DFG Research Center for Experimental Biomedicine,
University of Würzburg
Annual Report
2010
Prof. Dr. Martin Lohse
Forewo rd
We are presenting here the 2010 Annual Report of the Rudolf Virchow Center, the DFG-Research Center for
Experimental Biomedicine of the University of Würzburg. The report describes our research on target
proteins – proteins that exert key regulatory functions in a cell. We investigate these proteins on multiple
levels, ranging from atomic resolution to their functions in health and disease. Because of their biological
importance, target proteins may serve diagnostic or therapeutic purposes, and here our interests are
focussed on cardiovascular diseases and cancer. Strong interdisciplinary collaborations in the Center itself,
in Würzburg and elsewhere enable us to analyze these proteins from different perspectives, ranging from
their molecular structure and biochemical mechanisms to their role in pathophysiological states.
The Rudolf Virchow Center is one of currently six centers of excellence funded by the German Research
Foundation, DFG. These centers are funded by the DFG for up to 12 years and represent an opportunity to
follow both, new venues of research and new university structures. The Rudolf Virchow Center was among
the first three of these centers and was funded in 2001. Since the concept and the performance of the
Center were considered “outstanding” at the last site visit of the DFG in 2009, the government of the State
of Bavaria, the „Ministerrat“, decided to continue its funding beyond the end of DFG funding in 2013.
We are happy to see that the Rudolf Virchow Center has by now evolved into a highly dynamic and
productive research center. The new building, which houses the Rudolf Virchow Center since 2009, offers
excellent conditions for research and cooperation. International visibility has been achieved through
scientific publications, through the organization of international symposia and conferences, membership
in scientific academies, organizations and boards, and many national and international awards.
The Rudolf Virchow Center intends to remain a young center, and this entails constant change. All
members of the first generation of group leaders have by now successfully moved into senior positions here
or elsewhere in Germany and abroad. We are very proud about the success of this first generation and look
forward to future collaborations with our alumni. The second generation of research groups has now
reached full productivity. 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. The research
focus continues to span the spectrum from structural biology to in vivo function. Two new group leaders
join these efforts: Shashi Bhushan, who comes from the Gene Center in Munich with a research program
using cryo electron microscopy to characterize protein complex structures, and Kathrin Heinze from the
IMP in Vienna, who develops optical microscopy techniques.
The Rudolf Virchow Center continues to play an active role in the research-oriented BSc/MSc-program
in Biomedicine as well as in the Graduate School of Life Sciences, both realized together with the
Faculties of Sciences and of Medicine. These programs consistently attract highly talented students from
Germany and abroad.
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
with a number of established and new programs, including a new fully integrated collaboration project
undertaken with four high schools, which is attended by high school students for two years.
I hope that you will enjoy reading our Annual Report.
Chairman Rudolf Virchow Center/
DFG Research Center for Experimental Biomedicine, University of Würzburg
Contents
The Rudolf Virchow Center
4
Overview
Research Program
Events
Output and Evaluation
4
7
12
14
Research Groups
Junior Research Groups
16
18
Shashi Bhushan
Heike Hermanns
Asparouh Iliev
Stephan Kissler
Alma Zernecke
18
20
22
24
26
Core Center
28
Caroline Kisker
Hermann Schindelin
28
30
Research and Senior Professorships, RVZ Network
32
Utz Fischer
Antje Gohla
Bernhard Nieswandt
32
34
36
Roland Benz
Martin Heisenberg
38
40
Martin Eilers
Manfred Gessler
Roland Jahns
Thomas Müller
42
44
46
48
Bio-Imaging Center
50
Gregory Harms
Manfred Heckmann
Martin Lohse
50
52
54
Early Independence Program
56
Ingrid Tessmer
56
Outlook
57
Katrin Heinze
57
Teaching & Training
Undergraduate & Graduate Program
BSc/ MSc Program Biomedicine
58
60
61
Graduate Training
62
Public Science Center
66
Kristina Kessler
66
Appendix
68
Executive Committees and Scientific Members
Academic Members and Supporting Staff
Teaching Committees and International
Graduate School Board
Bachelor and Master theses of the
Undergraduate Program in Biomedicine
PhD theses of the Virchow Graduate Program
Publications 2010
68
69
74
76
78
Imprint
87
73
3
Rudolf Virchow Center
An Overview
Research and Senior Professorships, RVZ Network
Background
Fig. 1:
Structure of the Rudolf Virchow Center.
Structurally, the Rudolf Virchow Center (RVZ) covers seven areas:
Junior Research Groups, five-year groups with a tenure-track option,
which are housed together to create a place of maximum freedom
and dynamics
Core Center, with long-term groups addressing mechanisms of protein
structure and function using advanced technologies (structural biology,
molecular microscopy, mass spectrometry and proteomics)
Research and Senior Professorships, offering established researchers
the support and freedom to concentrate on a 5-year high-risk project;
this area includes the RVZ Network, which offers 2-year support
for collaborative projects within the RVZ, or with other researchers
in Würzburg
Bio-Imaging Center, a more recent addition of research groups in
molecular biological imaging, funded by the State of Bavaria and
the University
Undergraduate Program in Biomedicine, a research oriented BSc/MSc
Program
Graduate Program in Biomedicine, that initiated the University-wide
creation of Graduate Schools and is now a part of the Graduate School
of Life Sciences
Public Science Center, promoting dialog with the public through press
and media work, several programs for children and high school students,
as well as public scientific debates
An international Scientific Advisory Board monitors scientific progress,
provides advice on the recruitment of Group Leaders, awards Research
Professorships, as well as monitors RVZ Network projects
4
The Rudolf Virchow Center is a research
center for experimental biomedicine funded by the German Research Foundation
(Deutsche Forschungsgemeinschaft - DFG).
The decision to establish the facility was
made by the DFG in 2001, and it was
founded in January 2002. The purpose of
the Center was research on target proteins
the crossroads between and medicine and
natural science. In 2009, the DFG reviewed the Center and acknowledged
its positive development into an entity,
that has not only become an inherent
part of the research community in Würzburg, but is also recognized by scientists
worldwide.
The malfunctioning of target proteins
which are important regulators of vital
cellular functions can lead to diseases.
Therefore, these proteins may serve as
targets for diagnostics or therapeutical
interventions. Among the multitude of
potential target proteins, the focus of the
Rudolf Virchow Center lies on receptors
and corresponding signal proteins and
on proteins, which bind nucleic acids.
These proteins, seem promising on account of their biomedical significance.
At the Center, target proteins are studied
at different levels of complexity, ranging
from their molecular structure to their
(mal-)functioning in animal models or
human diseases. The goal is to unravel
the way in which these proteins act and
thereby to provide the foundations for
combatting disease. The different research
groups deal with inflammatory diseases,
cancer, autoimmune diseases, and cardiovascular diseases.
The Center aims to recruit outstanding
scientists, who are offered a predetermined time-frame (5-year appointment,
with tenure track option) and a maximum
amount of freedom. Such groups are
established as junior groups (Nachwuchsgruppen) or as research professorships (Forschungsprofessuren). This enables junior as well as established
researchers to work on high-risk projects.
In recent years, the “RVZ-Network” was
added. This network supports risky
projects, which are carried out by local
research groups in collaboration with
groups from the Rudolf Virchow Center.
In 2010, senior professorships were
introduced in order to enable renowned
emeriti to continue their research efforts.
Besides trying to facilitate dialog between
the generations, the Center hopes to
benefit from the valuable knowledge of
the experienced researchers. Although the
content of the research programs of the
senior professors is based on the work of
the Rudolf Virchow Center, their projects
are financed mainly through external
funds.
The Core Center, which consists of groups
with a long-term perspective, develops and
uses new and expensive technology in the
fields of molecular microscopy, proteomics,
and structural biology. These groups follow
their own projects and provide the technological core for joint projects within the
Center as well as for related biomedical
research at the University of Würzburg.
The Bio-Imaging Center at the Rudolf
Virchow Center develops and applies the
latest technologies in the innovative field
of imaging and integrates them into
current biomedical research. The groups are
funded by the State of Bavaria and the
University of Würzburg.
These structures have created a dynamic
and inspiring environment for state-of-theart research within the Center as well as for
national and international cooperations.
The scientific success of the junior group
leaders has catapulted them into independent careers. The entire cohort of the “first
generation” successfully acquired leading
positions here, elsewhere in Germany, or
abroad. Hence, the research groups have
changed considerably even though the
structures have remained mainly the same
since the foundation of the center.
Bernhard Nieswandt, one of the first
junior group leaders who was promoted to
research professor in 2007, took over the
chair of vascular medicine in 2010. This
chair was newly created by the Rudolf
Virchow Center together with the Medical
Faculty.
Current junior group leaders have
also made considerable achievements.
In 2010, Stephan Kissler was the first
scientist working in Germany to be
honored with the Career Development
Award of the Juvenile Diabetes Research
Foundation International, one of the
most important foundations for diabetes
research. He can now look forward to
funds of $750,000 for his work over the
next five years.
Alma Zernecke´s work focuses on the role
of immune cells and how their mobility
is directed by chemokines. Zernecke, a
medical scientist, was awarded the Albert
Fraenkel Award in 2010 by the German Association for Cardiology, Heart and Cardiovascular Research (Deutsche Gesellschaft
für Kardiologie – Herz- und Kreislaufforschung e.V.) for her work on the role
of cytokines in artheriosclerosis.
New group leaders have come to the
Rudolf Virchow Center. Roland Benz and
Martin Heisenberg received the first senior
professorships. Roland Benz is interested
in the action of prokaryotic membrane toxins and the transport mechanisms of bacterial toxins into eukaryotic target cells.
What controls the behavioral patterns of
animals? What role does the brain play?
Fig. 2:
Academic members of the Rudolf Virchow Center at the annual Retreat 2010 in Monastery Schöntal.
5
Fig. 3:
In laboratory courses, the students get a genuine insight into the laboratory routine.
Martin Heisenberg, a world renowned neurogeneticist, started his research project at
the Center in an effort to answer such
questions. His work is being funded by the
DFG (German Research Foundation) with
1.2 million Euros for the next five years.
Prior to their current work, Benz and
Heisenberg were both conducting their
research at the Biocenter of the University
of Würzburg.
Shashi Bhushan began his work as a new
junior group leader. He uses cryo-electron
microscopy (cryo-EM) in order to investigate the structure of functional states of
ribosomes during various stages of translation. Previously, he worked as a postdoc
in the group of Roland Beckmann at
the Gene Center and Department of
Chemistry and Biochemistry at the Ludwig
Maximilians University in Munich.
In 2001 the Rudolf Virchow Center is
looking forward to welcoming Katrin
Heinze, from the Institute of Molecular
Pathology (IMP), Vienna into the BioImaging Center. Her group develops apparatuses and probes that enhance the
spatial and temporal resolution of multidimensional fluorescence imaging.
6
The next generation of scientists
One of the major goals of the Center is to
find, recruit, and support talented people
of all ages; ranging from high school, university, and PhD students to established
scientists.
A central element is the close connection
between research and training of undergraduates and PhD students. This sets the
Center apart from comparable facilities
that lack the connection to a university.
The purpose of these efforts is to skilled students who can work here with
state-of-the-art technology early in their
careers. Thus a main goal of the Rudolf
Virchow Center is the teaching and
training of scientists of the next generation. For that purpose, a research oriented
Undergraduate Program and a Graduate
School of Biomedicine were established.
The Graduate School of Biomedicine
was the prototype for the Graduate
School of Life Sciences and later
became part of it. The Graduate School
of Life Sciences was founded in 2006
with funds from the Excellence Initiative
(Exzellenzinitiative).
The concept of the Rudolf Virchow Center,
attracting the best people to science, starts
with the very young. Attracting children
and their parents to science is an integral
part of the overall idea of the Center.
Therefore, the “Public Science Center” created several new programs for children and
high school students: “Rudis Forschercamp”
for children aged 8 to 12, “Virchowlab” for
high school students aged 13 and older,
and the recently started project “Gemeinsam Forschen” for the higher classes.
Furthermore, the Public Science Center
is committed to promoting the dialog
between science and society.
Rudolf Virchow Center
In all its elements, the Rudolf Virchow
Center is guided by the successful principle
of giving 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 support
the very best scientists.
The research pursued at the Center deals
with target proteins – proteins that exert
key regulatory functions, and are therefore
good candidates for understanding the
etiology of diseases, and potentially also
for their diagnosis and treatment. Major
questions regarding these proteins address
their folding and mobility, the ways they
are modified, and how they are ultimately
degraded. Recognition between proteins
and small ligands is essential to understand
how the function of signaling proteins
Research Program
such as receptors can be regulated, and
how activation switches work. Similarly,
recognition between proteins and nucleic
acids is fundamental for signaling to the
nucleus and for using and maintaining genetic information. The research pursued at
the Center can therefore be grouped into
four Research Fields: (1) Protein Structure
and Function, (2) Proteins in Cellular Signaling, (3) Nucleic Acid Binding Proteins,
and (4) Proteins in Cell-Cell Interactions
and Motility. The main projects reflect the
focus on cell surface proteins and their
signaling proteins, and on nucleic acid
binding proteins. While each group studies
its own set of proteins, as indicated in
the figure, many projects are carried out in
collaborations involving two or more
groups providing different technologies or
complementary biomedical expertise.
Fig. 4:
Target proteins investigated at the Rudolf Virchow Center.
7
Research Field 1 - Protein Structure and
Function
Cellular proteins adopt defined structures
that are necessary for their specific functions. The fundamental mechanisms of protein folding, modification, and degradation
are intricately linked to their molecular
function, and also ultimately to their roles
in physiology and pathophysiology. In this
research field scientists utilize a variety of
techniques to study structural aspects of
biological macromolecules, which allow
visualization of these players at different
levels of resolution, their identification, as
well as characterization of different modifications. X-ray crystallography, high-resolution microscopy and mass spectrometry
are key methods in this research field.
These approaches are used to gain insights
into how newly synthesized proteins are
folded in the endoplasmic reticulum, how
they are targeted for degradation, and how
large protein complexes are assembled in
order to perform cellular signaling.
A common goal of this research field is
understanding specificity and affinity in
bio-molecular recognition processes.
Groups in Research Field 1:
Shashi Bhushan
Gregory Harms
Manfred Heckmann
Fig. 5:
Adaptor protein binding to the AAA ATPase p97.
Ribbon representations of three adaptor proteins
together with the molecular surface of the p97 Ndomain colored according to electrostatic potential.
(Haenzelmann et al., 2011)
Caroline Kisker
Thomas Müller
Hermann Schindelin
Ingrid Tessmer
Research Field 2 – Proteins in Cellular
Signaling
Signaling between and within cells is the
key to coordinated functions in all living
organisms. The proteins that are involved
in such signaling processes are of fundamental importance for life. Their dysregulation often causes diseases, and therapeutic
drugs often target these signaling proteins.
Receptors, which are most often localized
at the cell surface, are the most important
class of signaling proteins. They receive
signals from other cells (hormones and
transmitters) and then activate signaling
processes in the cell interior that ultimately cause cellular reactions.
Various signaling pathways are investigated at the Rudolf Virchow Center; for
example those that are triggered by
G-protein-coupled receptors and receptor
tyrosine kinases. These systems are investigated at various levels of complexity,
ranging from the molecular understanding of receptor/ligand binding interfaces
and the receptor activation process, to
studying complex physiological responses.
Key molecular questions addressed in
these model systems concern mechanisms
of recognition in signaling systems and
how intracellular signals are patterned in
space and in time. These molecular mechanisms are linked to (patho)physiology,
with a special focus on molecular mechanisms (Fig. 6) that underlie major cardiovascular diseases.
Fig. 6:
CCL17 is exclusively expressed by a myeloid-related
mature subset of dendritic cells (DCs). Using mice
with a targeted replacement of the Ccl17 gene by the
enhanced green fluorescent protein gene (Egfp, Ccl17E/
E), here we could show that EGFP+ DCs accumulate
within atherosclerotic lesions. The image shows maximum intensity projection of a z-stack with EGFP+ DCs
(bright green cytoplasmic staining, see arrows) in the
atherosclerotic aortic root of a Ccl17EGFP/+ apolipoprotein E-deficient mouse. Nuclei are counter-stained with
propidium iodide (yellow/red); collagen is visible due
to second harmonic generation (blue).
Groups in Research Field 2:
Gregory Harms
Manfred Heckmann
Martin Heisenberg
Heike Hermanns
Roland Jahns
Stephan Kissler
Martin Lohse
Thomas Müller
Bernhard Nieswandt
Alma Zernecke
8
Research Field 3 – Nucleic Acid Binding
Proteins
Interactions between proteins and nucleic
acids (DNA and RNA) are central to all aspects of the maintenance and realization of
genetic information. Again, recognition
processes represent a fundamental aspect
of these interactions. Several pathways
that rely on protein-nucleic acid interactions are studied at the Rudolf Virchow
Center through a combination of structural,
biophysical and biochemical techniques.
They encompass the DNA damage recognition pathway (Fig. 7), which involves sophisticated mechanisms to excise pieces
of damaged DNA and replace them with the
original sequence; the p53 tumor suppressor family, which initiates transcriptional
programs that ultimately arrest proliferation and prevent the generation of genetically altered cells; several transcription
factors, for example Hey, Nab and Myc,
which regulate growth and differentiation
in the cardiovascular system and in cancer; the spliceosome, which catalyzes the
removal of non-coding sequences from
pre-mRNAs; and finally the so-called TOP
response, which regulates the level of
translation in response to the nutritional
status of a cell via proteins that bind
to specific sequences in RNA (so-called
TOP motifs).
Analysis of these pathways will provide
an understanding of how the intricate
interactions of individual proteins or
of multi-protein complexes with nucleic
acids lead to the formation of higher
order complexes required to maintain
the genomic integrity and to carry out
genomic programs in the cell.
Fig. 7:
FIONA-AFM image of quantum dot conjugated
DNA repair proteins (red fluorescence probability
signals) bound to UV-damaged DNA fragments.
This super-resolution approach allows us to
overlay fluorescence and atomic force microscopy
data with high (< 10 nm) accuracy, to pinpoint
specific, labeled protein molecules in heterogeneous protein-DNA samples. (Fronzcek et al.,
2010).
Groups in Research Field 3:
Martin Eilers
Utz Fischer
Manfred Gessler
Caroline Kisker
Ingrid Tessmer
Research Field 4 – Proteins in Cell-Cell
Interaction and Motility
Interactions of cell surface proteins with
the extracellular space and regulation of
the cytoskeleton determine cellular adhesion and motility. Cell adhesion and migration are central to diverse homeostatic
processes – for example the mounting of an
effective immune response or the repair of
injured tissues. Failure of cells to migrate,
or migration of cells to aberrant locations,
is intricately involved in many pathologies, including vascular and inflammatory
diseases as well as tumor formation and
metastasis (Fig. 8). Several target proteins
were identified at the Rudolf Virchow
Center that regulate the interactions of a
cell with its micro-environment. The Center
has also developed methods to visualize,
analyze, and manipulate these dynamic
processes from single molecules to protein
complexes, and from cultured cells to living animals. To assess the potential importance of candidate proteins that may
provide keys to the prevention or treatment
of major diseases, in vivo mouse models
of cardio- and cerebrovascular diseases,
(auto)immune disorders, and malignant
tumors have been established. Together,
this multidisciplinary approach has not
only provided fundamental insights into
the biology of cell-cell interactions, but
has also identified target proteins that
hold promise for the development of new
therapeutic strategies.
