our ∆Np73 transgenic mice provide a first
glance at the putative phenotype of a homozygous compound knockout of all three
p53 family members. The embryonic lethality observed upon inhibition of the p53
family by ∆Np73 reveals that the p53 family not only functions as a family of tumor
suppressors but also plays essential roles
in mammalian development. As the phenotype observed in our ∆Np73 transgenic
mice is more severe than any knockout of a
single p53 family member, our studies pro-
vide the first experimental support for the
hypothesis of functional redundancy within
the p53 family in coordinating embryonic
development.
∆Np73 inhibits multiple differentiation
processes
In order to better define the role of the
p53 family in development we analyzed the
effects of deregulated ∆Np73 expression
using cell culture models for cellular differentiation. Whereas myoblasts induced to
differentiate activate all three p53 family
members and mature to form multinuclear
myotubes, ∆Np73 expressing myoblasts fail
to differentiate (Fig. 2).
Fig. 2:
Inhibition of myogenic differentiation by ∆Np73α.
Importantly, ∆Np73 not only interferes
with myogenic differentiation but also
effectively inhibits bone morphogenetic
protein 2 (BMP2) induced conversion of
myoblasts to the osteoblast lineage, and
efficiently blocks all-trans retinoic acid
(ATRA) induced neuronal differentiation
of neuroblastoma cells. ∆Np73 is therefore a potent repressor of differentiation in
multiple experimental settings, including
myogenic, osteoblastic and neuronal differentiation, suggesting that ∆Np73 levels
control the onset of differentiation during
development and tissue regeneration.
p53 family members
differentiation
in
Extramural Funding
Krebshilfe (10-1884-St1), (10-2075/St2)
DFG (TR 17)
Selected Publications
Fig. 3:
p73-p57-RB pathway alterations in rhabdomyosarcoma. Immunohistochemical staining of a
normal muscle and a ∆Np73-positive rhabdomyosarcoma tissue for p57 and the inactive,
phosphorylated form of RB.
myogenic
The molecular details of ∆Np73‘s function
in differentiation control were revealed by
genome-wide expression profiling of the
myogenic differentiation pathway. These
analyses indicated that the individual p53
family members control retinoblastoma (RB)
protein activity, which is known to be
essential for both the differentiation-associated cell cycle exit and the transactivation of muscle-specific genes. Whereas p53
transactivates the RB gene, p63 and p73 are
required to upregulate the cyclin dependent
kinase inhibitor p57, which maintains RB in
an active, hypophosphorylated state. The
p53 family members therefore have different, but complementary functions in controlling the muscle differentiation program.
∆Np73 functions as an oncogene in
rhabdomyosarcoma
By inhibiting these p53 family functions
in RB activity control, ∆Np73 blocks the
muscle differentiation program. In the
presence of cooperating oncogenes such as
Pax3:Fkhr or IGF2 this results in the malignant transformation of non-tumorigenic
myoblasts to tumor cells. Induction of cellular differentiation is therefore a key tumor suppressor activity of the p53 family.
The importance of this finding is underlined by the high frequency of p53 family alterations in patients suffering from
malignant skeletal muscle tumors (called
rhabdomyosarcomas) - the most frequent
soft tissue sarcomas in children. More than
85% of all patients express elevated levels
of the ∆Np73 oncogene in the tumor tissue. Compared to normal muscle tissues,
this results in a downregulation of p57
levels and a concomitant increase in the
amount of inactive RB (Fig. 3).
When the ∆Np73 protein is depleted from
these tumors by in vivo RNA interference,
p57 levels increase, and the tumors stop
growing and regress. Depending on the mutational status of p53, this growth arrest
is due to a reversible cell cycle arrest or
differentiation into mature muscle fibers as
a permanent barrier to proliferation. Since
expression of ∆Np73 is essential for rhabdomyosarcoma growth in vivo, it represents
a suitable target protein for novel anticancer therapies.
In summary, our results indicate that p53‘s
relatives and evolutionary predecessors,
p63 and p73, possess essential functions in
embryonic development and differentiation
control, which are in part shared by p53.
The findings further suggest that these ancestral functions in differentiation control
contribute to the tumor suppressor activity
that the p53 family is famous for (Fig. 4).
Beitzinger, M., Oswald, C., BeinoraviciuteKellner, R., and Stiewe, T. (2006) Regulation of telomerase activity by the p53 family member p73. Oncogene, 25, 813-826.
Cam, H., Griesmann, H., Beitzinger, M.,
Hofmann, L., Beinoraviciute-Kellner, R.,
Sauer, M., Hüttinger-Kirchhof, N.,
Oswald, C., Friedl, P., Gattenlöhner, S.,
Burek, C., Rosenwald, A., and Stiewe, T.
(2006) p53 family members in myogenic
differentiation and rhabdomyosarcoma
development. Cancer Cell, 10, 281-293.
Hüttinger-Kirchhof, N., Cam, H., Griesmann, H., Hofmann, L., Beitzinger, M.,
and Stiewe, T. (2006) The p53 family inhibitor ∆Np73 interferes with
multiple
developmental
programs.
Cell Death Differ, 13, 174-177.
Stiewe, T. (2007) p53 family in differentiation and tumorigenesis. Nature Rev
Cancer, in press.
Fig. 4:
Differentiation control - a new aspect of p53 family functions in tumor suppression.
19
Immune Tolerance-Stephan Kissler
Email: [email protected]
Phone: +49(0)931 201 487 00
Fax:
+49(0)931 201 487 02
http://www.rudolf-virchow-zentrum.de/forschung/kissler.html
Stephan Kissler will join the Rudolf Virchow Center as
Junior Group Leader in early 2007.
The primary function of the immune system is to recognize and eliminate pathogens. This task requires
immune cells to be reactive to a wide range of antigens. While mechanisms are in place to prevent
immune activation by innocuous antigens, including self-antigens, a significant percentage of the
population develops autoimmune diseases. Our laboratory seeks to understand the genetic polymorphisms that predispose individuals to autoimmunity and the regulatory pathways that fail during onset
of disease. Our main approach is the genetic manipulation of model organisms by RNA interference
(RNAi). We employ lentiviral transgenesis to generate animals with target genes constitutively silenced
by RNAi. After pioneering this strategy in the most widely used model for type 1 diabetes, we are now
refining lentiviral technology to make it more versatile and specific for studying of immune tolerance.
Understanding genetic factors and functional pathways involved in autoimmunity should ultimately
help develop new therapeutic approaches.
Immune tolerance and autoimmunity
The immune system evolved to protect
us against pathogenic viruses, bacteria,
parasites and fungi. Due to the diversity
of existing pathogens, the immune system
developed complex mechanisms to ensure
that it would be capable of recognizing
virtually any invading pathogen. This high
degree of reactivity, however, required the
simultaneous evolution of regulatory pathways to ensure immune tolerance of selfantigens, or antigens whose presence is innocuous, particularly on mucosal surfaces
(airways and digestive system). A prime
example of such a regulatory process is the
thymic selection of T lymphocytes, where
developing T cells are subject to a stringent
quality control that eliminates cells whose
reactivity to self-antigen is too strong. In
addition, a specialized population of selfreactive T cells is generated whose role it is
to attenuate inadequate immune responses
against self.
Despite these regulatory mechanisms, a
significant percentage of the population
20
develops autoimmunity, in most cases
characterized by immune responses against
a specific tissue or organ (e.g. type 1 diabetes, multiple sclerosis). Many of these
autoimmune diseases can be studied in
animal models. This is particularly true for
type 1 diabetes, for which the non-obese
diabetic (NOD) mouse strain constitutes
a widely studied and very relevant experimental model. Over the last two decades,
many genetic loci in the NOD mouse have
been shown to be associated with disease
susceptibility. Several susceptibility genes
in the mouse have now been found to correspond to human candidate genes, reinforcing the validity of this animal model.
Over 20 susceptibility loci have been
identified in the NOD mouse by congenic
breeding and positional cloning strategies.
While several of these intervals have been
narrowed down to a few candidate genes,
or in a few instances to single genes, many
loci are found in gene-dense chromosomal
regions and the exact genes associated
with disease are still unclear. The NOD
mouse poses additional problems, since the
generation of embryonic stem (ES) cells
from this strain has so far been unsuccessful. The use of gene knockout (KO) technology therefore requires the backcrossing
of KO alleles from other strains onto the
NOD background. This process is very time
consuming and is intrinsically flawed since
donor-derived chromosomal regions flanking the KO allele may influence disease
outcome, thereby obscuring the effect of
the deleted gene.
Fig. 1:
Lentiviral transgenic NOD mouse expressing
the green-fluorescent protein (GFP) marker.
Studying disease genetics by RNA interference
To circumvent the difficulties of gene-deletion in the NOD mouse, we have pioneered
lentiviral transgenesis in conjunction with
RNAi in this disease model. The generation
of transgenic mice by single-cell embryo
infection with lentivirus has proven highly
efficient and technically simpler than conventional transgenesis methods. In addition, this method allows the rapid generation of transgenic mice directly in the NOD
background (Fig. 1). We have determined
in this system that lentiviral transgenes
are inherited with the expected Mendelian distribution, and that their expression
remains consistent both over time in individual animals, and over several generations after extensive breeding (>300 mice
and 7 generations). Lentiviral transgenesis
is therefore adequate for studying type 1
diabetes in the NOD model, which requires
the generation of large cohorts (50-100
mice/group) and the monitoring of animals
for periods of up to 6 months.
Using short-hairpin RNA (shRNA)-encoding constructs, we have successfully
silenced the expression of the candidate
gene Nramp1 in NOD mice and determined
this gene to be associated with disease
(Fig. 2). The positive outcome of this first
study has now led us to initiate several
new projects investigating further candidate genes associated with type 1 diabetes
in this mouse model. These genetic studies
are being carried out in close collaboration
with Linda Wicker (Cambridge University,
UK) and Larry Peterson (Merck), whose
extensive studies of the NOD model have
resulted in the identification of several
more promising candidate genes awaiting
to be evaluated.
Expanding the use of lentiviral RNAi
In our first study involving lentiviral RNAi,
we employed a construct, where shRNA
expression is driven by the ubiquitous
U6 promoter. While this led to consistent
and effective gene silencing, we have
now adopted a new strategy that enables
shRNA expression to be restricted to particular cell types. Using tissue-specific promoters and a new shRNA design, our laboratory has engineered lentiviral vectors to
silence genes exclusively in T cells or in
beta islet cells of the pancreas.
We have also recently made use of the
unique features of RNAi to modulate gene
expression in ways not previously possible
by conventional KO technology. Many genes
are subject to alternative splicing, and
while KO studies are informative, the respective roles of different splice variants
are often difficult to determine. By targeting exon-boundary sequences, we have
generated transgenic animals, where single
splice variants are silenced by RNAi without affecting other isoforms that share exons with the targeted mRNA (Fig. 3). We
aim to use this new strategy to dissect the
contributions of all isoforms of several susceptibility genes.
Overall, our novel approach to studying
disease genetics and pathways by RNAi
in the NOD model of type 1 diabetes
should help elucidate parameters that
influence the onset and development
of autoimmunity.
Fig. 3:
Design of shRNA sequences targeting unique exon-boundaries allows the selective silencing
of single splice variants (fl: full-length - exons 1/2/3/4; s: soluble - exons 1/2/4;
li: ligand-independent - exons 1/3/4). Isoform silencing was tested in vitro by luciferasereporter assay.
Selected Publications
Kissler, S., Stern, P., Takahashi, K.,
Hunter, K., Peterson, L., and Wicker, L.S.
(2006) In vivo RNA interference demonstrates a role for Nramp1 in modifying susceptibility to type 1 diabetes.
Nature Genetics, 38, 479-483.
Fig. 2:
The Nramp1 gene is silenced by RNAi, resulting in decreased NRAMP1 protein levels and reduced type 1 diabetes frequency in lentiviral transgenic NOD mice.
21
Molecular Microscopy-Gregory Harms
E-mail: [email protected]
Phone: +49(0)931 201 487 17
Fax:
+49(0)931 201 487 02
http://www.rudolf-virchow-zentrum.de/forschung/harms.html
Our group, Molecular Microscopy, studies molecular interactions in cell signaling of membrane proteins
and cytosolic messengers by fluorescence resonance energy transfer (FRET) microscopy, single-molecule
microscopy and dynamic confocal microscopy. These approaches are made possible by constructing custom wide-field and confocal microscopes capable of ratiometric FRET, fluorescence recovery after photobleaching (FRAP), fluorescence correlation spectroscopy (FCS) and single-molecule tracking. These
microscopes allow detection of low, endogenous levels of proteins in and on living cells. Over the past
year, we have focused on Bone Morphogenetic Protein (BMP) signaling mechanisms, on interleukin-5
(IL-5) clustering on living eosinophils and on Ca2+ signaling. Other projects include ion channels, platelet signaling, G-Protein coupled receptors, and model membrane systems. We also develop microscopes
and methods to study single-molecule interactions in living organisms.
Quantum Dots
Our research focuses on cell signaling
and kinetics and the role of natural compartments in cells. Optical microscopy
allows us to observe these aspects and is
further applied to dynamic techniques to
determine their timing. FRET microscopy
allows us to measure the dynamics and
observe cellular localization of protein
conformational changes and proteinprotein interactions with improved interpretation due to both anisotropy and
fluorescence lifetime. We can measure the
diffusion dynamics of lipids and proteins
by long-range techniques such as FRAP,
complementary to short-range methods
like FCS. We can also track single-molecules by wide-field imaging and TIRF,
since we now have the latest technologies
to investigate long and short diffusion
ranges with tracing, FRET, and co-localization events. We are extending our range to
observe the signaling effects in multicellular systems in living organisms by using
other techniques.
The localization, tracking, and stoichiometry of endogenous levels of receptors in
vivo are important extensions to structural
and systems biology. For many receptors,
the level is so low they cannot be detected
by standard light microscopy due to low
sensitivity, limited excitation power, and
high background noise. The detection of
single-quantum dots (QDots) targeted to
such receptors on living primary cell lines
and the maximized use of QDot detection
in living systems is of key importance.
Quantum Dots are semi-conductor nanocrystals of CdSe with a bio-inert coating
that are easily conjugated to biological
molecules. Single QDots, in contrast to
standard organic fluorophores, can easily
be discriminated above the high autofluorescent background of primary cells, which
is ideal for fine positioning. Moreover, they
are stable for long periods of times. We
have demonstrated the maximized use of
QDots in living systems, avoiding the traits
of blinking and non-fluorescence.
Interleukin-5 Receptor
Fig. 1:
Diagram of the custom built single molecule microscopes.
22
Targeting asthma by cytokine networks
interference is a promising approach for
prevention. IL-4, -5 and -13 expressed by
eosinophilic granulocytes play a key role in
the TH2-type immune response that domi-
nates the exacerbation of atopic diseases.
The low endogenous expression of IL-receptors (~1000 receptors/cell) on eosinophils requires extremely sensitive imaging,
and bright QDots are essential due to high
eosinophilic background autofluorescence.
IL-5 receptor common β-chain (IL-5Rbc)
dynamics were visualized on the surface of
HL-60 cells (differentiated to eosinophils)
by QDots coupled to specific antibodies.
IL-5Rbc’s on these cells demonstrated two
patterns of confined motion due to membrane flow and cytoskeletal restructuring.
The receptors showed a high complexation
stoichiometry indicating that although the
crystallographic picture of IL-5Rbc as a stable intertwined homodimer is applicable to
complexes on the surface of living eosinophils, receptor complexes of higher orders
might be necessary for signaling in vivo.
bile and immobile diffusion that correspond
to preformed complexes. These studies also
indicate that membrane micro-domains and
the cytoskeleton are involved in regulating
of complex formation of receptors.
Smad 1/4 signaling
Extramural Funding
The Smad signaling pathway, an intracellular mediator of BMP signaling, influences
cell growth, differentiation, adhesion,
migration, and also carcinogenesis and
immune responses. Smads transduce the
signal from the membrane into the nucleus. Smad1 is phosphorylated upon Bone
Morphogenetic Protein (BMP) stimulation
of the receptor and interactions to form a
complex with Smad4. This complex translocates into the nucleus and regulates transcription of target genes. Smad Biosensors
created by fusions of cyan and yellow fluorescent proteins to Smad1 and Smad4 allow
us to determine the rate-limiting steps of
this signaling cascade by FRET microscopy.
We were able to visualize rate-limiting
delays between BMP-4 activation, Smad1
phosphorylation, and complex formation.
Further experiments indicated that the delays are influenced by the MH1 domain of
Smad1. The Smad biosensors provide new
insights into the BMP–Smad1/4 signaling
process and provide powerful tools for rapid evaluation of Smad activation.
European Union Marie Curie (FP6-2006-Mobility-7 – 022327)
DFG (GK 1342), (Ob 137/3-1)
Selected Publications
Friedl, P., Wolf, K., von Andrian, U., and
Harms, G. (2007) Biological second and
third harmonic generation microscopy.
Curr Prot Cell Biol, 4.15.1-4.15.21.
Fig. 2:
IL-5RBC´s on eosinophil moving in a 3D
collagen matrix.
Bone Morphogenetic Protein signaling
The BMP signaling system regulates growth
and differentiation and is important for
tissue engineering. BMP and BMP receptors are implicated in diseases such as
preliminary pulmonary hypertension, juvenile polyposis, breast cancer, colon
cancer, and other forms of cancer. BMPs,
members of the transforming growth factor
(TGF-β) super-family, initiate a regulatory
response through two types of receptors, BRI
and BRII. The BMP ligand binding event
controls signaling and regulates by either
binding to preformed homodimer complexes of BRI, signaling the p38 MAP kinase
pathway or to preformed heteromeric (BRI
and BRII) complexes for the Smad dependent pathway. A more precise picture of the
receptor signaling can now be described by
observing individual receptors on the surface of the biologically relevant cells. Individual BRII receptors show patterns of mo-
Fig. 3:
A: BMP signaling system. B: Single BRII receptors and tracking. C: Smad signaling pathway.
D: FRET from Smad 1/4 complexation.
23
Structural Biology: DNA Repair and Structure
based Drug Design-Caroline Kisker
E-mail: [email protected]
Phone: +49(0)931 201 483 00
Fax:
+49(0)931 201 487 02
http://www.rudolf-virchow-zentrum.de/forschung/kisker.html
Mutations are the primary cause of hereditary diseases, as well as cancer, and it has been shown
that 80 to 90% of all human cancers are ultimately due to DNA damage. Nucleotide excision repair (NER) is unique in its versatility to repair a broad range of damages including carcinogenic cyclobutane pyrimidine dimers induced by UV radiation, benzo[a]pyrene-guanine adducts caused by
smoking, as well as guanine-cisplatinum adducts formed during chemotherapy. We aim to understand the fundamental mechanisms of the bacterial and mammalian NER machinery. Since damage
can accumulate and may not be repaired prior to replication, we also analyze how DNA polymerases accomplish trans-lesion DNA synthesis. A second focus is structure-based drug design to identify
new therapeutics against Mycobacterium tuberculosis. Currently, more than two million people die
from tuberculosis each year. We are in the process of identifying new inhibitors against essential
M. tuberculosis proteins.
Nucleotide excision repair
The importance of this repair mechanism
is reflected by three severe inherited diseases in humans that are due to defects in
NER: xeroderma pigmentosum, Cockayne’s
syndrome and trichothiodystrophy. We are
using structural, biochemical and biophysical methods to characterize the individual components of NER and their cognate
complexes, which are vital to the reaction
cascade. Three major aspects in the “recognition” and “repair” events of NER are still
not understood: (1) What are the structural determinants of the DNA required
for damage recognition? (2) How are
damage-induced conformational changes
in the DNA perceived by a DNA repair protein complex, and how does the recognition of protein-DNA contacts translate into
high binding affinities? (3) How does the
recognition process lead to incision?
sis. In eubacteria, NER is mediated by the
products of the uvrA, uvrB and uvrC genes.
UvrA is involved in damage recognition and
forms a heterotrimeric UvrA2/UvrB complex
with UvrB. This complex identifies conformational perturbations induced by DNA lesions. After the damage has been identified, UvrA dissociates, while UvrB remains
bound to the DNA in a stable pre-incision
complex. UvrC binds to this complex and
triggers the incision four nucleotides 3’ to
the damaged site, followed by an incision
seven nucleotides 5’ to the damaged site.
This cascade of events and the involvement
of several proteins in damage recognition
and repair ensures discrimination between
damaged and non-damaged DNA.
UvrB, the central component in prokaryotic NER, plays an essential role in
damage recognition. Several crystal structures of UvrB in the absence of DNA have
been solved. We recently solved the first
structure of a UvrB-DNA complex, thereby
providing insights into the mechanism of
how UvrB binds to DNA and forms the preincision complex.
Structural basis for DNA recognition and
processing by UvrB
Eukaryotic and eubacterial cells share the
same basic mechanisms in the NER process
and recognize the same DNA damages.The
well-characterized eubacterial proteins are
therefore ideal targets for structural analyFig. 1:
Structure of the UvrB-DNA complex. The surface of UvrB is color coded by domains.
24
UvrB contains five domains: 1a, 1b, 2, 3
and 4. Domain 2 has been shown to interact with UvrA, while domain 4 interacts
with both UvrA and UvrC. Domains 1 and 3
share high structural similarity with DNA
helicases and bind ATP at their domain
interface. UvrB contains all the structural
properties of a helicase. However, it has
only limited strand-separating activity. It
is likely that domain movements are an essential requirement for damage recognition
and formation of the preincision complex.
The most prominent feature of UvrB is a
highly conserved and flexible β-hairpin
connecting domains 1a and 1b, which is
rich in aromatic and hydrophobic residues.
We used a DNA hairpin (hpDNA) with a
three base-pair stem, an eleven-nucleotide
loop and a three-nucleotide 3’ overhang as
a substrate for UvrB (Fig. 1). Our structure
revealed that the DNA strand containing the
3’ overhang threads behind the β-hairpin,
indicating that this motif inserts between
double stranded DNA, thereby locking down
either the damaged or the non-damaged
strand. The nucleotide directly behind the
β-hairpin is flipped out and is inserted into
a small, highly conserved pocket in UvrB,
which only provides space for a planar molecule (Fig. 2). The second DNA strand must
pass in front of the β-hairpin and re-anneal
with the clamped DNA strand as it emerges
from the binding pocket.
tide would be occluded due to steric hindrance, thus arresting translocation. Subsequent ATP hydrolysis by UvrB, which is
required prior to the first incision reaction,
would further distort the DNA rather than
separating its base pairs in the preincision
complex, thus priming the DNA for cleavage by UvrC.
Development of new inhibitors against
M. tuberculosis
Based on estimates from the World Health
Organization about a third of the world’s
population is infected with M. tuberculosis and about 10% of these individuals will
develop an active infection. Critical issues
in the treatment and control of tuberculosis include emergence of multi-drug resistant strains of this organism and the role
of this disease as a major opportunistic
pathogen in patients with HIV/AIDS. The
World Health Organization, the Global Partnership to Stop Tuberculosis and the Millennium Development Project recently defined new goals for the tuberculosis control
programs, since previous strategies have
not been sufficient to control the disease.
In collaboration with Peter Tonge (Stony
Brook University) and Richard A. Slayden
(Colorado State University) and by means
of structure-based drug design, we are analyzing proteins that are essential for the
viability of this organism. We are focusing
on three different pathways: fatty acid biosynthesis, vitamin K biosynthesis and mycobactin synthesis and aim to develop new
inhibitors against drug-sensitive and multidrug resistant strains of M. tuberculosis.
Fig. 2:
Interactions between UvrB and the DNA Position
18 indicates the hydrophobic nucleotide-binding
pocket.