Groups in Research Field 4:
Roland Benz
Fig. 8:
Crystal structure of the heptameric alphahemolysin of Staphylococcus aureus. The image
depicts the side view of the heptamer with the
membrane spanning beta-barrel cylinder
composed of 14 amphipatic beta-strands (lower
part of the oligomer) and the mushroom-like
water-soluble part of the complex (upper part
of the oligomer). The Figure was designed using
the program Rasmol (http://rasmol.org/) and
the PDB data bank 7AHL.
Antje Gohla
Asparouh Iliev
Bernhard Nieswandt
Alma Zernecke
9
Biomedical focus
Like any protein, target proteins are often
expressed in many different cells and tissues, and may therefore be involved in
several, very different (patho)physiological
functions. While their basic structural and
functional mechanisms may be the same in
all these situations, the study of a given
protein can provide insights into a whole
variety of physiological processes and diseases, and can lead into very different
biomedical fields. This is where the interdisciplinary nature of the Rudolf Virchow
Center comes into play, as well as the fact
that it is embedded in a large network of
biomedical research.
The many implications that research
on a given target protein may have can
best be illustrated with an example. A protein currently studied by several Rudolf
Virchow Center groups is called “stromal
interaction molecule 1” (STIM1). STIM1 appears to represent the long postulated
“missing link” that connects depletion of
intracellular Ca2+ stores to the opening of
a specific group of calcium channels at
the cell surface, in order to “refill” the
cell with calcium.
Some years ago, the group of Albert
Sickmann observed by mass spectrometry
that STIM1 and closely related STIM2
are abundantly expressed in platelets.
This prompted the group of Bernhard
Nieswandt to investigate their in vivo
functions, and this then catalyzed studies
on other functions of these proteins by
several groups at the Rudolf Virchow Center. Mouse lines deficient in STIM1 or STIM2
were generated and analyzed for defects in
different cell systems. STIM1-deficient
platelets showed major functional defects
in vivo, revealing that this pathway of
Ca2+ entry is of paramount importance for
thrombus stabilization. Subsequent collaborations with the Department of Neurology in Würzburg showed that the lack of
STIM1-dependent Ca2+ entry protected mice
from developing experimental ischemic
stroke. Shortly after, mice lacking STIM2
were also found to be strongly protected
from neurological damage, although platelet function and thrombus formation is not
altered in these animals. Closer analysis
revealed that this protection is based on
increased resistance of STIM2-deficient
neurons to ischemic damage, a process
known to involve Ca2+ influx through the
plasma membrane. Further studies showed
for the first time that store-operated
Ca2+ entry is indeed involved in this process
and that this is mediated by STIM2, but
not STIM1.
Investigations on the role of STIM1 and
STIM2 in immune cell function were undertaken in collaboration with the Institute of
Immunology and the Neurological Clinic in
Würzburg, as well as partners outside Würzburg. These studies showed that STIMdependent Ca2+ regulation is an essential
determinant of immune cell activation in
the settings of Ig-dependent inflammation,
autoimmune CNS inflammation, and IgEdependent anaphylaxis.
The important role of STIMs in the regulation of Ca2+ entry in platelets, T-cells, and
neurons has prompted collaborations of
several groups at the Rudolf Virchow Center
to study their function in various organs
and biological systems. The aim is to investigate their biochemical and cellular regulation and to search for cellular proteins
that interact with STIMs.
Fig. 9:
STIM2 regulates store-operated Ca2+ entry in neurons and plays a key role in hypoxic neuronal cell death.
(A) Immunofluorescence staining of STIM2 (red) in cultured hippocampal neurons (MAP2a/b, green) of wild-type and Stim2-/- mice. Cell nuclei are counterstained with DAPI (blue). Scale bars, 10 µm. (B) Effect of combined oxygen-glucose deprivation (OGD) on intracellular Ca2+ levels. Cultured neurons (DIV1214) were exposed for 1 h (WT) or 2 h (Stim2-/-) to a glucose-free solution continuously bubbled with N2. Representative curves and mean values ± SD; *
P<0.05. (C) Wild-type and Stim2-/- mice were subjected to transient middle cerebral artery occlusion (tMCAO) and analyzed after 24 h. Representative TTC
stains of corresponding coronal brain sections. Infarcted areas are white, while healthy tissue is red. (Berna-Erro et al., Sci. Signal., 2009).
10
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 most commonly only
nance energy transfer (FRET) and fluorescence
lifetime
imaging
(FLIM)
measurements, stimulated emission depletion (STED) microscopy, and single molecule fluorescence microscopy permit the
analysis of protein-protein interactions and
Fig. 10:
Technologies used for visualizing and imaging target proteins at the Rudolf Virchow Center.
achieved in Germany by non-university institutions. In order to study target proteins
at various levels of complexity, this meant
establishing a whole range of technologies
and methods. The special framework of the
Center, with its wide spectrum of cutting
edge analytical technologies under one
roof, strong collaborations between the
research groups, and a close connection to
clinical research, offers excellent opportunities to analyze these proteins at different
levels of complexity, ranging from their
molecular structure and function to biochemical mechanisms, cellular responses,
and (patho)physiological roles. A particular focus is the visualization of biological
macromolecules at different levels of resolution – ranging from atomic structures to
imaging in the whole body. The technologies that have been established and are
now used are depicted in Figure 10.
They range from methods for investigating
individual proteins and the complexes they
form – analyzed by X-ray crystallography
and atomic force microscopy – to methods
that assess complex biomedical functions,
such as cardiac catheterization and ultrasound, in vivo platelet function microscopy,
and also generating the respective genetically modified mouse models. Optical
methods have gained a particularly prominent status, with the establishment of
several technologies that elucidate information at the nm scale: fluorescence reso-
conformational changes, as well as the
direct optical resolution of larger proteins
and protein complexes in living cells.
Other techniques include various mass
spectrometry technologies that are essential for identifying proteins and analyzing
their modifications; transgenic mouse technologies by classical as well as lentiviral
transgenic, knock-out and RNAi-technologies; and a DNA array unit was established in collaboration with the Interdisciplinary Center for Clinical Research
(IZKF), providing access to custom-made
as well as commercial array analyses.
All of these technologies are not only
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. Groups receive specific funding
from companies, mostly in the context
of projects funded by the Federal Ministry
of Research and Education (BMBF) and
similar sources, but also through direct
collaborations.
Several new microscopic instruments were
co-developed in formal collaborations with
optical companies. For example, a multi-
photon platform for optical imaging in vivo
was constructed with LaVision BioTec
(Bielefeld) by the group of Gregory Harms
and by the former microscopy group of
Peter Friedl. New rapid FLIM detectors for
this microscope have been developed as
an RVZ Network project. A new type of microscopy platform, called iMIC, has been
engineered with Till Photonics (Gräfelfing),
in a BMBF-funded collaboration. Formal
collaborations with Leica concerned the
development of a TIRF-microscope that is
suitable for multi-color detection and
FRET measurements. A second collaboration with Leica, an extension of a collaboration between Stephan Sigrist and Stefan
Hell at the Max Planck Institute for Biophysical Chemistry in Göttingen, was the
development of a commercial high-resolution STED microscope, which is now used
by Manfred Heckmann as well as several
other groups. This type of fluorescence
microscopy allows resolution below the
diffraction limit and is used at the Rudolf
Virchow Center most extensively in imaging
of receptors and synaptic proteins.
Some of our research projects have led to
patents, which are usually held by the University; in some instances, patents were
applied for together with biotech, pharmaceutical or technology companies. The high
number of patents was especially mentioned at the last site visit of the German
Research Foundation.
A transfer project that received major
funding for technology transfer is carried
out in collaboration with the Institute of
Pharmacology and Toxicology and the Department of Medicine. This project, led by
Roland Jahns, intends to develop new
therapies against receptor autoantibodies
in heart failure. It successfully obtained
funding as one of twelve projects in the
“Exist GoBio“ competition of the BMBF,
and led to the foundation of a new spin-off
company called CorImmun.
11
Rudolf Virchow Center
Events
Rudolf Virchow Lecture
As a part of the “Virchow Lecture” series
Meinrad Busslinger was honored in 2010
with the Virchow Medal, the highest award
of the Medical Faculty of Würzburg University. During the ceremony, Meinrad
Busslinger presented his work in a talk
about “Lineage Commitment and Plasticity
in the Hematopoietic System”.
Rudolf Virchow was head of the Department of Pathology at Würzburg University
from 1849 to 1856. These seven years are
regarded as the most fruitful period of
his scientific career. He also established
himself as an excellent lecturer and managed to captivate his audience with an extraordinary body of knowledge about his
field and latest scientific discoveries. Accordingly, his talks and courses were very
popular. The Rudolf Virchow Lecture Series
is devoted to this outstanding researcher.
In his honor, scientists who have produced
results of extraordinary importance are
invited to give a lecture and are awarded
the Virchow Medal.
Fig. 11:
Representatives of the Rudolf Virchow Center (Caroline Kisker), the Medical Faculty (Thomas Hünig)
and the Pathology Department/Faculty (Edgar Serfling) (from left to right) awarded Meinrad Busslinger
(second to the right) the Virchow Medal. During the ceremony he gave a lecture about “Lineage
Commitment and Plasticity in the Hematopoietic System” at the historical lecture hall of the Rudolf
Virchow Center.
5th Symposium for Students of
Biomedicine
“Medical Needs - Biomedical Solutions:
Promise and Challenge of Modeling Human
Disease” was the motto of a symposium,
which was organized by biomedical students in Würzburg in October 2010. They
were able to invite and welcome students
from all over Germany at the Rudolf Virchow Center. The goal of the meeting was
to give the students a perspective of the
wide range of topics and opportunities
that are offered in research after graduation. Many of Würzburg’s best and most
ambitious scientists participated in the
event. They used captivating presentations in order to point out the promises
and challenges of their field and to spark
interest in their research programs.
Fig. 12:
During the 5th Symposium for Students of Biomedicine a diversified program consisting of workshops, keynote talks and networking events was organized by
biomedical students from Würzburg for students from all over Germany.
12
Exhibition “MenschMikrobe“
The exhibition “MenschMikrobe” was displayed at the Rudolf Virchow Center for approximately five weeks. Numerous visitors
took the opportunity to gain insights into
current knowledge about bacteria, viruses,
and parasites; as well as the social and historical dimensions of diseases.
The Würzburg “MenschMikrobe” team was
able to welcome over 5,300 visitors in
these five weeks. The exhibition was especially popular among local schools. About
150 classes and groups joined free tours
through the exhibition as a vivid and
fresh addition to their biology class. In
addition to high schools, numerous technical schools also found their way to the
exhibition.
The exhibition “MenschMikrobe – The
legacy of Robert Koch and the modern
research of infections” is a joint project of
the German Research Foundation (Deutsche
Forschungsgemeinschaft, DFG) and the
Robert Koch Institute (RKI). The exhibition
was based around on the discoveries of
Robert Koch, who died 100 years ago.
Fig. 13:
The foyer of the Rudolf Virchow Center was the perfect place for
the exhibition “MenschMikrobe” topically as well as spatially.
200 invited guests from the DFG, the Robert Koch Institute (RKI),
the Rudolf Virchow Center and local politicians were the first visitors
during the opening ceremony.
13
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 funding by the State of Bavaria
and the University of Würzburg, the main
source of support is the core funding by
the DFG. In 2010 the Center received a
direct grant of the DFG of about 5.9
million Euro (plus overhead costs about
1.2 million Euro). This funding is complemented by grants from various sources
(Fig. 14) totaling more than 5.9 million
Euro in 2010.
DFG
BMBF
EU
Foundations
other
IZKF
BayStMWIVT
Industry
Publications
Fig. 14:
Sources of extramural funding at the Rudolf Virchow Center with a total amount of 5.9 million
Euro in 2010. (DFG: German Research Foundation; BMBF: Federal Ministry of Education and
Research; EU: European Union; IZKF: Interdisciplinary Center for Clinical Research; BayStMWIVT:
Bavarian Ministry of Economic Affairs and Technology.
Publications are the main reflection of an
academic research center. The total number
of peer-reviewed publications by groups
and members of the Rudolf Virchow Center
since the establishment was 1579, with
631 originating directly from funding by
the Center. The scientific quality of these
publications is evidenced by the fact that
15% of our publications appeared in the
top 1% journals, and almost 81% appeared
in the top 10% journals. The normalized
relative field impact, a size-independent
measure of scientific impact developed by
the Center for Science and Technology
Studies, Leiden, was 2.99, which is considered excellent. This indication is supported
by looking for highly cited papers via percentile analysis. 6% of the publications
produced by Virchow researchers were
ranked in the 99th percentile in their subject categories. Approximately 38% of their
publications were ranked in the 90th percentile in those same categories. Members
of the Rudolf Virchow Center have been
cited more than 69.451 times for over 2523
papers published since 2002. Finally,
benchmarking shows citation rates similar
to renowned institutes of comparable orientation and size in Germany and in the
US. (Fig. 15)
Fig. 15:
Benchmarking of citations with institutes of comparable orientation in Germany and in the US
shows similar results. (RVZ: Rudolf Virchow Center; MPI Heidelberg: Max Planck Institute for
Medical Research, Heidelberg; MPI Göttingen: Max Planck Institute of Experimental Medicine,
Göttingen, Beckman Ctr: Beckman Center, Stanford University, USA).
14
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 fulfills 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. 16).
To foster collaborations with other groups
in Würzburg, we initiated the RVZ Network
in 2006. Following review by the Scientific
Advisory Board, high risk projects are funded and 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
flexible allocation of resources.
Fig. 16:
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;
continued BMBF projects: yellow).
Science Careers
Many of our members, including all junior
group leaders, have received offers for professorial positions in Germany and abroad,
and the first generation of group leaders
has now successfully moved into senior
positions. In 2010, responding to a competing offer, Bernhard Nieswandt, one of
the first three junior group leaders, was
offered and accepted the newly created
Chair in Vascular Medicine.
Several group leaders of the Rudolf
Virchow Center accepted attractive offers
from leading institutions in Germany
and abroad and left the Center in 2008.
Meanwhile, the second generation of group
leaders has grown together into a closely
collaborating unity.
It is too early for a formal evaluation of
the alumni of our teaching programs, but
we do stay in contact with them and
follow where they go and how they fare.
It is a good sign that most undergraduate
as well as graduate students have stayed
in science and are continuing their research
in institutions all over the world.
15
Research Groups
Junior Research Groups
Shashi Bhushan
Heike Hermanns
Asparouh Iliev
Stephan Kissler
Alma Zernecke
Core Center
Caroline Kisker
Herrmann Schindelin
Research and Senior Professorships, RVZ Network
Utz Fischer
Antje Gohla
Bernhard Nieswandt
Roland Benz
Martin Heisenberg
Bio-Imaging Center
Gregory Harms
Manfred Heckmann
Martin Lohse
16
Martin Eilers
Manfred Gessler
Roland Jahns
Thomas Müller
Page 18 - 27
Page 28 - 31
Page 32 - 49
Page 50 - 55
17
Shashi Bhushan
Email: [email protected]
Phone: +49(0)931 31 83230
Fax:
+49(0)931 31 83255
http://www.rudolf-virchow-zentrum.de/forschung/bhushan.html
Ribosomes are large macromolecular particles that synthesize proteins from the substituent amino acids. In
simple terms they are the protein-producing factories of a cell. They are composed of both proteins and RNA.
Ribosomes are very important; first they make all the proteins required in a cell or organism. Secondly, they
are also targets for several antibiotics. Electron microscopy (cryo-EM) in combination with single particle
reconstruction is our main method to study different functional states of the translating ribosomes. Besides
ribosomes, we are also very interested in determining sub-nanometer resolved structures of other macromolecular complexes such as the protein translocation machinery, DNA repair complex, etc..
Cryo-EM and single particle reconstruction
Cryo-EM is a very useful technique to
determine the sub-nanometer resolved
structure of big macro-molecular complexes such as the viruses, ribosomes,
etc. The potential of Cryo-EM is further
evident by the recent progress where
atomic resolution was also achieved using
Cryo-EM for symmetrical particles such
as viruses.
We have set up a Spirit 120 Kv FEI electron
microscope for collecting the Cryo-EM data.
This microscope has enabled us to screen
the potential complexes for their suitability to be analyzed further for higher resolution. For higher resolution we have
access to a Polara 300 Kv FEI microscope at
the MPI for Molecular Genetics in Berlin.
For data processing with single particle
reconstruction, we have installed a dedicated Linux cluster consisting of more
then 200 CPUs.
Fig. 1:
General principle of Cryo-EM and single particle reconstruction.
18
We are currently focusing on two different
projects:
1. Investigating the molecular mechanism of antibiotic-induced stalling of
bacterial ribosomes
Although the function of the ribosomes is
similar in bacteria and higher organisms
including humans, there are many differences in their composition and structure.
These differences allow some antibiotics
to target only the bacteria by inhibiting
Fig. 2:
A low-resolution preliminary structure of an
Erythromycin-stalled ribosome indicating the
presence of P-tRNA.
their ribosomes, while leaving human ribosomes unaffected. During the last decade
much progress has been made in understanding the molecular mechanisms of the
action of antibiotics on bacterial ribosomes. We now know the binding sites
in ribosomes of most of the antibiotics.
Despite understanding a lot about the binding sites of antibiotics, the mechanisms by
which these antibiotics exerts their inhibitory action on the ribosomes remain poorly
understood. This is also reflected by the
facts that there is no structure of translating ribosomes inhibited by an antibiotic.
We are trying to understand the molecular
mechanisms of such antibiotics by determining the structures of translating bacterial ribosomes inhibited by them. We
have collected a large data set of erythromycin-stalled ribosomes to determine
the sub-nanometer resolved structure of
antibiotic stalled ribosomes. Preliminary
reconstruction shows the presence of Peptidyl-tRNA indicating the stalled complex
(Figure 2). We are currently in the process
of reconstructing sub-nanometer resolved
structures of this complex, which should
enable us to determine the molecular
mechanism of the inhibition.
2. Structural investigations of a pathogenic bacteria, Borrelia porin P66
Borrelia are pathogenic bacteria that use
an arthropod vector as a carrier and cycles
between the vector and a mammalian host.
Borrelia cause Lyme disease, which is
known to affect different organs such as
skin, joints and the nervous system. The
main way of catching the disease is a bite
by a Borrelia carrying tick. Bacterial porins
are involved in transport of nutrients and
other molecules between host and bacteria. Roland Benz´s group has been
investigating the pore forming properties
of p66. We have been able to generate a
preliminary 3D model of the p66 complex
provided by Prof R. Benz´s group from
negative stained EM images. The structure revealed a trimer with three visible
channels. We are currently trying to improve the resolution in order to determine
the exact size of the channels.
Selected Publications
Armache, J. P., Jarasch, A., Anger,
A. M., Villa, E., Becker, T., Bhushan, S.,
Jossinet, F., Habeck, M., Dindar, G.,
Franckenberg, S., Marquez, V., Mielke,
T., Thomm, M., Berninghausen, O.,
Beatrix, B., Söding, J., Westhof, E.,
Wilson, D. N., and Beckmann, R. (2010).
Localization of eukaryote-specific ribosomal proteins: in a 5.5-Å cryo-EM map
of the 80S eucaryotic ribosome. PNAS,
107, 19754-9.
Armache, J. P., Jarasch, A., Anger, A.
M., Villa, E., Becker, T., Bhushan, S.,
Jossinet, F., Habeck, M., Dindar, G.,
Franckenberg, S., Marquez, V., Mielke,
T., Thomm, M., Berninghausen, O.,
Beatrix, B., Söding, J., Westhof, E.,
Wilson, D. N., and Beckmann, R. (2010).
Cryo-EM structure and rRNA model of a
translating eukaryotic 80S ribosome at
5.5 Å resolution. PNAS, 107, 1974853.
Bhushan, S., Gartmann, M., Halic, M.,
Armache, J. P., Jarasch, A., Mielke, T.,
Berninghausen, O., Wilson, D. N., and
Beckmann, R. (2010). Alpha-helical
nascent polypeptide chains visualized
within distinct regions of the ribosomal
exit tunnel. Nat Struct Mol Biol, 17,
313-7.
Bhushan, S., Hoffman, T., Seidelt, B.,
Frauenfeld, J., Mielke, T., Berninghausen, O., Wilson, D. N., and Beckmann, R.
(2011). SecM-stalled ribosomes adopt
an altered geometry at the peptidyl
transferase center. PLOS Biology, 9,
e10000581.
Fig. 3:
Reconstruction of Borrelia porin P66 from
negative stained EM images.
Bhushan, S., Mayer, H., Mielke, T., Berninghausen, O., Sattler, M., Wilson, D. N.,
and Beckmann, R. (2010). Structural
basis for translational stalling by human
cytomegalovirus and fungal arginine attenuator peptide. Mol Cell, 40, 138-46.
19
Heike Hermanns
Email: [email protected]
Phone: +49(0)931 31 80362
Fax:
+49(0)931 31 83255
http://www.rudolf-virchow-zentrum.de/forschung/hermanns.html
The inflammatory response is a highly coordinated answer of our body to trauma, tissue injury or infection. It
involves a complex interplay of different cell types releasing cytokines that act in an auto- or paracrine manner
to induce the so-called acute phase response. This reaction starts with the release of pro-inflammatory cytokines
such as interleukin (IL)-1β, tumour necrosis factor (TNF)α or oncostatin M (OSM). Later, the major mediator of
hepatic acute phase protein expression, IL-6, is expressed and exerts pro- as well as anti-inflammatory activities.
It is mandatory that the function of these cytokines is stringently regulated, since dysregulated cytokine signaling, involving the JAK/STAT-, NFκB-, MAPK- as well as PI3K/Akt-pathway, leads to severe inflammatory
diseases such as rheumatoid arthritis, inflammatory bowel disease or multiple sclerosis, as well as cardiovascular disorders and cancer. Our laboratory studies the molecular mechanisms controlling cytokine signaling at
different levels of complexity.
Pro-inflammatory cytokines restrict IL-6
signaling through receptor internalization and degradation
Interleukin-6-mediated signal transduction
is controlled by the IL-6-induced feedback
inhibitor SOCS3 and the protein tyrosine
phosphatase SHP2. Apart from this, several lines of evidence indicate a negative
cross-talk with pro-inflammatory cytokines.
TNFα induces SOCS3 in macrophages and
additionally leads to binding of the protein tyrosine phosphatase SHP2 to gp130.
IL-1β counteracts signal transduction of
IL-6-type cytokines at different levels: It
affects IL-6-induced gene expression
through acting on target gene promoters or
by enhancing the IL-6-induced expression
of the SOCS3 feedback inhibitor.
Besides these rather late acting mechanisms, it has been recognized for some
time that pro-inflammatory cytokines also
affect the initial STAT3 activation by IL-6;
however, the underlying molecular mechanism remained unknown. We identified a
novel negative cross-talk mechanism for
cytokine signaling, where cytokine receptor turnover is rapidly accelerated by proinflammatory cytokines and stress stimuli
to coordinate the inflammatory response.
Using a classical internalization assay we
could show that the IL-6 signaling receptor
subunit gp130 is rapidly internalized in
response to IL1 (Fig. 1A), TNFα or stress
signals in a variety of cell lines and primary
20
Fig. 1:
(A) IL-1β treatment of hepatoma cells
accelerates gp130 endocytosis. (B) Upon IL-1β
treatment gp130 colocalizes with the early
endosome marker EEA-1. (C) Inhibition of p38
MAPK activity restores full STAT3 tyrosine
phosphorylation in response to IL-6/sIL-6R
despite presence of IL-1β.
cells. We identified the internalized
gp130 in early endosomes by co-localization with the early endosomal marker EEA1
(Fig. 1B). Live cell imaging in confocal
laser scanning microscopy delineated the
fate of internalized gp130. Although a few
anti-gp130 positive vesicles co-localize
with transferrin-positive vesicles a much
larger number co-localizes at later time
points with a lysosomal marker. From
these data we excluded that IL-1 merely
increases overall endocytosis but rather
specifically leads to gp130 translocation
into the lysosome.
We characterized the signaling cascade
required for the accelerated internalization
of gp130 and identified a serine residue in
the cytoplasmic region of gp130 which
needs to be phosphorylated to start the
endocytosis process. This serine residue is
phosphorylated by the p38 MAPK target kinase MK2. As hypothesized, this enhanced
internalization of gp130 is crucially involved in the early inhibitory activity of
pro-inflammatory cytokines on IL-6-mediated signal transduction, since inhibition
of endocytosis restores full STAT activation
(Fig. 1C) and induction of acute phase
protein expression by IL-6 despite the
presence of IL-1β.
We believe that internalization and degradation of gp130 plays an important role
in limiting inflammation and we are currently in the process of verifying this hypothesis by generation of transgenic mice.
Crosstalk- and ligand-mediated internalization of the interleukin-6-type cytokine common receptor gp130 differ in
their molecular mechanism
Gp130 is not only subject to endocytosis
mediated via cross-regulation, it is also internalized in a ligand-mediated fashion.
Comparing the various interleukin-6-type
cytokines we could clearly show that the
strength of gp130 internalization differed
significantly. IL-6 and LIF hardly induce
gp130 internalization; OSM on the other
hand was a strong inducer of gp130 endocytosis (Fig 2A).
Fig. 2:
(A) OSM induces a much stronger internalization
of gp130 than IL-6 or LIF. (B) IL-1β-, but not
OSM-induced internalization of gp130 is
inhibited by abrogation of p38 MAPK activity,
Intriguingly, while blocking p38 MAPK
activation completely blocks gp130 endocytosis by pro-inflammatory cytokine crosstalk it has no effect on OSM-mediated
internalization (Fig. 2B). This led us to hypothesize that the molecular mechanisms of
internalization, and potentially the fate of
internalized gp130 as well as the consequences for signaling differ substantially in
response to cytokine crosstalk and natural
ligands. We are currently characterizing the
molecular mechanisms and consequences
underlying each internalization process.
Identification of novel signaling components in Oncostatin M-mediated signal transduction
Our laboratory has a long-standing interest
in the physiological relevance and signal
transduction of the interleukin-6-type cytokine Oncostatin M (OSM). Its name originates from the growth inhibitory effect
OSM exerts on many solid tumors. Meanwhile, however, it is well-accepted that
OSM represents an important regulator of
the inflammatory response. As a pro-inflammatory cytokine it is released by neutrophils, monocytes and activated T cells in
the early phase of inflammation. Like its
close relatives IL-6 or leukemia inhibitory
factor (LIF), OSM can regulate the expression of acute phase proteins. These proteins are essential for innate immunity
since they regulate coagulation and activate the complement system. Latest studies in OSM-deficient mice indicate a function for this cytokine in suppression of
autoimmune diseases.
Al though OSM and IL-6 share the common signaling receptor subunit gp130,
their physiological effects differ substantially. Our laboratory elucidates differences
in the signaling pathways responsible for
specific gene expression. To identify novel
proteins involved in OSM signal transduction we performed immunoprecipitations of
the adapter protein Shc, which we identified as a specific signaling component in
OSM-mediated signal transduction comparable to IL-6 or LIF. In collaboration with
Prof. Dr. Albert Sickmann (ISAS, Dortmund)
co-immunoprecipitated proteins were identified by mass spectrometry. One of the
proteins identified was the inositol-5’phosphatase SHIP2 (Fig. 3). This protein
is involved in the tight control of levels of
phosphoinositides, particularly PI(3,4,5)P3
and PI(4,5)P2, in the plasma membrane by
dephosphorylating the 5’ position on the
inositol ring. Recent studies indicate that
PI(3,4,5)P3 and PI(4,5)P2 are important for
coat assembly in clathrin-dependent endocytosis. Therefore, SHIP2 could play an
important role in negative regulation of
internalization processes. Investigations
on its exact role in OSM-mediated signal
transduction are under way.
Fig. 3:
(A/B) Upon OSM stimulation Shc and SHIP2
coimmunoprecipitate in HepG2 hepatoma cells.
Extramural Funding
SFB 487, TP B9 (from 01/2009)
Selected Publications
Radtke, S., Wüller, S., Yang, X. P., Lippok, B. E., Mütze, B., Mais, C., SchmitzVan de Leur, H., Bode, J. G., Gaestel, M.,
Heinrich, P. C., Behrmann, I., Schaper,
F.* and Hermanns, H. M.* (2010). Crossregulation of cytokine signalling: proinflammatory cytokines restrict IL-6
signalling through receptor internalisation and degradation. J Cell Sci, 123,
947-59.
(*equal contribution).
21
Asparouh Iliev
E-mail: [email protected]
Phone: +49(0)931 201 48997
Fax:
+49(0)931 201 48539
http://www.rudolf-virchow-zentrum.de/forschung/iliev.html
Funded by the Emmy Noether Program of the DFG. Since February 2008, Asporouh Iliev
is associated to the Rudolf Virchow Center as a Junior Research Group leader.
Evolutionary, pathogenic bacteria developed various adaptive mechanisms to improve their ability to invade
hosts. Bacterial toxins represent one of these adaptation approaches. While some toxins destroy cells (poreforming toxins), other modulate cellular functions. One of the evolutionary-favored approaches is the modulation of the small GTPase activity and other actin cytoskeletal components by bacterial toxins, leading to massive
cytoskeleton remodeling. Pneumolysin, a major virulence factor of S. pneumoniae, leads to cell lysis at high
concentrations and non-lytic changes at low concentrations. Pneumolysin produces strong actin remodeling,
small GTPase activation and microtubule stabilization, although the exact molecular steps in this process, as well
as its pathophysiological meaning remain unclear. Last year we succeeded in clarifying some major aspects of
these questions, involving the importance of pore formation for these changes to occur, although ion influx as
a factor was completely excluded. Our recent experimental evidence suggests a direct interaction between
pneumolysin and the submembranous actin fragments. All these changes were apparent in brain tissue in the
form of brain swelling and synapto-dendritic damage. Expanding the knowledge of pneumolysin to other
members of the cholesterol-dependent cytolysin group will be of critical importance for characterizing this group
of toxins.
Bacteria have developed specific mechanisms to facilitate their interaction with
host organisms during the course of their
co-evolution. One of these is the poreforming capacity of cholesterol-binding
cytolysins. Other toxins modulate intracellular signaling cascades allowing bacteria
to overcome the pathogen defense of the
host. Among these, the modulation of the
Rho GTPases is of particular interest. Rho
GTPases belong to the large superfamily of
Ras-proteins, monomeric GTP-binding proteins with a molecular mass between 20
and 30 kDa. Rho, Rac, and Cdc42 are
the most intensively studied Rho GTPase
members. They mediate cell adhesion, motility, endo-/exocytosis, and phagocytosis
through the regulation of actin cytoskeleton dynamics. Several other bacterial and
fungal toxins (e.g. phalloidin) target and
modify actin directly. All these show that
actin is a very suitable target for improving
the virulence of the pathogens in the
course of evolution. A surprising finding of
our previous work was the discovery of
small GTPase (specifically Rac1 and RhoA)
activation by sub-lytic amounts of the cholesterol-dependent cytolysin pneumolysin,
known as a classical pore-forming toxin.
Surprisingly, despite the early astrocyte
shape actin-dependent changes, the modu-
22
lation of small GTPase activity succeeded
the actin changes, but did not precede
them. Thus, we had evidence that the toxin
could cause primary cytoskeletal changes,
which subsequently followed by small
GTPases modulation.
Over the last year, we have succeeded in
pinpointing the actin changes within the
first minute after challenge with pneumolysin as the critical initiator of cellular
Fig. 1:
Cell displacement and retraction following
exposure to pneumolysin. Primary astrocytes
demonstrated retraction and displacement only
after exposure to 0.1 µg/ml WT-PLY, but not
when challenged with an equivalent amount of
the mutant variants (WT – WT-PLY, d6 – delta6PLY, W433F – W433F-PLY).
shape changes. High-resolution live cell
imaging allowed us to demonstrate an
early toxin interference with the membrane/actin structures, leading to temporary detachment of the membrane and
the subcellular actin structures from each
other. In this context, the activation of
the small GTPases was confirmed to play
only a secondary role. We are further defining the exact molecular parameters of these
changes, since the perspective is to finish
the study within the second funding period
(2011) of the Emmy Noether Funding programme. As an important step, we use an
artificial giant unilamellar vesicle (GUV)
model system, which allows us to study the
toxin/membrane itneractions in a smaller,
very well defined biochemical system.
Although the effects of pneumolysin
on primary astrocytic cytoskeleton were
achieved at sublytic concentrations, the
pore forming capacity of the toxin
remained critically important for actin remodeling to occur. Application of non-pore
forming mutants (delta6 and W433F) failed
to remodel actin cytoskeleton even at high
concentrations (Fig. 1). Despite the need
for pore forming capacity, the remodeling
remained calcium and sodium-independent,
as well as independent of the membrane
depolarization phenomena (Fig. 2). All this
implies that the toxin pore configuration,
even if not lytic, might be an important
component in the reorganization of the cytoskeleton (Förtsch et al., Toxins, 2011).
We have found that the pore formation
by pneumolysin is a phenomenon occurring
with exponential kinetics and reaching a
saturation level within 30-40 min after exposure, meaning that the rest of the cells,
which we normally analyse, remain intact
within a longer time-frame, thus none of
them dies (Fig. 3). Such a definition is essential, since many seemingly important
phenomena (such as cytoskeleton reorganization) might occur in the course of cell
death (thus being not relevant in tissues,
where pneumolysin’s lytic toxicity is weaker). This is not the case with the non-permeabilized population of glial cells in our
experiments.
As a major pathogenic factor of Streptococcus pneumoniae, pneumolysin con-
tributes substantially to the neurological
symptoms in the course of pneumococcal
meningitis. The serious patient disability
contrasts with the relatively limited neuronal cell damage. Our experiments confirm
the presence of dendritic spine reduction
and dendrite varicosity formation. Currently, we have pinpointed the exact molecular
steps leading to these changes, involving
not only actin, but also specific mediator
signaling cascades.
Outlook
Our milestones for the next year include:
• Continuing current studies on the role
of specific toxin domains in transmembrane actin reorganization.
• Using a GUV model system for studying
transmembrane phenomena in a defined
biochemical environment.
• Further characterizing the toxin’s micropores by a combination of electrophysiological measurements in artificial
lipid bilayers (in collaboration with the
laboratory of Roland Benz) and fluorescent FRAP microscopy.
• Characterizing the toxin turnover and
internalization in glial cells.
• Establishing a bacterial brain slice model,
which would be much closer to real bacterial meningitis.
• Correlating the morphological synaptic
and dendritic changes with brain slice
electrophysiological properties.
Fig. 2:
(A)Membrane depolarization (curve increase above 1 (measurement by the fluorescent dye
DiBAC(4)) by pneumolysin and membrane hyperpolarization by exposure to high sucrose. (B)
Preserved actin-dependent cell displacement by pneumolysin after membrane hyperpola rization
with sucrose. (C) Preserved cell displacement when calcium is absent from the medium (calcium
independence). (D) Preserved cell displacement when sodium is absent from the medium
(sodium independence).
Extramural Funding
Emmy Noether Program (DFG ENP IL
151/1-1)
Selected Publications
Fig. 3:
Establishing a permeabilized population of cells (using propidium iodide staining) by pneumolysin in
a dose-dependent manner. After the first 30-40 min, the permeabilized population reaches a plateau.
Förtsch, C., Hupp, S., Ma, J., Mitchell,
T. J., Maier, E., Benz R., and Iliev, A. I.
(2011). Changes in astrocyte shape induced by sublytic concentrations of the
cholesterol-dependent cytolysin pneumolysin still require pore-forming capacity. Toxins, 3(1), 43-62.
23
Stephan Kissler
E-mail: [email protected]
Phone: +49(0)931 31 80367
Fax:
+49(0)931 31 83255
http://www.rudolf-virchow-zentrum.de/forschung/kissler.html
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 the immune system is tightly regulated to prevent activation by innocuous antigens, including self-antigens, a significant number of people develop 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 use lentiviral transgenesis to generate animals in which
target genes are silenced by RNAi, either constitutively or in an inducible and reversible manner. This strategy
allows us to directly assess the contribution of individual genes to autoimmune disease. Furthermore, the
inducible silencing of selected target genes will enable us to assess the therapeutic potential of intervention in
specific pathways.
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 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 self-antigens,
or antigens whose presence is innocuous,
particularly on mucosal surfaces (airways
and digestive system).
Despite these regulatory mechanisms,
autoimmunity does occur 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 nonobese
diabetic (NOD) mouse strain is the most
widely-studied and relevant experimental
model. Numerous genetic loci have been
associated with type 1 diabetes. While a
number of causal genes have been identified, the exact functional contribution to
disease of individual susceptibility gene
variants is unknown.
24
Studying disease genetics by RNA interference
To facilitate the manipulation of gene-expression in the NOD mouse, we have pioneered lentiviral transgenesis in conjunction with RNAi in this disease model.
Generating 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.
In previous work, we successfully silenced
the expression of the candidate gene
Nramp1 in NOD mice and determined that
this gene is associated with disease. The
positive outcome of this first study led us
to initiate several new projects to investigate genes that associate with type 1
diabetes in humans using the mouse model.
These genetic studies are being carried out
in collaboration with Linda Wicker (Cambridge University, UK), whose extensive
characterization of disease genetics in the
NOD mouse helps us choose the most
appropriate targets. Importantly, many
disease-associated gene polymorphisms in
humans do not lead to the complete absence of a gene product, but instead result
in altered splicing and/or reduced expression. RNAi is exquisitely suited to model
more subtle variations in gene expression
compared to conventional KO technology.
For example, mutation of the gene CTLA4
that associates with type 1 diabetes in humans is known to alter splicing and reduce
expression of a shorter form of the gene
called sCTLA-4. We have now generated
NOD animals where this shorter splice variant, but not the full-length isoform, is reduced by RNAi. The reduction of sCTLA-4 in
the NOD mouse mimics the human CTLA-4
polymorphism associated with autoimmunity. We found that the loss of this splice
variant reduces the potency of regulatory T
cells both in vitro and in vivo. For example,
regulatory T cells from transgenic mice, in
constrast to wild-type (wt) regulatory T
cells, failed to reduce the severity of colitis
induced by pathogenic CD4+CD45RBhi T
cells in immunodeficient recipients (Figure
1). Our study shows that CTLA4 splicing
can directly affect regulatory T cell function, and provides a likely explanation for
the association of CTLA4 gene variation
with autoimmunity.
Fig. 1:
Loss of sCTLA-4 impairs the suppressive
activity of regulatory T (Treg) cells in
a colitis model. Purified CD4+CD45RBhi
cells were transfered into NOD.SCID
mice with Treg cells from wild-type
(wt) or sCTLA-4 knockdown mice
(sCTLA-4 KD), and colitis was scored
Six weeks later by histological analysis.
Inducible and reversible silencing of a
potential therapeutic target gene
In our first studies of the NOD model using
lentiviral RNAi, we employed constructs
that only allowed constitutive expression
of shRNA, leading to permanent gene
silencing. The gene PTPN22 is associated
with multiple autoimmune diseases, and is
considered a promising therapeutic target.
To evaluate the therapeutic potential of
this gene, we decided to employ a doxycyclin-regulated system. We have now generated two transgenic lines in which Ptpn22
expression can be downregulated by simple addition of the antibiotic doxycyclin
to the drinking water of transgenic mice.