Our structure clearly shows that the inner
DNA strand fits tightly behind the β-hairpin and bases are rotated one by one into a
conserved, shape complementary (planar)
pocket after passing a patch of highly conserved, charged residues. Damage recognition of structurally unrelated adducts may
therefore be achieved by allowing only undamaged nucleotides to rotate behind the
β-hairpin, whereas the damaged nucleo-
Fig. 3:
Structure of the FabI-ACP complex. Two ACP
molecules (pink) are bound to the FabI tetramer.
We recently solved the structure of a
complex between the acyl carrier protein
covalently attached to an acyl chain (acylACP) bound to the fatty acid biosynthesis
enoyl reductase (FabI). FabI is a proven
drug target and our structure provides insights into the interaction between these
two proteins during fatty acid biosynthesis,
and thus a foundation for developing novel
FabI inhibitors that antagonize the interaction of FabI with its natural substrate.
The structural data are substantiated by
mutagenesis, and reveal that interactions
between ACP and FabI are largely electrostatic in nature. Our structure suggests
that the substrate is delivered from the ACP
molecule to the active site of FabI between
a flexible loop and an α-helix of FabI.
Extramural Funding
NIH R01 GM070873
NCI 5PO1 1CAO4799514
DFG (SFB 630)
Selected Publications
Truglio, J.J., Karakas, E., Rhau, B.,
Wang, H., DellaVecchia, M.J.,
Van Houten, B., and Kisker, C.
(2006) Structural basis for DNA recognition and processing by UvrB.
Nat Struct Mol Biol, 13, 360-364.
Karakas, E., Truglio, J., Croteau, D.,
Rhau, B., Wang, L., Houten, B.V.,
and Kisker, C. (2006) Structure of the
C-terminal half of UvrC reveals an
RNase H endonuclease domain with an
Argonaute-like catalytic triad. EMBO J,
in press.
Sullivan, T.J., Truglio, J.J., Boyne,
M.E., Novichenok, P., Zhang, X.,
Stratton, C.F., Li, H.-J., Kaur, T.,
Amin, A., Johnson, F., Slayden,
R.A., Kisker, C., and Tonge, P.J.
(2006) High affinity InhA inhibitors
with activity against drug-resistant
strains of Mycobacterium tuberculosis.
ACS Chemical Biology, 1, 43-53.
Rafi, S., Novichenok, P., Kolappan, S.,
Zhang, X., Stratton, C.F., Rawat, R.,
Kisker, C., Simmerling, C., and Tonge,
P.J. (2006) Structure of acyl carrier protein bound to Fabi, the FASII
enoyl reductase from Escherichia coli.
J Biol Chem, 281, 39285-39293.
Yakubovskaya, E., Chen, Z., Carrodeguas, J.A., Kisker, C., and Bogenhagen, D.F. (2006) Functional human
mitochondrial DNA polymerase gamma
forms a heterotrimer. J Biol Chem,
281, 374-382.
25
Structural Biology: Protein Folding, Function and
Degradation-Hermann Schindelin
E-mail: [email protected]
Phone: +49(0)931 201 483 20
Fax:
+49(0)931 201 483 09
http://www.rudolf-virchow-zentrum.de/forschung/schindelin.html
Our general aim is to understand the detailed functions of functionally important proteins. At present
the research is focused on two general topics: Firstly, protein folding in the endoplasmic reticulum
(ER) and degradation of misfolded proteins via the ubiquitin-dependent protein degradation pathway.
Secondly, we are interested in the structure and function of inhibitory neuronal receptors and the
mechanism of their anchoring at the postsynaptic membrane (see Kim et al., EMBO J 25, 1385-1395,
2006). Our intention is to understand these proteins and the processes they participate in at the
molecular level. We are, therefore, using a combination of different techniques including X-ray
crystallography, biochemical and biophysical methods. The results of our projects have direct medical
relevance, for instance it is known that protein misfolding and aggregation lead to a variety of diseases
such as neurodegenerative disorders.
Protein folding and maturation in the
endoplasmic reticulum
Secretory proteins are translocated into the
endoplasmic reticulum where a sophisticated machinery assists them in achieving
their native conformations. Many of these
newly synthesized proteins are N-glycosylated and/or contain disulfide bonds, which
stabilize their three-dimensional structures. We are studying the enzymes oligosaccharyl transferase and protein disulfide
isomerase (PDI), which are essential for the
folding and maturation of proteins passing
through the ER. Oligosaccharyl transferase
is an integral membrane protein consisting of nine subunits in yeast, which transfers high-mannose carbohydrates from the
sugar donor dolichol phosphate onto one
or more asparagine side chains of the target glycoprotein. In collaboration with the
group of Dr. Lennarz (SUNY Stony Brook)
we are optimizing the expression conditions for this multi-subunit membrane protein and are screening detergents for effective solubilization.
Protein disulfide isomerase catalyzes both
the formation of disulfide bonds in newly
synthesized proteins and the isomerization
of incorrectly formed disulfide bonds.
We have recently determined the crystal
structure of yeast protein disulfide isomerase. The enzyme features a modular architecture with four thioredoxin domains, a, b,
b’ and a’, and a C-terminal tail arranged in
the shape of a twisted “U“. The active sites
of the catalytic domains, a and a’, face each
26
other across a large cleft on the arms of the
“U”, with the rigid b and b’ domains at the
bottom of the “U“. The inside surface of the
“U“ is enriched in hydrophobic residues
which facilitate interactions with partially
folded substrates. In a second crystal form
(unpublished data) a pronounced conformational change was observed, which leads
to the rotation of the a domain by almost
100° so that the two active sites no longer
face each other. As a result of this structure
we have introduced disulfide bonds which
reduce the flexibility of the a and a’ domains relative to the b/b’ core and discovered that conformational flexibility is required for PDI activity. We are also in the
process of studying the flexibility of the
catalytic domains by FRET experiments.
Fig. 1:
Structure of the yeast E1 enzyme (color coded
by domains with the active site cysteine in
orange) in complex with ubiquitin (yellow).
Protein degradation
Ubiquitin dependent protein degradation is
a key cellular pathway by which proteins are
removed in a highly selective manner. The
activation and transfer of ubiquitin is catalyzed by an enzyme cascade consisting of
a single ubiquitin activating enzyme (E1),
several ubiquitin conjugating enzymes (E2)
and multiple ubiquitin ligases (E3), which
allow the specific recognition of a large
number of target protein substrates.
The ubiquitin activating enzyme catalyzes
the ATP dependent activation of ubiquitin
in a two-step reaction: First, a ubiquitinacyl adenylate is formed in which the Cterminus of ubiquitin is covalently linked
to AMP. Subsequently, the activated ubiquitin is transferred to an active site cysteine
leading to the formation of a high-energy
thioester linkage. We have determined the
first crystal structure of a ubiquitin activating enzyme, namely the yeast E1 in complex with ubiquitin. The crystal structure
reveals a multi-domain architecture with
an active (cyan) and an inactive (magenta)
adenylation domain, a 6-stranded β-barrel
domain (green), a catalytic domain featuring the active site cysteine (blue) and a Cterminal ubiquitin-like (Ubl) domain (red).
The C-terminus of ubiquitin is bound at
the adenylation site at a distance of more
than 35 Å from the active site cysteine.
How this large gap is bridged is currently
not known. We hypothesize that the ubiquitin C-terminus slides in between two
loops connecting the catalytic domain and
the adenylation domain to reach the active
site cysteine. A comparison of the two copies of the enzyme present in the asymmetric unit indicates a 10° rotation of the Ubl
domain, which is presumably responsible
for bringing the E2 enzyme, which binds to
the Ubl domain into close spatial proximity
of the E1 active site cysteine from which
ubiquitin is transferred onto the active site
cysteine in the E2 enzyme.
Proteins that pass through the endoplasmic reticulum (ER), but fail to fold are
retrotranslocated to the cytosol, ubiquitinated and degraded by the proteasome in
a process referred to as ER associated protein degradation (ERAD). If they are glycoproteins the carbohydrate chains have to be
removed prior to proteasomal degradation,
and this reaction is carried out by peptide
N-glycanase (PNGase). PNGase interacts
with a variety of binding partners including the proteasome and the AAA ATPase
p97 (Cdc48). We have now determined the
crystal structures of the core domain in
complex with the xeroderma pigmentosum
group C domain of HR23B, the C-terminal
domain and the N-terminal domain of the
mouse enzyme and characterized the functions of these domains through biochemical and biophysical techniques.
Extramural Funding
Fig. 2:
Ribbon diagram of the mPNGase C-terminal domain with bound mannotetraose.
The crystal structure of the C-terminal domain revealed a jelly-roll fold with structural homologies to carbohydrate-binding proteins. Based on this homology we
hypothesized that this domain assists in
carbohydrate binding. This activity was
confirmed by biochemical experiments and
a complex structure with bound mannopentaose illustrated the binding.
The N-terminal domain of mPNGase interacts with p97, and we identified the last
10 residues of p97 as the binding site. The
crystal structure of the N-terminal domain
revealed a compact molecule with a highly
helical architecture. A complex structure
with the p97 C-terminus demonstrated that
the peptide binds to a positively charged
groove which contains a tightly fitting
pocket for the side chain of p97’s highly
conserved penultimate tyrosine residue.
Phosphorylation of this tyrosine, the main
phosphorylation site during T cell receptor
stimulation, completely blocks binding of
PNGase to p97. We are currently investigating whether phosphorylation of this residue modulates ERAD activity.
Fig. 3:
Surface representation of the mPNGase Nterminal domain (pink and gold: strictly and
partially conserved residues) with the p97 Cterminus in all-bonds representation.
NIH R01 DK-54835
NIH R01 NS-48605
Selected Publications
Hanzelmann, P., and Schindelin, H.
(2006) Binding of 5‘-GTP to the Cterminal FeS cluster of the radical
S-adenosylmethionine enzyme MoaA
provides insights into its mechanism.
PNAS U S A, 103, 6829-6834.
Kim, E.Y., Schrader, N., Smolinsky, B.,
Bedet, C., Vannier, C., Schwarz, G.,
and Schindelin, H. (2006) Deciphering the structural framework of glycine receptor anchoring by gephyrin.
EMBO J, 25, 1385-1395.
Tian, G., Xiang, S., Noiva, R.,
Lennarz, W.J., and Schindelin, H.
(2006) The crystal structure of yeast
protein disulfide isomerase suggests
cooperativity between its active
sites. Cell, 124, 61-73.
Zhao, G., Zhou, X., Wang, L., Li,
G., Kisker, C., Lennarz, W.J., and
Schindelin, H. (2006) Structure of
the mouse peptide N-glycanase-HR23
complex suggests co-evolution of the
endoplasmic
reticulum-associated
degradation and DNA repair pathways.
J Biol Chem, 281, 13751-13761.
Zhou, X., Zhao, G., Truglio, J.J.,
Li, G., Wang, L., Lennarz, W.J., and
Schindelin, H. (2006) Structural and
biochemical studies of the C-terminal
domain of mouse peptide-N-glycanase identify it as a mannose-binding
module. PNAS U S A, 103, 17214-9.
27
Functional Proteomics-Albert Sickmann
E-mail: [email protected]
Phone: +49(0)931 201 487 30
Fax:
+49(0)931 201 481 23
http://www.rudolf-virchow-zentrum.de/forschung/sickmann.html
In the post-genomic era, mass spectrometry has evolved to the key technology for proteomic characterization of biological systems. Besides basic identification of minute protein and peptide amounts, the
recent focus of proteomic approaches has shifted towards the analysis of posttranslational modification
of proteins. Current estimations indicate that every second protein is phosphorylated or glycosylated with
numerous implicated functions, e.g. in regulation of protein activity and cell-cell interactions. Determination of these modifications requires the utmost sensitivity, which is achieved by miniaturization and
continuous method development of chromatographic purification technologies and mass spectrometry.
As a logical consequence of many biological issues, direct quantification of protein species including their
modification status has also become a further hot topic in proteome research. This can provide insights
into dynamic phosphorylation events, e.g. during signaling cascades leading to platelet aggregation.
Platelet (membrane) proteomics
Platelets are of central importance for primary and secondary hemostasis, and in this
context are involved in a range of related
cardiovascular diseases such as stroke and
myocardial infarction. These are among the
leading causes of death in Western countries and therefore a major aim of platelet research is to understand the distinct
role of platelets in healthy conditions and
disease. Since they represent the interface
between platelets and their interaction
partners, the analysis of proteins within
the plasma membrane is a current focus
of our group. Proteins localized to the exterior of the cell mediate platelet adhesion,
rolling as well as initiation of aggregation by
signaling cascades.
A first survey of plasma membrane components by traditional purification methods revealed several new potential membrane receptors such as the immunoglobulin receptor G6B (Moebius et al., Mol Cell
Prot, 2005). Furthermore, characterization
of microdomains on the cell surface is
currently in progress, providing information about coordination of proteins within distinct domains of the membrane. To
further optimize the analysis of plasma
membranes, aqueous two-phase partitioning for the isolation of membranes
has been adapted for platelets as well as
other samples (Schindler et al., Mol Cell
Prot, 2006). This led to the identification
of an even larger number and proportion
of membrane proteins, providing access to
low abundant membrane receptors (Fig. 1).
28
Fig. 1:
Aqueous two-phase partitioning for platelet membranes.
The developed methodology permits more
rapid and efficient enrichment of membrane
proteins than comparable techniques. It
can be adapted to small sample amounts
and also be used for functional assays,
since proteins may be isolated in their native state. Furthermore, it allows analysis
of posttranslational modifications of membrane proteins. Results from both approaches
surpass every proteomic study conducted on
platelets so far with respect to the number
of identified membrane components. While
the results obtained are currently undergoing validation in cooperation with other
groups, further separation techniques such
as combined fractional diagonal chromatography are in preparation. This will establish a unique repository of methionine/
cysteine and N-terminal peptides.
Posttranslational modifications
The addition of posttranslational modifications to proteins, such as glycosylation and
phosphorylation, has a major impact on
protein function and activity. For both research fields, specific enrichment of
modified peptides from the highly complex
set of total peptides is a prerequisite for
successful analysis. Due to suppression effects and limitations of mass spectrometric
analysis, glycosylated and phosphorylated
peptides are hardly accessible among the
superior number of non-modified peptides.
For N-glycosylation research, an initial survey of platelet glycoproteins has been conducted using hydrazide- and lectin-affinity
capturing (Lewandrowski et. al., Mol Cell
Prot, 2006). This approach has now been
enhanced by combining aqueous two-phase
partitioning with a novel technique for specific enrichment of sialylated glycopeptides.
It is possible to discover over 150 glycosylation sites, also comprising low abundant
protein species such as G-protein coupled
receptors, due to system-inherent reduction
of sample complexity (Fig. 2). Based solely
on liquid chromatography steps, the prob-
lematic class of transmembrane proteins
can be targeted without the limitations
known from gel-based techniques. The need
for first hand data on N-glycosylation of
platelet proteins is evident considering the
fact that over two thirds of the identified
sites have not been confirmed previously.
This enables, for example, improvements
regarding the fragmentation of phosphopeptides by MS3-sequencing. Among the
numerous identified proteins of known
signaling cascades during platelet aggregation, we could also elucidate phosphorylation sites within mitochondrial proteins.
Extramural Funding
DFG (Si 835/2-1), (Transregio 17),
(SFB 688)
BMBF (QuantPro Project C)
Mitochondria
Selected Publications
Fig. 2:
MS2 sequencing of a novel glycosylation site
for the putative membrane receptor G6B.
Phosphorylation events are known to trigger a whole variety of cellular functions.
Despite their major importance, no large
scale analysis of phosphorylation sites in
platelets has been performed so far. By a
multifaceted approach the determination
of over 500 phosphorylation sites in human platelets was possible. This involved
different enrichment techniques such as
immobilized metal ion affinity chromatography as well as using several metaloxide complexes. In addition, very low
abundant phosphotyrosine sites could be
identified despite the unfavorable ratio of
1800:200:1 for pS:pT:pY of general phosphorylation levels. Moreover, the development of sensitive, phosphorylation-specific
mass spectrometric scan events (precursor
ion scanning, neutral loss scanning etc.)
is of major interest for our group (Fig. 3).
Fig. 3:
Enhancement of phosphopeptide signals
by specific neutral loss scanning events.
Besides being the power plants of cells,
mitochondria are vital for a number of
biological processes including apoptosis
and metabolism of lipids, amino acids and
metal ions. Even after the primary proteomic analysis of mitochondria by our
group, which resulted in identification of
749 proteins, a further survey yielded an
additional 102 identifications by enhanced
multidimensional separation techniques
(Reinders et al., J Prot Res, 2006). Thereby,
the so far most complete molecular characterization of mitochondria was possible.
In addition, detailed analysis of the outer
mitochondrial membranes by 2D-BAC/SDSPAGE resulted in elucidation of a subclass
of preproteins localized to the outer membrane compartment prior transport to the
inner compartments of mitochondria (Zahedi et al., Mol Biol Cell, 2006). The experiments showing an accumulation of preproteins destined for internal mitochondrial
compartments at the outer membrane were
performed with cell growth on fermentable
(YPD) or non-fermentable (YPG) medium,
i.e., under conditions of high mitochondrial activity and high synthesis rates for
mitochondrial proteins (Fig. 4). The value
in brackets represents the mitochondrial
localization of mRNA from 1 (no mitochondrial association) to 100 (mitochondrial
association). A possible hypothesis for the
accumulation of preproteins at the outer
membrane would be an overflow of the presequence import pathway mainly used by a
conserved class of proteins.
Lewandrowski, U., Moebius, J., Walter,
U., and Sickmann, A. (2006) Elucidation
of N-glycosylation sites on human platelet proteins: a glycoproteomic approach.
Mol Cell Proteomics, 5, 226-233.
Reinders, J., Zahedi, R.P.,Pfanner,N.,
Meisinger, C., and Sickmann, A. (2006)
Toward the complete yeast mitochondrial
proteome:
multidimensional
separation techniques for mitochondrial
proteomics. J Proteome Res, 5, 15431554.
Schindler, J., Lewandrowski, U., Sickmann, A., Friauf, E., and Nothwang,
H.G. (2006) Proteomic analysis of brain
plasma membranes isolated by affinity
two-phase partitioning. Mol Cell Proteomics, 5, 390-400.
Zahedi, R.P., Begonja, A.J., Gambaryan,
S., and Sickmann, A. (2006) Phosphoproteomics of human platelets: a quest
for
novel
activation
pathways.
Biochem Biophys Acta, 1764, 1963-76.
Zahedi, R.P., Sickmann, A., Boehm,
A.M., Winkler, C., Zufall, N., Schonfisch,
B., Guiard, B., Pfanner, N., and Meisinger, C. (2006) Proteomic analysis of the
yeast mitochondrial outer membrane reveals accumulation of a subclass of preproteins. Mol Biol Cell, 17, 1436-1450.
Fig. 4:
2D-BAC/SDS-PAGE of outer mitchondrial membrane vesicles.
29
Molecular Cell Dynamics-Peter Friedl
E-mail: [email protected]
Phone: +49(0)931 201 267 31
Fax:
+49(0)931 201 267 00
http://www.rudolf-virchow-zentrum.de/forschung/friedl.html
The Molecular Cell Dynamics Group focuses on visualizing cell-matrix interactions and dynamic cell
patterning during immune cell interactions and tumor invasion using 3D ECM-based cell culture models
and, more recently, intravital imaging. Previously, the group has provided insights into the serial dynamics of T cells scanning across antigen-presenting cells (serial encounter model), the diversity of
tumor invasion mechanisms (individual, collective), as well as novel escape responses in tumor cell
migration (mesenchymal-amoeboid transition, collective-amoeboid transition).
Protease function in tumor cell invasion
During cancer progression, proteases serve
to degrade the extracellular matrix, and to
process growth factors and cell surface receptors, thereby contributing to tumor invasion, metastasis and enhanced survival.
Besides conventional biochemical assays in
test solutions, the reconstruction of enzyme function in time and space in livecell cultures allows to integrate subcellular-localized protease functions in
pathologic cell-tissue interactions. Using
3D and 4D confocal microscopy of cancer
cells expressing membrane-type matrix metalloproteinase-1 (MT1-MMP), an important
collagenase implicated in cancer invasion,
we have integrated sequential steps of proteolytic processing of the extracellular matrix (ECM) into the mechanisms of cell migration during single cancer cell invasion.
Whereas the leading edge protrudes and attaches to the ECM to generate anterior
force and traction (Fig. 1, zone 1), individual collagen fibers are cleaved 5-10 micrometers posterior to the leading edge
(Fig. 1, zone 2).
Loose fiber ends then become realigned
in parallel, generating small matrix defects
and tissue micropatterning (Fig. 1, asterisks). Thus, although MT1-MMP foci are
found near many ECM attachment sites,
only selective regions support its engagement and fiber cleavage. These findings
suggest that proteolytic digestion of the
ECM and tissue micropatterning are critical
steps in the cell migration program.
Fig. 1:
Four-channel confocal resonstruction of pericellular proteolysis (cyan) in invasive fibrosarcoma cell.
Transition from individual to collective
cell invasion and migration
Besides individually disseminating cells,
most cancers produce groups of cells moving as cell strands or clusters within the
adjacent tumor stroma, which is also called
collective cell migration. To date, collective
invasion is arguably one of the most underrated mechanisms in progressive cancer
diseases. We visualized the cellular and mo-
lecular mechanisms underlying the onset of
collective invasion using a 3D in vitro model of tumor cell spheroids invading collagen
lattices. Microtracks generated by individual cells were sequentially filled by following
cells and further widened, resulting in
strand-like multicellular invasion at the expense of the surrounding ECM (Fig. 2).
Fig. 2:
Transition from single cell to the collective invasion of cell strands.
30
MT1-MMP clustering and collagen degradation
occur at the border between cell strands and
the ECM. This causes large-scale regression of ECM, track widening and multicellular strand progression. Inhibition of
pericellular proteolysis using a combination of MMP, serine and cysteine protease inhibitors resulted in complete
abrogation of collective invasion and
causedacon-version to individual am-
oeboid dissemination (collective-amoeboid
transition) (Fig. 3).
Thus, protease engagement is essential
for multicellular invasion patterns and invasive growth, but not for individual cell
dissemination, suggesting that protease
inhibition alone may be insufficient to
prevent or inhibit cancer cell motility and
systemic dissemination leading to distant
metastasis.
Fig. 3:
Different forms and interconversion of cancer invasion and metastasis programs.
Intravital microscopy of cancer progression
Multiphoton microscopy has emerged as a
standard approach to noninvasive imaging
of thick specimens and intravital microscopy with subcellular resolution. Higher
harmonic generation microscopy, based
on nonlinear multiphoton excitation, is a
contrasting mechanism for the structural
and molecular imaging of native samples
in cell culture, as well as in fixed and live
tissues for both 3D and 4D reconstructions.
In collaboration with Gudrun Köhl and Ed
Geissler, Institute of Experimental Surgery,
University of Regensburg, we have set up
a tumor xenograft invasion model of fluo-
rescent HT1080 fibrosarcoma spheroids injected into the mouse dermis in the dorsalskin folder chamber (Fig. 4A).
Within 2 weeks of observation, individual
and collective invasion as well as substantial neoangiogenesis are apparent (Fig.
4B). This xenograft model thus allows the
simultaneous detection of growth, vessel
perfusion, cell invasion and mitotic activity from the same sample and will be instrumental in future studies on the molecular
mechanisms underlying different invasion
programs and microenvironmental control
of cancer metastasis.