Figure 2 shows a significant decrease in
PTPN22 protein following treatment of
transgenic, but not wt mice, with the
antibiotic doxycyclin, which inteferes with
the TetR repressor that prevents gene
silencing in the non-induced state (Figure
2). Initial experiments suggest that the
acute inhibition of this gene can lead
to the expansion of regulatory T cells. Experiments are now underway to assess
the protective potential of this expansion
within the context of spontaneous autoimmune diabetes.
Extramural Funding
Juvenile Diabetes Research Foundation
International:
Career Development Award 2-2010-383
Selected Publications
Fig. 2:
Inducible silencing of Ptpn22 in lentiviral transgenic mice. Expression of Ptpn22, beta
actin, and tetracyclin repressor (TetR) was measured by Western blotting in cells from
wt or inducible-Ptpn22 knockdown (tg) mice with or without doxycyclin treatment.
Acharya, M., Mukhopadhyay, S., Païdassi,
H., Jamil, T., Chow, C., Kissler, S.,
Stuart, L. M., Hynes, R. O., and LacyHulbert, A. (2010). αv Integrin expression by DCs is required for Th17
cell differentiation and development of
experimental autoimmune encephalomyelitis in mice. J Clin Invest, 120,
4445-52.
25
Alma Zernecke
E-mail: [email protected]
Phone: +49(0)931 31 80373
Fax:
+49(0)931 31 83255
http://www.rudolf-virchow-zentrum.de/forschung/zernecke.html
Atherosclerosis, with its clinical manifestations of myocardial infarction, stroke and peripheral artery disease, is
imminently becoming the leading cause of death worldwide. Inflammation has emerged as a crucial force driving
the initiation and progression of atherosclerotic lesion formation. Initiated by the activation and dysfunction of
endothelial cells, leukocyte subsets are recruited and accumulate in atherosclerotic lesions. Details regarding the
involvement of different leukocyte subpopulations in the pathology of this disease are emerging. While mononuclear cells found in the lesions are predominantly comprised of monocyte-derived macrophages, which transform into foam cells characteristic for fatty-streak lesions, T-lymphocytes and dendritic cells have also been revealed in close proximity. Moreover, immune responses are described to participate in all phases of
atherosclerosis, and pro-atherogenic and atheroprotective cytokines and T cell subpopulations have been defined. However the delicately adjusted two-edged immune balance and the exact function of these cell types
remain elusive.
We previously identified several adhesion
molecules and chemokines/receptors and
their regulation that are important in the
accumulation of leukocytes at sites of
inflammation. Recently, we examined the
contribution of the C5a receptor (C5aR)
to neointima formation in apolipoprotein
E-deficient mice employing a C5aR antagonist (C5aRA) and a C5aR-blocking monoclonal antibody. Compared with controls,
neointima formation was significantly
reduced in mice receiving C5aRA or antiC5aR-blocking monoclonal antibody for
one week but not for three weeks, attributable to an increased content of vascular
smooth muscle cells. In contrast, a marked
decrease in monocyte and neutrophil content
was associated with reduced vascular cell
adhesion molecule-1 (Shagdarsuren et al.).
On the other hand, recruitment of cultured angiogenic early outgrowth cells
(EOCs) may be beneficial for repair mechanisms of the injured vessel, and accelerate
endothelial regeneration and attenuate
neointimal remodelling. We have recently
revealed that cultured human peripheral
blood-derived angiogenic early outgrowth
cells (EOCs) strongly expressed CD40 mRNA
and protein. After preincubation with recombinant human sCD40L, EOC adhesion to
fibronectin, fibrinogen, intercellular adhesion molecule-1, and vascular cell adhesion
molecule-1 under flow conditions, as well
as their transmigration toward stromal cellderived factor-1alpha, was dose-dependently reduced. Notably, CD40-/- mice dis-
26
Fig. 1:
Platelet CD40L promotes leukocyte adhesion to the atherosclerotic arterial wall. Images show intravital
microscopy of adhering, rhodamine-labeled leukocytes in carotid arteries of Apoe–/– mice treated with
activated Cd40l+/+Apoe–/– or Cd40l–/–Apoe–/– platelets, or vehicle.
played reduced neointima formation and
improved re-endothelialization after carotid wire injury compared with wild-type
mice. Thus, endothelial dysfunction due to
persistently elevated plasma levels of
sCD40L may be attributable to an impairment of EOC function (Hristov et al.).
CD40 ligand (CD40L), identified as a costimulatory molecule expressed on T cells,
is also expressed and functional on platelets. We therefore recently investigated the
thrombotic and inflammatory contributions
of platelet CD40L in atherosclerosis. Although CD40L-deficient platelets exhibited
impaired platelet aggregation and thrombus stability, the effects of platelet CD40L
on inflammatory processes in atherosclerosis were more remarkable. Repeated injec-
tions of activated CD40L-/- platelets into
Apolipoprotein E-deficient (ApoE-/-) mice
strongly decreased both platelet and leukocyte adhesion to the endothelium (Figure
1) and decreased plasma CCL2 levels compared with wild-type platelets. Moreover,
CD40L-/- platelets failed to form proinflammatory platelet-leukocyte aggregates. Expression of CD40L on platelets was required
for platelet-induced atherosclerosis since
injection of CD40L-/- platelets in contrast
to CD40L+/+ platelets did not promote
lesion formation (Figure 2). Remarkably,
injection of CD40L+/+, but not CD40L-/platelets transiently decreased the amount
of regulatory T cells (Tregs) in the blood
and spleen. Depletion of Tregs in mice
injected with activated CD40L-/- plate-
lets abrogated the athero-protective effect, indicating that CD40L on platelets
mediates the reduction of Tregs, leading to
accelerated atherosclerosis. We thus concluded that platelet CD40L plays a pivotal
role in atherosclerosis, not only by affecting platelet-platelet interactions but
especially by activating leukocytes, thereby
increasing platelet-leukocyte and leukocyte-endothelium interactions (Lievens &
Zernecke et al.).
The CD40-CD40 ligand signaling axis also
plays an important role in immunological
pathways. We could moreover show that
deficiency in hematopoietic CD40 reduces
atherosclerosis and induces features of
plaque stability, a clinically favorable plaque phenotype that is low in inflammation
and high in fibrosis. To further elucidate
the role of CD40-tumor necrosis factor receptor-associated factor (TRAF) signaling
in atherosclerosis, we examined disease
progression in mice deficient in CD40 and
its associated signaling intermediates.
Absence of CD40-TRAF6, but not CD40TRAF2/3/5 signaling abolishes atherosclerosis and conferred plaque fibrosis in
ApoE-/- mice. Mice with defective CD40TRAF6 signaling displayed a reduced blood
count of Ly6C(high) monocytes, an impaired recruitment of Ly6C(+) monocytes
to the arterial wall, and polarization of
macrophages toward an antiinflammatory
regulatory M2 signature. These data unveiled a role for CD40-TRAF6, but not CD40TRAF2/3/5, interactions in atherosclerosis
and established that targeting specific
components of the CD40-CD40L pathway
harbors the potential to achieve therapeutic effects in atherosclerosis (Lutgens et al.).
Inflammatory cytokines are also well-recognized mediators of atherosclerosis. In a
recent study we further demonstrated that
the type I interferon IFNbeta enhances
macrophage-endothelial cell adhesion and
promotes leukocyte attraction to atherosclerosis-prone sites in mice in a chemokine-dependent manner, accompanied by
increased macrophage accumulation within
plaques and accelerated lesion formation
in different mouse models of atherosclerosis. Concomitantly, absence of endogenous
type I IFN signaling in myeloid cells inhibited lesion development and protected
against lesional accumulation of macrophages (Goossens et al.).
Less is known about the recruitment
and function of T cells and dendritic cell
subsets in atherosclerosis. By targeting
specific chemokines/cytokines and their
receptors in Ldlr-/- mice, we will address
the functions of different immune cell subpopulations in atherosclerosis. A particular
focus will be on their interactions at sites
of inflammation, and also within lymphatic
tissue, and their role in shaping specialized
immune responses that control the development of atherosclerosis. Furthermore, we
will investigate the localization of these
cells in the vessel wall and their routes of
entry during lesion formation.
Given the remarkable role of adaptive
and innate immunity in atherosclerosis,
targeting of its cellular constituents and
understanding the complex equilibrium and
interplay between immune cell subpopulations that contribute to the process of atherosclerosis will be important to identify
new therapeutic approaches for treating
this disease.
Extramural Funding
DFG (SFB 688 TP A12, ZE 827/1-2,
ZE827/4-1)
Selected Publications
Goossens, P., Gijbels, M. J., Zernecke, A.,
Eijgelaar, W., Vergouwe, M. N., van der
Made, I., Vanderlocht, J., Beckers, L.,
Buurman, W. A., Daemen, M. J., Kalinke,
U., Weber, C., Lutgens, E., and de Winther, M. P. (2010). Myeloid type I interferon signaling promotes atherosclerosis
by stimulating macrophage recruitment
to Lesions. Cell Metab, 12, 142-153.
Hristov, M., Gümbel, D., Lutgens, E.,
Zernecke, A., and Weber, C. (2010). Soluble CD40 ligand impairs the function
of peripheral blood angiogenic outgrowth cells and increases neointimal
formation after arterial injury. Circulation, 121, 315-24.
Lievens, D.*, Zernecke, A.*, Seijkens, T.,
Soehnlein, O., Beckers, L., Munnix, I.,
Wijnands, E., Goossens, P., van Kruchten,
R., Thevissen, L., Boon, L., Flavell, R. A.,
Noelle, R. J., Gerdes, N., Biessen, E. A.,
Daemen, M. J., Heemskerk, J. W., Weber,
C., and Lutgens, E. (2010). Platelet
CD40L mediates thrombotic and inflammatory processes in atherosclerosis.
Blood, 116, 4317-27. *equal contribution.
Lutgens, E., Lievens, D., Beckers, L., Wijnands, E., Soehnlein, O., Zernecke, A.,
Seijkens, T., Engel, D., Cleutjens, J.,
Keller, A. M., Naik, S.H., Boon, L.,
Oufella, H. A., Mallat, Z., Ahonen, C.L.,
Noelle, R. J., de Winther, M. P., Daemen,
M. J., Biessen, E. A., and Weber, C.
(2010). Deficient CD40-TRAF6 signaling
in leukocytes prevents atherosclerosis
by skewing the immune response toward
an antiinflammatory profile. J Exp Med,
207, 391-404.
Fig. 2:
Platelet CD40L promotes atherosclerosis initiation. Plaque area of the aortic arch, including the main
branch points (brachiocephalic trunk [BCT], left common carotid artery [LCC], left subclavian artery)
of ApoE–/– mice, injected with activated CD40L+/+ApoE–/– or CD40L–/–ApoE–/– platelets, or vehicle;
representative longitudinal sections (hematoxylin and eosin staining). Scale bar represents 200 µm.
Shagdarsuren, E., Bidzhekov, K., Mause,
S. F., Simsekyilmaz, S., Polakowski, T.,
Hawlisch, H., Gessner, J. E., Zernecke,
A., and Weber, C. (2010). C5a receptor
targeting in neointima formation after
arterial injury in atherosclerosis-prone
mice. Circulation, 122, 1026-36.
27
Caroline Kisker
E-mail: [email protected]
Phone: +49(0)931 31 80381
Fax:
+49(0)931 31 87320
http://www.rudolf-virchow-zentrum.de/forschung/kisker.html
Our DNA is constantly damaged by endogenous and exogenous sources such as reactive oxygen species produced
as by-products of oxidative metabolism, or by environmental factors such as UV radiation. It has been estimated
that approximately 200,000 damage events occur daily in each human cell; thus, organisms require efficient DNA
damage response pathways to maintain their genomes in a functional state. Nucleotide excision repair (NER) is
one of these repair mechanisms and recognizes damage such as the carcinogenic pyrimidine dimers induced
by UV radiation, benzo[a]pyrene-guanine adducts caused by smoking, as well as guanine-cisplatinum adducts
formed during chemotherapy. It is our goal to obtain a general understanding of the sequential process of
damage recognition followed by damage excision using a combination of structural, microscopic and biochemical studies involving the discrete steps of the NER cascade. The second focus in our laboratory is on
structure-based drug design to identify new therapeutics for infectious diseases. Our main targets are essential
bacterial enzymes involved in fatty acid biosynthesis to combat diseases such as tuberculosis or nosocomial
infections due to multidrug resistant Staphylococcus aureus strains.
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 (XP), Cockaynes syndrome (CS) and trichothiody-strophy (TTD). 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 initial
binding event affect subsequent binding of
additional proteins, and thereby allow the
cascade of events to proceed?
The XPD protein
In the eukaryotic NER pathway a general
disruption of Watson-Crick base-pairing
created in the vicinity of the damaged
nucleotide is recognized by the combined
action of XPC and HR23B. Both proteins
subsequently recruit the 10-subunit transcription factor TFIIH to this site. XPD,
which comprises one of the subunits of
TFIIH, utilizes its helicase activity to verify
the damage and ensure that the backbone
distortion is not the result of an unusual
DNA sequence. Unlike many other helicases, XPD contains an auxiliary domain
28
with an 4Fe4S cluster that is essential for
its function as a helicase, and as such
makes it a family member of related super
family 2 (SF2) helicases. The common feature of the XPD-like SF2 helicases is the FeS
cluster domain that is present in various
family members such as bacterial DinG
(damage-inducible G) and the eukaryotic
XPD paralogues FancJ (Fanconi’s anaemia
complementation group J), RTEL (regular
of telomere length) and Chl1 (chromatid
cohesion in yeast) and therefore can be
found in all kingdoms of life.
We and two other laboratories solved XPD
structures from different archaeal organisms, namely Sulfulobus tokodaii (stXPD),
Sulfolobus acidocaldarius (saXPD) and Thermoplasma acidophilum (taXPD). Two of the
three structures showed a similar four-domain organization, with two RecA-like domains (HD1 and HD2), an α-helical domain
and the FeS cluster containing domain.
HD1 together with the FeS-cluster domain
and the α-helical domain form a donut-like
structure, but there are differences in the
relative arrangement of the domains that
can be used to derive functional implications. TaXPD and saXPD share significant
homology in their tertiary structure, although the overall sequence identity is
relatively low at only 19%. Within this
19%, however, almost all of the diseaserelated mutations leading to XP, XP/CS and
TTD are included, emphasizing their functional significance. One of the major differences between the two structures is the
size of the pore formed by the three
domains (Fig. 1); with ~ 8 Å in diameter
in the saXPD structure it is much smaller
than the hole in taXPD with a diameter
of ~ 13 Å.
The difference in pore size is mainly
mediated by different positioning of the
α-helical domain and the FeS domain relative to each other. The two domains could
A
B
C
D
Fig. 1:
Overall structures of taXPD (left) and saXPD
(right). The surface is shown in grey and
the domains are shown in yellow and red for
the two RecA-like domains (HD1 and HD2)
and cyan and green for the FeS cluster- and
α-helical-domains. Below the overall structures
is shown a zoom into the pore.
act in tandem with a gentle push from
helix α22 in the second RecA-like domain
to facilitate the dynamics of the pore
(Fig. 2). A structural flexibility analysis of
taXP supports this notion, indicating a
linked movement between the two RecA
domains that also affects the pore and thus
provides a dynamic model of how the pore
size can vary upon the expected movement of the two RecA domains during ATP
hydrolysis. The functional consequences of
this mechanism could be to mediate translation along the DNA using the pore as
a clasp. Furthermore, for XPD to act on
a double-stranded DNA substrate the ring
of the pore has to open up to allow the
passage of one DNA strand through the
pore. This ring opening could be achieved
by a transient separation of the α-helical
domain and the FeS cluster domain.
A
FAS II pathway (Fig. 3). Isoniazid, a front
line drug for treating tuberculosis that
targets the mycobacterial enoyl ACP reductase InhA, and the antimicrobial agent
triclosan are the most successful inhibitors
of the FabI proteins.
S. aureus is the only known organism that
uses NADPH instead of NADH as a reducing
agent for the reduction catalyzed by FabI.
The diphenylether inhibitor triclosan and
the cofactor NADP+ were used for co-crystallization trials with S. aureus FabI and
Structure-based drug design
Methicillin-resistant Staphylococcus aureus
(MRSA) is a major problem in hospitals all
over the world and causes 19,000 deaths
per year in the United States alone.
64.4% of the S. aureus infections are currently not treatable with β-lactam antibiotics due to their resistance against these
drugs. In 2002 the problem became even
more severe due to the emergence of Vancomycin-resistant S. aureus (VRSA) strains,
thus preventing the successful use of the
“last resort antibiotic ”. The increasing
occurrence of community-acquired MRSA
infections further substantiates the fact
that there is an urgent need for novel antibiotics against S. aureus.
The essential type II fatty acid biosynthesis (FAS II) of bacteria, which shares only
low homology with the mammalian FAS I
system, is a validated target for developing of new drugs. FabI, the trans-2-enoyl
ACP reductase, catalyzes the last and ratelimiting step of each fatty acid elongation
cycle. Due to this additional regulatory
role, the FabI enzyme is the most extensively explored drug target of the
DFG KI-562/2 PI Caroline Kisker
DFG SFB630, B7, PI Caroline Kisker
Selected Publications
Basu, A., Broyde, S., Iwai, S., and Kisker,
C. (2010). DNA damage, mutagenesis,
and DNA repair. J Nucleic Acids, 2010,
182894.
B
Fig. 2:
Two views of the superposition of taXPD and
saXPD. The FeS- and α-helical domain of taXPD
and saXPD are colored in green and darkgreen
and cyan and blue, respectively. Helix α22 of
domain 4 is colored in red and salmon, and is
highlighted by an arrow. The FeS cluster of taXPD
is shown in orange. Domains 3 and 4 are colored
in darkgrey (taXPD) and grey (saXPD).
Extramural Funding
Fig. 3:
Structure of the functional unit of S. aureus
FabI. Each monomer within the tetramer is
depicted in a different color.
we solved the structure of the ternary
saFabI/NADP+/triclosan complex at a resolution of 2.8 Å (Fig. 4). The overall structure comprises the typical Rossmann-fold
of nucleotide binding proteins. The crystal
structure of the ternary complex shows
that three residues stabilize the additional
2’-phosphate of NADP+, thus explaining
the different cofactor use of NADPH
instead of NADH.
Our structure also supports the hypothesis of a substrate-binding loop enclosing
the triclosan molecule upon binding to
the protein. Additionally the observed interactions between protein, cofactor and
inhibitors provide the first insights to help
Fig. 4:
Structure of the S. aureus FabI monomer in
complex with its cofactor NADP+ and the broad
spectrum antibacterial agent triclosan.
understand triclosan resistance mechanisms
in S. aureus. The structure of the ternary
complex thus forms the basis for future drug
development efforts in search of a novel
remedy for MRSA and VRSA-infections.
Breuning, A., Degel, B., Schulz, F., Buchold, C., Stempka, M., Machon, U.,
Heppner, S., Gelhaus, C., Leippe, M.,
Leyh, M., Kisker, C., Rath, J., Stich, A.,
Gut, J., Rosenthal, P.J., Schmuck, C.,
and Schirmeister, T. (2010). Michael
acceptor based antiplasmodial and
antitrypanosomal cysteine protease inhibitors with unusual amino acids. J
Med Chem, 53, 1951-63.
Luckner, S. R., Liu, N., am Ende, C. W.,
Tonge, P. J., and Kisker, C. (2010). A
slow, tight binding inhibitor of InhA,
the enoyl-acyl carrier protein reductase
from Mycobacterium tuberculosis. J Biol
Chem, 285, 14330-37.
Machutta, C. A., Bommineni, G. R.,
Luckner, S. R., Kapilashrami, K., Ruzsicska, B., Simmerling, C., Kisker, C.,
and Tonge, P. J. (2010). Slow onset inhibition of bacterial beta-ketoacyl-acyl
carrier protein synthases by thiolactomycin. J Biol Chem, 285, 6161-69.
Qiu, J. A., Wilson, H. L., Pushie, M. J.,
Kisker, C., George, G. N., and Rajagopalan, K. V. (2010). The structures of the
C185S and C185A mutants of sulfite oxidase reveal rearrangement of the active
site. Biochemistry, 49, 3989-4000.
Schlereth, K., Beinoraviciute-Kellner,
R., Zeitlinger, M.K., Bretz, A.C., Sauer,
M., Charles, J.P., Vogiatzi, F., Leich, E.,
Samans, B., Eilers, M., Kisker, C., Rosenwald, A., and Stiewe, T. (2010). DNA
binding cooperativity of p53 modulates
the decision between cell-cycle arrest
and apoptosis. Mol Cell, 38, 356-68.
Wolski, S.C., Kuper, J., and Kisker, C.
(2010). The XPD helicase: XPanDing archaeal XPD structures to get a grip on
human DNA repair. Biol Chem, 391,
761-65.
29
Hermann Schindelin
E-mail: [email protected]
Phone: +49(0)931 31 80382
Fax:
+49(0)931 31 87320
http://www.rudolf-virchow-zentrum.de/forschung/schindelin.html
The general aim of our research is to understand the functions of biologically important proteins. We therefore investigate two general topics: (1) Protein folding in the endoplasmic reticulum (ER) and degradation of misfolded
proteins via the ubiquitin-dependent protein degradation pathway. (2) The structure and function of inhibitory
neurotransmitter receptors and the mechanism of their anchoring at the postsynaptic membrane. Our intention
is to understand these proteins and the processes they participate in at the molecular level. Besides X-ray
crystallography, we are use a combination of complementary techniques for the biochemical and biophysical
characterization of these target proteins. These studies have direct medical relevance. For example, misfolding
and aggregation due to deficiencies in the endoplasmic reticulum associated protein degradation (ERAD)
pathway lead to a variety of pathophysiological states such as the neurodegenerative disorders Alzheimer’s and
Parkinson’s disease.
Endoplasmic reticulum associated protein degradation
Endoplasmic reticulum associated protein
degradation (ERAD) is an essential cellular
pathway which prevents the accumulation
of misfolded (glyco)proteins in the ER and
entails their recognition, retrotranslocation to the cytosol, ubiquitylation and
degradation by the proteasome. The AAA+
ATPase p97 (Cdc48 in yeast) plays a crucial
role in the retrotranslocation of misfolded
proteins by providing the energy required
to extract them from the ER. The N-terminal domain of p97/Cdc48 interacts with a
variety of partner proteins that are classified as either substrate-recruiting, or substrate-processing cofactors. In the past we
have focused on substrate-processing cofactors interacting with p97’s C-terminus,
however, during the last year we started to
also investigate cofactors interacting with
the N-domain.
Ufd2 is a substrate-processing cofactor
that has both ubiquitin protein ligase
(E3) and ubiquitin chain elongation (E4)
activities; it binds to the C-terminus of
Cdc48 with micromolar affinity. At the same
time Ufd2 in yeast also binds to two proteasomal shuttle factors, Rad23 and Dsk2,
which both feature a modular architecture
including a ubiquitin-like (UBL) and a
ubiquitin-associated (UBA) domain. Via
their UBA domains these proteins bind to
ubiquitylated substrates, and at the same
time, their UBL domains are recognized by
different subunits located in the lid of the
proteasome. The interaction of Ufd2 with
30
Fig. 1:
The Ufd2-UBL interface. (A) Close-up view of the
Ufd2-Rad23 interface between Ufd2 (orange) and
Rad23 (green). (B) Structure-based sequence
alignment of Rad23-UBL, Dsk2-UBL, hHR23A-UBL,
and Ub. Residues involved in the Ufd2•Rad23-UBL
interaction are labeled with green stars.
Rad23 and Dsk2 is also mediated via their
N-terminal UBL domains. The crystal structures of Ufd2 in complex with the UBL domain of either protein were determined.
Ufd2 interacts via its α-helical N-terminal
domain with three segments of the UBL
(Fig. 1), including a hydrophobic core region corresponding to the well-characterized hydrophobic patch in ubiquitin that is
crucial for binding. Both binary complexes
are highly stable with dissociation constants of ~0.1 μM.
The interface can be subdivided into
three regions (I, II and III) including a
central area (II) which harbors most of the
contacts. A detailed structure-function
analysis revealed the contribution of individual residues to complex stability. The
majority of Rad23-UBL single mutants revealed reduced binding to Ufd2, with
Rad23-I45A displaying the most prominent
binding defect, a 130-fold reduction in
binding. However, none of the other mutants displayed a more than 10-fold reduction in binding. The most significant effect
for Ufd2 was observed for all residues located in the hydrophobic UBL pocket. Mutation of Val100 and Phe107 to Ala completely abolished binding and the Y97A
variant strongly reduced binding (1900fold). Interestingly, the corresponding mutations in the Dsk2-UBL reveal a less severe
phenotype, a surprise given the high degree of structural similarity between the
two complexes (Fig. 2)
Fig. 2:
Superposition of the Ufd2•Rad23-UBL (orange
and green) and Ufd2•Dsk2-UBL (gray and
yellow) complexes.
The N-terminus of budding yeast Ufd2
shares only limited sequence homology
with the human Ufd2s, E4A and E4B, and
other Ufd2s from higher eukaryotes. In
agreement with this finding, there are
no reports that hHR23A/B interacts with
either of the human homologs of Ufd2.
Apparently, the high affinity interaction of
the UBL domains of Rad23 and Dsk2 has
been lost during the evolution of this
domain. The N-terminus of Ufd2 therefore
represents a unique and conserved UBLbinding domain, which further enhances
the growing list of protein modules interacting with ubiquitin or ubiquitin-like
proteins.
mutations reduce synaptic clustering of
GABAARs. ITC analysis revealed that the
phosphomimetic mutation to glutamate
decreases the affinity for gephyrin, an effect not present in the alanine mutant
(Fig. 4). In vivo this substitution leads to
a reduced number of receptors at inhibitory
synapses, resulting in a weakened inhibitory synaptic transmission. Taken together,
these results provide a dynamic mechanism
to explain the selective accumulation of
specific GABAARs at inhibitory synapses,
which modulates the strengths of inhibitory synaptic transmissions.
Neurotransmitter receptor anchoring
The neurotransmitter receptor anchoring
protein gephyrin is essential for the proper
location of inhibitory neuronal glycine
and GABAA receptors. Gephyrin presumably
forms a hexagonal scaffold below the postsynaptic membrane, thus allowing it to
cluster glycine and GABAA receptors while
bridging them to cytoskeletal elements. In
the past we have focused on the interaction between gephyrin and glycine receptors GlyRs, which is mediated by the C-terminal E-domain of gephyrin and the large
cytoplasmic loop located in between transmembrane segments 3 and 4 of the receptor β-subunit. Conflicting results have been
published, however, on the role of gephyrin
in GABAAR clustering. Gephyrin is enriched
at postsynaptic inhibitory specializations
throughout the brain and does co-localize
with α1-3, β2/3 and γ2 subunit containing
GABAARs. Thus it remained to be determined how the majority of GABAARs are
selectively enriched at inhibitory synapses.
We addressed this question in close collaboration with the group of Steve Moss at
Tufts University.
The interaction between the E-domain of
gephyrin and the intracellular domain of
the α1 subunit was analyzed by isothermal titration calorimetry (ITC). To gain
further information on the binding site(s)
for gephyrin within the α1 subunit, an
additional α1 expression construct was
employed where 16 residues were exchanged for the corresponding region of
the α6 subunit, which does not bind to
gephyrin (α1/6-chimera). ITC revealed that
these residues of the α1 subunit are essential in mediating the direct binding to
gephyrin in vitro (Fig. 3).
Fig. 3:
Comparative binding affinities of gephyrin’s
E-domain to the cytoplasmic loop of the
GABAAR α1 subunit (top) and the α1/6
chimera (bottom). The corresponding
dissociation constants (KD) are indicated
in the inset, N.D.: not detectable.
Intriguingly, the gephyrin affinity and stoichiometry to the GABAAR α1 subunit is very
similar to the low affinity binding site
found for the GlyR β subunit. The lack of a
high affinity binding site on GABAAR α1
subunits is a possible explanation for why
the direct binding of these proteins remained hidden for a long time. Moreover
the lower affinity of GABAARs for gephyrin
compared to GlyRs has profound implications for the relative stability of these two
receptors at inhibitory synapses and may
explain the rapid rates of endocytosis seen
for GABAARs. Significant structural conservation exists between the GABAAR α1 and
α2 subunits, which suggests of a common
mechanism for their binding to gephyrin;
however, there appears to be little conservation with the GlyR β subunit and hence it
is unclear whether both receptors interact
with gephyrin in an analogous fashion.
With the aid of X-ray crystallography we
are currently analyzing how gephyrin
mediates GABAAR and GlyR binding at the
molecular level.
Regulatory mechanisms that may influence the interaction of the α1 subunit with
gephyrin were examined focusing on the
phosphorylation of a threonine residue in
the α1 subunit. It was demonstrated by
our collaborators that this residue can be
phosphorylated and that phosphomimetic
Fig. 4:
Binding affinities of the wild-type (WT, ■),T→A
(☐) and T→E ( ) variants of the GABAA receptor
α1 subunit as determined by ITC are shown
together with the corresponding KD values.
Extramural Funding
DFG Schi 425/3-1, PI
DFG SFB487, C7, PI Hermann Schindelin
Selected Publications
Hänzelmann, P., Stingele, J., Hofmann,
K., Schindelin, H., and Raasi, S. (2010).
The yeast E4 ubiquitin ligase Ufd2 interacts with the ubiquitin-like domains of
Rad23 and Dsk2 via a novel and distinct
ubiquitin-like binding domain. J Biol
Chem, 285, 20390-20398.
Völler, D., and Schindelin, H. (2010).
And yet it moves: active site remodeling in the SUMO E1. Structure, 18,
419-21.
31
Research Professorships
Utz Fischer
Theodor-Boveri-Institute, Biochemistry
E-mail: utz.fi[email protected]
Phone: +49(0)931 31 84029
Fax:
+49(0)931 31 84026
http://www.biochem.biozentrum.uni-wuerzburg.de
The generation of mature mRNAs and their translation into proteins depends on the elaborate interplay of a large
number of trans-acting factors in eukaryotes. These factors are often organized in functional units (called macromolecular machines), which catalyze the sequential steps in mRNA metabolism and timely coordinate their
progression. Using a combination of biochemistry and structural biology (single particle cryo-electron microscopy and X-ray crystallography) our group studies the functional dynamics of key complexes acting on mRNA,
and how their malfunction causes human diseases.
U snRNP assembly and pre-mRNA processing
The cell has developed intricate strategies
in order to generate the highly complex
RNPs required for mRNA processing (UsnRNPs and spliceosome, see Fig. 1 for an
overview of the assembly pathway of snRNPs). Formation of U snRNPs starts with
the transport of newly transcribed snRNA
to the cytoplasm. A set of common (Sm)
proteins are then loaded onto U snRNAs,
leading to the formation of a ring-shaped
Sm core domain. In a next step, the monomethyl-guanosine cap of the snRNA is converted to a trimethyl-guanosine cap. Even-
tually, the assembled snRNP particle is
imported into the nucleus, where specific
proteins are also added to complete the
biogenesis pathway. In a series of experiments we have shown that assembly of U
snRNPs requires assisting factors that are
united in the SMN complex, consisting of
the SMN protein and eight additional proteins called Gemin2-8 and unrip. This macromolecular unit recruits Sm proteins as
well as U snRNAs and mediates the formation of the Sm core domain. An additional
unit called the PRMT5 complex cooperates
with the SMN-complex in snRNP assembly. The PRMT5 complex functions as an
assembly chaperone by forcing Sm proteins
into higher order structures required for
their subsequent transfer into the SMNcomplex (Chari et al., Cell 135, 497-509).
Once U snRNPs have been assembled and
imported to the nucleus, they can integrate into the spliceosome. Spliceosome
formation likewise requires specific proteins. We could show a role of the SMNhomologous protein SMNrp (also termed
SPF30), in the integration of the tri-snRNP
into the pre-spliceosome. It is hence possible that spliceosome formation requires
assembly factors that are functionally
and/or structurally related to components
of the SMN-complex (Chari and Fischer,
2010, Fig. 1).
Fig. 1:
The assembly pathway of splicesomal U snRNPs.
Most snRNAs are transcribed by polymerase II
and transported for their maturation to the
cytoplasm. After import of the assembled
particle, they transit through the Cajal Body
(CB) for further maturation before they become
incorporated into spliceosomes.
32
Splicing and disease
It has been appreciated in the past that
mutations in cis-acting regulatory sequences of mRNAs as well as in trans-acting factors of mRNA metabolism are a
major cause of genetic diseases. Disease
mutations in cis-elements of pre-mRNAs
often interfere with the appropriate processing of the affected primary transcript,
and hence with the generation of translation-competent mRNAs. However, the
situation is more complicated when factors
implicated in general mRNA metabolism
are mutated. While in the former case only
the fate of the mutated mRNA is affected,
genes affect mRNA metabolism. To gain
insights into the question why defects in
these general factors evoke highly tissue
specific phenotypes (motorneuron degeneration in the case of SMA and photoreceptor-degeneration in RP), we have recently
established a zebrafish model for RP (Fig.
2). Despite reducing the level of the constitutive splicing factor prp31, no global
defects in gene expression were found.
Instead, retinal genes were selectively
affected, providing the first in vivo link
between mutations in splicing factors and
the RP-phenotype (Linder et al., 2011).
Extramural Funding related to project
DFG Forschergruppe FOR 855
Selected Publications
Chari, A., and Fischer, U. ( 2010). Cellular strategies for the assembly of molecular machines. Trends Biochem Sci,
35(12), 676-83.
Fig. 2:
Mutations in the spliceosomal tri-snRNP proteins PRP3, PRP4 and PRP31 cause the eye disease
Retinitis pigmentosa. We have established a zebrafish model of this disease. These blind fish are being
analyzed by means of behavioral tests, histology and gene expression profiling to understand the
etiology of the disease.
a more general defect is anticipated in the
latter case. Our group is engaged in the
analysis of diseases that are caused by mutations in general trans-acting factors of
mRNA splicing. The first disease that falls
into this group is the monogenic neuromuscular disorder spinal muscular atrophy
(SMA), which is caused by reduced expression of the U snRNP assembly factor SMN.
The second disease of this group is Retinitis pigmentosa (RP). Mutations in genes
encoding spliceosomal tri-snRNP proteins
(prp3, 8 and 31) evoke the RP phenotype,
providing an outstanding example of a disease caused by defects in general splicing
factors. In both cases it can be anticipated
that mutations in the respective disease
Guderian, G., Peter, C., Wiesner, J., Sickmann, A., Schulze-Osthoff, K., Fischer,
U., and Grimmler, M. (2010). RioK1, a
new interactor of protein arginine methyltransferase 5 (PRMT5), competes with
plCln or binding and modulates PRMT5
complex composition and substrate
specificity. J Biol Chem, 286(3), 197686.
Schäffler, K., Schulz, K., Hirmer, A., Wiesner, J., Grimm, M., Sickmann, A., and
Fischer, U. (2010). A stimulatory role
for the La-related protein 4B in translation. RNA, 16, 1488-99.
Linder, B., Dill, H., Hirmer, A., Brocher,
J., Lee, G. P., Mathavan, S., Bolz, H. J.,
Winkler, C., Laggerbauer, B., and Fischer, U. (2011). Systemic splicing factor
deficiency causes tissue-specific defects: a zebrafisch model for retinitis
pigmentosa. Hum Mol Genet, 20(2),
368-77.
33
Research Professorships
Antje Gohla
E-mail: [email protected]
Phone: +49(0)931 201 489 77
Fax:
+49(0)931 201 485 39
http://www.rudolf-virchow-zentrum.de/forschung/gohla.html
Cell adhesion and migration are essential for development and homeostasis. Adhesion to the extracellular matrix
and between cells occurs at specialized plasma membrane domains where transmembrane adhesion receptors,
signaling proteins such as kinases and phosphatases, and a large number of adaptor proteins interact with the
cytoskeleton. These adhesions operate as dynamic structural and signaling hubs to transmit information in and
out of cells. Cell migration is based on the coordinated formation and remodeling of adhesions, leading to spatiotemporally controlled changes of the cytoskeleton and of cell morphology. Whereas altered cell adhesion and
migration are known to be important in pathologies such as cardiovascular diseases and malignant tumors, the
target proteins and molecular interactions that regulate these complex processes remain incompletely understood. Our group employs biochemical and cell biological methods to study key regulators of Rho-GTPase dependent cytoskeletal dynamics. During the past year, the functions of a newly emerging family of human phosphatases for cell adhesion and migration have turned into the focus of our research.
Identification and characterization of
Chronophin regulators
The actin depolymerizing factor (ADF) and
cofilin are closely related proteins that
control actin cytoskeletal remodeling, adhesion and migration in virtually all cells.
Their actin-reorganizing activities are
tightly controlled by interaction with lipids
and by an inhibitory phosphorylation of a
single serine residue. Using an activitybased biochemical purification approach
coupled with mass spectrometry and cell
biological studies, we have previously identified Chronophin as a novel ADF/cofilinactivating phosphatase (Gohla et al., Nature Cell Biol, 2005). The depletion of
Chronophin by RNA interference in various
cell types results in altered cell division
and cell motility, suggesting that Chronophin may function as a key regulator of
cytoskeletal dynamics. Chronophin belongs
to the emerging family of over 20 human
DXDX(T/V) phosphatases, a ubiquitous and
evolutionarily conserved class of serine/
threonine- or tyrosine-directed phosphatases whose functions for eukaryotic biology are only beginning to emerge.
Chronophin is a small (~32 kDa) protein
with no discernible protein-protein interaction, regulatory or targeting domains.
As a first step towards deciphering its
regulation in response to extracellular
34
cues, we conducted a screen for Chronophin interactors using the yeast two-hybrid
system in combination with biochemical
and cellular protein-protein interaction
studies. We found that Chronophin directly
interacts with and is activated by two
calcium-binding proteins, the Ca2+- and
integrin-binding protein 1 (CIB1) and
Calmodulin, and that Ca2+ functions as a
master regulator to shift between Chronophin/CIB1 and Chronophin/Calmodulin
complex formation (Fig. 1). Furthermore,
we identified a regulatory domain in Chronophin that may control the access of
substrates to the catalytic pocket of the
phosphatase.
Fig. 1:
Ca2+ determines the composition of Chronophin regulatory complexes. Crosslinking experiments were
performed with purified proteins in the presence of different Ca2+ concentrations. Chronophin
associates with CIB1 under basal conditions, whereas Ca2+ triggers the formation of a Chronophin/
Calmodulin complex. Abbreviations: CIN, Chronophin; CIB1, Ca2+ and integrin binding protein 1, CaM,
Calmodulin; MWM, molecular weight marker.
These results also provide a model for the
local coordination of cofilin kinase and cofilin phosphatase signaling networks that
regulate cellular actin dynamics in a Ca2+dependent manner (Fig. 2).