Other projects
Other projects of the group address the
mechanisms of cell-cell communication between immune cells during migration, particularly T cells interacting with dendritic
cells (“dynamic immunological synapse”)
and cytolytic T cells engaging with multiple target cells, such as virus-infected or
transformed tumor cells (“serial killing concept”). The molecular mapping of different
types of cell-cell communication shows
how cell motility can support a membrane
platform for receptor engagement during
leukocyte trafficking along cell and tissues
scaffolds, allowing for the sequential sampling of molecular information from a local microenvironment. Such dynamic signal
acquisition from resident tissue cells may
contribute to the regulation of immune
defense, autoimmunity as well as hyporesponsiveness. Other projects devoted to
cell motility focus on the function of integrin and non-integrin receptors, as well as
Ras/Raf pathways in different forms of cancer cell invasion. A translational project for
tissue-engineered skin repair is the in vitro
generation of skin using provisional fibrin
scaffolds, together with keratinocytes, fibroblasts and endothelial cells. Lastly, in
collaboration with Hannes Baumann and
Otthein Herzog, Center for Computing Technologies, University of Bremen, we have
developed a fully automated cell tracking platform allowing near high-throughput analysis of cells migrating within 3D
collagen lattices.
Extramural Funding
IZKF Würzburg (D-21)
DFG (FR 1155/6-1, 6-2, 7-1, 7-2, 8-1),
(SFB TR 17; P A3), (SPP 1090)
NoE EMIL – LSHC-CT – 2004-503569; (P 45)
Dt. Krebshilfe (AZ 106950)
Selected Publications
Friedl, P., Wolf, K., von Andrian, U.H.,
and Harms, G. (2007) Biological second and third harmonic generation microscopy. Curr Prot Cell Biol, 4.15.14.15.21.
Fig. 4:
A: Overview and B: detail of dual-color HT1080 fibrosarcoma xenograft.
Wolf., K., and Friedl., P. (2006) Molecular mechanisms of cancer cell invasion and plasticity. Br J Dermatol,
154 (Suppl. 1), 11-15.
31
Inflammation and Tumor Biology-Michael P. Schön
E-mail: [email protected]
Phone: +49(0)931 201 489 77
Fax:
+49(0)931 201 487 02
http://www.rudolf-virchow-zentrum.de/forschung/schoen.html
Adhesion molecules mediate tissue-specific recruitment of leukocytes, a key step in the pathogenesis
of inflammatory disorders. We investigate the role of adhesion molecules and whether they can be
exploited as therapeutic target structures in inflammation-related disorders.
In a second focus, we investigate how tumors progress, why they are resistant to chemotherapy and
how novel therapeutic compounds can overcome mechanisms of resistance.
Novel anti-tumor therapies
Modulation of inflammatory responses
Since the 5-year-survival of patients
with metastasized melanoma is < 5 % ,
such tumors represent a major therapeutic
challenge.
We have identified anti-tumor properties
of a novel small-molecule inhibitor of IκBkinase-β (IKKβ). It prevented phosphorylation of IκB, thereby diminishing activation
and nuclear translocation of NF-κB, a transcription factor implicated in tumor progression and inducible chemoresistance.
This resulted in down-regulation of many
anti-apoptotic and proliferation-related
gene products, but did not affect proliferation or apoptosis of melanoma cells. However, significant inhibition of proliferation
was observed when it was combined with
doxorubicin at suboptimal concentrations.
In vivo, IKKβ inhibition or doxorubicin at
low doses had no therapeutic effect, but
a combination of the two significantly
diminished pulmonary metastases (Fig. 1).
Moreover, since cultures established from
metastases of treated and control mice
responded similarly to cytostatic treatment, IKKβ inhibition did not induce
chemoresistance.
Thus, the novel therapeutic principle of
selective IKKβ inhibition may enhance the
efficacy of anti-tumor therapies and prevent chemoresistance.
Tissue-specific recruitment of lymphocytes
is pivotal for their proper functions. The
integrin αEβ7 has been implicated in epithelial localization of T cells through binding to E-cadherin.
We have shown that αE-deficient mice
have significantly fewer dendritic epidermal
T cells (DETC) than wild type mice. Moreover, dendrites of wildtype DETC spanned
an area that was significantly larger than
that covered by αE-deficient DETC (Fig. 2).
When a lymphoblastic cell line was doubly
transfected with the integrin subunits αE
and β7, time-lapse microscopy revealed
significantly increased active movement
on E-cadherin. When the cells were transfected with a “locked open“ point-mutated
αE in a constitutively active conformation,
the difference was even more pronounced.
In contrast, an inactive “locked closed“
mutant did not result in enhanced motility. Antibodies against αE or cytochalasin
D abrogated the enhanced motility of the
transfectants.
When we coupled YFP to the constructs,
significantly longer dendrites spanning
larger areas became visible in wild type as
compared to mock or “locked closed“ transfectants. This difference was even greater
after transfecting the “locked open“ species
and resembled the difference seen between
wildtype and αE-deficient DETC in vivo.
In an animal model of psoriasis, a common inflammatory skin disorder in humans,
blocking of αE resulted in abrogation of
disease development. In addition, when
32
Fig. 1:
Pulmonary melanoma metastasis in mice treated
with doxorubicin alone (left panels) or a
combination of doxorubicin and a novel inhibitor
of IKKβ (right panels).
In another project, we have shown that
some melanoma cells induce expression of
E-selectin in cultured endothelia, either
through direct contact or soluble factors.
In vivo, selectin-blocking antibodies reduced pulmonary metastasis. Likewise, a
small-molecule inhibitor of selectin functions (efomycine M), resulted in significantly reduced metastasis. These findings
were corroborated by P-selectin-deficient
mice, which showed significantly diminished melanoma metastasis compared to
wildtype mice.
Given that tumor metastasis depends
on direct or indirect adhesion to vascular
endothelium, we propose that blocking of
selectin functions may contribute to the
reduction of tumor metastasis.
we generated double mutant mice overexpressing TGFβ within the epidermis and
lacking expression of αE, the psoriasis-like
phenotype of TGFβ transgenic mice was
markedly alleviated.
Overall, we propose that αEβ7 not only
plays a role in retention of epithelial T
cells, but is also involved in their locomotion and morphogenesis. In addition, it
may serve as a therapeutic target.
Thus, it is possible to diminish T cell-mediated allergic reactions through interference with L-selectin functions during the
sensitization phase.
Extramural Funding
Deutsche Krebshilfe/Dr. Mildred Scheel-Stiftung
(10-1765 Schö1) (10-2196 Schö2)
DFG (Scho 565/5-1) (Scho 565/5-2)
EU (Angioskin, LSH-2003-512127)
IZKF
Industrie (Bayer 8251290) (3M Medica)
(Serono GmbH)
Awards
Paul-Langerhans-Preis
Deutscher Hautkrebspreis
International Patent Application
Tumor treatment with IKKβ inhibitors
Selected Publications
Fig. 2:
Dendritic epidermial T cells (DETC) visualized by
fluorescent staining of CD3 in whole epidermal
sheets from a wildtype (left) and an αE integrindeficient mouse (right).
Selectins have been implicated in disorders whose initial steps depend on interactions between circulating blood cells and
endothelial cells. Such disorders include
common inflammatory and cardiovascular diseases rendering selectins attractive
targets for specific therapies. Using ex vivo
isolated lymphocytes as well as an L-selectin transfected lymphocyte line in dynamic
flow chamber experiments, we have shown
that efomycine M significantly blocks Lselectin-mediated adhesion on sialylated
LewisX, an action that was confirmed at
the molecular level by plasmone resonance
spectroscopy.
Since recruitment of naive T cells to peripheral lymph nodes depends on L-selectin,
intravital microscopy revealed a significant
reduction of lymphocyte rolling in lymph
nodes of mice treated with efomycine
M. Since the recruitment of naive lymphocytes is a prerequisite for lymphocyte sensitization in allergic reactions, mice were
treated with efomycine M or an L-selectin-specific antibody during contact sensitization. We could demonstrate that the
capacity of their T cells to induce contact
hypersensitivity responses after adoptive
transfer into non-sensitized recipients was
significantly reduced (Fig. 3).
Fig. 3:
Contact hypersensitivity (cellular infiltrate and
dermal edema) to an obligate allergen in mice
adoptively transferred with sensitized T cells
isolated from a syngeneic donor mouse treated
with vehicle (upper panel) or the selectin
inhibitor efomycine M (lower panel) during the
sensitization phase (Oostingh et al., J Invest
Dermatol, 2006)
P-selectin has been implicated in important platelet functions, but its suitability
as therapeutic target is not entirely clear.
We have demonstrated that antibody or
small molecule-mediated inhibition of Pselectin functions significantly reduced
platelet aggregation and platelet-neutrophil adhesion in vitro. Established aggregates were degraded, via detachment of
either single platelets (efomycine M) or
of multi-cellular clumps (anti-P-selectin
Fab-fragments). In vivo, selectin inhibition resulted in > 50% reduction of platelet rolling on cutaneous venules, similar to
the rolling fractions observed in P-selectin
deficient mice. Moreover, selectin inhibition significantly decreased the thrombus
size in arterial thrombosis in mice. In an
ischemia/reperfusion injury model, small
molecule-mediated selectin inhibition significantly reduced myocardial infarct size
and reperfusion injury.
Thus, inhibition of P-selectin functions
reduced platelet aggregation and alleviated platelet-related disorders in diseaserelevant preclinical settings.
Wienrich, B.G., Krahn, T., Schön, M.,
Rodriguez,
M.L.,
Kramer,
B.,
Busemann, M., Boehncke, W.H., and
Schön, M.P. (2006) Structure-function
relation of efomycines, a family of smallmolecule inhibitors of selectin functions.
J Invest Dermatol, 126, 882-889.
Schön, M.P., Schön, M., and Klotz, K.N.
(2006) The small anti-tumoral immune
response modifier imiquimod interacts
with adenosine receptor signaling in
a TLR7- and 8-independent fashion.
J Invest Dermatol, 126, 1338-1347.
Oostingh, G.J., Ludwig, R.J., Enders, S.,
Grüner, S., Harms, G., Boehncke, W.H.,
Nieswandt, B., Tauber, R., and Schön,
M.P. (2007) Diminished lymphocyte
adhesion and alleviation of allergic responses by small-molecule- or antibodymediated inhibition of L-selectin functions. J Invest Dermatol, 127, 90-97
Schön, M., and Schön, M.P. (2006)
The antitumoral mode of action of imiquimod and other imidazoquinolines.
Curr Med Chem, in press.
Oostingh, G.J., Pozgajova, M., Ludwig, R.J., Krahn, T., Boehncke, W.H.,
Nieswandt, B., and Schön, M.P. (2006)
Diminished thrombus formation and alleviation of myocardial infarction and
reperfusion injury through antibody- or
small-molecule-mediated inhibition of
selectin-dependent platelet functions.
Haematologica Hematol J, in press.
33
Project: Translational regulation: TOP-response
Proteins-Utz Fischer
Email: utz.fi[email protected]
Phone: +49(0)931 888 402 9
Fax:
+49(0)931 888 402 8
http://www.biochem.biozentrum.uni-wuerzburg.de
To cope with the increasing demand of protein synthesis during cell growth and proliferation, factors of
the translational apparatus, such as ribosomal proteins and translation factors, need to be produced in
large quantities. However, this situation changes when nutrition becomes limiting (starvation) or when
proliferation is arrested. Under these conditions, production of translational machinery components is
reduced to save energy and resources for producing other proteins required for the functioning of the
cell. Adjustment of the translational machinery to growth conditions has been shown in recent years
to occur in vertebrates, and even in insects, at the level of translation in a process collectively termed
“TOP-response”.
The mRNAs encoding these proteins contain a unique sequence motif at their 5’ends, called the TOP motif, which is the
core cis-regulatory element of all TOP
mRNAs. As a cellular reponse to insufficient nutrition, TOP mRNAs are prevented
from being translated and instead stored
in mRNP-particles of unknown function and
composition. Despite intense efforts to unravel the details of this regulatory event,
neither its underlying mechanism nor the
trans-acting factors that bind to the TOP
motif are known. In this project we aim to
identify factors involved in the regulation
of TOP-mRNAs and to elucidate their mode
of function.
Identification of the TOP motif-binding
factor TBF
We developed a UV-crosslinking strategy
to identify factor(s) interacting with the
TOP-motif. A radioactively labeled RNA
oligonucleotide containing the TOP-sequence of ribosomal protein S6 was incubated with extracts derived from starved
or non-starved cells and irradiated with UV
light. The crosslinked proteins were subsequently identified by autoradiography of
34
Fig. 1:
Identification of TBF. A: Crosslink of a 70kDa protein to a TOP-RNA oligonucleotide. B: Schematic
drawing of tobramycin-tag affinity chromatography used to isolate TBF. The domain structure of
TBF is shown in the lower part of the figure.
conventional SDS-polyacrylamide gels. As
shown in Figure 1A, this approach identified a 70kDa protein that crosslinks to the
RNA oligonucleotide in starved but not in
non-starved cell extracts.
The crosslinked factor was subsequently purified by means of tobramycin-tag
affinity chromatography and sequenced
by mass spectrometry. These studies revealed a so far unknown factor containing
two RNA-recognition motifs (RRMs) and a
La motif (Fig. 1B).
TBF associates with TOP-mRNAs
Anti-TBF antibodies were raised to investigate whether cellular TOP-mRNAs specifically bind to TBF upon serum starvation.
Extracts from cells cultivated under either
non-starvation or starvation conditions
were immunoprecipitated with anti-TBF
antibodies and the co-precipitated mRNAs
were identified by RT-PCR (Fig. 2). These
studies revealed a specific association of
TOP-mRNAs (S6-mRNA), but not mRNAs
lacking this motif (FeLc or hnRNP E2-mRNA)
with TBF in extracts from starved cells.
Our data suggest that TBF plays a crucial
role in the regulation of TOP-mRNAs. Since
the TOP-motif is always located at the very
5’-end of the message, we speculate that
TBF interferes with translation of TOP-
mRNAs at the level of initiation, presumably by preventing eIF4E binding to the
cap structure (Fig. 4). The precise mode of
function of TBF in the regulation of TOPmRNAs in vivo is currently been addressed.
Fig. 2:
TOP-mRNA S6 associates with TBF. TBF was
immunoprecipitated (IP) and bound mRNAs
(S6, FeLc and hnRNP C) were analyzed by
RT-PCR. Precipitated TBF was detected by
western blot (lower panel).
Given its specific association with TOPmRNAs under starvation conditions, we
next tested whether this factor would interfere with translation of reporter mRNAs
carrying this motif. To address this issue,
we generated recombinant TBF in E. coli.
This protein bound specifically to the TOPmotif but not to a mutant version thereof
containing a single base substitution and
is known to disrupt the function of this
motif (Fig. 3A). Strikingly, recombinant
TBF efficiently inhibited translation of
a TOP-mRNA reporter but failed to interfere with translation of a non-TOP mRNA
(Fig. 3B). Thus, TBF acts as a negative regulator of TOP-mRNA translation in vitro.
Fig. 4:
A possible mode of action of TBF in the translational repression of TOP-mRNAs.
Extramural Funding related to project
GIF
Selected Publications
Otter, S., Grimmler, M., Neuenkirchen,
N., Chari, A., Sickmann, A., and Fischer,
U. (2007) A comprehensive interaction map of the human SMN-complex.
J Biol Chem, in press.
Fig. 3:
Functional characterization of TBF in vitro. A: Recombinant TBF binds to the TOP-motif
but not to an inactive mutant thereof. B: Specific inhibition of TOP-mRNA translation by
recombinant TBF in vitro. Translation of mRNAs containing either a mutated TOP-motif
(muTOP-mRNA) or lack the TOP-motif (Unrip-mRNA) are unaffected.
35
Project: Hey Factors in Cardiac
Development-Manfred Gessler
Email: [email protected]
Phone: +49(0)931 888 415 9
Fax:
+49(0)931 888 703 8
http://www.biozentrum.uni-wuerzburg.de/pc1/gessler
Development of complex biological structures such as the human heart and vascular tree depends on
the coordinate interplay of multiple signaling pathways that control cellular differentiation. We are
interested in understanding the role of the Notch signaling pathway in these processes.
The Notch pathway represents a key molecular switch determining cell fate in many
cell types and tissues. During heart development signaling through Notch1 is essential for forming the septum and valves.
Loss of the Notch target gene Hey2 in mice
results in similar fatal cardiac septum and
valve defects, suggesting that Hey2 transmits Notch signals in cardiac development.
In addition, Hey2 cooperates with Hey1
during angiogenesis to control vascular
remodeling and the decision to form arteries as opposed to veins. By elucidating
the function of Hey proteins we hope to
reach a better understanding of embryonic
developmental processes and to gain novel
insights into the pathogenesis of cardiovascular diseases.
the embryonic AV cushions. We found that
Notch1 and Notch2 receptors are co-expressed with the Jag1 ligand and all three
Hey genes (Hey1, Hey2 and HeyL) in the
endocardium of the AV cushion region,
which represents the primary source of
mesenchymal cells that subsequently give
rise to the membraneous septum and the
valves. Histological and 3D MRI analysis
of embryonic hearts revealed very similar
phenotypic alterations in Notch1, Hey2 and
HeyL/1 mutants.
Hey1/HeyL loss leads to cardiovascular
defects
A loss of Hey1 or HeyL does not lead to
obvious developmental alterations or defects in mice. However, combined loss of
both genes results in cardiac septum and
atrioventricular valve defects essentially
similar to those seen in Notch1 and Hey2
knockout mice. In a C57BL/6 background
this leads to early postnatal lethality with
massive cardiac enlargement. The affected
membraneous part of the septum and the
tricuspid valve leaflets are derived from
36
Fig. 1:
HeyL/1 double knockout embryo: AV valve
(arrow) and ventricular septum defect (*)
at E15.5 (upper row) and postnatal day 7
(lower row).
Fig. 2:
3D-MRI reconstruction of a HeyL/1 mutant
E15.5 heart. Septum defect is marked by a
white arrow (abbreviations: rv/lv, right/left
ventricle, ra/la, right/left atrium).
Loss of HeyL/Hey1 impairs EMT
Upon explant culturing of AV cushion tissue we found that epithelial-mesenchymal
transformation (EMT) of endocardial cells
was initiated normally, but full transformation into mesenchymal cells failed as
judged by morphology and actin staining.
This was most pronounced in Notch1 mutants and attenuated in HeyL/1 deficient
embryos. Time-lapse imaging in collaboration with Peter Friedl revealed that mutant
cells exhibit amoeboid-type motility, while
wild-type cells were elongated and spindlelike during migration. The mutant phenotype was accompanied by reduced levels
of matrix metalloproteinase-2 (Mmp2) and
Snail-1 expression, which may explain some
of the defects observed.
Fig. 4:
Hey gene family shows overlap in controlling Notch induced endocardial EMT,
a process critical for AV valve and septum formation.
Fig. 3:
Impaired EMT in Hey2 and HeyL/1 mutant
AV cushion explants. Cells were stained with
DAPI and FITC-Phalloidin to visualize nuclei
(blue) and actin (green).
Combinatorial functions of Hey genes
Hey protein interaction
While Notch1 null mutations completely
abolish EMT, we could show that loss of
Hey2 or HeyL/1 leads to very similar, but
less severe deficiencies with almost normal
initiation of EMT, but a lack of full transformation. Due to the dynamic nature of
septum formation and presumably a narrow time window the final results are then
rather similar. The overlapping phenotypic
effects of loss of Hey2 and HeyL/1 clearly
point to a common pathway. This is further supported by the efficient formation
of heterodimers between the different,
but highly related Hey proteins. Thus,
there seems to be a combinatorial action
of Notch pathway target genes to control
cardiovascular development.
To gain insights into the transcriptional
activity of Hey proteins we searched for
potential binding partners. A yeast-twohybrid screen identified Bre as an interacting protein. We found nuclear co-localization of Bre with Hey, but not Hes proteins.
A short motif of 7 amino acids within the
carboxy-terminal third of Hey proteins
was identified as a binding site. Although
Bre was shown to stimulate the E3-ligase
activity of the BRCA1/2 complex, we did
not find evidence of any alterations in Hey
protein half-life or localization. Experiments to measure potential transcriptional
regulation under the influence of Bre are
currently under way.
Selected Publications
Diez, H., Fischer, A., Winkler, A.,
Hu, C. J., Hatzopoulos, A. K., Breier,
G., and Gessler, M. (2007) Hypoxia-mediated activation of Dll4-Notch-Hey2
signaling in endothelial progenitor
cells and adoption of arterial cell fate.
Exp Cell Res, 313, 1-9.
Rutenberg, J. B., Fischer, A., Jia, H.,
Gessler, M., Zhong, T. P., and Mercola, M.
(2006) Developmental patterning of the
cardiac atrioventricular canal by Notch
and Hairy-related transcription factors.
Development, 133, 4381-4390.
37
Project: T Cell Surface Proteins-Thomas Hünig
Email: [email protected]
Phone: +49(0)931 201 499 51
Fax:
+49(0)931 201 492 43
http://www.virologie.uni-wuerzburg.de/
One of the main goals of immunology is to understand why autoimmune disorders occur. Ten years ago,
studies into a lethal autoimmune disease in a strain of mice known as `scurfy‘ led to the discovery of
a unique population of T lymphocytes that negatively regulate immune responses. These regulatory T
cells (Treg cells or suppressor T cells) have since been confirmed to play an essential role in both selftolerance and in preventing exaggerated immune responses to foreign antigens. However, significant
gaps in our understanding of Treg cell biology remain, partly because of the lack of suitable markers
for purifying these cells. All of the cell surface proteins that are currently used to identify Treg cells,
such as CD4, CD25, CD152, GITR, LAG-3, and neuropilin-1 are also expressed on other T cell subsets.
The most reliable marker of Treg cells is the transcription factor Foxp3, but as a nuclear protein it is
unfortunately not a suitable marker for the purification and manipulation of viable Treg cells. Our aim
is to identify unique Treg cell surface markers through two approaches, namely via the generation of
monoclonal antibodies against Treg cells and by comparing the membrane proteome of Treg cells with
conventional T cells.
Production of large numbers of highly
activated Treg cells
Previous attempts at identifying Treg-specific proteins have relied on using DNA
microarrays to detect genes that are expressed in Treg cells but not other T cell
sub-populations. While this approach has
the advantage of only requiring a small
amount of sample material, it has not been
entirely successful. Molecules identified
by this approach, such as GITR and neuropilin-1, are not restricted to Treg cells.
Furthermore, it has recently been shown
that Treg development is controlled at a
post-transcriptional level by the ribonuclease III enzyme Dicer and a unique profile
of micro-RNAs. These findings, combined
with the inability of RNA-based approaches
to detect posttranslational modifications
that may make a protein cell-type specific,
suggest that comparative proteomics may
be a more promising approach to identify
Treg-specific proteins. This approach is
only feasible, however, if large numbers
of – preferentially activated – Treg cells
are available as a source of membrane
proteins to be analyzed, and moreover in
38
collaboration with a highly experienced
proteomics group.
We previously developed stimulatory
CD28-specific monoclonal antibodies that
dramatically expand and activate Treg cells
in rodents. Administration of these `superagonistic‘ antibodies to rats and mice
in vivo increased the percentage of CD4+
CD25+ Foxp3+ cells from around 5-10%
of CD4+ T cells to over 45%, providing
us with a rich source of starting material
(Fig. 1). Furthermore, functional assays
have revealed that the suppressive activity of these Treg cells expanded in vivo is
approximately 10-fold greater than that of
resting Treg cells, increasing the likelihood
that functionally relevant molecules are
present in membrane preparations.
Fig. 1:
Treg cells are expanded in vivo using CD28 superagonistic mAbs.