Identification of AUM, a novel human
tyrosine phosphatase
By database mining and phylogenetic analysis of human DXDX(T/V) enzymes, we
have discovered a previously unidentified
phosphatase. We cloned and characterized
this novel protein and called it AUM (actin
remodeling, ubiquitous magnesium-dependent phosphatase). AUM is broadly expressed in all major human and mouse tissues, and acts as a specific protein tyrosine
phosphatase. At a cellular level, AUM-depleted cells are characterized by accelerated cell adhesion and spreading due to altered tyrosine phosphorylation in focal
adhesions (Fig. 3).
Outlook
Major future aims are to elucidate the regulation and functions of Chronophin and
AUM to develop a better understanding
of these elusive phosphatases that have
emerged as key regulators of fundamental
cellular processes. In addition to biochemical and cellular studies, we intend
to analyze the in vivo roles of these
phosphatases using conditionally AUMand Chronophin-deficient mouse models.
Fig. 2:
Model of Ca2+-dependent cellular cofilin
phosphoregulation.
Extramural Funding
DFG (SFB 728, TP A3)
DFG (SFB 688, TP A11)
Selected Publications
Stan, A., Pielarski, K. N., Brigadski, T.,
Wittenmayer, N., Fedorchenko, O., Gohla, A., Lessmann, V., Dresbach, T., and
Gottmann, K. (2010). Essential cooperation of N-cadherin and neuroligin-1 in
the transsynaptic control of vesicle accumulation. Proc Natl Acad Sci USA,
107, 11116-21.
Bender, M., Eckly, A., Hartwig, J. H., Elvers, M., Pleines, I., Gupta, S., Krohne,
G., Jeanclos, E., Gohla, A., Gurniak, C.,
Gachet, C., Witke, W., and Nieswandt, B.
(2010). ADF/n-cofilin-dependent actin
turnover determines platelet formation
and sizing. Blood, 116, 1767-75.
Fig. 3:
AUM regulates tyrosine phosphorylation in focal adhesions. AUM was depleted by RNA interference,
cells were spread on fibronectin for 10 minutes, and tyrosine phosphorylated proteins were stained
using 4G10 antibodies.
von Holleben, M.*, Gohla, A.*, Janssen,
K. P., Iritani, B. M., and Beer-Hammer,
S. (2011). Immunoinhibitory adapter
protein Src homology domain 3 lymphocyte protein 2 (SLy2) regulates actin
dynamics and B cell spreading. J Biol
Chem, 286(15), 13489-501.
* contributed equally
35
Research Professorships
Bernhard Nieswandt
E-mail: [email protected]
Phone: +49(0)931 31 80405
Fax:
+49(0)931 31 61652
http://www.rudolf-virchow-zentrum.de/forschung/nieswandt.html
Platelet activation and subsequent formation of fibrin rich thrombi at sites of vascular injury is not only crucial
for hemostasis but is also a major pathomechanism underlying myocardial infarction and stroke. Although major
advances in understanding basic platelet functions such as adhesion, activation, aggregation, secretion, and
procoagulant acitvity have been made during the last few years, the mechanisms of how platelets orchestrate
hemostasis and inflammation are only partly understood. We work on the role of platelet membrane glycoproteins, their signaling pathways, and their inteaction with the coagulation system in hemostasis, thrombosis,
and ischemia-reperfusion injury. Using genetically modified mouse lines we study these mechanisms in vitro
by cell biological techniques and in vivo by intravital microscopy-based models of thrombosis and inflammation.
A new area of reseach that we are now interested in is the process of platelet production from their bone
marrow precursor cells, the megakaryocytes (MK).
Factor XIIa inhibitor recombinant human
albumin Infestin-4 abolishes occlusive
arterial thrombus formation without affecting bleeding.
Upon vascular injury the plasma coagulation system becomes activated and acts
in concert with blood platelets to form a
fibrin- and platelet-rich clot. Blood coagulation is a tightly regulated process of sequentially activated proteases that can be
induced by the extrinsic or the intrinsic
pathway. The factor XII (FXII)-induced intrinsic pathway, however, was long consid-
ered to be irrelevant for physiological clot
formation. Recent studies with FXII-deficient mice changed this view and revealed
that the FXII-induced pathway is essential
for pathological thrombus formation but
dispensable for hemostasis, which proposed FXII as a promising target for safe
antithrombotic therapy. We could confirm
the high potential of this therapeutic strategy by the activated factor XII (FXIIa) inhibitor rHA-Infestin-4 (recombinant human
albumin Infestin-4). This fusion protein,
cloned from the hematophagus insect Triatoma infestans, specifically inhibits FXIIa
and consequently causes prolonged aPTT
(activated partial thromboplastin time) in
mice, rat and human plasma. Intravenous
injection of rHA-Infestin-4 in mice or rats
resulted in completely abolished pathological thrombus formation, whereas it did
not influence bleeding times. Additionally,
rHA-Infestin-4 protects mice from ischemic
stroke (Fig.1). These results identify rHAInfestin-4 as a promising agent to achieve
powerful protection from ischemic cardioand cerebrovascular events without affecting hemostasis (Hagedorn et al., Circulation, 2010).
Phospholipase D1 (PLD1) is required for
glycoprotein (GP) Ib–dependent aggregate formation under high shear conditions.
Fig. 1:
(A) rHA-Infestin-4 completely blocks arterial thrombus formation in mice. Endothelial damage
was induced by topical application of FeCl3 on mesenteric arterioles and thrombus formation
was monitored using intravital fluorescence microscopy. An asterisk indicates full vessel occlusion.
Bar=50 µm. (B) rHA-Infestin-4-treated mice are protected from ischemic stroke. Representative
coronal sections from control and rHA-Infestin-4-treated mice stained with TTC 24 h after transient
middle cerebral artery occlusion. Arrows indicate infarct areas (white tissue). (C) Normal bleeding
times in rHA-Infestin-4-treated mice in a tail bleeding model. Each symbol represents one
individual. Ctrl=control; Inf-4=rHA-Infestin-4.
36
Platelet activation triggers phospholipasemediated cleavage of membrane phospholipids to generate lipids and soluble messengers. An essential role in platelet
signaling has been established for phospholipase C (PLC), but not for PLD and its
products choline and phosphatidic acid
(PA). Platelets express two PLD isoforms,
PLD1 and PLD2, which translocate to the
plasma membrane during platelet activation, and both isoforms were suggested to
be involved in platelet degranulation. To
reveal the role of PLD1 in platelet activation we generated Pld1-/- mice. Platelets
from these mice displayed no defects in
granule release, but integrin αIIbβ3 activation was impaired in response to thrombin receptor or GPVI stimulation and this
defect could be ascribed to the lack of
PLD1-generated PA. The most intriguing
phenotype of PLD1-deficient platelets was
however a selective adhesion defect on
immobilized vWF at high shear rates (Fig.
2), indicating a central function of the enzyme in GPIb-triggered signaling events.
This defect resulted in severe thrombus
instability in Pld1-/- mice and protection
from occlusive arterial thrombus formation
and ischemic brain infarction (Fig. 2). Remarkably, tail bleeding times of the mice
were unaffected, indicating that this pathway is dispensable for normal hemostasis
(Elvers et al., Sci Signal, 2010).
have the capacity to generate platelets.
While microtubules are the main structural
component of proplatelets and microtubule
sliding is known to drive proplatelet elongation, the role of actin dynamics in the
process of platelet formation has remained
elusive. Actin-depolymerizing factor (ADF)
and cofilin are actin-binding proteins that
coordinate actin dynamics by actin depolymerization and severing. We tailored a
mouse model lacking all ADF/cofilin-mediated actin dynamics in MK in order to specifically elucidate the role of actin filament
turnover in platelet formation. We demonstrated for the first time that in vivo actin
filament turnover plays a critical role in the
late stages of platelet formation from MK,
and the proper sizing of platelets in the
Extramural Funding
DFG (SFB 688 TP A1; TP B1; SFB 487 TP
C6; Ni 556/8-1)
Selected Publications
Bender, M., Eckly, A., Hartwig, J. H., Elvers, M., Pleines, I., Gupta, S., Krohne,
G., Jeanclos, E., Gohla, A., Gurniak, C.,
Gachet, C., Witke, W., and Nieswandt B.
(2010). ADF/n-cofilin-dependent actin
turnover determines platelet formation
and sizing. Blood, 116(10), 1767-75.
Fig. 2:
(A) Pld1-/- platelets fail to firmly adhere to vWF
under flow. Whole blood was perfused over
immobilized murine vWF with the indicated shear
rates, then washed with Tyrode’s buffer for a
period equal to the perfusion time. Bar graphs
depict mean values ± SD of firmly adhered
platelets on the vWF-coated surface (n ≥ 3 mice
each). (B). Reduced thrombus stability of Pld1-/platelets in vivo. The right carotid artery of the
indicated mice was injured by topical application
of 15% ferric chloride and time to irreversible
occlusion was determined with a Doppler flowmeter. Each symbol represents one individual
mouse. (C) Pld1-/- mice are protected from
cerebral ischemia. Representative images of
coronal sections from wild-type and Pld1-/- mice
stained with TTC 24 hours after transient middle
cerebral artery occlusion.
ADF/n-cofilin-dependent actin turnover
determines platelet formation and size
Blood platelets originate from bone
marrow megakaryocytes (MK). The cellular
and molecular mechanisms orchestrating
the complex process by which MK form
and release platelets remain poorly
under-stood. Mature MK generate long cytoplasmic extensions, proplatelets, which
periphery (Fig. 3). Our results provide the
genetic proof that platelet production from
MK strictly requires dynamic changes in the
actin cytoskeleton mediated by ADF and
cofilin (Bender et al. Blood, 2010).
Fig. 3:
Cofilin-null platelets are markedly increased in
size, whereas ADF/cofilin-null platelets display
striking variability in size and morphology, and
an abnormal platelet ultrastructure. Transmission electron microscopical analysis of resting
platelets. Scale bar: 2 µm (upper panel).
Visualization of the cytoskeleton of resting
platelets on poly-L-lysine. Scale bar: 1 µm
(lower panel). Note: ADF/cofilin-null platelets
display a proplatelet-like structure.
Bender, M., Hofmann, S., Stegner, D.,
Chalaris, A., Bösl, M., Braun, A., Scheller, J., Rose-John, S., and Nieswandt, B.
(2010). Differentially regulated GPVI
ectodomain shedding by multiple platelet-expressed
proteinases.
Blood,
116(17), 3347-55.
Elvers, M., Stegner, D., Hagedorn, I.,
Kleinschnitz, C., Braun, A., Kuijpers,
M. E., Boesl, M., Chen, Q., Heemskerk.
J. M., Stoll, G., Frohman, M. A. and
Nieswandt, B. (2010). Impaired alpha(IIb)beta(3) integrin activation and
shear-dependent thrombus formation in
mice lacking phospholipase D1. Sci Signal, 3(103), ra1.
Hagedorn, I., Schmidbauer, S., Pleines,
I., Kleinschnitz, C., Kronthaler, U., Stoll,
G., Dickneite, G., and Nieswandt, B.
(2010). FXII inhibitor rHA-Infestin-4
abolishes arterial us formation without affecting bleeding. Circulation,
121(13), 1510-17.
Pleines, I., Eckly, A., Elvers, M., Hagedorn, I., Eliautou, S., Bender, M., Wu,
X., Lanza, F., Gachet, C., Brakebusch, C.,
and Nieswandt, B. (2010). Multiple
alterations of platelet functions dominated by increased secretion in mice
lacking Cdc42 in platelets. Blood,
115(16), 3364-73.
37
Senior Professorships
Roland Benz
E-mail: [email protected]
Phone: +49(0)931 201 48903
Fax:
+49(0)931 201 48123
http://www.rudolf-virchow-zentrum.de/forschung/benz.html
We are interested in the biophysics of membranes and membrane components. Of special interest in recent years
was the interaction of toxins with biological and artificial membranes. This can lead to the formation of pores in
the membranes followed by a collapse of membrane structure and the dissipation of membrane potential caused
by the rapid leak of ions out of the cell. Other toxins act as enzymes on intracellular targets. The toxins have to
be transported across membranes, otherwise the toxic activity cannot be developed inside the target cells.
Translocation of prokaryotic toxins into eukaryotic cells is a rather simple process that requires only one or two
proteins and no energy. The transport of toxins into target cells is very often combined with the formation of
pores in the target cell membrane. Besides studying the interaction of toxins with membranes and the
transport of toxins across membranes, we are interested in the permeability properties of bacterial cell walls.
The matrix space of gram-negative and certain gram-positive bacteria is surrounded by two membranes, or a
membrane and a mycolic acid layer. Outer membrane and mycolic acid layers act as specific molecular sieves.
Small, water-soluble molecules permeate these barriers with high velocity through aqueous pores. The study of
their permeability properties is of considerable interest. All substrates essential for growth of these bacteria
or the export of proteins or harmful substances out of the cells have to pass the cell wall through channels,
which we study in artificial lipid bilayers.
Binary toxins
Binary toxins are among the most potent
bacterial protein toxins performing a cooperative mode of translocation and exhibit
fatal enzymatic activities in eukaryotic
cells. Anthrax toxin of Bacillus anthracis
and C2 toxin of Clostridium botulinum are
the most prominent examples for the AB7
type of toxins, which are composed of an
enzymatic unit A and a binding component
B, and which forms heptamers on the
surface of target cells (see Figure 1).
Fig. 1:
Water-soluble form of the PA heptamer of
B. anthracis. The white parts form β-hairpins,
responsible for the formation of a heptameric
transmembrane channel.
38
The A-component of C2-toxin has ADP-ribosyltransferase activity inside the eukaryotic
target cells and enters them via a heptamer
of the binding component B. Bacillus anthracis produces a similar A1A2-B toxin. In
this case the toxic activity is caused by a
receptor binding moiety B called protective antigen (PA) and two enzymatically
active components A1 and A2. One of them
is the edema factor (EF; a calcium and
calmodulin-dependent adenylate-cyclase).
The other one is the lethal factor (LF; a
highly specific zinc metalloprotease). To
investigate the mechanism of translocation
of these toxins into target cells and possible cross-reactivity of toxin binding and
translocation, we performed various in vitro
and in vivo experiments by interchanging
the respective A and B components of
Anthrax and C2. Although the binding
and translocation components Anthrax
protective antigen (PA63) and C2II of C2
toxin share sequence homology of about
35%, our results revealed clear functional
differences. In vitro black lipid bilayer
measurements demonstrated that Anthrax
edema factor (EF) and lethal factor (LF)
bind to channels formed by C2II with
higher affinities than C2 toxin’s C2I
binds to Anthrax protective antigen (PA63).
Furthermore, we could demonstrate in vivo
that PA in high concentrations has the
ability to transport the enzymatic moiety
C2I into target cells, causing actin modification and cell rounding, whereas C2II is
not able to efficiently transport Anthrax EF
or LF. Our findings support the commonly
accepted mode of translocation of AB7 type
toxins. In addition, we present the first
evidence that a heterogenic combination
of enzymatic and translocation components
of different AB7 toxins exhibit toxicity to
primary human endothelial cells (HUVECs).
Porins of Borrelia
Porins in the outer membrane of different
gram-negative bacteria were also studied
in recent years. Special emphasis was
given to porins from the outer membrane
of Borrelia. The genus Borrelia causes two
human diseases: Lyme disease (LD) and relapsing fever (RF). Both LD and RF Borrelia
species are obligate parasites and depend
on nutrients provided by their hosts. The
first step of nutrient uptake across the
outer membrane is accomplished by waterfilled pores. Knowledge about porins in
the outer membranes of the different
pathogenic Borrelia species was limited.
Only one porin has been described in
relapsing fever spirochetes, whereas four
porins are known to be present in Lyme
disease agents. Of these, the Borrelia
burgdorferi outer membrane channel P66
is known to act as an adhesin and a porin.
To investigate whether the P66 porin is
expressed and similarly capable of pore
formation in other Lyme disease and relapsing fever Borrelia, three LD species
(B. burgdorferi, B. afzelii, B. garinii) and
three RF species (B. duttonii, B. recurrentis
and B. hermsii) were investigated for outer
membrane proteins homologous to P66
(Figure 2).
A search in published RF ge-nomes, of
B. duttonii, B. recurrentis and B. hermsii,
indicated that they all contain P66 homolog. The P66 homolog of the six Borrelia
species were purified to homogeneity and
their pore-forming abilities, as well as
the biophysical properties of the pores
were analyzed using the black lipid bilayer
assay (Figure 2).
The cell wall of Nocardia farcinica contains
a cation-selective channel, which may be
responsible for the limited permeability of
the cell wall of N. farcinica for negatively
charged antibiotics. Based on partial
sequencing of the protein responsible
for channel formation, derived from
N. farcinica ATTC 3318, we were able to
identify the corresponding genes (nfa15890
and nfa15900) within the known genome
of N. farcinica IFM 10152. The corresponding genes of No farcinica ATTC 3318 were
separately expressed in E. coli BL21 DE3
Omp8 and the N-terminal His10-tagged proteins were purified to homogeneity. The
pure proteins were designated NfpANHis and
NfpBNHis, for Nocardia farcinica porin A and
Nocardia farcinica porin B. The proteins
were checked separately for channel formation in lipid bilayers. Our results clearly
indicate that only together could the
proteins NfpANHis and NfpBNHis expressed
Fig. 3:
Upper panel: Prospective structure of the NfpA/
NfpB oligomeric cell wall channel of Nocardia
farcinica. It is assumed that four NfpA–NfpB
subunits (shown in the four colors) form the
cell wall channel. The channel is seen from the
surface side. Lower panel: Side view of the
prospective structure of the NfpA/NfpB oligomeric cell wall channel of Nocardia farcinica.
The graphics were designed using rasmol.
Extramural Funding
DFG GK 1048
DFG GK 1342
SFB 487 TP A5
Selected Publications
Fig. 2:
Current steps observed with diphytanoyl phosphatidylcholine/n-decane membranes shown for P66
of three LD species (B. burgdorferi, B. afzelii and B. garinii) and two RF species (B. duttonii and
B. recurrentis).
Cell wall channels from mycolata
Many years ago we were able to demonstrate the presence of channels in the cell
walls of two gram-positive bacteria, Mycobacterium chelonae and Mycobacterium
smegmatis. Recently, we could show that
other actinomycetes, such as Corynebacterium glutamicum, Nocardia farcinica, Nocardia asteroides and Rhodococcus erythropolis
also contain cell wall channels.
in E. coli form a channel in lipid bilayer
membranes, suggesting that the cell wall
channel of N. farcinica is formed by a heterooligomer (Figure 3). Together NfpA and
NfpB form a channel structurally related to
MspA of Mycobacterium smegmatis based
on amino acid comparison.
Bárcena-Uribarri, I., Thein, M., Sacher,
A., Bunikis, I., Bonde, M., Bergström,
S., and Benz, R. (2010). P66 porins are
present in both Lyme dis-ease and relapsing fever spirochetes: a comparison
of the biophysical prop-erties of P66
porins from six Borrelia species. Biochim Biophys Acta, 1798(6), 1197203.
Barth, E., Barceló, M.A., Kläckta, C.,
and Benz, R. (2010). Reconstitution experiments and gene deletions reveal the
existence of two-component major cell
wall channels in the genus Corynebacterium. J Bacteriol, 192(3), 786-800.
Kläckta, C., Knörzer, P., Rieß, F., and
Benz, R. (2010). Hetero-oligomeric cell
wall channels (porins) of Nocardia farcinica. Biochim Biophys Acta, 1808(6),
1601-10.
39
Senior Professorships
Martin Heisenberg
E-mail: [email protected]
Phone: +49(0)931 31 84451
Fax:
+49(0)931 31 83255
http://www.rudolf-virchow-zentrum.de/forschung/heisenberg.html
One of the great challenges in natural sciences is the basic organization of the brain. What is needed is a model
describing the basic processes and states of the brain generating the stream of behaviors of an animal or human
throughout their life. We need a model that accounts for the amazing precision, and at the same time the remarkable error-tolerance of behavior, which explains how experience from the past can affect the future, how the
pressure of demands and constraints can be mitigated in advance, and how the continuity and identity of an
individual can be preserved over many decades.
Brain and behavior
Our brain is 10-20 million-fold larger than
the brain of a fruitfly (Drosophila melanogaster). Yet all the basic functions our
brain also performs for us, the fly brain
performs for the fly. The fly is not only a
favourite model organism of genetics, it
also offers unique opportunities in brain
research. So much smaller, means so much
simpler. Moreover, the fly provides genetic
tools to have its brain manipulated in
unprecedented detail for analysis. It may
well be with the Drosophila brain that a
functional model is first established. We
have started to investigate the endogenous brain processes that control the fly‘s
behavior.
Since the discovery of the spinal reflex
by C. S. Sherrington a large fraction of
the neuroscience community still considers
that the primary task of the brain is to
integrate the sensory stimuli and transform
them into motor commands. They maintain
that the basic constituents of brain function, the neurons and glia cells, signalling
molecules and ion channels are sufficiently
well known, and only the details remain to
be filled in.
We adhere to the alternative view that a
functional model of the brain has to be
based on the autonomy of the organism.
Animals and humans are able to generate
behavior of their own accord (‘self’). This
is a crucial aspect of the functional organization of brains. We have unequivocal
evidence that behavior can originate in
the fly. The behavioral output may be
guided, but is often not initiated by
the sensory input.
An animal or human can initiate behavior because their brain is active. Brain
40
activity occurs by itself. The term “by
itself” is justified, because in the search
for the right behavioral module at the
appropriate moment the brain may rely on
the catalytic element of chance. If behavior was fully determined by sufficient
causes and their causes, etc., back to the
beginning of the Universe, no behavior
would be one‘s own. The “self”, the subject
would not exist as a meaningful concept.
We record and characterize the behavioral
activity of single animals during, for instance, visual orientation or operant conditioning, in order to analyze its temporal
structure, such as the stochastic properties
of turning manoeuvres or the temporal distribution of errors. How are these patterns
influenced by time of day, diet, age, other
sensory stimuli, prior experience, etc. How
does a fly distinguish self-induced from externally derived sensory stimuli? Can genes,
neurons and tissues be identified that are
specifically required for the control of activity? From comparing activity in different
behaviors we expect to get a glimpse of the
general rules that constitute the basic organization of behavior. The brain continuously adjusts the outcome expectations for
the behavioural options and eventually selects the right behaviour. We expect activity to play a major role in this process. A
better understanding of activity in behaviour would have considerable clinical
(ADHS; M. Parkinson; depression) and practical (robotics) relevance.
behaviour. A fly tethered at a torque meter
(Fig.1) spontaneously generates yaw torque
to the left and right. If it is now heated by
a laser beam when ever it tries to turn left
(or right), it will reduce the frequency of
intended left (or right) turns. The fact that
this works for intended left as well as right
turns, shows that the turns cannot be responses to the stimulus (heat). The fly tries
out ways of how it can escape from the
heat. It detects the coincidence between
the change in temperature and its behaviour. This enables the fly to influence the
temperature. Its behavior is called operant
conditioning, if after some practice it suppresses intended left (or right) turns as a
Operant conditioning
In operant conditioning a fly activates behavioural modules and changes the frequency of activation for a particular module according to the consequences of this
Fig. 1:
Drosophila at the flight simulator. Turning in
the horizontal plane is simulated by rotating the
virtual panorama on the LED display in the
opposite direction. (Yaw torque proportional to
angular velocity.)
precaution even after the laser is switched
off permanently.
We compare this learning task to other
paradigms of operant conditioning such as
learning to cope with inverted feedback in
the flight simulator and learning not to
rest in the heat box. Do the time traces of
the behaviour reveal a discrete moment at
which the fly discovers how to solve the
problem? To what extent do flies vary in
the delay until they discover the solution?
Does this delay get shorter if flies have
gone through one of these experiments
before? Do flies go through a sequence of
behavioural adjustments? Is this sequence
the same from fly to fly, or do flies have
distinct strategies?
Visual attention
In visual attention brain activity organizes
the relation between the visual stimulus
and the behaviour. At the torque meter the
fly often restricts its visual behavior to
only a fraction of its visual field. Rarely
does it just respond to the sum of all visual
stimuli. On the one hand, it can actively
shift its focus of attention, on the other,
its attention can be steered to a certain
position by external stimuli, visual and
non-visual (Wolf and Heisenberg 1980;
Heisenberg and Wolf 1984). Visual attention has long been known in humans and
other primates.
In an on-going study we investigate the
guidance of visual attention by external visual stimuli. We have identified stimuli
that steer the focus to the side where
they occur and others that steer it away
from that side. Their steering effect is independent of the yaw torque responses
they elicit. Steering is restricted to the
lower half of the visual field (P. Sareen, in
prep.). This finding should help to identify
the neural substrate of visual attention in
the fly.
Learned helplessness
We study a special kind of behavioral
plasticity called “learned helplessness”.
This kind of default learning has been
found throughout the animal kingdom
and is considered an animal model of clinical depression. It is the safety switch of
operant behavior. If an animal tries everything to avoid a noxious stimulus, but to
no avail, there must be a time point when
it is better to stop trying.
We have built a small chamber in which a
single fly can walk back and forth; its
position is recorded and the temperature in
the chamber can be acutely regulated (heat
box, Fig. 2). Flies are treated in this chamber for 10 minutes by noxious heat pulses
That they can not control. Subsequently,
they are tested in place learning and
perform significantly worse than control
flies that received the same sequence of
heat pulses but under their own control
(in a different learning task). Surprisingly,
learned helplessness is more pronounced
in females than in males and it can be
“cured” by three common anti-depressant
drugs that are supposed to increase the
serotonin concentration at serotonergic
synapses (F. Bertolucci, Doctoral Thesis,
Würzburg 2008).
We want to better understand the role of
serotonin in learned helplessness. Is the
gender difference a property of the brain?
Do female brains contain less serotonin
than male brains? Would reduction of serotonin cause males to give up earlier? The
abundant genetic tools available for this
project promise unique insights into the
neural basis of stress and perseverance in
flies.
In addition to the molecular and cellular
studies further investigations will address
the nature of the learning deficit. How long
does helplessness last? Does it generalize
to other operant learning tasks? Does it
affect other behaviours such as aggression,
courtship, sleep, feeding and circadian
rhythms? Animal models of psychoses are
in demand.
Fig. 2:
The heat box. Single flies are confined to small
boxes where they can walk back and forth in
complete darkness. Their position in the box is
continuously recorded, and the boxes can be
instantaneously heated (for conditioning).
Extramural Funding
DFG GK 1048
DFG GK 1342
Selected Publications
Yamaguchi, S., Desplan, C., and Heisenberg, M. (2010). Contribution of photoreceptor subtypes to spectral wavelength preference in Drosophila. Proc
Natl Acad Sci, 107, 5634-39.
Heisenberg, M. (2010). Von Natur aus
frei – Die Organisation menschlichen
und tierischen Verhaltens ermöglicht
Freiheit. Theologie und Glaube, 100,
208–15.
Schmid, B., Schindelin, J., Cardona, A.,
Longair, M., and Heisenberg, M. (2010).
A high-level 3D visualization API for
Java and ImageJ. BMC Bioinformatics,
11, 274.
41
RVZ Network Project
Martin Eilers
Theodor-Boveri-Institute, Physiological Chemistry II
E-mail: [email protected]
Phone: +49(0)931 31 84111
Fax:
+49(0)931 888 4113
http://pch2.biozentrum.uni-wuerzburg.de/
Cancer is a genetic disease caused by the accumulation of mutations in proto-oncogenes and tumor suppressor
genes. Epigenetic mechanisms also contribute to the silencing of tumor suppressor genes. Individual cancers
differ form each other in the set of genetic or epigenetic alterations that drive cancer development.
The proto-oncogene MYC and two closely related genes, MYCN and MYCL, are key factors that drive the
development of most human tumors. With the exception of lymphomas, mutations in MYC are relatively
rare. However, expression of one of the three MYC genes is enhanced and deregulated in the majority of
human tumors. A large number of transgenic experiments show that deregulated MYC is a major promoter
of tumorigenesis.
Fig. 1:
Sections of T-cell lymphomas developing in transgenic mice that express either wild type Myc or a point mutant of Myc
(MycV394D) that does not bind to Miz1. The panels show that Myc-induced apoptosis is decreased, but that lymphoma cells
expressing MycV394D also accumulate K9-methylated histone H3, a histone modification that is typical for cells undergoing
senescence (see van Riggelen et al., 2010).
42
Our group aims to understand how the Myc
oncoprotein contributes to tumorigenesis.
Myc is a transcription factor that can form
a transcriptional activator complex with a
partner protein called Max. My group has
shown that Myc can also form a ternary
complex that in addition to Max contains a
zinc finger protein called Miz1 to repress
transcription. During 2010, we published
evidence that complex formation with
Miz1 is critical to suppress a form of
terminal growth arrest called senescence
during Myc induced lymphomagenesis (van
Riggelen et al., 2010; Müller et al., 2010).
We also cooperated with Tark Möröy to
continue the analysis of Miz1-knockout
animals, and together published the analysis in B-cells (Kosan et al., 2010). Current
projects look at the role of the Myc/Miz1
complex in the self renewal of neuronal
stem cells and during the biogenesis of
glioblastomas; this work is part of the
SFB581 (“Molecular models for diseases of
the neuronal system”).
As one technically new approach, we have
established high throughput sequencing
and used it to determine all binding sites
for Myc and Miz1 in the human genome.
Surprisingly, this has lead to the identification of thousands of genes that are jointly
bound by Myc and Miz1, and to the realization that many biological processes may be
controlled by these factors. Most of our
current work focuses on cell migration and
invasion, two processes that are tightly
linked to the development of metastases.
We also use high throughput sequencing
in combination with lentiviral shRNA
screening to screen thousands of individual
shRNAs in a single experiment in order to
find genes that are required to maintain
the transformed states of Myc-expressing
cells. These screens are currently running
in neuroblastoma and, as a close collaboration with the group of Ralf Bargou at
the university clinic, in multiple myeloma.
We also use it to identify genes that
mediate chemoresistance genes in myeloid
leukemia.
Activation of single oncogenes in primary
cells does not cause transformation, but
induces mechanisms that eliminate the
affected cell and thereby protect the organism. One of these mechanisms is apoptosis, which is triggered by deregulated
expression of Myc. This involves induction
of a small tumour suppressor protein, called
Arf, that is specifically induced by very
high, but not physiological levels of Myc.
We also found that Arf in turn binds to Myc
and Miz1, induces them to repress a group
of genes encoding cell adhesion proteins to
trigger cell detachment and anoikis (Herkert et al., 2010). How precisely Arf acts to
control Myc and Miz1 function is currently
unclear, and we have evidence that Arf-mediated sumoylation may play a key role.
This hypothesis is being actively pursued.
We also study other factors that control the
assembly of the Myc/Miz1 complex.
In a separate line of experiments, we
continued our analysis of Myc turnover
control and analysed the interaction of
the β-TrCP ubiquitin ligase with Myc. In
non-tumor cells, Myc is rapidly turned
over by the Fbw7 ubiquitin ligase. However, in specific situations, turnover is
disrupted, one being the recovery of cells
from arrest in the S-phase of the cell
cycle. We have now found that this is
due to competitive ubiquitination, where
β-TrcP can ubiquitinate the same acceptor
site in Myc as in Fbw7, but assembles an
ubiquitin chain that is fairly inefficient in
triggering Myc degradation (in contrast to
that assembled by Fbw7), causing transient stabilization of the protein (Popov
et al., 2010). In order to gain a better
insight into how Myc stability is controlled in vivo, we have generated a conditional knockout mouse for Usp28, a deubiquitinating enzyme that counteracts
degradation by Fbw7. We have found that
several targets of Fbw7 are destabilized in
Usp28 knockout mice, arguing that Usp28
has an important physiological function in
counteracting Fbw7 function in vivo.
Selected Publications
Kosan, C., Saba, I., Godmann, M., Herold, S., Herkert, B., Eilers, M., and
Möröy, T. (2010). Transcription factor
miz-1 is required to regulate interleukin-7 receptor signaling at early
commitment stages of B cell differentiation. Immunity, 33, 917-28.
Popov, N., Schülein, C., Jaenicke, L.
and Eilers, M. (2010). Ubiquitylation
of the amino-terminus of Myc by
SCF(beta-TrCP) antagonizes (Fbw7)mediated degradation. Nat Cell Biol,
12, 973-81.
van Riggelen, J., Müller, J., Otto, T.,
Beuger, V., Samans, B., Yetil, A., Tao, J.,
Choi, P., Kosan, C., Möröy, T., Felsher,
D., and Eilers, M. (2010). The interaction between Myc and Miz1 is required
to
antagonize
TGFbeta-dependent
atutocrine signaling during lymphoma
formation and maintenance. Gene Dev,
24, 1281-94.
Herkert, B., Dwertmann, A., Herold, S.,
Naud, J. F.,Finkernagel, F., Harms, G. S.,
Wanzel, M., and Eilers, M. (2010). The
Arf tumor suppressor protein inhibits
Miz1 to suppress cell adhesion and induce apoptosis. J Cell Biol, 188 (6),
905-18.
Herkert, B., and Eilers, M. (2010). Transcriptional Repression: the dark side of
Myc. Genes and Cancer, 1, 580-86.
43
RVZ Network Project
Manfred Gessler
Theodor-Boveri-Institute, Developmental Biochemistry
E-mail: [email protected]
Phone: +49(0)931 31 84159
Fax:
+49(0)931 31 87038
http://www.biozentrum.uni-wuerzburg.de/pc1/gessler
The Notch signaling pathway is an evolutionary highly conserved signaling pathway involved in a multitude of
developmental decisions. Typical scenarios for such signals are binary cell fate decisions, although also inductive
signals and border formation. This holds true for not only embryonic development, but also in later life. The
pathway conveys many of its effects through the Hey and Hes bHLH transcription factors, which represent key
transcriptional targets. We have previously shown that Hey genes are critical for correct development of the
heart, as reflected by the fact that Hey2-/- as well as Hey1-/- / HeyL-/- mice suffer from severe congenital heart
defects. Hey1 and Hey2 are also essential for angiogenesis and arterial fate decision, since combined knockout
in the mouse leads to early embryonic lethality due to hemorrhage. We have since been able to extend this work
to other organ systems (e.g. neural, thymus and bone development). In addition it has become obvious that Hey
factors not only interact with themselves and complement each other, but they also form complexes with the
related Hes proteins in vitro. Our first in vivo evidence was again a vascular phenotype when a Hey2 deletion is
combined with Hes1 knockout alleles. The latter was previously implicated primarily in neurogenesis and lymphocyte development, but apparently this view was too narrow.
Targets of Hey and Hes factors
Using our well established inducible vector
system we overexpressed Hey1, Hey2 and
Hes1 in human HEK293 cells. Microarray analysis showed an overlap in target
genes between the three bHLH transcription factors, with the two Hey factors sharing the majority of their regulated targets.
This has its roots in the similarity of the
DNA-binding basic region that apparently
leads to quite related or identical target
sites. On the other hand, a significant fraction of regulated genes appears to be specific for Hey or Hes factors. We have been
able to validate a large set of regulated
genes by quantitative real-time RT-PCR and
in several cases in vivo validation seems
possible as well. The predominant mode of
action is repression of gene expression, but
the magnitude of regulation tends to be
limited to a range of up to only 4-fold.
inducible transgenes. An almost complete
overlap of target sites could be seen for
Hey1 and Hey2 in human and mouse cells
(Fig. 1). For many genes the profiles of Hey
binding are quite similar between human
and mouse DNA, suggesting a strongly
conserved mode of action for Hey proteins
in both species.
ChIP analysis of Hey and Hes proteins
To provide evidence for direct regulation of
targets and identify additional candidates, we extended our ChIPseq analyses
(next-generation sequencing of DNA from
chromatin IP) of Hey1 and Hey2 from
HEK293 cells to include mouse embryonic
stem (mES) cells that harbor doxycyclin-
44
Fig. 1:
Hey1 and Hey2 target genes strongly overlap. 57% of the top 1000 binding sites observed in ChIPseq
experiments overlap between the two proteins. The remaining targets are mostly contained within the
top 5000 sites of the other Hey factor.
Comparing the effects of Hey1/2 and
Hes1 again revealed more substantial
divergence. While a significant set of genomic Hey targets is also bound by Hes1,
there are several examples where Hes1 does
not bind to the corresponding sites. Thus,
there is clear evidence for shared as well as
unique modes of action for Hey and Hes
proteins. this may explain the different
phenotypic effects seen in knockout mice
that cannot be attributed to differences
in expression patterns alone.
Gene ontology analysis classified target
genes of Hey and Hes proteins as being
part of developmental control and organogenesis circuits. This is in line with the
proposed functions of both gene families.
Their inherent propensity to form heterodimers as shown by co-immunoprecipitation as well as mass spectrometry further
points to overlaps in target site recognition and also partial functional redundancy,
but this will only be fully understood using
in vivo systems.
mouse development, we generated a whole
series of allelic combinations in mice and
characterized these mice in collaboration
with several expert collaboration partners.
This has led to a model of multiple
organs or cell types depending on singular
or specific combinations of Hey or Hes
genes for their proper development and or
subsequent homeostasis (Fig. 2). Some of
these phenotypes will need further charac-
terization to fully understand their molecular basis. Elucidating the function of
Hey and Hes proteins in cells and animal
models will allow us to better understand
their importance for embryonic developmental processes, as well as to gain
insights into the pathogenesis of cardiovascular and other diseases and the novel
interplay of these genes in neural development and immunological processes.
Cooperation between Hey2 and Hes1
To search for in vivo cooperation we intercrossed Hey2 and Hes1 knockout lines and
detected an early embryonic lethality that
apparently is due to vascular defects. To
validate the vascular system as being the
critical site, we employed mice with a
floxed Hes1 allele in combination with a
tie2-cre deleter line. Similar early lethality
points to endothelial cells as the critical
site of overlap. The type of defects is
currently under study using histology
and multiple marker staining of embryo
sections. A floxed Hes1 allele also allows
us to analyze postnatal vascular functions tested using a tamoxifen-inducible
VE-Cadherin-cre allele. The corresponding
mouse line is currently being expanded
to study adult vascular regeneration in
the hind limb ischemia model, where
arteriogenesis restoring regional blood
flow can be quantified by laser Doppler
measurements.
Functional interaction of Hey and Hes
proteins
To further define the extent of redundancy
between Hey and Hes genes and delineate
the effects of combined deletions during
Fig. 2:
Phenotypic effects of Hey and Hes gene deletions. Either the heart, blood vessels, thymus, or neural
and neuroendocrine tissues are affect by single or combined (see brackets) deletions of the corresponding genes.
Extramural Funding
SFB 688, TP A16
Selected Publications
Bielesz, B., Sirin, Y., Si, H., Niranjan, T.,
Gruenwald, A., Ahn, S., Kato, H., Pullman, J., Gessler, M., Haase, V. H., and
Suzstak, K. (2010). Epithelial Notch
signaling regulates interstitial fibrosis
development in the kidneys of mice and
humans. J Clin Invest 120, 4040-54.