Generation of monoclonal antibodies
It is our aim to induce mouse anti-mouse
Treg-specific monoclonal antibodies by
immunizing Foxp3-deficient (scurfy) mice
with membrane proteins derived from
mouse Treg cells. Since Foxp3-deficiency
results in a lethal inflammatory disease, it
is necessary for us to introduce additional
transgenes to protect the mice from early
death. Breeding of an appropriate mouse
line is currently under way.
In the meantime, we have initiated a
program to generate mouse anti-rat Treg
monoclonal antibodies. Treg cells obtained
from CD28 superagonist-stimulated rats are
used to prepare plasma membranes as an
immunogen. Screening of the first fusions
is currently in progress.
Proteomics approach
We have developed a protocol for enriching the plasma membrane proteins from
in vivo activated rat and mouse Treg cells
(Fig. 2). As confirmed by René Zahedi and
Albert Sickmann, our collaborators at the
RVZ, numerous T cell-specific membrane
proteins are readily identified by nano-LC
ESI-MS/MS following the one-dimensional
separation of such a preparation. It is our
plan to compare activated Treg cells with
conventional CD4 T cells by differential
labeling of the membrane proteins with
the fluorescent dyes Cy3 and Cy5, followed
by 2D BAC/SDS PAGE and image analysis
to identify Treg-specific proteins. These
will then be excised and analyzed by mass
spectrometry.
Fig. 2:
Enrichment of T cell plasma membrane proteins.
Selected Publications
Beyersdorf, N., Balbach, K., Hunig, T.,
and Kerkau, T. (2006) Large-scale expansion of rat CD4 CD25 T cells in the
absence of T-cell receptor stimulation.
Immunology, 119, 441-449.
Dennehy, K.M., Elias, F., Zeder-Lutz, G.,
Ding, X., Altschuh, D., Luhder, F., and
Hunig, T. (2006) Cutting edge: monovalency of CD28 maintains the antigen
dependence of T cell costimulatory responses. J Immunol, 176, 5725-5729.
Dennehy, K.M., Elias, F., Na, S.-Y.,
Fischer, K.-D., Hunig, T., and Lühder, F.
(2007) Mitogenic CD28 signals require the
exchange factor Vav1 to enhance TCR signaling at the SLP-76-Vav-Itk signalosome.
J Immunol, in press.
Kerstan, A., Armbruster, N., Leverkus,
M., and Hunig, T. (2006) Cyclosporin A abolishes CD28-mediated resistance to CD95-induced apoptosis via superinduction of caspase-3.
J Immunol, 177, 7689-7697.
39
Project: Ligand-Receptor RecognitionThomas D. Müller
Email: [email protected]
Phone: +49(0)931 888 410 0
Fax:
+49(0)931 888 411 3
http://www.biozentrum.uni-wuerzburg.de/pc2
Analysis of the human genome has clearly shown that the complexity of an organism is more than just
the sum of its genes. It is therefore necessary to understand how signaling diversity is achieved with
a relatively small number of genes. One important aspect in this investigation is to analyze how biomolecules interact with each other. In the past, protein-protein interactions were interpreted strictly
via a key-and-lock mechanism. More and more new data however reveal that proteins often interact
with more than one binding partner. This constrained specificity might not only be useful to enhance
redundancy in important signaling cascades, but it could also enhance signaling diversity. Using structural biology as a tool we are studying two protein families of secreted factors: the cytokines IL-4, -5
and -13, which are involved in the development and progression of allergic diseases and asthma, and
the bone morphogenetic proteins (in collaboration with W. Sebald), which are important regulators in
embryonal development as well as organ and tissue homeostasis. Both protein families represent prime
examples of ligand-receptor promiscuity. Understanding how a ligand can interact with various different receptors will yield important insights into how proteins generate and modulate binding specificity
at the molecular level.
IL-4 and IL-13 as key regulators of allergies
Interleukin 4 (IL-4) is a pleiotropic cytokine that plays a major regulatory role in
the immune system, e.g. it induces differentiation of T helper cells to a TH2 type
and is involved in class switching to IgE
and IgG4. Activated TH2 cells trigger the
activation and/or recruitment of IgE antibody-producing B cells, mast cells and eosinophils, which are all involved in allergic
inflammation. Thus, IL-4 plays a key role
in the development and progression of allergic hypersensitivity. Signal transduction
of IL-4 is mediated by a sequential binding
process, initiated first by IL-4 binding to
its high-affinity receptor subunit IL-4Rα.
This intermediate complex then recruits
one of two low-affinity receptor subunits,
the common gamma (γc) or the IL-13Rα1
chain, into the functional hetero-trimeric
complex to initiate signaling. The γc receptor subunit is shared among cytokines of
40
the IL-2 family, whereas the IL-13Rα1 subunit is exclusively used by IL-4 and -13.
Fig. 1:
Sequential binding mechanism of interleukin-4
and -13 to their cognate receptor.
IL-13 is described as the alter ego of IL-4,
but both cytokines share only 25% identity
at the amino acid sequence level. Despite
this moderate homology, IL-13 and IL-4 use
an identical cellular receptor formed by the
same IL-4Rα and IL-13Rα1 subunits. However, the order of binding events and affinities to the individual receptor subunits
differ markedly between the two cytokines.
In contrast to IL 4, IL-13 first binds to the
IL-13Rα1 subunit with high affinity and
subsequently recruits the IL-4Rα chain as
the low-affinity receptor subunit into the
complex. Thus both receptors, IL-4Rα and
IL-13Rα1, can alter their affinities in the
context of the bound ligand over a range of
almost 1000-fold.
To determine the molecular mechanism
by which a protein can “switch” its affinity
without compromising its binding specificity, we have used a combined mutagenesis/structure analysis approach. Analysis
of the IL-4:IL-4Rα complex has revealed a
new type of protein-protein interface. The
epitope consists of three independently
acting interaction clusters, which are characterized by hydrogen-bonding networks
that only act within a cluster. No H-bonds
occur in between the different clusters. For
the high-affinity binding of IL-4 to IL-4Rα,
two (cluster I and II) of the three clusters
are fully active, while cluster III is only
used to a minor extent. Two mutations in
cluster III, T13D and F82D, activate this
cluster, yielding IL-4 variants that bind
IL-4Rα with increased affinity. The mechanism mediating such superagonist activity
is due to stabilization of the H-bond network in cluster III.
Fig. 2:
The modular architecture of the IL-4:IL-4Rα
interface.
Mutagenesis studies of IL-13 show that the
low-affinity binding of IL-13 to IL-4Rα is
achieved by using only two of these interaction clusters; cluster III is completely
inactive, resulting in a low affinity interaction. This novel interaction interface
presents a possible mechanism by which
proteins can alter binding affinity independently from binding specificity. A modular
or clustered interface acts as a low-affinity epitope if only a few (or one) clusters
are active, while all clusters are switched
on for high-affinity binding. High binding
specificity is ensured since the non-active
cluster(s), although not contributing to
binding affinity, would not act in a repulsive manner. Understanding the recognition mechanism will finally allow the design of highly specific growth factors that
are able to distinguish between different
receptor combinations.
IL-5 receptor IL-5Rα shows a novel cytokine receptor architecture
Interleukin-5 (IL-5) is the key cytokine
directly involved in the onset and progression of asthma. Its main target cells are eosinophils. IL-5 is involved in the early differentiation process from CD34+ progenitor
cells. It is responsible for eosinophil migration into lung tissue and activates the
eosinophils in this tissue, leading to the
known asthma symptoms such as airway remodeling and airway hyper-responsiveness.
Hence, IL-5 represents a highly interesting drug target for the treatment of asthma
diseases. High-throughput screening has
helped develop the first small organic
drugs, which block the high-affinity IL-5
receptor IL-5Rα. Knowledge of the structure of the IL-5 ligand-receptor complex
would certainly further facilitate development of drugs by rational drug design.
After many unsuccessful attempts we
have finally been able to obtain crystals of
the binary ligand-receptor complex of IL-5
bound to the extracellular part of IL-5Rα.
Receptor activation is quite similar to IL-4.
IL-5 binds first to its high-affinity receptor
subunit IL-5Rα; this intermediate complex
then recruits a second receptor subunit, the
common beta (βc) chain into the complex.
The latter subunit is shared with cytokines
IL-3 and GM-CSF. Besides these similarities,
IL-5 also has a number of unique features.
First, IL-5 forms a disulfide-linked dimer;
one helix of the four-helix bundle is part of
the other chain. Despite its dimeric nature
IL-5 binds only one IL-5Rα receptor molecule per dimer. Second, mutagenesis studies have identified three (instead of two as
for classical cytokines) binding regions in
IL-5 that are important for binding to its
high-affinity receptor IL-5Rα, suggesting a
novel interface architecture. The extracellular part of IL-5Rα consists of three fibronectin type III (FNIII) modules instead
of two as for other cytokine receptors. Deletion variants of IL-5Rα have confirmed
that all three fibronectin modules are required for binding.
Fig. 3:
Hydrogen bonding network in cluster III is responsible for superagonist activity.
Preliminary structural analysis is now
yielding the first insights into this new
cytokine ligand-receptor interface. The
three FNIII modules wrap around IL-5 like
a wrench, and all regions of IL-5 identified
as binding regions are in contact with the
receptor molecule. The location of the receptor molecule on the ligand also explains
the unexpected stoichiometry. The putative second binding site in the IL-5 dimer
is partially blocked by the first receptor
molecule bound. Currently, we are trying to
analyze the data to obtain a higher resolution picture of the IL-5:IL-5Rα complex
structure. The final high-resolution structure will then be used for the rational design of more potent IL-5 inhibitors, which
could be used to treat diseases like asthma
and hypereosinophilia.
Fig. 4:
Preliminary structure analysis of the IL-5:IL-5Rα complex.
Extramural Funding related to project
DFG (SFB 487, TP B2), (MU1095/3-1),
(GK520 TP C1)
Selected Publications
Kraich, M., Klein, M., Patino, E.,
Harer, H., Nickel, J., Sebald, W., and
Mueller, T.D. (2006) A modular interface of IL-4 allows for scalable affinity
without affecting specificity for the IL-4
receptor. BMC Biol, 4, 13.
Meierjohann, S., Mueller, T.D.,
Schartl, M., and Buehner, M. (2006) A
structural model of the extracellular domain of the oncogenic EGFR variant Xmrk.
Zebrafish, 3, 359-369.
41
Project: Posttranslational Gene RegulationManfred Schartl
Email: [email protected]
Phone: +49(0)931 888 414 8
Fax:
+49(0)931 888 415 0
http://www.pch1.biozentrum.uni-wuerzburg.de/
Tumor modifier genes are genetic factors involved in determining the malignant phenotype of the tumor and the course of the cancerous disease, but not in the primary steps of neoplastic transformation
and initiation of the tumorigenesis process. Such genes have been identified in various organisms but
knowledge about their molecular identity and their biochemical functions is almost lacking. We use
melanoma developing transgenic fish as model systems for identifying and isolating such tumor modifier genes by a mutagenesis screen. We search for genes that lead to either a more benign or a more
malignant phenotype of the melanoma.
Xmrk as candidate
Pigment cell specific promoters
It is expected that tumor modifier genes
are involved in processes such as transition from the benign to the malignant
state, tumor cell migration, invasion, and
metastasis. In molecular terms, it is anticipated that these genes are involved in
modulating the intracellular signals elicited by the activity of the melanoma-inducing transmembrane receptor.
Our strategy is based on previous knowledge about the Xmrk oncogene of Xiphophorus. Xmrk is the mutated version
of the epidermal growth factor receptor. It
is known that overexpression of the Xmrk
gene alone is sufficient to induce highly
malignant melanoma in fish. As Xiphophorus fish are inaccessible to transgenic approaches, we attempted to transfer the
Xmrk melanoma system to medaka, a small
aquarium fish species comparable to zebrafish. Most importantly, large-scale mutagenesis screens have been successfully
performed in this fish and the full genome
sequence is available.
First, using GFP reporter constructs we
searched for a promoter that directs transgene expression exclusively in the pigment
cell lineage, and is strong enough to drive
the appropriate level of overexpression
necessary for Xmrk-mediated transformation. The 300 bp fragment from the proximal promoter of the medaka tyrosine gene
was found to be unsuitable, since the
promoter becomes leaky in adult fish, leading to unspecific widespread expression
of the transgene. We then identified a
1000 base pair fragment of the fish mitf
(microphtalmia transcription factor) promoter, which met the required criteria in
stable transgenic medaka lines. Expression of the transgene is restricted to the
pigment cell lineage (Fig. 1). The promoter is active in the non-pigmented
precursor cells as well as in differentiated
pigment cells. Interestingly, expression
occurs not only in melanin synthesizing
cells but also in the erythrophore/xanthophore sub-lineage.
Fig. 1:
Pigment cell specific expression of GFP under control of the mitf promoter in transgenic
medaka. A: Embryo with green fluorescing cells showing the branched morphology of
pigment cell precursors. B: Stellate terminally differentiated pigment cells in the
epidermis of an adult fish.
42
Melanoma developing transgenic medaka
Extramural Funding related to project
An mitf Xmrk-SV40polyA construct flanked
by meganuclease (Sce I) sites was then
constructed and injected into one-cell
stage medaka embryos, together with Sce
I restriction enzyme. A large number of
the F0 animals developed highly malignant
pigment cell tumors (Figures 2 and 3).
DFG GK 1048
Selected Publications
Meierjohann, S., and Schartl, M. (2006)
From mendelian to molecular genetics: the Xiphophorus melanoma model.
Trends Genet, 22, 654-61.
Volff , J.N., Nanda, I., Schmid, M.,
and Schartl, M. (2006) Governing
sex determination in fish: regulatory putsches and ephemeral dictators.
Sex Dev, in press.
Fig. 2:
Medaka fish expressing the Xmrk transgene under the control of the medaka mitf promoter. Upper: fish
with a non-malignant hyperpigmentation, a large area of the tail fin is covered by melanocytes. This
type of pigmentation abnormality is a precursor lesion that eventually can develop into melanoma.
Lower: Fish with highly malignant melanoma showing metastasis and invasion at various sites.
F0 animals were intercrossed to obtain F1
fish with stable integration of the transgene. Two independent transgenic lines
have been obtained so far, and these show
pigment cell specific overexpression of
Xmrk and spontaneously develop tumors.
The tumor phenotype within each line is
stable and comparable between individuals, while the two lines are different from
each other with respect to growth characteristics and malignancy of the pigment
cell lesions. Melanocytic tumors are mostly
derived from extracutaneous pigment cells
and show highly invasive growth, preferentially in the trunk muscular compartment
and in abdominal organs. Tumors from the
erythrophore/xanthophore lineage show
exophytic growth with only a low tendency
towards invasion and metastasis. The different types of developing tumors are currently being characterized on the gene
expression and biochemical level and compared to the situation in Xiphophorus. So
far strong activation of the ras/raf/MAP kinase pathway was detected in the tumors,
which is in line with in vivo and in vitro
data from Xiphophorus and mammalian
melanomas.
Fig. 3:
Histological sections of two different medaka, developping malignant melanoma due to
the mitf::Xmrk transgene. Left: Section through the anterior abdomen. The melanoma
(T) is developing around the foregut and the spinal chord (SC). Tumor cells are invading
the musculature (M) and metastasis is seen in the liver (L). Right: Section through the
posterior abdomen. A melanoma (T) consisting mainly of unpigmented lowly differentiated cells (except in the area of the interstine) is filling the whole body cavity and
shows invasive growth into the ovary (O).
43
Project: BMP Receptors Structure and FunctionWalter Sebald
Email: [email protected]
Phone: +49(0)931 888 411 1
Fax:
+49(0)931 888 411 3
http://www.biozentrum.uni-wuerzburg.de
Bone morphogenetic proteins (BMPs) and BMP-like proteins are key regulators of organ development
and tissue regeneration. Dysregulation of BMP signaling results in tumor formation and among others
in cardiovascular, musculoskeletal and urogenital diseases. To understand how BMPs bind and activate
their receptors and how they are regulated by extracellular modulator proteins, we are studying the
structure of ligand receptor complexes and the energetics and kinetics of BMP interactions with receptors and modulator proteins. We are generating BMPs that mimic mutations in familial disorders and
which have useful properties for applications in regenerative medicine and musculoskeletal diseases.
BMP signaling
proteins or binary complexes. It is also seen
that no direct contacts exists among the
ECDs. They only bind to BMP-2. Thus, the
ligand serves as a rigid scaffold, which assembles the ECDs in a defined orientation.
BMPs are a family of dimeric extracellular
proteins, which signal into cells by initiating transactivation among two types of
single-span serine/threonine kinase receptors in the plasma membrane. BMP-2 is a
prototypical BMP that determines multiple
steps during embryonal development and
which regulates bone regeneration in the
adult organism. The type I chains (BMPRIA, BMPR-IB) bind BMP-2 with high affinity
and initiate the intracellular Smad-dependent pathways. The type II chains (BMPRII, ActR-II, ActR-IIB), which bind BMP-2
with low affinity, transactivate the type I
chain by phosphorylation at their gylcine-/
serine-rich GS box. Dimeric BMP-2 can bind
two type I and two type II receptors. The
receptors exist in the membrane in monomeric as well as pre-assembled homo- and
heterodimeric forms.
Modulator proteins (ligand traps)
The activity of BMPs is regulated by many
modulator proteins in the extracellular
space. Some of them can inhibit or support
BMP functions depending on the context;
they have pro- and anti-BMP activity. Many
of these proteins contain one or several
VWCs (Von Willebrand factor C), domains
which at least in some instances bind BMPs
directly. Some members of this group seem
to be involved in diseases such as fibrosis
(CTGF, kielin, crossveinless-2) or osteoarthritis (chordin-like 2).
The activated state of the BMP receptor
The affinity of BMP-2 for its BMPR-IA receptor, which has a KD of 20nM for a 1:1 interaction, is strongly increased to a KD <1nM if
two chains are present and a 2:1 interaction
is possible. Such a situation exists in the
membrane or on a biosensor during a biacore experiment. The ectodomains (ECDs)
bind to BMP-2 independently, in particular
the binding affinity for type II ECDs is not
influenced by the type I ECD. This is shown
by means of biacore experiments where
ActR-IIB ECD exhibits the same low affinity
(9-10 µM KD) during the interaction with
BMP-2 alone or with BMP-2 in the binary
complex with BMPR-IA ECD (Fig. 1).
44
Fig. 1:
BMPR-IA and ActR-IIB receptor ectodomains bind
independently to BMP-2.
The crystal structure of the ternary complex consisting of BMP-2 and two BMPR-IA
and two ActR-IIB ECDs shows that during
complex formation the proteins retain their
conformation in comparison with the free
Fig. 2:
Modulator protein Crossveinless-2 (CV2) has proand anti-BMP activity.
Crossveinless-2 (cvl2) in zebrafish has ventralising (pro-BMP) and dorsalising (antiBMP) activities (Fig. 2). We could show
that the N-terminal segment of cvl2 (cvl2N), which consists of five VWC domains in
series, binds BMP-2 like the complete protein. It competes with chordin for binding
to BMP-2, but does not interact with Tsg
(twisted gastrulation). Since the affinity
of cvl2 for BMP-2 is comparable to that of
the high affinity BMPR-IA receptor it seems
possible that it can act as a transport vehicle for BMP-2. Remarkably, an uncleavable
mutant form of cvl2 (cvl2-CM), which in
contrast to a processed cvl2 binds strongly
to proteoglycans in the extracellular matrix
via a heparin-binding epitope has strong
anti-BMP activity.
Loss-of-function and gain-of-function
mutations in human GDF-5
GDF-5 uses BMPR-IB as the main type I
receptor; its affinity for BMPR-IA is much
lower. This differs from BMP-2, which binds
to BMPR-IA and BMPR-IB with similar high
affinities. The affinity and specificity of
type I receptor binding is determined in
both in GDF-5 and BMP-2 by the so-called
pre-helix loop (Fig. 4).
Receptor-dead Noggin blocker
We could generate BMP-2 variants which
are inactive in receptor activation but efficiently block modulator proteins like noggin, gremlin, chordin, chordin-like 2, and
crossveinless-2 (Fig. 3).
Fig. 4:
Gain-of-function mutant R52L of GDF-5 leads to
symphalangism in men.
Extramural Funding related to project
DFG (SFB 487 TP1), (KFO103, C (Se 438/
8-3, Se 438/8-4))
Industry
Selected Publications
Fig. 3:
BMP-2 mutant L51P releases Noggin-inhibition of
BMP-2 signaling.
The critical position for receptor affinity is
the residue at position 51, which can form
a functional hydrogen bond in the contact.
A “receptor-dead” L51P BMP-2 variant is
presently being studied in animal disease
models for fracture repair, osteoporosis, fibrosis, and osteoarthritis.
The residue at position 52 of GDF-5 (position 438 in the proprotein) is critical for
the discrimination between IA and IB BMP
receptors. An Ala52 and also a Leu52 give
high IA affinity, whereas Arg52 disrupts
IA binding. Affinity for the IB subtype
is equally high in both GDF-5 variants.
Interestingly, a gain-of-function mutant
R52L in human GDF-5 results in missing
joints (symphalangism, SYM1). In contrast,
a loss-of-function GDF-5 mutant L55P
(position 441 in the proprotein) causes
shortened or missing digits (brachydactyly, BDA2).
Rentzsch, F., Zhang, J., Kramer, C.,
Sebald, W., and Hammerschmidt, M.
(2006) Crossveinless 2 is an essential
positive feedback regulator of Bmp signaling during zebrafish gastrulation.
Development, 133, 801-811.
Kraich, M., Klein, M., Patino, E., Harrer,
H., Nickel, J., Sebald, W., and Mueller,
T.D. (2006) A modular interface of IL-4
allows for scalable affinity without affecting specificity for the IL-4 receptor.
BMC Biol, 4, 13.
45
Receptor-Cyclic Nucleotide Signaling -Martin Lohse
Email: [email protected]
Phone: +49(0)931 201 484 01
Fax:
+49(0)931 201 484 11
http://www.rudolf-virchow-zentrum.de/forschung/bioimagingcenter/lohse.html
Cyclic Nucleotides – cyclic AMP (cAMP) and cyclic GMP (cGMP) – belong to the most ubiquitous intracellular messengers. The discovery of cAMP, cGMP and their signaling pathways in the 1950s and 1960s led
to the concepts of second messengers and intracellular signaling. For both cAMP and cGMP it was then
shown that they were produced in response to multiple stimuli, that they acted on several intracellular
targets, and that they regulated a vast array of biological functions.
However, in spite of the fundamental importance of these signaling systems, very little is known about
the temporal and spatial patterns of their production and action. In fact, space and time seem to play
almost no role in concepts of intracellular signaling. To gain an insight into these dimensions, we began a project in 2004, which is funded since 2006 within the Bio-Imaging Center. The aim of this project is to develop methods to create images of these second messengers in intact cells, and to resolve
these intracellular signals in space and in time.
Generation of cyclic nucleotide sensors
The basic strategy to create sensors for
cAMP and cGMP is based on the observation that they act by binding to specific
sites in proteins and induce a conformational change. This conformational change
is then picked up by a technique called
fluorescence resonance energy transfer
(FRET). FRET is the transfer of energy from
one fluorescent moiety to another close by.
If these moieties are, for example, cyan
(CFP) and yellow (YFP) fluorescent proteins, then FRET can cause a YFP to emit
yellow light when a nearby CFP is excited.
Fig. 1:
Structure of a sensor which responds
to cAMP with a decrease in FRET.
46
FRET is exquisitely sensitive to changes in
the distance between the two fluorescent
moieties: if they are attached to a protein
that changes its conformation, even small
changes in their distance will lead to a significant loss in FRET (seen in this example
as a loss in yellow light).
By fusing cAMP- and cGMP-binding domains
from various proteins to CFP and YFP, we
have succeeded in generating a number of
sensors for these cyclic nucleotides. They
respond to binding of cAMP or cGMP with a
decrease (or in some instances an increase)
in FRET (Fig. 1). Such changes in FRET can
be monitored by either simply measuring
the signal intensities of the CFP- and YFPemissions (and their ratio), or generating
the corresponding images with a CCD-camera
(Fig. 2). This allows one to monitor both the
spatial and the temporal patterns of changes
in cyclic nucleotide concentrations. It is also
possible to combine these measurements
with determining of intracellular free calcium with fluorescent dyes (e.g. fura-2). These
measurements have revealed reciprocal oscillations between cAMP and calcium.
Extramural Funding related to project
BMBF-Verbundprojekt: LiveCell Screening
Selected Publications
Fig. 2:
Imaging of cGMP in primary mesangial cells with a cGMP FRET-sensor. FRET-ratio
images at different times after addition of NO-donor (sodium nitroprusside, 10 µM)
are presented.
Harbeck, M.C., Chepurny, O., Nikolaev,
V.O., Lohse, M.J., Holz, G.G., and Roe,
M.W. (2006) Simultaneous optical
measurements of cytosolic Ca2+ and
cAMP in single cells. Science STKE,
353, pl6.
Lohse, M.J. (2006) GPCRs – too many
dimers? Nature Meth, 3, 972-973.
Nikolaev, V.O., Gambaryan, S., and
Lohse, M.J. (2006) Fluorescent sensors for rapid monitoring of intracellular cGMP. Nature Meth, 3, 23-25.
Nikolaev, V.O., Bünemann, M., Schmitteckert, E., Lohse, M.J., and Engelhardt,
S. (2006) Cyclic AMP imaging in adult
cardiac myocytes reveals far-reaching
β1-adrenergic but locally confined β2adrenergic receptor-mediated signaling. Circ Res, 99, 1084-1091.
Local cAMP signals and their propagation
In response to local stimuli, cAMP and
cGMP are, in general, synthesized by
membrane-bound enzymes, and they then
diffuse into the cell interior. We have begun
to analyze the local generation as well as
the diffusion of cAMP, first in transfected
cells and then also in cardiomyocytes from
mice with transgenic expression of a cAMPsensor (in collaboration with Stefan Engelhardt). Initial data reveal that indeed local
generation of cAMP appears to be followed
by its almost free diffusion within the
cell. However, the spatial pattern of cAMP
diffusion in cardiomyocytes depends on
the type of receptors that initiate cAMP
production: while the β1-adrenergic receptor initiates a wide-spread “cAMP-wave“,
the β2-adrenergic receptor causes a very
local signal (Fig. 3). The reasons for this
discrepancy remain to be solved – however,
the data show that there are indeed spatial
signatures in cAMP signaling.
These experiments are at the limit of
current sensitivity and resolution. We are,
therefore, attempting to increase the sensitivity by various means. The two most
important strategies will be the generation of sensors with greater amplitudes,
and measurement of localized signals by
total internal reflection (TIRF) microscopy.
For the latter approach, together with the
group of Gregory Harms we are collaborating with Leica to build a TIRF microscope
capable of creating temporally resolved
FRET images.
Nikolaev, V.O., and Lohse, M.J.
(2006) Monitoring of cAMP synthesis and degradation in living cells.
Physiology, 21, 86-92.
Fig. 3:
cAMP signals generated by local stimulation (at the right edge) of a cardiomyocyte
expressing a cAMP sensor. Stimulation of
β1- (top) vs. β2-adrenergic receptors results
in a generalized vs. a local cAMP signal.
47
Synapse Architecture -Stephan Sigrist
Email: [email protected]
Phone: +49(0)931 201 440 50
Fax:
+49(0)931 201 440 59
http://www.rudolf-virchow-zentrum.de/forschung/bioimagingcenter/sigrist.html
At synaptic contacts between neurons, the presynaptic active zone organizes Ca2+-mediated release of
neurotransmitters to activate neurotransmitter receptors localized at the postsynaptic specialization.
How these synaptic compartments assemble and control their function is under intense investigation.
Genetic analysis in the fruit fly Drosophila allowed us to identify a master organizer of presynaptic
active zones, a protein we called Bruchpilot. At synapses lacking Bruchpilot, clustering of presynaptic
Ca2+-channels is defective, and efficiency of neurotransmitter release is dramatically reduced. Thus, this
protein might well organize changes in synaptic performance in vivo. We are now addressing the architecture of active zones by systematically analyzing synapses in two models, flies and mice. To this end,
genetic and biochemical analyses are combined with a recent advance in light microscopy, i.e. stimulated emission microscopy (STED). STED drastically increases the resolution of fluorescence microscopy,
uncovering so far unseen substructures in the molecular architecture of synapses. Our results are relevant in the context of learning and memory as well as degenerative diseases of the nervous system.
Model system: glutamatergic synapses
easily accessible for genetics and imaging
Synaptic plasticity, meaning changes in
structure and/or function of the synaptic
connections between neurons, is a cellular basis of learning and memory processes
in the brain. Particularly, the formation of
new, additional synapses within neuronal
circuits is considered to be a primary mechanism for long-term synaptic plasticity.
However, the elementary mechanisms controlling functional and structural assembly
of synapses remain very poorly understood.
The aim of our laboratory is to study synaptic assembly and remodeling processes in
intact living preparations, particularly concentrating on the relation between structural and functional organization.
Glutamate is the dominant excitatory
neurotransmitter in our brain. The primary
model of our group are glutamatergic synapses of Drosophila neuromuscular junctions (NMJs). While similar to mammalian
glutamatergic CNS synapses in terms of
ultrastructure and molecular composition,
NMJs combine a comparatively simple architecture (Fig. 1) with straightforward
genetic accessibility. In addition, our
group recently devised protocols to allow
in vivo imaging over days of identified NMJ
48
synapse populations in living larvae, using confocal and two-photon-microscopy
(Rasse et al., Nature Neuroscience, 2005).
This enabled us to directly visualize the
protein dynamics organizing the synapse
assembly process in living animals, e.g. by
in vivo fluorescence recovery experiments.
Our approach is finally complemented by
biochemical, ultrastructural and electrophysiological analysis.
Fig. 1:
Morphological organization of the Drosophila Neuromuscular Junction (NMJ): Shown is a larval muscle
(orange) innervated by motor neurons (green) branching into numerous boutons (left panel). 10-20
synapses consisting of postsynaptic glutamate receptor fields and the associated presynaptic active
zone (right panel) are found per bouton.
The presynaptic active zone: bruchpilot-like proteins as major organizers of
structure and function
The current focus of our research is the presynaptic compartment organizing release
of neurotransmitter filled vesicles. Synaptic vesicles fuse at active zone membranes,
characterized by a specific membrane protein composition and electron dense specializations. Active zones are characterized
by voltage-gated Ca2+-channels, docked
vesicles, and electron-dense structures (Tbars). The molecular organization of presynaptic active zones is the focus of intense investigation. In collaboration with
the groups of Erich Buchner and Manfred
Heckmann (University of Würzburg) we
could identify and characterize the first
active zone component (Bruchpilot, BRP)
conserved between flies and mammals
(Wagh et al., Neuron, 2006; Kittel et al.,
Science, 2006). BRP is a large coiled-coil
domain protein with homology to a largely
uncharacterized family of mammalian active zone proteins. Mutants were produced
in brp showing defective active zone membranes, a complete loss of presynaptic specializations, and severely depressed vesicle
release. Particularly, electrophysiology and
in vivo imaging showed that Ca2+-channels
no longer cluster appropriately at synapses
lacking BRP.
We are now further addressing the exact functional and structural role of BRP.
To identify BRP we initially used Nc82, a
monoclonal antibody specifically recognizing the C-terminal end of the BRP protein.
Notably, however, the signal of an antibody
directed against the N-terminus of BRP is
about 150 nanometers distant from the
Nc82 signal (Fig. 2 left). Thus, BRP may
well be an elongated filamentous protein,
associating with the active zone in an oriented fashion: the N-terminal head associating with the active zone membrane, the
tail converging into the cytoplasm (Fig. 2
right). We speculate that this arrangement
might have evolved to direct synaptic vesicles to their site of fusion. Biochemical and
genetic analysis are currently being used to
further work out BRP function.
Extramural Funding
DFG (Si849/2-1 and 2-2), (SFB 554), (SFB
581; TP 25)
MPI
Tom-Wahlig-Stiftung
Selected Publications
Ataman, B., Ashley, J., Gorczyca, D.,
Gorczyca, M., Mathew, D., Wichmann, C.,
Sigrist, S.J., and Budnik, V. (2006)
Nuclear trafficking of Drosophila
Frizzled-2 during synapse development requires the PDZ protein dGRIP.
PNAS U S A, 103, 7841-6.
Kittel, R. J., Hallermann, S., Thomsen, S.,
Wichmann, C., Sigrist, S.J., and
Heckmann, M. (2006) Active zone
assembly and synaptic release.
Biochem Soc Transact, 34, 994-947.
Kittel, R., Wichmann, C., Rasse, T.,
Fouquet, W. , Schmid, A., Wagh, D.,
Buchner,E. Heckmann, M., and Sigrist, S.J. (2006) The Drosophila
Bruchpilot protein is needed for presynaptic active zone assembly and
calcium channel clustering to ensure
efficient, fast neurotransmission.
Science, 312, 1051-1054
Schmid, A., Qin, G., Wichmann, C.,
Kittel, R., Mertel, S., Fouquet, W.,
Schmidt, M., Heckmann, M., and
Sigrist, S.J. (2006) Non-NMDA type
glutamate receptors are essential for
maturation but not for initial assembly of synapses at Drosophila NMJs.
J Neurosci, 26, 11267-11277.
Sigrist, S.J. (2006) Neurobiology tools: flashdancing worms.
Curr Biol, 16, R100-2.
Fig. 2:
Simultaneous labeling (left image) of N- and C-terminus (Nc82) suggests that BRP associates
with the active zone membrane in an oriented fashion to cluster Ca2+-channels (right image).
Outlook: supramolecular architectures
for fast and efficient vesicle release
Synapses are typically only about 300 nm
in diameter or less. This fact has inhibited
studying synaptic substructures with conventional light microscopy (diffraction limited and thus only allowing optical resolution below down to 250 nanometer). STED
(stimulated emission depletion microsocopy) is a novel advance in fluorescence
microscopy with a spatial resolution conceptually no longer limited by diffraction,
allowing resolution of substructures below
100 nanometers. Visualized with STED,
Bruchpilot forms a donut-like distribution
centered at active zones of NMJ synapses
(Kittel et al., Biochem Soc Transact, 2006).
To boost our understanding of active zone
function, a full picture of the molecular
synaptic architecture on the subsynapselevel is critically needed. Thus, we are also
Swan, L.E., Schmidt, M., Schwarz,
T., Ponimaskin, E., Prange, U.,
Boeckers, T., Thomas, U., and
Sigrist, S.J. (2006) Echinoid and Drosophila GRIP organize Drosophila muscle guidance via a complex interaction.
EMBO J, 25, 3640-51.
studying the distribution of other proteins
involved in organizing active zone structure or function, such as Liprin. STED analysis revealed that Liprin forms discrete
“quantal” spots surrounding the active
zone center labeled by BRP. Combining
these novel forms of imaging with genetic
and biochemical analysis we are seeking to
extend our understanding of synapse assembly in both the healthy and diseased
nervous system.
49
Teaching & Training
Undergraduate and Graduate Programs
Coordinator: Bw. (VWA) Carmen Dengel
E-Mail: [email protected]
Phone: +49(0)931 201 487 13
Fax:
+49(0)931 201 489 78
http://www.rudolf-virchow-zentrum.de/ausbildung/ausbildung.hml
In addition to the central role of research, the Rudolf Virchow Center is actively involved in many educational programs for both undergraduate and graduate students. For example, the Center hosts the undergraduate BSc/MSc program “Biomedicine“ and a graduate program “Target Proteins“ that, together
with several other graduate programs, form the core of training in the International Graduate School.
This structure continues the Humboldt tradition by encompassing research and teaching as one functional unit. Both training programs are fully integrated into studies offered by the university and,
furthermore, have become development grounds and pilot projects for reforms of undergraduate and
graduate teaching. Thus, the Rudolf Virchow Center has developed into an open and stimulating environment for specialists, students and young researchers.
As in research, the Rudolf Virchow Center strives to achieve excellence in biomedicine in its undergraduate and graduate training programs. Both programs aim specifically at future researchers at the
interface between the natural sciences and medicine. The new undergraduate program was initiated in
the winter term 2001/2 and run jointly by the natural sciences and the medical faculties. This program
is characterized by a research-oriented and demanding curriculum in biomedicine leading to the award
of BSc and MSc degrees.
Structured graduate training has always been a key focus at the Rudolf Virchow Center as demonstrated
by the establishing of the graduate program “Target Proteins“ right from the beginning of the center.
Together with several other DFG-funded graduate programs, it has become the nucleus for a large-scale
reform of graduate training at the University of Würzburg, which culminated in the foundation of the
“Graduate School for Life Sciences“. This School won approval in the context of the national “Excellence
Initiative of the Federal and State Government“ in the fall of 2006.
Teaching at the Rudolf Virchow Center means undergraduate and
graduate training. This year, the first students of the biomedicine
program finished their Master theses:
Julia Pagels, Doreen Haase, Alexandra Reckewell, Stephanie
Alexander, Julia Pfrang, Markus Bender, Thomas Premsler, Kathrin
Mandery, Kathrin Fischer, Christine Schultheiss (from left) with
Parliamentary State Secretary of the Federal Ministry of Education
and Research Andreas Storm at the Graduate Day. (not on foto:
Theresia Kress, Claudia Leikam, Lilia Leisle, Sonja Ortman)
50
Teaching Activities
Undergraduate Program in Biomedicine
The undergraduate program in biomedicine is a small, research-oriented program for 24 students
per year. Its main focus is research-oriented training at the interface between the natural sciences
and medicine. Members of the Rudolf Virchow Center carry more than half of the teaching load and
also provide opportunities for and supervision of the majority of the theses.
Bachelor Program (BSc – 6 semesters)
Admission
Demand for this program is exceptionally high, with about 500600 applications for 24 places. Admissions are based on excellent results in the final high school examination.
Structure and content
The three-year BSc curriculum combines elements of undergraduate programs in biology and natural sciences with key courses
of the first years in medicine: biology, physics, chemistry, (bio-)
mathematics, anatomy, physiology, biochemistry, cell biology,
pathology, pharmacology, virology, immunology, and microbiology. Many of these courses were newly developed, while
others were adapted from the curricula of the Faculties of
Biology and Medicine.
The curriculum has a strong focus on practical laboratory work
in order to prepare the students for active participation in research. The subjects are weighted according to the needs in
most biomedical research laboratories. The curriculum also includes courses in scientific administrative matters in order to
provide the qualifications necessary to meet legal regulations
with respect to animal experimentation, chemical safety, radioactive compounds and biosafety/gene technology.
To facilitate international exchange, the curriculum complies
with the European Credit Transfer System (ECTS). Credit points
(CP) and corresponding marks count and put toward the final
examination. Each module ends with an examination: written
tests, practical tests, presentations of research results, interpretation and completion of a scientific paper. The students
have done particularly well in the latter, more research-oriented
types of examinations. The organized nature of the curriculum
allows rapid progress of the students. A thesis based on laboratory work written in English in the format of a scientific paper
concludes the studies with the final examination being a public
defense of the thesis.
Master Program (MSc – 1,5 years)
Admission
Admission to the MSc program is based either on a Bachelor degree in Biomedicine from the University of Würzburg or a degree
from other universities with respective curriculum equivalent
to that one.
Structure and contents
The MSc curriculum permits much more freedom than the structured BSc program. All students start with a six-week laboratory course on model organisms used in biological and medical
research (Arabidopsis, viruses, E. coli, Candida, S. cerevisiae,
Drosophila, zebrafish, mouse/rat).
This is followed by two laboratory rotations of six weeks. The
students are free to choose their subjects and can also do the
Prof. Dr. Werner Lutz (Chairman Study Committee)
Institute of Pharmacology and Toxicology
course elsewhere, in particular abroad. Accompanying lectures
cover molecular pathology, biomaterials, neurobiology and cardiovascular biology.
After this, the students start their 9-month MSc–research
project. At the end of this period, a MSc thesis is written and
publicly defended. The MSc program leads directly into doctoral
training and the thesis can be credited to the PhD.
Management of the Programs
Two committees with members of the Faculties of Biology and of
Medicine and a coordinator, based at the Rudolf Virchow Center,
share the responsibility for the content and the organization of
the BSc/MSc program:
The examination committee supervises the organization
of examinations and decides on individual cases of admission, transfer and approval of courses taken at other universities or research institutions. The committee is chaired by
Prof. Manfred Schartl. The study committee is responsible for the
study program and supervizes quality and content of teaching.
The committee is chaired by Prof. Werner Lutz. Carmen Dengel
coordinates both the BSc and the MSc program.
Results
Since 2001, the BSc curriculum has accepted one new class every winter term. The number of applications has remained high
with about 500-600 applications per year. Currently, there are
87 BSc students and 28 MSc students, from all over Germany
(77% female). Some students of the BSc program elected to
switch to medicine, which is possible within the first two terms.
These places were rapidly filled with new students. A few students elected to study biomedicine in parallel with medicine or
physics.
The first classes of BSc and MSc students obtained their degrees in the summers of 2004 and 2006 respectively. The results
of the examinations were in general excellent. Two key features
of these structured training programs may be responsible for
their popularity and success: The first is that our students acquire a particular ability to address, summarize and present a
research project. The second is that about half take the opportunity to spend parts of their study abroad, with a particular
preference for top universities in England.
Most BSc graduates decided to continue their studies with the
MSc program, while a few started exchange programs or enrolled
for a PhD position abroad.
51
Training Activities
Graduate Program
Right from its start, it was a main goal of the Rudolf Virchow Center to offer high-level structured
graduate training. These efforts were based on earlier experiences with structured graduate training
at the University of Würzburg, most notably in the context of several DFG-funded graduate programs
(Graduiertenkollegs). A prime example of graduate training is also the MD/PhD-program, which was
initiated by the Faculties of Biology and of Medicine in 1996/7 as the first such program in Germany.
These programs with several generations of basic and clinical scientists have shown the effectiveness
of such structured graduate training.
Therefore, the Rudolf Virchow Center has not only developed his own graduate training program but
also acted as a catalyst for the large-scale introduction of structured graduate training at the whole
university by proposing key elements and by building up new structures. The last years, and most
notably 2006, have seen major steps towards this goal, in particular the foundation of separate graduate schools and the funding of the “Graduate School for Life Sciences” in the “Excellence Initiative of
the Federal and State Government”.
Foundation of the International Graduate School
(2003-2005)
Discussions in the entire university on modern forms of graduate training culminated in the foundation of the “International
Graduate School” (IGS) by the University Senate in December
2003. This “International Graduate School” was initiated to encompass the whole university, with separate sections (“Klassen”) to cover the specific scientific and training needs and
cultures of their diverse disciplines.
Key elements of training in the Graduate Schools
The traditional single advisor (“Doktorvater“) is replaced
by a three-person committee.
A schedule of training activities is offered, and an individual, obligatory program is tailored for each graduate
student from these activities.
Graduate students actively participate in the program by
offering and organizing courses and symposia.
A set of requirements has to be met to establish a common quality standard.
Section Biomedicine
As a first step, a Section Biomedicine was formed in the IGS
in 2003 by unifying several programs and their graduate
students:
The graduate program “Target Proteins” of the
Rudolf Virchow Center
The graduate program of the Research Center for
Infectious Diseases
The MD/PhD program of the Interdisciplinary Center
for Clinical Research
Four DFG-funded graduate programs (GK520 “Immunomodulation”, international GK587 “Gene regulation
in and by microbial pathogens”, GK639 “Molecular and
structural basis of tumor instability”, and GK1048
“Molecular basis of organ development in vertebrates”)
These programs came together to find and develop common
structures and curricula, to share activities and to set common
standards (see box). In 2006, the first graduate students received their PhD from this common program.
52
Mentoring System
Each student has an individual supervisory committee. The graduates report about the status of their expertise within the research
groups in monthly meetings. As a result of these meetings, the
graduates have the opportunity to become familiar with the different research topics of the others. The connection of both, the
structured training and the personal, thematic and organizational
relationships guarantees high quality training.
Training Activities
The training activities total a minimum of 150 hours per year and
consist of laboratory seminars, journal clubs, program-seminars,
methods courses and transferable skills workshops as well as retreats and international conferences.
Common Graduation Committee
Participating faculties form a new common graduation committee
within the Graduate School. This structural novelty will be the first
such committee in Germany. As the committee will confer formally
the PhD on every graduate student not only the development of
common standards across disciplines will be facilitated but also
interdisciplinary graduate training will be fostered.
Training Activities
The growing Graduate School – Developments in 2006
Increases in size and scope resulting from the progressive integration of further programs and the discussions in the context of the national “Excellence Initiative” called for a number
of changes within the International Graduate School in 2006.
These changes concerned both the internal structure and the
formal status. Under the roof of the IGS, separate Graduate
Schools were formed in order to better accommodate the needs
of their respective fields and to grant greater independence;
these are the Graduate Schools for Life Sciences, for Science and
Technology, and for the Humanities.
A new legal status was conferred to graduations in the International Graduate School by the new graduation regulations
(“Promotionsordnung”) passed by the University Senate in May
2006. These regulations contain a set of general rules as well
as specific regulations for the individual schools. The general
structure of the programs as well as key elements will remain
those that were established in previous years, including the
mentoring system as well as rules for admissions and formal
standards (see box).
In 2006, Dr. Stefan Schröder-Köhne joined the International
Graduate School as administrative director to create a professional administration and to mediate its growth process.
The future: International Graduate School and Graduate
School for Life Sciences (GSLS)
From 2006 on, the International Graduate School will represent the common roof of three individual graduate schools
(see Figure). Each of these schools will handle most of their
specific affairs, while the IGS will assure adherence to common
rules and provide a general service to the individual schools. As
before, the schools will be subdivided into thematically oriented
sections (“Klassen”) that contain the individual programs.
The Graduate School for Life Sciences will initially house the
graduate students of all collaborative research programs – such as
DFG-funded collaborative research centers (“Sonderforschungsbereiche”), graduate training groups (“Graduiertenkollegs”) and
research groups, but also of other collaborative programs funded
by the Federal Ministry of Education and Research (BMBF), the
European Union and other sources. This is a total of more than
300 graduate students. The school will, therefore, be divided
into five separate sections. In addition to the already existing
Section Biomedicine, there will be sections of Infection and
Immunity, Neurosciences, Integrative Biology plus the MD/PhD
program. Each section will comprise different programs of about
15 to 25 graduate students. These programs – like the graduate
program “Target Proteins” of the Rudolf Virchow Center – are the
scientific as well as social “home” of the graduate students.
The Graduate School for Life Sciences successfully applied for
funding in the “Excellence Initiative “ and was awarded support
in October 2006. Fellowships for 30 fellows were announced immediately and resulted in about 400 applications from all over
the world. The selection process, involving written submissions
as well as interviews in Germany and abroad, is ongoing.
Structure of the International Graduate School
53
Training Activities
Program Section Biomedicine
Meetings and Events
Besides many international scientific events that
graduate students could attend, a number of
events were organized specifically for the graduate students. Highlights were:
Prof. Dr. Caroline Kisker
(Chairperson Section Biomedicine)
Rudolf Virchow Center
The Section Biomedicine provides a structured training program in biomedicine for all graduate students. As the section does not only serve as an interdisciplinary link between
the different graduate programs but also between scientific
research and practical experience, the graduate students
have the possibility to work in international teams with scientists from other fields.
Training activities
In addition to the training activities offered by the individual programs and their research groups, a number of activities
were organized for all graduate students in biomedicine and
the life sciences. Training activities and events, organized by
Carmen Dengel, coordinator at the Rudolf Virchow Center,
included 2006:
Lecture series “Clinical medicine for graduate students”
The ongoing weekly lecture series is intended to introduce graduate students with a background in the natural sciences into the problems and approaches of modern medicine. Topics in 2006 included nuclear medicine,
nephrology, gastroenterology, abdominal surgery,
plastic surgery, dermatology, allergology, hematology,
cardiology, and orthopedics.
Workshops “Effective scientific writing”
Four separate three-day workshops were held by a professional science writer to provide tools to organize
structure and write research papers.
Workshops “Oral presentation”
Two separate two-day workshops provided opportunities
to learn and to experiment with strategies for effective
and concise oral presentations.
Workshops “Poster presentations”
A two-day workshop focused on the key elements of effective poster design.
54
Junior Faculty Lectures “Hottest Life Science”
(July 14th-15th, 2006)
This two-day-event brought together graduate
students and some of the university’s top young
scientists in the life sciences. The lectures were
embedded in a barbeque with plenty of time for
networking.
Graduate Day (July 27th-28th, 2006)
At the end of each academic year a Graduate Day
is organized to celebrate the achievements of
the graduate students. Parliamentary State Secretary of Andreas Storm from the Federal Ministry
of Education and Research was this year’s keynote
speaker. At the eve of this year’s Graduate Day,
a screening of the movie “Sleeper“ was arranged
followed by a discussion with its director Benjamin
Heisenberg about competition and ethics of conduct in science. The Graduate Day was rounded off
by company contacts, a new format for networking
between graduate students and companies as potential future employers.
Graduate student-organized activities
Graduate students organized a number of informal laboratory
courses as well as seminars. This year’s graduate student symposium “From Bench to Bedside - Molecular Approaches for Novel
Therapies” was held on October 23rd, 2006. Again, it was fully
organized, including the acquisition of sponsors, by students of
the Section Biomedicine and the MD/PhD programs.
The lectures were given by several graduate students as well
as distinguished scientists like James P. Allison (New York),
Alexander von Gabain (Vienna), Richard Marais (London), Axel
Ullrich (Martinsried / Singapore), and Denisa Wagner (Boston).
Training Activities
Graduate Program Target Proteins
In order to enhance the education of young scientists in
disease-oriented research, the Graduate Program “Target
Proteins“ was initiated at the Rudolf Virchow Center as part
of the Section Biomedicine. Research on target proteins regulating key cellular functions, and in particular on “druggable” proteins has a long-standing interdisciplinary tradition
in Würzburg.
In 2006, the Graduate Program “Target Proteins” comprises 10
graduate students funded by the Rudolf Virchow Center and
15 associated students. Most projects include training in
cutting-edge technology, strategies to identify molecular
mechanisms of disease, as well as tools to develop strategies
to monitor, inhibit, or even abrogate pathologic cell behavior.
The experimental strategies range from the analysis of single
molecules to complex models on molecular mechanisms of cell
cycle and growth regulation, and computer modeling of protein
and cell function.
Prof. Dr. Helga Stopper
Coordinator Graduate
Program “Target Proteins“
Institute of Pharmacology and Toxicology
Events
Annual Retreat
This year’s annual retreat was held in Gemünden on November
6th and 7th and combined again social and scientific activities.
Graduate students presented their projects and discussed the
topic “Career in Germany or abroad“, based on talks of senior
scientists with divergent personal paths.
All key technological platforms provided by the groups of the
Rudolf Virchow Center are fully integrated into the Graduate
Program and used by students for their thesis work on a daily
basis. These include:
State-of-the-art proteomic approaches to protein
content and function in cells, including
quantitative mass spectrometry, phosphoproteomics
and interactomics
Advanced molecular imaging of molecules and cells
in vitro and in vivo, including biosensors, single
molecule and multiphoton microscopy, and optical
whole-mouse imaging
Cutting-edge approaches to resolve protein structure
and conformation
The generation and analysis of transgenic and knockout
mouse models of disease
To provide easy access to other technologies available on the
Würzburg campus, individualized hands-on training programs
are defined and realized within practical training units, tutorials and small workshops. The aim is to provide a view beyond
too narrow borders of lab work and reach into related aspects of
clinical medicine, critical review of research articles, communication skills, and designing and presenting own projects.
Student activities
Graduate students of the program “Target Proteins” assumed
their share in the student-organized symposium (“From Bench
to Bedside“, see left). The coordinator of the program, Peter
Friedl, served as tutor for this event. Furthermore, graduate
students organized their own seminar, with presentations of
ongoing theses and discussions of topics of broader interest.
Numerous informal laboratory rotations were done by individual
graduate students or in small groups. All students participated
in the activities of the Section Biomedicine described on the
preceding page.
55
Public Science Center
Public Science Center-Sonja Jülich
E-mail: [email protected]
Phone: +49(0)931 201 487 14
Fax:
+49(0)931 201 487 02
http://www.rudolf-virchow-zentrum.de/public/public.html
The main purpose of the Public Science Center is not only to explain the Center´s research and teaching
activities in a transparent way but also to enhance the direct dialog between scientists and public as
well as stakeholders such as politicians, thereby raising awareness of both the excitement but also of
the problems of biomedicine and biomedical science as well as building up trust. In addition to this,
we are also attracting new generations of students to research science by involving young students in
experimental projects highlighting the fun and creative side of research and also by informing highschool students about the interesting and extensive range of scientific career options open to them. To
achieve our goals, we have established and maintain contacts with all interested parties including journalists as important partners in facilitating better science communication through different projects.
Politicians
First, politicians need to have access to
information about the achievements and
advances made in biomedical research.
Second, the Rudolf Virchow Center has implemented a new and innovative structure
within the traditional university setting in
order to promote scientific excellence with
special emphasis on promotion of better
overall education and opportunities for
high potentials. Both politicians and decision makers are interested to know about
the benefits and pitfalls of such a system.
To this end, members of the Junge Union
Bayern, the Bavarian Minister for Research
Dr. Thomas Goppel and the Parliamentary
State Secretary of the Federal Ministry of
Education and Research (BMBF) Andreas
Storm have recently visited the Center.
quests to participate in this project have
been very high and are still continuing.
To date, over 350 children have taken
part with the upshot of making the Center a more accessible sort of place as well
as promoting the concept of research for
both children and parents alike. To build
on this success and to satisfy the public’s
demands, we will now establish yet another
camp for the thirteen to sixteen year age
group. The two key aims of this project are
General Public
Rudis Forschercamp(Rudi`s Research Camp)
In April 2004, the Public Science Center set
up a special one-month laboratory course
for children aged between eight and twelve
from which they can learn more about the
exciting side of the natural and biological
sciences (Fig. 1). Right from the start, re-
56
Fig. 1:
From Biology to Medicine – every day in
Rudis Forschercamp is an adventure.
to maintain and generate further interest
in science through a range of research-oriented project activities.
Future Students
Rudolf Virchow Paten for Jugend forscht
With this project, we support high school
students who want to participate in the
“Jugend forscht” competition. Students
can realize their own research projects using the facilities available at the Rudolf
Virchow Center as well as discussing their
questions with an allocated scientific mentor. This year, Constanze Rieckmann was
placed first in the biological category of
the Bavarian competition “Jugend forscht/
Schüler experimentieren”. Prof. Dr. Michael
P. Schön and PD Dr. Margarete Schön from
the Rudolf Virchow Center and Gregor
Wienrich and Dr. Charis Papavassilis from
the Dermatology Clinic at the University
Hospital supported her. Constanze is the
second winner in series. Feedback from
this scheme has been highly positive, with
participants reporting that the project enforced their decision to study a scientific or
medical subject.
burg has been built up, and this will act as
an excellent resource for further projects.
Adults
Fig. 2:
Constanze Rieckmann: “Biosurgery – how
fly grub makes wounds healing”.
ForscherReporter
In the context of our general concept to
attract future generations of students, in
September 2006 we established a monthly
course for A-level students. The aim is to
provide students with the possibility to
orient themselves in the occupational field
of “research”. By carrying out a biochemical experiment, these students learn more
about the practical aspects of science. At
the same time, we motivate them to slip
into the role of a journalist so that they
can gain insight into how scientists work
from a different perspective and also improve their ability to communicate this. To
this end, we collaborate with the local radio station from the Bayerischer Rundfunk
from which the reporter Irina Hanft supports the students. In addition, we also inform the students about teaching programs
in science.
So far, five schools have participated, and
“ForscherReporter” was booked out right
from the start for a year. Finally, a spin off of
this project is that a strong network of contacts to teachers and students around Würz-
Our aim is to enable the general public to
learn more about biomedical research by
supplying them with information and entering into dialog, thereby allowing them
to form their own opinions. To attract also
those who usually do not go to special
seminars or lectures, we want to establish
a Café Scientifique in an already existing
café in the city center of Würzburg as a
quarterly institution. The idea behind such
a café is that for the price of a cup of coffee, anyone can come to explore science
and that such an informal setting will allow
the public to access the more human side
of science. In addition, it is envisaged that
scientists will explain and discuss their research with the general public in this relaxed atmosphere. The Café Scientifique is
start in Spring 2007.
enhance our contact, we actively participated in large international meetings like
the “AAAS Annual Meeting” and the “European Science Open Forum”. Our press clippings of 2006 show a good response (see
Media Reports).
To promote scientific communication
competence to the general public and to
journalists, our scientists had the opportunity to participate in media training held
by Dr. Markus Lehmkuhl and Dr. Nikolas
Westerhoff from the Freie Universität,
Berlin, last July.
Science Community
Communication to the science community
is usually through publication in internationally recognized scientific journals
along with attendance of meetings, symposia and workshops, several of which have
taken place at the Rudolf Virchow Center.
In addition, the Public Science Center
publishes the Annual Report in which research results as well as teaching and public relations activities are reported.
Journalists
The development and maintenance of an
extensive network with those working in
the media is a main focus of the Public
Science Center since we regard journalists
as our most important partners in facilitating better science communication. For
example, compared to direct activities with
the general public, our press releases can
reach a far wider audience. Therefore, to
Extramural Funding
BMBF: Learning Location Laboratory (LeLa),
Center for Advice and Quality Development
for Extracurricular Activities at the Leibniz Institute for Science Education in Kiel.
The Public Science Center attended LeLa at
the German education trade fair exhibition
„didacta“ of the BMBF in Hannover
Media Reports
“Rudis Forschercamp“ Frankenschau,
Bayerischer Rundfunk 12th Feb., 2006
“Die Insel der Seeligen“, Süddeutsche
Zeitung 20th April, 2006
“Die Herrin der Fliegenmaden“, Mainpost 11st May, 2006
“Die Inventur im Kraftwerk der HefeZellen ist abgeschlossen“, Forschung
aktuell, Deutschlandfunk 06th June,
2006
“Die ersten Master Deutschlands in
Biomedizin“, Mainpost 04th Aug., 2006
“Rudolf-Virchow-Zentrum startet “das
etwas andere“ Schülerlabor”, P.M.
Magazin online 26th Sept., 2006
“Forschergeist und Schreibtalent“,
Mainpost 28th Sept., 2006
Fig. 3:
Students produce a radio report for publication on the “ForscherReporter”
web site: www.forscherreporter.de and for presentation at their school.
“Universität Würzburg: Forschen wie
die Bienen“, Focus 12nd Sept., 2006
57
Executive Committees and Scientific Members
Chairman:
Vice-Chairs:
Prof. Dr. Martin Lohse, Institute of Pharmacology and Toxicology
Prof. Dr. Jörg Hacker, Institute of Molecular Infection Biology
Prof. Dr. Manfred Schartl, Theodor-Boveri-Insitute, Physiological Chemistry I
Members:
Prof. Dr. Peter Friedl, Rudolf Virchow Center
Prof. Dr. Caroline Kisker, Rudolf Virchow Center
Prof. Dr. Bernhard Nieswandt, Rudolf Virchow Center
Prof. Dr. Michael Sendtner, Institute of Clinical Neurobiology
Scientific Advisory Board
Chairman:
Members:
Prof. Dr. Fritz Melchers, Basel Institute for Immunology/Biocenter Basel
Prof. Dr. Ueli Aebi, Biocenter, University of Basel
Prof. Dr. Volkmar Braun, University of Tübingen
Prof. Dr. Sabine Werner, Eidgenössische Technische Hochschule Zürich
Prof. Dr. Heiner Westphal, Laboratory of Mammalian Genes and Development, NICHD, Bethesda, MD, USA
Prof. Dr. Alfred Wittinghofer, MPI for Molecular Physiology, Dortmund
Prof. Dr. Claes Wollheim, University of Geneva
I. Funded Members
Prof. Dr. Dr. Stefan Engelhardt, Rudolf Virchow Center (funded by Sanofi-Aventis, Procorde, Bavarian
Ministry of Economic Affairs)
Prof. Dr. Utz Fischer, Theodor-Boveri-Institute, Biochemistry
Prof. Dr. Peter Friedl, Rudolf Virchow Center
Prof. Dr. Manfred Gessler, Theodor-Boveri-Institute, Physiological Chemistry I
Prof. Dr. Gregory Harms, Rudolf Virchow Center
Prof. Dr. Thomas Hünig, Institute of Virology and Immunobiology
Prof. Dr. Caroline Kisker, Rudolf Virchow Center
Prof. Dr. Martin Lohse, Institute of Pharmacology and Toxicology, Bio-Imaging Center
Dr. Thomas D. Müller, Theodor-Boveri-Institute, Physiological Chemistry II
Prof. Dr. Bernhard Nieswandt, Rudolf Virchow Center
PD Dr. Andreas Rosenwald, Institute of Pathology
Prof. Dr. Manfred Schartl, Theodor-Boveri-Institute, Physiological Chemistry I
Prof. Dr. Hermann Schindelin, Rudolf Virchow Center
Prof. Dr. Michael P. Schön, Rudolf Virchow Center
Prof. Dr. Walter Sebald, Theodor-Boveri-Institute, Physiological Chemistry II
Dr. Albert Sickmann, Rudolf Virchow Center
Prof. Dr. Stephan Sigrist, Clinical Neurobiology, Bio-Imaging Center
Dr. Thorsten Stiewe, Rudolf Virchow Center
II. Non-funded Members
Prof. Dr. Gerhard Bringmann, Institute of Organic Chemistry
Prof. Dr. Matthias Frosch, Institute of Hygiene and Microbiology
Prof. Dr. Werner Goebel, Theodor-Boveri-Institute, Microbiology
Prof. Dr. Jörg Hacker, Institute of Molecular Infection Biology
Prof. Dr. Martin Heisenberg, Theodor-Boveri-Institute, Genetic and Neurobiology
Prof. Dr. Bert Hölldobler, Theodor-Boveri-Institute, Zoology II
Prof. Dr. Peter Jakob, Institute of Physics, Biophysics
Prof. Dr. Hans-Konrad Müller-Hermelink, Institute of Pathology
Prof. Dr. Ulf Rapp, Institute of Medical Radiation and Cell Research
Prof. Dr. Markus Riederer, Theodor-Boveri-Institute, Botany II
Prof. Dr. Michael Sendtner, Institute of Clinical Neurobiology
Prof. Dr. Volker ter Meulen, Institute of Virology and Immunobiology
Prof. Dr. Klaus V. Toyka, Clinic of Neurobiology
Prof. Dr. Ulrich Walter, Institute of Clinical Biochemistry and Pathobiochemistry
58
Academic Members and Supporting Staff
Junior Research Groups
Group Molecular Tumor Biology
Group Cardiac Target Proteins
Group leader:
Dr. Thorsten Stiewe
Group leader:
Prof. Dr. Dr. Stefan Engelhardt
Postdocs:
Dr. Robert Frost
Dr. Francesca Rochais
Scientific Staff:
Lydia Vlaskin
Graduate Students:
Carina Gross
Claudia Jentzsch
Sabine Merkle
Master Student:
Andrea Ahles
Technicians:
Ursula Keller
Silke Oberdorf-Maass
Julia Schittl
Nadine Yurdagül-Hemmrich
Postdocs:
Dr. Rasa Beinoraviciute-Kellner
Dr. Michaela Beitzinger
Dr. Nicole Hüttinger-Kirchhof
Graduate Students:
Hakan Cam
Heidi Griesmann
Claudia Oswald
Markus Sauer
Master/Diploma Students:
Anne-Catherine Bretz
Claudia Dornhöfer
Simone Reeb
Katharina Schlereth
Postdocs:
Dr. Attila Braun
Dr. Margitta Elvers
Dr. Miroslava Pozgajova
Dr. Ulrich Sachs
Graduate Students:
Markus Bender
Alejandro Berna Erro
Irina Pleines
Rastislav Pozgaj
Amrei Strehl
Dávid Varga-Szabó
Bachelor/Diploma Students:
Frauke May
Silke Mühlstedt
Group leader:
Prof. Dr. Caroline Kisker
Postdoc:
Dr. James J. Truglio
Graduate Students:
Akna Bonsra
Zhixin Cheng
Jennifer Doebbler
Erkan Karakas
Sylvia Luckner
Margaret Luk-Parszyk
Heidi Roth
Master/Diploma Students:
Uwe Dietzel
Matthias Leyh
Stefanie Wolski
Core Center
Group Molecular Microscopy
Group leader:
Prof. Dr. Gregory Harms
Group Vascular Biology
Group leader:
Prof. Dr. Bernhard Nieswandt
Group Structural Biology: DNA Repair
and Structure-Based Drug Design
Postdoc:
Dr. Andrey Noskov
Scientific Staff:
Revaz Nozadze
Graduate Students:
Kira Gromova
Geoffrey Lambright
Martin Vielreicher
Diploma Student:
Benjamin Klasczyk
Technician:
Wiebke Buck
Technicians:
Sabine Erhard
Liqun Wang
Group Structural Biology: Protein
Folding, Function and Degradation
Group leader:
Prof. Dr. Hermann Schindelin
Postdocs:
Dr. Petra Hänzelmann
Dr. Daniela Schneeberger
Dr. Geng Tian
Dr. Gang Zhao
Graduate Students:
Juma Daniels
Eun Young Lee
Imsang Lee
Kelvin Luther
Xiaoke Zhou
Diploma Student:
Florian Sauer
Technician:
Silvia Scheuring
Technicians:
Azer Achmedov (Animal Care Taker)
Sylvia Hengst
Sonja Kraus-Katzenberger
59
Group Functional Proteomics
Research Professorships
RVZ Network
Group leader:
Dr. Albert Sickmann
Group Molecular Cell Dynamics
Project: Translational Regulation:
TOP-response Proteins
Postdoc:
Dr. Katrin Denker
Scientific Staff:
Andreas Böhm
Stefanie Wortelkamp
Graduate Students:
Urs Lewandrowski
Jan Moebius
Stephanie Pütz
Juliane Schröter
Julia Wiesner
René Zahedi
Bachelor/Master/Diploma Students:
Beate Eyrich
Thomas Premsler
Oliver Simon
Technicians:
Claudia Berger
Christiane Winkler
Group leader:
Prof. Dr. Peter Friedl
Postdocs:
Dr. Annemieke den Boer (collaboration
NCMLS, Nijmegen)
Dr. Matthias Reinhard
Dr. Katarina Wolf
Clinical Associate:
Dr. Anke Hartmann
Graduate Students:
Stephanie Alexander
Volker Andresen
Katrin Bührle
Julian Storim
Bachelor/Master/Diploma Students:
Stefanie Liedel
Christin Luft
Technicians:
Hannes Baumann (Software Engineer; TZI,
Bremen)
Markus Hirschberg
Martina Jossberger
Monika Kuhn
Margit Ott
Andrea Staudigel
Group Inflammation and Tumor Biology
Group leader:
Prof. Dr. Michael P. Schön
Postdocs:
PD Dr. Margarete Schön
Dr. Kai Michaelis
Dr. Gertie Janneke Oostingh
Dr. Stephanie Schlickum
Dr. Katrin Wallbrecht
Gregor Wienrich
60
Group leader:
Prof. Dr. Utz Fischer
Graduate Student:
Julia Wiesner
Project: Hey Factors in Cardiac
Development
Group leader:
Prof. Dr. Manfred Gessler
Graduate Student:
Julia Schneider
Project: T Cell Surface Proteins
Group leader:
Prof. Dr. Thomas Hünig
Postdoc:
Dr. Kirsty McPherson
Project: Ligand-Receptor Recognition
Group leader:
Dr. Thomas Müller
Project: Posttranslational Gene Regulation
Group leader:
Prof. Dr. Manfred Schartl
Project: BMP Receptors Structure
and Function
Group leader:
Prof. Dr. Walter Sebald
Graduate Students:
Stefan Harth
Marianne Rattel
Graduate Student:
Katharina Amschler
Luise Erpenbeck
Bio-Imaging Center
Technicians:
Elisabeth Axt
Bianca Schlierf
Helga Sennefelder
Group leader:
Prof. Dr. Martin Lohse
Receptor - Cyclic Nucleotide Signaling
Postdoc:
Dr. Viacheslav Nikolaev
Group Synapse Architecture
Teaching and Training
Group leader:
Prof. Dr. Stephan Sigrist
Coordinator:
Carmen Dengel
Postdoc:
Dr. Carolin Wichmann
Assistant:
Elke Drescher
Graduate Students:
Frauke Christiansen
Wernher Fouquet
Robert Kittel
Sara Mertel
David Owald
Andreas Schmidt
Manuela Schmidt
Tobias Schwarz
Public Science Center
Sonja Jülich
Christiane Weber
Technicians:
Jens Hörl
Christine Quentin
Franziska Zehe
Central Technologies
Transgene Technologies
Veterinarians:
Dr. Bettina Holtmann
Dr. Eva Schmitteckert
Technicians:
Melanie Seider
Antje Barthel (Animal Care Taker)
DNA Arrays
PD Dr. Thomas Rosenwald
Administration
Administrative Director:
Prof. Dr. Karl-Norbert Klotz
Administrative Assistants:
Eva Albero
Bianca Klotz
Barbara Zahn
Petra Lütke
System Administrator:
Joachim Baumeister
61
Visiting Scientists
Group Bernhard Nieswandt
From August to September 2006, Dr. Harald Schulze (Laboratory for Pedriatic Molecular Biology,
Charité - Berlin) worked together with Bernhard Nieswandt on in vitro megakaryocyte differentiation and proplatelet formation from fetal liver cells of mice expressing an EF hand mutant variant
of Stim1.
Group Gregory Harms
In 2006, Prof. Dr. Carey Johnson (University of Kansas, USA) collaborated with the group of Gregory
Harms and observed and described the dynamic motions of single fluorescence-labeled Calmodulin
by fluorescence by FRAP, FCS, TIRF and single-molecule tracking in living cells and also the binding
of single fluorescence labelled-Calmodulin of fluorescence-labeled membrane protein candidates by
single-molecule co-localization.
Jelena Drazic (University of Toronto, Canada) created a high-resolution, low interference TIRF and
FRET (fluorescence resonance energy transfer) microscope for high-throughput screening of FRET
based pharmacological targets in living cell culture and accessible to easily add pharmacological
agents during a summer internship in 2006.
Group Martin Lohse
Prof. Dr. Peter Friedman (University of Pittsburgh, USA) conducted further experiments with Fluorescence Resonance Energy Transfer (FRET) and Fluorescence Recovery After Photobleaching (FRAP)
with the PTH receptor to determine effects of binding to the NEHRF protein.
Group Peter Friedl
Stefania Berton (Centro di Riferimento Oncologico, Instituto Nazionale Tumori, IRCCS Aviano)
worked two months on microtubule stability in cancer cell invasion.
Sarah Bernd (Université de Liege, Belgium) analyzed during her five week stay dynamics of antigenisis from aortic ring culture.
Group Albert Sickmann
Elena Wiederhold (University of Groningen) worked on a collaborative project with the group of
Albert Sickmann characterizing vacuolar proteins in S. cerevisiae. During her two-week stay at the
Rudolf Virchow Center, she did a quantitative characterization of different purification states of
vacuoles by MDLC-MS/MS using the iTRAQ (TM) technology.
62
Teaching Committees
BSc/MSc Study committee
Chairman:
Members:
Prof. Dr. Werner Lutz, Institute of Pharmacology and Toxicology
Prof. Dr. Wolfgang Rößler, Theodor-Boveri-Institute, Zoology II
Dr. Ursula Rdest, Theodor-Boveri-Institute, Microbiology
Prof. Dr. Michael Gekle, Institute of Physiology
Prof. Dr. Manfred Gessler, Theodor-Boveri-Institute, Physiological Chemistry
BSc/MSc Examination committee
Chairman:
Members:
Prof. Dr. Manfred Schartl, Theodor-Boveri-Institute, Physiological Chemistry
Prof. Dr. Werner Goebel, Theodor-Boveri-Institute, Microbiology
Prof. Dr. Ulrich Scheer, Theodor-Boveri-Institute, Cell & Developmental Biology
Prof. Dr. Michael Sendtner, Institute of Clinical Neurobiology
Prof. Dr. Helga Stopper, Institute of Pharmacology and Toxicology
Prof. Dr. Ulrich Zimmermann, Theodor-Boveri-Institute, Biotechnology
International Graduate School
Board of Directors (University of Würzburg)
Director:
Members:
Prof. Dr. Martin Lohse, Faculty of Medicine (Pharmacology)
Prof. Dr. Gerhard Bringmann, Faculty of Chemistry & Pharmacy (Organic Chemistry)
Prof. Dr. Rainer Hedrich, Faculty of Biology (Molecular Plant Physiology)
Prof. Dr. Ulrich Konrad, Faculty of Philosophy II (Musicology)
Prof. Dr. Werner Riedel, Faculty of Philosophy II (German Literature)
Graduate School for Life Sciences (since 09/2006)
Dean:
Vice Deans:
Prof. Dr. Markus Riederer
Prof. Dr. Jörg Hacker, Institute of Molecular Infection Biology
Prof. Dr. Martin Lohse, Institute of Pharmacology and Toxicology
Section Biomedicine (since 2003)
Chairperson:
since 9/2006:
Vice-Chair:
Prof. Dr. Jörg Hacker, Institute of Molecular Infection Biology,
Prof. Dr. Caroline Kisker, Rudolf Virchow Center
Prof. Dr. Helga Stopper, Institute of Pharmacology and Toxicology
Program coordinators Section Biomedicine
Prof. Dr Peter Friedl, Grad. Progr. Rudolf Virchow Center “Target Proteins“
Prof. Dr. Manfred Gessler, Grad. Coll. “Tumor Instability“
Prof. Dr. Thomas Hünig, Grad. Coll. “Immunomodulation“
Prof. Dr. Joachim Morschhäuser, Grad. Coll. “Gene Regulation in and by Microbial Pathogens“
Prof. Dr. Manfred Schartl, Grad. Coll. “Organ Development in Vertebrates“
Prof. Dr. Helga Stopper, Grad. Progr. Rudolf Virchow Center “Target Proteins“
63
Undergraduate program in Biomedicine
Bachelor theses 2006
The theses have been written in the context of the undergraduate program in Biomedicine.
“Mechanismus der Glukokortikoid-induzierten Apoptose”
Alb, Miriam Constanze
“Prionpathogenese in Mausscrapie”
Becker, Juliane Claudia
“Inducible and cardiac specific overexpression of the neuronal NO-synthase (nNos or NOS1) enzyme
in a new transgenic mouse model”
Burkard, Natalie Ilse
“Regulation of cardiac growth through Egr-1”
Burkert, Cornelia
“Cell genetic studies of the Fanconi anemia/Breast cancer pathway”
Depner, Harald
“Analysis of the function of WT1 genes in sex determination and gonadal development”
Driessle, Julia Heidrun Serena
“Antimicrobial activity of human colonocytes against Enterobacter aerogenes ”
Engelmann-Pilger, Kerstin Corinna
“Neue Möglichkeiten zur Inhibition des Calcineurin Signalweges”
Gebhardt, Claudia
“Molekulare Charakterisierung einer ERK-Kinase aus Echinokokkus multilocularis”
Graf, Martin
“Kandidatengen-Analyse bei Angst/ADHS/Schizophrenie”
Haderlein, Julia Katharina
“Analysis of microRNA expression in cardiac hypertrophy”
Lauer, Andrea
“Verification of specific proteins and mRNAs in platelet preparations”
Leierseder, Simon Johannes
“Tumorcellinvasion: Role of fibroblasts and endothelial cells”
Liedel, Stefanie Martina
“Serielle Interaktionen zytotoxischer T Lymphozyten mit Antigen-beladenen Zielzellen:
Molekulare Rekonstruktion der Interaktionszone”
Luft, Christin
“Histological analysis of embryonic development in mice lacking functional stromal interactor molecule 1 (STIM1)“
Mühlstedt, Silke
“The co-inhibitory molecule B7H4 in ovarian carcinoma- more than just a biomarker?”
Opitz, Elisa
“Molekulare Mechanismen der Interleukin-7 Rezeptoraktivierung”
Regneri, Janine
64
“Characterization of platelet membranes using peptide centric proteomics”
Simon, Oliver
“Neue Ansätze zur frühzeitigen Detektion nicht-gentoxischer Kanzerogene”
Synwoldt, Peggy
“Establishment of an inducible differentiation system for medakafish embryonic stem cells. - Pluripotent
spermatogonia can be induced to directed differentiation”
Thoma, Eva Christina
“Molekulare Marker des “oxidativen Stresses“ als Frühindikatoren für chronische Entzündungen
und den Kanzerogeneseprozess”
Troppens, Stefan
“Studies on the regulation of agn43 expression in Escherichia coli”
Vögtle, Timo Stephan
”Determination of light-emission in organs of live mice”
Vollmers, Christopher Stephan
“Genetic and molecular biological analysis of the anti-invasive activity of Escherichia coli strain Nissle 1917:
Effects on the cytoskeleton of the host cell ”
Wegehaupt, Marko
“The role of Synapsin and Sap47 for associative learning in Drosophila larvae”
Wegener, Stephanie
“mRNA- and protein-examinations of the NOS-interactome in human platelets”
Winnebeck, Eva Charlotte
65
Master theses published 2006
The theses have been written in the context of the undergraduate program in Biomedicine.
“Physical limits of tumor cell migration: Molecular and biochemical characterization of proteolytic
and nonproteolytic invasion strategies”
Alexander, Stephanie
“Targetingvektoren zur Generierung von human GPVI transgenen Mäusen”
Bender, Markus
“Rolle der Mismatch-Repair und der Cytosin-Methylierung in der Sensitivität von
Zellen für gentoxische Agentien”
Fischer, Kathrin
“Charakterisierung der Expression und Regulation von im NSCLC differentiell regulierten Genen”
Haase, Doreen
“Profiling of signaling proteins in a panel of colorectal carcinoma cells”
Kress, Theresia
“Analysis of the Glycosylation pattern of ACID-Sensing ion Channel (ASIC) 1”
Leisle, Lilia
“Genomweite Analyse der Genregulation durch Secreted Frizzled – Related Protein 4
in Mesenchymalen Stammzellen”
Mandery, Kathrin
“Charakterisierung von Tumorstammzellen in Melanomen und kutanen T Zellymphomen”
Ortmann, Sonja
“Molekulare Epidemiologie und funktionelle Spezifität der O-Acetyltransferasen von
Escherichia coli K1 und Neisseria meningitidis”
Pagels, Julia
„Characterization of highly purified platelets and platelet-derived microparticles for the effects
of insulin on these preparations“
Pfrang, Julia
“Isolierung und Charakterisierung der humanen Monozyten Membran”
Premsler, Thomas
“Genomewide analysis of genregulation of FGF 23 in human kidney cells and mesenchymal stem cells”
Reckewell, Alexandra
“Rekombinante Expression und Immunogenität von partikulären Strukturen auf der Basis
des murinen Polyomavirus”
Schultheiß, Christine
66
PhD theses of the Graduate Program “Target proteins” of
the Section Biomedicine
“New approaches in nonlinear microscopy: applications in
biomedicine”
Andresen, Volker
“Bedeutung der Proteinphosphorylierung bei der Thrombozytenaktivierung”
Wiesner, Julia
“Funktion von Stromal Interaction Molecule 2 (STIM2) in
Hämostase und Thrombose“
Berna Erro, Alejandro
“Phosphoproteomics of human platelets“
Zahedi, René
“Analysing the oncogenic potential of ∆Np73 in vivo“
Griesmann, Heidi
“SMAD fluorescent biosensors“
Gromova, Kira
“The role of miRNAs in cardiac disease“
Groß, Carina
“Toxins in renal disease: genotoxic potential and
mechanism action“
Fink, Kristin
“Membrane biophysics giant unilamallar vesicles as biosensors with ultra-sensitive, dynamic microscopy
instrumentation“
Lambright, Geoffrey
“Analysis of glycosylation pattern in human platelets“
Lewandrowski, Urs
“Towards the development of high affinity InhA inhibitors
with activity against drug-resistant strains of Mycobacterium tuberculosis”
Luckner, Sylvia
“Role of interleukin-converting enzyme in heart failure“
Merkle, Sabine
“Characterization of the platelet membranes“
Moebius, Jan
“Differential and Quantitative Proteome Analysis of a Cell
Culture Model for Malignant Transformation”
Pütz, Stephanie
Concluded in 2006:
“Regulation of telomerase activity by the p53-homologous p73“
Beitzinger, Michaela
“Methoden zur effizienten Proteinidentifizierung anhand
von Massenspektrometrie am Beispiel des mitochondrialen Außenmembran-Proteoms der Bäckerhefe Saccharomyces cerevisiae“
Böhm, Andreas
“Role of p73 isoforms in cellular differentiation“
Cam, Hakan
“Mechanism of action of the cytomegalovirus M/UL45
proteins“
Gehrke, Claudia
“Role of p73 in the malignant transformation“
Hofmann, Lars
“Molecular basis of the cytomegalovirus species specificity”
Jurak, Igor
“Mechanisms of thrombus stabilization“
Pozgajova, Miroslava
“Analysis of the mitochondrial proteome of Saccharomyces cerevisiae“
Reinders, Jörg
“Functional analysis of p53 via fluorescence microscopy“
Sauer, Markus
“Identification and characterization of the centrosomal
proteome Dictyostelium discoideum“
Reinders, Yvonne
“Cooperation of platelet adhesion receptors in activation
and coagulant activity“
Strehl, Amrei
“Functional analysis of the cytomegalovirus immediateearly proteins m142 and m143“
Valchanova, Ralitsa
67
Publications
Junior Research Groups
Group Stefan Engelhardt
Hallhuber, M., Burkard, N., Wu, R., Buch, M.H.,
Engelhardt, S., Hein, L., Neyses, L., Schuh, K.,
and Ritter, O. (2006) Inhibition of nuclear
import of calcineurin prevents myocardial
hypertrophy.
Circ Res, 99, 626-35.
Hein, P., Rochais, F., Hoffmann, C.,
Dorsch, S., Nikolaev, V.O., Engelhardt, S.,
Berlot, C.H., Lohse, M.J., and Bunemann, M.
(2006) Gs activation is time-limiting
in initiating receptor-mediated signaling.
J Biol Chem, 281, 33345-51.
Merkle, S., Frantz, S., Schön, M.P., Bauersachs,
J.,Buitrago, M., Frost, R.J.A., Schmitteckert,
E., Lohse, M.J., and Engelhardt, S. (2007) A role
for caspase-1 in heart failure. Circ Res, in press.
Nikolaev, V.O., Bunemann, M., Schmitteckert, E.,
Lohse, M.J., and Engelhardt, S. (2006)
Cyclic AMP imaging in adult cardiac
myocytes reveals far-reaching β1-adrenergic but locally confined β2-adrenergic
receptor-mediated signaling. Circ Res, 10,
1084-91.
Rochais, F., Vilardaga, J.-P., Nikolaev, V. O.,
Bünemann, M., Lohse, M.J., and Engelhardt,
S. (2007) Real-time optical recording of
β1-adrenergic receptor activation and
signaling reveals supersensitivity of the
Arg-389 variant to carvedilol.
J Clin Invest, 117, 229-35.
Seeland, U., Selejan, S., Engelhardt, S.,
Muller, P., Lohse, M.J., and Bohm, M.
(2007) Interstitial remodeling in
β1-adrenergic receptor transgenic mice.
Basic Res Cardiol, 102, 183-93.
Group Bernhard Nieswandt
Johne, J., Blume, C., Benz, M.B., Pozgajova, M.,
Ullrich, M., Schuh, K., Nieswandt, B.,
Walter, U., and Renne, T. (2006) Platelets
promote coagulation factor XII-mediated
proteolytic cascade systems in plasma.
Biol Chem, 387, 173-178.
68
Kleinschnitz, C., Stoll, G., Bendszus, M.,
Schuh, K., Pauer, U., Burfeind, P., Renne, C.,
Gailani, D., Nieswandt, B., and Renne, T.
(2006) Targeting coagulation factor XII
provides protection from pathological
thrombosis in cerebral ischemia without
interfering with hemostasis.
J Exp Med, 203, 513-518.
Oostingh, G.J., Ludwig, R.J., Enders, S.,
Gruner, S., Harms, G., Boehncke, W.H.,
Nieswandt, B., Tauber, R., and Schon, M.P.
(2006) Diminished lymphocyte adhesion
and alleviation of allergic responses by
small-molecule- or antibody-mediated
inhibition of L-Selectin functions.
J Invest Dermatol, 127, 90-97.
Oostingh, G.J., Pozgajova, M., Ludwig,
R., Krahn, T., Boehncke, W.H., Nieswandt,
B., and Schön, M.P. (2006) Diminished
thrombus formation and alleviation of
myocardial reperfusion injury through
antibody- or small-molecule-mediated
inhibition of selectin-dependent platelet
functions. Haematologica, in press.
Pozgajova, M., Sachs, U., Hein, L., and
Nieswandt, B. (2006) Reduced thrombus
stability in mice lacking the α2A adrenergic
receptor. Blood, 108, 510-514.
Rabie, T.,Varga-Szabo, D., Bender, M.,
Lanza, F., Saito, T., Watson, S.P., and
Nieswandt, B. (2007) Diverging signaling
events control the pathway of GPVI downregulation in vivo. Blood, in press.
Sachs, U. and Nieswandt, B. (2006) In vivo
thrombus formation: what can we learn
from murine models?
Circ Res, in press.
Arterioscler Thromb Vasc Biol,
26, 1640-1647.
Strehl, A., Munnix, I.C.A., Kuijpers,
M.J.E., Feijge, M.A.H., Cosemans,
J.M.E.M., van der Meijden, P., Nieswandt,
B., and Heemskerk, J.W.M. (2006) Dual
role of platelet protein kinase C in thrombus formation: stimulation of proaggregatory and suppression of procoagulant
activity in platelets. J Biol Chem, in
press.
Group Thorsten Stiewe
Beitzinger, M., Oswald, C., BeinoraviciuteKellner, R., and Stiewe, T. (2006)
Regulation of telomerase activity by the
p53 family member p73. Oncogene, 25,
813-826.
Cam, H., Griesmann, H., Beitzinger, M.,
Hofmann, L., Beinoraviciute-Kellner, R.,
Sauer, M., Hüttinger-Kirchhof, N., Oswald,
C., Friedl, P., Gattenlöhner, S., Burek, C.,
Rosenwald, A., and Stiewe, T. (2006) p53
family members in myogenic differentiation and rhabdomyosarcoma development.
Cancer Cell, 10, 281-293.
Hüttinger-Kirchhof, N., Cam, H., Griesmann, H., Hofmann, L., Beitzinger, M. and
Stiewe, T. (2006) The p53 family inhibitor
DNp73 interferes with multiple developmental programs.
Cell Death Differ, 13, 174-177.
Stiewe, T. (2007) p53 family in differentiation and tumorigenesis.
Nature Rev Cancer, in press.
Group Stephan Kissler New Group
Sayeh, E., Crow, M., Webster, M.L.,
Nieswandt, B., Freedman, J., and Ni, H.
(2006) Distinctive efficacy of IVIG in
ameliorating thrombocytopenia induced by
anti-platelet GPIIbIIIa versus anti-GPIbα
antibodies. Blood, 108, 943-946.
Schulte, V., Reusch, P., Pozgajova, M., Varga-Szabó, D., Gachet, C., and Nieswandt,
B. (2006) Two-phase antithrombotic protection after anti-GPVI treatment in mice.
Kissler, S., Stern, P., Takahashi, K., Hunter, K.,
Peterson, L. and Wicker, L.S. (2006). In
vivo RNA interference demonstrates a role
for Nramp1 in modifying susceptibility to
type 1 diabetes.
Nature Genetics, 38, 479-483.
Core Center
Group Gregory Harms
Friedl, P., Wolf, K., von Andrian, U., and
Harms, G. (2007) Biological second and
third harmonic generation microscopy.
Curr Prot Cell Biol, 4.15.1-4.15.21.
Group Caroline Kisker
Karakas, E., Truglio, J., Croteau, D., Rhau, B.,
Wang, L., Houten, B.V., and Kisker, C.
(2006) Structure of the C-terminal half
of UvrC reveals an RNase H endonuclease
domain with an Argonaute-like catalytic
triad. EMBO J, in press.
Kisker, C. (2006) Structural Features of
Bypass Polymerases.
Marcel-Dekker, Inc., New York.
Kolappan, S., Zwahlen, J., Zhou, R.,
Truglio, J.J., Tonge, P.J., and Kisker, C.
(2006) Lysine 190 is the catalytic base
in MenF, the menaquinone-specific
isochorismate synthase from Escherichia
coli: implications for an enzyme family.
Biochemistry, in press.
Rafi, S., Novichenok, P., Kolappan, S.,
Zhang, X., Stratton, C.F., Rawat, R.,
Kisker, C., Simmerling, C., and Tonge, P.J.
(2006) Structure of acyl carrier protein
bound to Fabi, the FASII enoyl reductase
from Escherichia coli.
J Biol Chem, 281, 39285-39293.
Sullivan, T.J., Truglio, J.J., Boyne, M.E.,
Novichenok, P., Zhang, X., Stratton, C.F.,
Li, H.-J., Kaur, T., Amin, A., Johnson, F.,
Slayden, R.A., Kisker, C., and Tonge, P.J.
(2006) High affinity InhA inhibitors with
activity against drug-resistant strains of
Mycobacterium tuberculosis.
ACS Chemical Biology, 1, 43-53.
Tonge, P.J., Kisker, C., and Slayden, R.A.
(2006) Development of Modern InhA
Inhibitors to combat drug resistant strains
of Mycobacterium tuberculosis.
Curr Top Med Chem, in press.
Truglio, J.J., Karakas, E., Rhau, B., Wang, H.,
DellaVecchia, M.J., Van Houten, B., and
Kisker, C. (2006) Structural basis for DNA
recognition and processing by UvrB.
Nat Struct Mol Biol, 13, 360-364.
Yakubovskaya, E., Chen, Z., Carrodeguas, J.A.,
Kisker, C., and Bogenhagen, D.F. (2006)
Functional human mitochondrial DNA
polymerase gamma forms a heterotrimer.
J Biol Chem, 281, 374-382.
Zhao, G., Zhou, X., Wang, L., Li, G., Kisker, C.,
Lennarz, W.J., and Schindelin, H. (2006)
Structure of the mouse peptide N-glycanase-HR23 complex suggests co-evolution
of the endoplasmic reticulum-associated
degradation and DNA repair pathways.
J Biol Chem, 281, 13751-13761.
Zwahlen, J., Kolappan, S., Zhou, R.,
Kisker, C., and Tonge, P.J. (2006) Structure
and mechanism of MbtI, the salicylate
synthase from Mycobacterium tuberculosis.
Biochemistry, in press.
Li, G., Zhao, G., Zhou, X., Schindelin, H.,
and Lennarz, W.J. (2006) The AAA ATPase
p97 links peptide N-glycanase to the endoplasmic reticulum-associated E3 ligase
autocrine motility factor receptor.
PNAS U S A, 103, 8348-8353.
Nichols, J.D., Xiang, S., Rajagopalan, K.V.,
and Schindelin, H. (2006) Mutational
analysis of Escherichia coli MoeA: Two
functional activities map to the active site
cleft. Biochemistry, 46, 78-86.
Suzuki, T., Hara, I., Nakano, M., Zhao, G.,
Lennarz, W.J., Schindelin, H., Taniguchi, N.,
Totani, K., Matsuo, I., and Ito, Y. (2006)
Site-specific labeling of cytoplasmic
peptide:N-glycanase by N,N‘-diacetylchitobiose-related compounds.
J Biol Chem, 281, 22152-22160.
Tian, G., Xiang, S., Noiva, R., Lennarz, W.J.,
and Schindelin, H. (2006) The crystal
structure of yeast protein disulfide isomerase suggests cooperativity between its
active sites. Cell, 124, 61-73.
Group Hermann Schindelin
Chavan, M., Chen, Z., Li, G., Schindelin, H.,
Lennarz, W.J., and Li, H. (2006) Dimeric
organization of the yeast oligosaccharyl
transferase complex.
PNAS U S A, 103, 8947-8952.
Hanzelmann, P., and Schindelin, H. (2006)
Binding of 5‘-GTP to the C-terminal FeS
cluster of the radical S-adenosylmethionine enzyme MoaA provides insights into
its mechanism.
PNAS U S A, 103, 6829-6834.
Kim, E.Y., Schrader, N., Smolinsky, B.,
Bedet, C., Vannier, C., Schwarz, G., and
Schindelin, H. (2006) Deciphering the
structural framework of glycine receptor
anchoring by gephyrin.
EMBO J, 25, 1385-1395.
Lawrence, S.H., Luther, K.B., Schindelin, H.,
and Ferry, J.G. (2006) Structural and
functional studies suggest a catalytic
mechanism for the phosphotransacetylase
from Methanosarcina thermophila.
J Bacteriol, 188, 1143-1154.
Xiang, S., Kim, E.Y., Connelly, J.J., Nassar, N.,
Kirsch, J., Winking, J., Schwarz, G., and
Schindelin, H. (2006) The crystal structure
of Cdc42 in complex with collybistin II, a
gephyrin-interacting guanine nucleotide
exchange factor.
J Mol Biol, 359, 35-46.
Zhao, G., Zhou, X., Wang, L., Li, G., Kisker, C.,
Lennarz, W.J., and Schindelin, H. (2006)
Structure of the mouse peptide N-glycanase-HR23 complex suggests co-evolution
of the endoplasmic reticulum-associated
degradation and DNA repair pathways.
J Biol Chem, 281, 13751-13761.
Zhou, X., Zhao, G., Truglio, J.J., Li, G.,
Wang, L., Lennarz, W.J., and Schindelin,
H. (2006) Structural and biochemical
studies of the C-terminal domain of mouse
peptide-N-glycanase identify it as a mannose-binding module.
PNAS U S A, 103, 17214-19.
Truglio, J.J., Croteau, D.L., Van Houten, B.,
and Kisker, C. (2006) Prokaryotic nucleotide excision repair: the UvrABC system.
Chem Rev, 106, 233-252.
69
Group Albert Sickmann
Boehm, A.M., and Sickmann, A. (2006)
A comprehensive dictionary of protein
accession codes for complete protein accession identifier alias resolving.
Proteomics, 6, 4223-4226.
Grunewald, T.G.P., Kammerer, U.,
Sickmann, A., Schindler, D., Winkler, C.,
and Butt, E. (2007) LASP-1 in human
ovarian cancer.
British Journal Cancer, 96, 296-305.
Hahlen, K., Ebbing, B., Reinders, J.,
Mergler, J., Sickmann, A., and Woehlke,
G. (2006) Feedback of the kinesin-1 necklinker position on the catalytic site.
J Biol Chem, 281, 18868-18877.
Koch, K.V., Reinders, Y., Ho, T.H., Sickmann, A., and Graf, R. (2006) Identification and isolation of Dictyostelium
microtubule-associated protein interactors
by tandem affinity purification.
Eur J Cell Biol, 85, 1079-1090.
Leuber, M., Orlik, F., Schiffler, B.,
Sickmann, A., and Benz, R. (2006)
Vegetative Insecticidal Protein (Vip1Ac)
of Bacillus thuringiensis HD201: Evidence
for Oligomer and Channel Formation.
Biochemistry, 45, 283-288.
Lewandrowski, U., Moebius, J., Walter, U.,
and Sickmann, A. (2006) Elucidation of
N-Glycosylation Sites on Human Platelet
Proteins: A Glycoproteomic Approach.
Mol Cell Proteomics, 5, 226-233.
Moebius, J., Zahedi, R., and Sickmann, A.
(2006) Platelet Proteomics: Essentials for
Understanding and Application. Transfus
Med Hemother, 33, 227-235.
Otter, S., Grimmler, M., Neuenkirchen, N.,
Chari, A., Sickmann, A., and Fischer, U.
(2006) A comprehensive interaction map
of the human SMN-complex.
J Biol Chem, in press.
Reinders, J., Meyer, H.E., and Sickmann,
A. (2006) Applications of highly sensitive
phosphopeptide derivatization methods
without the need for organic solvents.
Proteomics, 6, 2647-2649.
70
Reinders, J., Zahedi, R.P., Pfanner, N.,
Meisinger, C., and Sickmann, A. (2006)
Toward the complete yeast mitochondrial
proteome: multidimensional separation
techniques for mitochondrial proteomics.
J Proteome Res, 5, 1543-1554.
Reinders, Y., Schulz, I., Gräf, R., and
Sickmann, A. (2006) Identification of
Novel Centrosomal Proteins in Dictyostelium discoideum by Comparative Proteomic
Approaches. J Proteome Res, 5, 589-598.
Schindler, J., Lewandrowski, U., Sickmann,
A., Friauf, E., and Gerd Nothwang, H.
(2006) Proteomic Analysis of Brain Plasma
Membranes Isolated by Affinity Two-phase
Partitioning.
Mol Cell Proteomics, 5, 390-400.
Schulz, I., Reinders, Y., Sickmann, A.,
and Graf, R. (2006) An improved method
for Dictyostelium centrosome isolation.
Methods Mol Biol, 346, 479-489.
Zahedi, R.P., Begonja, A.J., Gambaryan, S.,
and Sickmann, A. (2006) Phosphoproteomics of human platelets: A quest for
novel activation pathways.
Biochim Biophys Acta, 1764, 1963-76.
Zahedi, R.P., Sickmann, A., Boehm, A.M.,
Winkler, C., Zufall, N., Schonfisch, B.,
Guiard, B., Pfanner, N., and Meisinger, C.
(2006) Proteomic analysis of the yeast
mitochondrial outer membrane reveals
accumulation of a subclass of preproteins.
Mol Biol Cell, 17, 1436-1450.
Research Professors
Group Peter Friedl
Cam, H., Griesmann, H., Beitzinger, M.,
Hofmann, L., Beinoraviciute-Kellner, R.,
Sauer, M., Huttinger-Kirchhof, N., Oswald, C.,
Friedl, P., Gattenlohner, S., Burek, C.,
Rosenwald, A., and Stiewe, T. (2006) p53
family members in myogenic differentiation and rhabdomyosarcoma development.
Cancer Cell, 10, 281-293.
Friedl, P., Wolf, K., von Andrian, U.H., and
Harms, G. (2007) Biological second and
third harmonic generation microscopy.
Curr Prot Cell Biol, 4.15.1-4.15.21
Hartmann, A., Boukamp, P., and Friedl, P.
(2006) Confocal reflection imaging of 3D
fibrin polymers.
Blood Cells Mol Dis, 36, 191-193.
Wolf., K., and Friedl., P. (2006) Molecular
mechanisms of cancer cell invasion and
plasticity.
Br J Dermatol, 154 (Suppl. 1), 11-15.
Merkle, S., Frantz, S., Schön, M.P., Bauersachs, J., Buitrago, M., Frost, R.J.A.,
Schmitteckert, E., Lohse, M.J., and Engelhardt, S. (2007) A role for caspase-1 in
heart failure. Circ Res, in press.
Mössner, R., Schön, M.P., Reich, K(2006)
TNFα inhibitors: Infliximab, etanercept
and adalimumab in the treatment of psoriasis. Clin Dermatol, in press.
Oostingh, G.J., Ludwig, R.J., Enders, S.,
Grüner, S., Harms, G., Boehncke, W.H.,
Nieswandt, B., Tauber, R., and Schön, M.P.
(2006) Diminished lymphocyte adhesion
and alleviation of allergic responses by
small-molecule- or antibody-mediated
inhibition of L-selectin functions.
J Invest Dermatol, in press.
Oostingh, G.J., Pozgajova, M., Ludwig, R.J.,
Krahn, T., Boehncke, W.H., Nieswandt, B.,
and Schön, M.P. (2006) Diminished
thrombus formation and alleviation of
myocardial infarction and reperfusion injury through antibody- or small-moleculemediated inhibition of selectin-dependent
platelet functions.
Haematologica Hematol J, in press.
Group Michael P. Schön
Benoit, S., and Schön, M.P. (2006) Psoriasis – klinisches Spektrum, Pathogenese
und neue Aspekte zur Therapie.
Therapiewoche, 3/4.06, 70-73.
Oostingh, G.J., Schlickum, S., Friedl, P.,
and Schön, M.P. (2006) Impaired induction of E-selectin expression in immortalized endothelial cells leads to functional
defects in dynamic interactions with
lymphocytes. J Invest Dermatol, in press.
Boehncke, W.H., and Schön, M.P. (2006)
Animal models and their value for psoriasis research. Clin Dermatol, in press.
Gesierich, A., Herzog, S., Grunewald, S.M.,
Tappe, D., Bröcker, E.B., and Schön, M.P.
(2006) Eosinophilic folliculitis in a caucasian patient: Association with toxocariasis? J Eur Acad Dermatol, 20, 1317-1321.
Rattenholl, A., Seeliger, S., Buddenkotte, J.,
Schön, M., Schön, M.P., Vergnolle, N.,
Ständer, S., and Steinhoff, M. (2006)
Proteinase-activated receptor-2 (PAR2):
A tumor suppressor in skin carcinogenesis.
J Invest Dermatol, in press.
Kerstan, A., Goebeler, M., Schmidt, E.,
Bröcker, E.B., and Schön, M.P. (2006)
Lupus erythematosus profundus in an
8-year-old child. J Eur Acad Dermatol,
21, 132-133.
Kerstan, A., and Schön, M.P. (2006) Viewpoint: Who is really in control of skin immunity under physiological circumstances
- lymphocytes, dendritic cells, or keratinocytes? Exp Dermatol, 15, 529-530.
Li, Y., Schön, M.P., and Zollner, T.M.
(2006) Targeting leukocyte recruitment.
Clin Dermatol, in press.
Schön, M., and Schön, M.P. (2006) The
antitumoral mode of action of imiquimod
and other imidazoquinolines.
Curr Med Chem, in press.
Schön, M.P. (2006) Cell adhesion molecules as therapeutic targets.
Expert Opin Ther Targets, 10, 799-802.
Schön, M.P. (2006) Die Behandlung der
Psoriasis.
Arzneiverordnung in der Praxis, 33, 99-101.
Schön, M.P. (2006) Efalizumab in the
treatment of psoriasis. Clin Dermatol, in
press.
Schön, M.P. (2006) Molekulare Mechanismen entzündlicher Erkrankungen als Grundlage für selektive Therapien. Jahrbuch
der Berlin-Brandenburgischen Akademie
der Wissenschaften, 138-145, AkademieVerlag, Berlin.
Schön, M.P. (2006) Pathophysiologie der
Psoriasis. Akt Dermatol, 32, 169-175.
Schön, M.P. (2006) Psoriasis in the limelight - a model disorder for chronic inflammation. Clin Dermatol, in press.
Schön, M.P., and Schön, M. (2006) The
small-molecule immune response modifier
imiquimod – Its mode of action and clinical use in the treatment of skin cancer.
Expert Opin Ther Targets, 10, 69-76.
Schön, M.P., and Schön, M. (2007) Topisch
applizierte antitumoral wirksame Medikamente – Imiquimod. In: Szeimies RM et
al., (Hg.): Tumoren der Haut, Thieme,
Stuttgart, 2007.
Schön, M.P., Schön, M., and Klotz, K.N.
(2006) The small anti-tumoral immune
response modifier imiquimod interacts
with adenosine receptor signaling in a
TLR7- and 8-independent fashion.
J Invest Dermatol, 126, 1338-1347.
Wachter, T., Murach, W.M., Bröcker, E.B.,
and Schön, M.P. (2006) Recalcitrant
lithium-induced psoriasis in a suicidal
patient alleviated by TNFα inhibition.
Br J Dermatol, in press.
Wienrich, B.G., Krahn, T., Schön, M.,
Rodriguez, M.L., Kramer, B., Busemann, M.,
Boehncke, W.H., and Schön, M.P. (2006)
Structure-function relation of efomycines,
a family of small-molecule inhibitors of
selectin functions.
J Invest Dermatol, 126, 882-889.
Wienrich, B.G., Oostingh, G.J., Ludwig,
R.J., Enders, S., Harms, G., Tauber, R.,
Krahn, T., Kramer, B., Boehncke, W.H.,
and Schön, M.P. (2006) Efomycine M:
an inhibitor of selections? (letter)
Nat Med, 12, 873-874.
Zollner, T.M., Asadullah, K., and Schön,
M.P. (2006) Targeting leukocyte trafficking
to the skin – Still an attractive therapeutic target? Exp Dermatol, 16, 1-12.
71
RVZ Network
Group Utz Fischer
Otter, S., Grimmler, M., Neuenkirchen, N.,
Chari, A., Sickmann, A., and Fischer, U.
(2007) A comprehensive interaction map
of the human SMN-complex. J Biol Chem,
in press.
Group Manfred Gessler
Diez, H., Fischer, A., Winkler, A., Hu, CJ.,
Hatzopoulos, A.K., Breier, G., and Gessler,
M. (2007) Hypoxia mediated activation of
Dll4-Notch-Hey2 signalling in endothelial
progenitor cells and adoption of arterial
cell fate. Exp Cell Res, 313, 1-9.
Group Thomas D. Müller
Kraich, M., Klein, M., Patino, E., Harrer,
H., Nickel, J., Sebald, W. and Mueller, T.D.
(2006) A modular interface of IL-4 allows
for scalable affinity without affecting
specificity for the IL-4 receptor.
BMC Biol, 4, 13.
Meierjohann, S., Mueller, T.D., Schartl, M.
and Buehner, M. (2006) A structural model
of the extracellular domain of the oncogenic EGFR variant Xmrk.
Zebrafish, 3, 359-369.
Group Manfred Schartl
Meierjohann, S., and Schartl, M. (2006)
From mendelian to molecular genetics:
the Xiphophorus melanoma model.
Trends Genet, 22, 654-61.
Rutenberg, J.B., Fischer, A., Haibo, J.,
Gessler, M., Zhong, T.P., and Mercola, M.
(2006) Developmental patterning of the
cardiac atrioventricular canal by Notch
and Hairy-related transcription factors.
Development, 133, 4381-4390.
Volff , J.N., Nanda, I., Schmid, M., and
Schartl, M. (2006) Governing sex determination in fish: regulatory putsches and
ephemeral dictators. Sex Dev, in press.
Group Thomas Hünig
Group Walter Sebald
Beyersdorf, N., Balbach, K., Hunig, T., and
Kerkau, T. (2006) Large-scale expansion
of rat CD4 CD25 T cells in the absence of
T-cell receptor stimulation.
Immunology, 119, 441-449.
Kraich, M., Klein, M., Patino, E., Harrer, H.,
Nickel, J., Sebald, W., and Mueller,
T.D.(2006) A modular interface of IL-4 allows for scalable affinity without affecting
specificity for the IL-4 receptor.
BMC Biol, 4, 13.
Dennehy, K.M., Elias, F., Na, S,Y.,
Fischer, K.D., Hunig, T., and Luhder, F.
(2006) Mitogenic CD28 signals require the
exchange factor Vav1 to enhance TCR signaling at the SLP-76-Vav-Itk signalosome.
J Immunol, in press.
Dennehy, K.M., Elias, F., Zeder-Lutz, G.,
Ding, X., Altschuh, D., Luhder, F., and
Hunig, T. (2006) Cutting edge: monovalency of CD28 maintains the antigen
dependence of T cell costimulatory
responses.
J Immunol, 176, 5725-9.
Kerstan, A., Armbruster, N., Leverkus, M.,
and Hunig, T. (2006) Cyclosporin A abolishes CD28-mediated resistance to CD95induced apoptosis via superinduction of
caspase-3. J Immunol, 177, 7689-7697.
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Rentzsch, F., Zhang, J., Kramer, C., Sebald,
W., and Hammerschmidt, M. (2006)
Crossveinless 2 is an essential positive
feedback regulator of Bmp signaling during zebrafish gastrulation.
Development, 133, 801-811.
Bio-Imaging Center
Group Martin Lohse
Harbeck, M.C., Chepurny, O., Nikolaev,
V.O., Lohse, M.J., Holz, G.G., and Roe,
M.W. (2006) Simultaneous optical measurements of cytosolic Ca2+ and cAMP in
single cells. Science STKE, 353, pl6.
Hein, P., Rochais, F., Hoffmann, C.,
Dorsch, S., Nikolaev, V.O., Engelhardt, S.,
Berlot, C.H., Lohse, M.J., and Bunemann,
M. (2006) GS activation is time-limiting in
initiating receptor-mediated signaling.
J Biol Chem, 281, 33345-51.
Lohse, M.J., (2006) GPCRs – too many
dimers? Nature Methods, 3, 972-973.
Lohse, M.J., Hoffmann, C., Vilardaga, J.P.,
and Bünemann, M. (2006) Kinetic analysis
of G-protein-coupled receptor signaling
using fluorescence resonance energy
transfer in living cells.
Adv Prot Chem, in press.
Nikolaev, V.O., Bunemann, M.,
Schmittecker,t E., Lohse, M.J., and Engelhardt, S. (2006) Cyclic AMP imaging in
adult cardiac myocytes reveals far-reaching β1-adrenergic but locally confined
β2-adrenergic receptor-mediated signaling.
Circ Res, 99, 1084-91.
Nikolaev, V.O., Gambaryan, S., and Lohse,
M.J. (2006) Fluorescent sensors for rapid
monitoring of intracellular cGMP.
Nature Methods, 3, 23-5.
Nikolaev, V.O., Hoffmann, C., Bunemann, M.,
Lohse, M.J., and Vilardaga, J.P. (2006)
Molecular basis of partial agonism at the
neurotransmitter alpha2A-adrenergic receptor and Gi-protein heterotrimer.
J Biol Chem, 281, 24506-11.
Nikolaev, V.O., and Lohse, M.J. (2006)
Monitoring of cAMP synthesis and degradation in living cells.
Physiology, 21, 86-92.
Group Stephan Sigrist
Ataman, B., Ashley, J., Gorczyca, D.,
Gorczyca, M., Mathew, D., Wichmann,
C., Sigrist, S.J., and Budnik, V. (2006)
Nuclear trafficking of Drosophila Frizzled-2
during synapse development requires the
PDZ protein dGRIP.
PNAS U S A, 103, 7841-6.
Kittel, R.J., Hallermann, S., Thomsen, S.,
Wichmann, C., Sigrist, S.J., and Heckmann, M. (2006) Active zone assembly
and synaptic release.
Biochem Soc Trans, 34, 939-41.
Kittel, R.J., Wichmann, C., Rasse, T.M.,
Fouquet, W., Schmidt, M., Schmid, A.,
Wagh, D.A., Pawlu, C., Kellner, R.R.,
Willig, K.I., Hell, S.W., Buchner, E., Heckmann, M., and Sigrist, S.J. (2006) Bruchpilot promotes active zone assembly, Ca2+
channel clustering, and vesicle release.
Science, 312, 1051-4.
Schmid, A., Qin, G., Wichmann, C., Kittel, R.J.,
Mertel, S., Fouquet, W., Schmidt, M.,
Heckmann, M., and Sigrist, S.J. (2006)
Non-NMDA-type glutamate receptors are
essential for maturation but not for initial
assembly of synapses at Drosophila neuromuscular junctions.
J Neurosci, 26, 11267-77.
Sigrist, S.J. (2006) Neurobiology tools:
flashdancing worms. Curr Biol, 16, R100-2.
Swan, L.E., Schmidt, M., Schwarz, T., Ponimaskin, E., Prange, U., Boeckers, T.,
Thomas, U., and Sigrist, S.J. (2006)
Complex interaction of Drosophila GRIP
PDZ domains and Echinoid during muscle
morphogenesis. EMBO J, 25, 3640-51.
Wagh, D.A., Rasse, T.M., Asan, E., Hofbauer, A., Schwenkert, I., Durrbeck, H.,
Buchner, S., Dabauvalle, M.C., Schmidt,
M., Qin, G., Wichmann, C., Kittel, R., Sigrist, S.J. and Buchner E. (2006) Bruchpilot, a protein with homology to ELKS/
CAST, is required for structural integrity
and function of synaptic active zones in
Drosophila. Neuron, 49, 833-44.
Rochais, F., Vilardaga, J.-P., Nikolaev, V.O.,
Bünemann, M., Lohse, and M.J., Engelhardt, S. (2007) Real-time optical recording
of β1-adrenergic receptor activation and
signaling reveals supersensitivity of the
Arg-389 variant to carvedilol.
J Clin Invest, 117, 229-35.
73
Central Technologies
Transgene Technologies
Oberle, S., Schober, A., Meyer, V., Holtmann, B., Henderson, C., Sendtner, M.,
and Unsicker, K. (2006) Loss of leukemia inhibitory factor receptor beta or
cardiotrophin-1 causes similar deficits in
preganglionic sympathetic neurons and
adrenal medulla.
J Neurosci, 26, 1823-32.
Kaiser, M., Maletzki, I., Hulsmann, S.,
Holtmann, B., Schulz-Schaeffer, W., Kirchhoff, F., Bahr, M., and Neusch, C. (2006)
Progressive loss of a glial potassium
channel (KCNJ10) in the spinal cord of the
SOD1 (G93A) transgenic mouse model of
amyotrophic lateral sclerosis.
J Neurochem, 99, 900-12.
Nikolaev, V.O., Bunemann, M.,
Schmitteckert, E., Lohse, M.J., and
Engelhardt, S. (2006) Cyclic AMP imaging in adult cardiac myocytes reveals
far-reaching β1-adrenergic but locally
confined β2-adrenergic receptor-mediated
signaling. Circ Res, 99, 1084-1091.
DNA Arrays
Cam, H., Griesmann, H., Beitzinger, M.,
Hofmann, L., Beinoraviciute-Kellner, R.,
Sauer, M., Huttinger-Kirchhof, N., Oswald, C.,
Friedl, P., Gattenlohner, S., Burek, C.,
Rosenwald, A., and Stiewe, T. (2006) p53
family members in myogenic differentiation and rhabdomyosarcoma development.
Cancer Cell, 10, 281-93.
Nagel, S., Burek, C., Venturini, L., Scherr, M.,
Quentmeier, H., Meyer, C., Rosenwald, A.,
Drexler, H.G., and MacLeod, R.A.F. (2006)
Comprehensive analysis of homeobox
genes in Hodgkin Lymphoma cell lines
identifies dysregulated expression of
HOXB9 mediated via ERK5 signaling and
BMI1. Blood, in press.
74
The Annual Report 2006 is a magazine providing information about the activities in Research, Teaching
and Public Relations at the Rudolf Virchow Center/DFG Research Center for Experimental Biomedicine of
the University of Würzburg.
I mprin t
Editor:
Rudolf Virchow Center/DFG Research Center for Experimental Biomedicine of the University of Würzburg
Editor in chief:
Sonja Jülich
Editorial:
Ruth Willmott, Avril Arthur Göttig, Christiane Weber
Design, Layout & Prepress:
Sascha Kreger
Email: info@sk-grafik.com
http://www.sk-grafik.com
Print:
Gruber Offset Druck
http://www.gruber-druck.de
Notice:
Neither the Rudolf Virchow Center, nor any person acting on its behalf may be held responsible for the
use to which information contained in this publication may be put, or for any errors which, despite
careful preparation and checking may appear.
© RVZ, 2007
Non-commercial reproduction authorized, subject to acknowledgement of source.
Edition: 1500
Editor`s office:
Rudolf Virchow Center
DFG Research Center for Experimental Biomedicine of the University of Würzburg
Public Science Center
Versbacher Str. 9
97078 Würzburg
Phone: +49(0)931 201 48714
Fax:
+49(0)931 201 48702
Email: [email protected]
http://www.rudolf-virchow-zentrum.de
Images:
Stephan Sigrist/Manfred Schartl/Peter Friedl/Caroline Kisker/Stefan Engelhardt (U1), Archiv Pathologie
Universität Würzburg (U4), Sascha Kreger/PSC/Stephan Sigrist/Caroline Kisker/Universitätsbaumt
Würzburg/Horst Pfrang/Beatrice Döge/PSC (p.4-11), Stephan Sigrist/Thorsten Stiewe/Hermann
Schindelin/ Manfred Schartl/Peter Friedl (p.12-13), Stefan Engelhardt (p.14-15), Bernhard Nieswandt
(p.16-17), Thorsten Stiewe (p.18-19), Stefan Kissler (p.20-21), Gregory Harms (p.22-23), Caroline Kisker
(p.24-25), Herrmann Schindelin (p.26-27), Albert Sickmann (p.28-29), Peter Friedl (p.30-31), Michael
P. Schön (p.32-33), Utz Fischer (p.34-35), Manfred Gessler (p.36-37), Thomas Hünig (p.38-39), Thomas
Müller (p.40-41), Manfred Schartl (p.42-43), Walter Sebald (p.44-45), Martin Lohse (p.46-47), Stephan
Sigrist (p.48-49), Carmen Dengel/Werner Lutz/Gunnar Bartsch/Stephan Schröder-Köhne/Monika Maier/
Peter Friedl/PSC (p.50-55), Christiane Weber/Sonja Jülich (p. 56-57)
Front Image:
Scientific pictures of (from left) the neuromucular junction (Stephan Sigrist), eye of a medaka fish
(Manfred Schartl), cancer cells around blood vessels (Peter Friedl), binding site for DNA in the protein
UvrB (Caroline Kisker), tissue of heart muscle (Stefan Engelhardt).
Back:
Excerpt from the section book of Rudolf Virchow at the Institute of Pathology, University of Würzburg.