Wiese, C., Heisig, J., and Gessler, M.
(2010). Hey bHLH factors in cardiovascular development. Pediatr Cardiol 31,
363-70.
45
RVZ Network Project
Roland Jahns
Department of Internal Medicine I/ Pharmacology
E-mail: [email protected]
Phone: +49(0)931 201 463 68
Fax:
+49(0)931 201 646 360
http://www.medizin1.uk-wuerzburg.de/
Research Program GoBio, Federal Ministry of Education and Research (BMBF)
Associated to the Rudolf Virchow Center.
Evidence for a pathophysiological role of autoimmunity in human heart disease has increased substantially over
the past decade. Conformational autoantibodies stimulating the cardiac beta1-adrenoceptor (beta1-aabs) are
thought to be involved in heart failure development. Such aabs allosterically activate the sympathetic signaling
cascade resulting in increased sarcoplasmatic cyclic adenosine-mono-phosphate (cAMP) concentrations that
are harmful for cardiomyocytes. We have developed a highly sensitive fluorescent cAMP-sensor that allows us to
assess cAMP changes directly in living cells by measuring intra-molecular fluorescence resonance energy transfer
(FRET). We used the FRET-method to determine the stimulatory potential of beta1-antibodies generated in rats.
In parallel, we tested the capacity of novel beta1-epitope mimicking cyclopeptides to block stimulatory antibody
effects both in vitro and after in vivo application.
Direct monitoring of a novel cyclopeptide-based therapeutic approach to neutralize cardio-noxious antibodies
Our GoBio research program aims to translate new therapeutic strategies to abolish
cardio-noxious beta1-aabs from (pathophysiologically relevant) animal models
into clinical practice.
We analyze the stimulating potential of
beta1-aabs generated in rats (where the
functionally important second extracellular
beta1-receptor loop is identical to humans)
using a novel fluorescence-based cAMP assay. The effects of antibody-neutralizing
cyclic peptides (a) in vitro and (b) after injection into immunized beta1-aab positive
animals are monitored using this assay.
It is suggested that in the presence of
stimulating beta1-aabs a low level of
sympathetic activity and thus continued
cytoplasmatic cAMP production and calcium load is chronically maintained.
Chronic activation of this pathway may
be deleterious for the heart, resulting in
slowly but steadily progressing myocyte
destruction, fibrotic repair, subsequent
heart muscle dysfunction, and, finally, a
cardiomyopathic phenotype.
While in the rat functional beta1-aabs
may be induced easily by immunization
with beta1/GST fusion proteins, the mechanisms triggering formation of endogenous
heart-directed (auto)antibodies in humans
are still unclear. Acute inflammatory or
ischemic cardiac damage is thought to represent the initial event, resulting in sudden
46
(or chronic) release of a “critical amount”
of potential self-antigens from the myocyte
surface or cytoplasm that were previously
hidden to the immune system. Exposure of
such antigens to the immune system may
then induce a host-directed (auto-)immune
response, which can result in perpetuation
of immune-mediated cardiac damage in-
volving either autoreactive T cells, B cells,
or co-activation of both the innate and
adoptive immune system.
As a model for human beta1-aabs, we
used beta1-ab positive sera from rats
immunized with beta1-ECII/GST fusion
proteins to analyze the in vitro blocking
capacity of cyclo-peptides mimicking the
Fig. 1:
Blocking capacity of beta1-ECII mimicking cyclic peptides (CP) in vitro, determined by ELISAcompetition assays. Pre-incubation with CP achieved ~60-70% signal reduction (P<0.0005).
same epitope (ECII-CP) by competitionELISA. Results obtained with IgG from five
representative beta1-ab positive rats revealed that ECII-CPs significantly reduce
immuno reactivity of rat beta1-abs with
linear receptor peptides by roughly 70%
(from 242±55 to 81±20 µg/ml; mean values ± SD, P<0.0005; Fig. 1). Next, we analyzed the inhibitory effect of ECII-CP on
antibody-induced activation of the adrenoceptor signalling cascade using our novel
functional FRET-assay. IgG from control
rats had only negligible effects on receptor
activity (~5%), whereas beta1-abs from
immunized animals activated the beta1-receptor by more than 20% (compared to the
full agonist (-)isoproterenol set at 100%;
Fig. 2a). In vitro pre-incubation of stimulating rat beta1-abs with ECII-CP efficiently blocked receptor- signalling and reduced
beta1-ab-induced cAMP-production by
more than 80% (from 22% to 3% ; Fig. 2a).
Subsequently, we checked whether in vivo
administration of ECII-CP into immunized
rats had an effect on the adrenoceptoractivating potential of their beta1-abs.
Indeed, comparison of the stimulating
potential of IgG from beta1-ab positive
rats before, and 24 hours after a single
injection of 1.0 mg/kg ECII-CP also
revealed a significant antibody-neutralizing effect of ECII-CP in vivo, yielding
about 60% reduction of the initially observed receptor activation (= FRET signals)
(Fig. 2b).
Our data demonstrate that stimulating
beta1-abs induced in rats can be neutralized efficiently by beta1-ECII-mimicking
peptides, both in vitro and transiently
in vivo.
Activating anti-beta1-aabs are supposed
to play an important role in human heart
failure, either as disease-causing agents or
as negative disease modifiers. Preliminary
clinical data even suggest that their presence clearly worsens the prognosis of patients suffering from idiopathic DCM. In
this context, initial pilot experiments with
functionally active beta1-aabs from DCMpatients indicate that antibody-induced
adrenoceptor activation might equally
be abrogated by incubation with beta1ECII-mimicking peptides – similar to those
used in the present study. Although clinical data with epitope-derived antibody
scavengers are still lacking, the findings
presented here should foster further devel-
Fig. 2:
Blocking capacity of beta1-ECII-CP (a) in vitro
and (b) in vivo monitored by functional FRET
assay. Arrows indicate addition of IgGantibodies (ab) or (-)isoproterenol (iso).
a: IgG from control rats had ~5% FRET activity
(left), whereas beta1-ab achieved ~22% FRET
activity (middle). After pre-incubation with CP
in vitro FRET signals were abolished (~3% FRET
activity, right).
b: Rat beta1-abs yielding ~25% FRET activity
(left). 24 h after application of 1 mg/kg ECIICP i.v., beta1-ab-induced signals were reduced
by 60% (~10% residual FRET activity, right).
Figs. reproduced from Jahns R et al., Semin.
Thromb. Hemost. (2010): 36, 212-218.
opment of specifically antibody-directed
therapeutic strategies.
Significant parts of the presented data
have been published in Semin. Thromb.
Hemost. (2010): 36, 212-218. In addition,
the University of Würzburg has filed for
patent protection of the cyclopeptides
described here.
Selected Publications
Jahns, R., Schlipp, A., Boivin, V., and
Lohse, M. J. (2010). Targeting receptorantibodies in immune-cardiomyopathy
Semin Thromb Hemost, 36, 212-18.
Deubner, N., Berliner, D., Schlipp, A.,
Gelbrich, G., Caforio, A. L. P., Felix, S.
B., Fu, M., Katus, H., Angermann, C. E.,
Lohse, M. J., Ertl, G., Störk, S., and
Jahns, R. (2010). Cardiac beta1-adrenoceptor autoantibodies in human heart
disease: rationale and design of the etiology, titre-course, and survival (ETiCS)
study - on behalf of the ETiCS-study
group. Eur J Heart Fail, 12, 753-62.
Jahns, R., Boivin, V., and Lohse, M. J.
(2010). Pathogenetical relevance of
autoantibodies in dilated cardiomyopathy. In: Inflammatory Cardiomyopathy – DCMi – Pathogenesis and Therapy.
Series Progress in Inflammation Research. Parnham MJ Ed., Birkhäuser
Verlag AG, Basel (Switzerland); pp 15972.
47
RVZ Network Project
Thomas Müller
Julius-von-Sachs-Institute, Department of Botany I
E-mail: [email protected]
Phone: +49(0)931 31 89207
Fax:
+49(0)931 31 86158
http://www.bot1.biozentrum.uni-wuerzburg.de
Bone morphogenetic proteins (BMPs) together with growth and differentiation factors (GDFs) represent the largest subfamily within the TGF-β superfamily. Besides their ability to induce bone growth at orthotopic and ectopic
sites, their actions encompass many aspects of proliferation and differentiation during embryonic development
as well as tissue homeostasis in the adult organism. BMP-receptor activation involves two different subsets of
receptors called type I and type II. Upon ligand binding, the Ser/Thr-kinase domains transactivate eachother
leading to phosphorylation and activation of downstream signaling cascades such as SMAD transcription factors
or the p38 MAP kinase. It is remarkable that only a very limited number of receptors exist, serving a considerably
larger number of ligands. About 18 BMP/GDF members signal through using only three different type I and three
different type II receptors. In addition, various ligands can bind and signal through several of these six receptors. This observation raises two main questions: firstly, what is the structural characteristic of the participating
epitopes allowing for these so-called promiscuous protein-protein interactions and secondly, do mechanisms
exist that enable BMPs to evoke ligand-specific responses.
Binding promiscuity is not restricted to the members of the TGF-β superfamily. Supported by an increasing
number of reports, in various protein families, promiscuous binding seems to be the rule, rather than a rare exception. Moreover, during the last ten years our general understanding of how biomolecules recognize and bind
each other has been expanded by several aspects, such as an inherent flexibility of the participating molecules
at the side and also main chain level, or the integration of solvent molecules into the binding interface creating
a high degree of adaptability, with protein interfaces being able to accommodate different partner surfaces,
chemically and geometrically. However, for the TGF-β superfamily promiscuous protein-protein interactions are a
long known characteristic thus this family can be regarded as a prime example for studying both the structural
plasticity underlying this phenomenon and the mechanisms used to generate specificity nonetheless.
Crystallization of a GDF-5:BMPR-IB complex
Growth and differentiation factor 5 (GDF-5)
represents a prototypic member of the
BMP/GDF-subfamily of ligands. The butterfly-shaped GDF-5 homodimer contains four
receptor binding sites, two, referred to as
knuckle-epitopes, binding type II receptors
and two so-called wrist-epitopes, which
present the binding sites for type I receptors. As mentioned above, GDF-5 signaling
occurs via an oligomerized complex containing Ser/Thr-kinase receptor chains of
both subsets, type I and type II. In vitro,
GDF-5 can equally use two different type I
receptors for signaling, BMPR-IA and IB.
However, the in vivo situation paints a
completely different picture. There, unlike
BMP-2, which binds and signals through
both type I receptors without noticeable
difference, GDF-5 exhibits a pronounced
specificity for BMPR-IB. The finding that
mutations in either GDF-5 or BMPR-IB lead
to similar phenotypes in respect to skeletal
malformation indicates that in chondrogenesis proper GDF-5 function requires
48
signaling exclusively mediated through
BMPR-IB. Since at the expression level
both type I receptors are present in zones
where GDF-5 is also expressed, the question emerges of how this specificity is
achieved at the molecular level.
In our recent studies we determined the
structure of the complex of GDF-5 bound to
its receptor BMPR-IB. The two BMPR-IB
ectodomains seem to be arranged in a
ligand-receptor assembly similar to that
found for the complex BMP 2:BMPR-IA.
However, detailed inspection reveals a local change concerning the β1β2-loop of
BMPR-IB, which presumably accounts for
the discrimination between BMPR-IA and
IB by GDF-5. In BMPR-IB a five-residue
segment within this loop is characterized
by high tension due to the presence of only
non-glycine residues in the segment, enforcing either one of two conformational
states, i.e. the “fully-open” and “open”
state. In both conformations of the β1β2loop BMPR-IB can dock to GDF-5, which
harbors the bulky side chain of arginine 57
in its binding site. In BMPR-IA a glycine is
present within this five-residue loop seg-
ment resulting in a “closed” conformation
for this β1β2-loop. This closed conformation of BMPR-IA’s β1β2-loop prevents its
binding to ligands with bulky amino acids
at the equivalent position of GDF-5’s Arg57.
Thus BMP-2 harboring a small alanine
residue at this position can bind equally
well to BMPR-IA and -IB, whereas GDF-5
binding to BMPR-IA will occur with
lower affinity than to BMPR-IB.
Another difference between the complexes GDF-5:BMPR-IB and BMP-2:BMPR-IA
is the orientation of the BMPR-IB and
BMPR-IA ectodomains, which differs by a
change in the angle of tilt by about 9°. Al
though this difference seems small, the
relative orientation of the cytoplasmic type
I and type II receptor kinase domains
might be altered to a remarkable extent:
Assuming that the complete receptor
chains behave like rigid arms, this possibly
amplifies the movements of the extracellular domains through lever actions. Since
these differences in receptor orientation
might result in differences in the transphosphorylation pattern, it possibly presents a mechanism by which a particular
ligand can transduce a specific signal despite using an identical receptor assembly
as used by other BMPs.
Crystal structure of BMPR-IA in a complex with the Fab AbD1556
As mentioned above, proteins engaging in
promiscuous protein-protein interactions
often show inherent protein flexibility at
the side chain as well as main chain level.
From previous studies we have learned that
the ligand-binding domain of BMPR-IA
suggested that the helix would form
regardless of the nature of the ligand.
Thus we tried to determine the structure of
BMPR-IA bound to a binding partner quite
distinct from BMP ligands. In collaboration with AbD Serotec we obtained antibody Fab fragments directed against the
BMPR-IA ectodomain. We could then determine the structure of BMPR-IA bound to
the Fab, AbD1556, which neutralizes BMP
activity due to binding to the same epitope on BMPR-IA as BMP-2. Comparison of
the structures of BMPR-IA in its un-
A possible answer would be that the importance of these residues is not a result of
direct non-covalent interactions with the
ligand but rather from allowing the folding
variability of the loop segment. Thus attenuating the loop’s ability to experience
all conformations, or altering the folding
kinetics impairs binding to both binding
partners regardless of the vast differences
between the final conformations.
Extramural Funding related to project
DFG GK 1048,
SFB 487 B2, DFG MU1093/3-2
Selected Publications
Fig. 1:
(A) Structure of the GDF-5 homodimer (blue and green) bound to the extracellular domain of BMP
receptor IB (red). (B) The 1β2 loop of BMPR-IB adopts two conformations, fully open and open,
providing sufficient space for the bulky residue Arg57 of GDF-5. (C) A comparison with BMPR-IA shows
the β1β2-loop adopting a closed conformation, which results in a steric clash with Arg57 of GDF-5.
undergoes a large conformational change
upon binding to BMP-2. Structure analysis
of unbound BMPR-IA by NMR spectroscopy
revealed that a large part of the BMP-2
binding epitope of BMPR-IA is disordered
and highly flexible before complex formation. Here, the β4β5-loop of BMPR-IA
adopts a defined conformation with a short
2-turn helix upon changes either in the
loop’s environment or upon binding to BMP
ligands. Most importantly, since this helix
element contains the main binding determinants for binding to BMPs, its folding
represents a prerequisite for acquiring
BMP binding capability. Our NMR studies
bound form and when bound to either
BMP-2 or Fab AbD1556 revealed that
the loop element can adopt different
structures, thereby adapting to different
surface geometries, when needed. Therefore promiscuous binding of BMPR-IA is
enabled by structural adaptability, which
is encoded in the inherently flexible
binding site of BMPR-IA. Mutagenesis
studies apparently revealed a paradox:
despite the structural differences seen
for BMPR-IA’s binding epitope, and
despite the different nature of the binding
partners the same set of residues account
for recognition.
Marcel D., Müller, T., Hedrich, R., and
Geiger, D. (2010). K+ transport characteristics of the plasma membrane tandem-pore channel TPK4 and pore chimeras with its vacuolar homologs. FEBS
Lett, 584(11), 2433-39.
Harth, S., Kotzsch, A., Hu, J., Sebald,
W., and Mueller, T. D. (2010). A selection fit mechanism in BMP receptor IA
as a possible source for BMP ligand-receptor promiscuity. PLoS One, 5(9),
e13049.
Harth, S., Kotzsch, A., Sebald, W., and
Mueller, T. D. (2010). Crystallization of
BMP receptor type IA bound to the antibody Fab fragment AbD1556. Acta
Cryst F, 66(Pt 8), 964-68.
Krause, C., Korchynskyi, O., de Rooij, K.,
Weidauer, S. E., de Gorter, D. J., van Bezooijen, R. L., Hatsell, S., Economides,
A.N., Mueller, T. D., Lowik, C. W., and
Ten Dijke, P. (2010). Distinct modes of
inhibition by sclerostin on bone morphogenetic protein and Wnt signaling
pathways. J Biol Chem, 285(53),
41614-26.
Piters, E., Culha, C., Moester, M., Van
Bezooijen, R., Adriaensen, D., Mueller,
T., Weidauer, S., Jennes, K., de Freitas,
F., Loewik, C., Timmermans, J. P., Van
Hul, W., and Papapoulos, S. (2010).
First missense mutation in the SOST
gene causing sclerosteosis by loss of
sclerostin function. Hum Mutat, 31(7),
E1526-43.
Fig. 2:
(A) Structure of the BMP receptor IA bound to AbD1556. (B) The β5β6-loop adopts different
conformations depending on the nature of the binding partner. The conformation of BMPR-IA bound to
BMP-2 (magenta) shows a short α-helix, the conformation bound to AbD1556 (green) is extended but
different from that seen for unbound BMPR-IA (cyan).
Sebald, W., Nickel, J., Zhang, J.L., and
Mueller, T.D. (2010). Molecular basis of
cytokine signalling--theme and variations. Febs J, 277(1), 106-18.
49
Gregory Harms
E-mail: [email protected]
Phone: +49(0)931 31 80357
Fax:
+49(0)931 201 48702
http://www.rudolf-virchow-zentrum.de/forschung/harms.html
The research group Biomedical Molecular Microscopy studies molecular interactions in cell signaling, membrane
proteins and cytosolic messengers. We apply a wide range of different techniques, such as fluorescence resonance
energy transfer (FRET) microscopy, single-molecule microscopy, and dynamic confocal microscopy. Such methods
use custom-built wide-field and confocal microscopes capable of ratiometric FRET, fluorescence recovery after
photo-bleaching (FRAP), fluorescence correlation spectroscopy (FCS), and single-molecule tracking (SMT). These
microscopes allow the detection of low, endogenous levels of proteins in and on living cells. Our key objectives
are the development of biosenors and imaging techniques to study different biological problems. We study the
biological aspects initially using optical microscopy and further apply the dynamic techniques to determine the
temporal distribution of cellular events.
Our research focuses on cell signaling and
kinetics as well as cell adhesion, migration
and cancer progression. Cell adhesion and
migration are investigated via growth and
development as well as molecular signaling
pathways, e.g. serine and tyrosine kinases.
Development and cancer progression are
studied with emphasis on the role of growth
factors, such as the Bone Morphogenetic
Protein (BMP)/Smad pathway. We have
also taken on new challenges such as
the imaging of neurodegeneration. FRET
microscopy allows us to observe both the
dynamics and cellular localization of protein conformational changes and proteinprotein interactions with improved interpretation based on both anisotropy and
fluorescence lifetime. We observe the diffusion dynamics of lipids and proteins by
long-range techniques such as FRAP, to
complement short-range methods such
as FCS. Single-molecules are measured by
wide-field imaging and total internal
reflection fluorescence microscopy (TIRF),
since we now have the latest technologies to monitor long and short diffusion
ranges with tracking, FRET, and co-localization events.
Quantum dots
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 noise. The detection of single-quantum dots (QDots) labeled to such receptors
on living primary cell lines and the maxi-
50
Fig. 1:
Microscopy systems in the RVZ.
mized 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 bioconjugated. Single QDots, in
contrast to standard organic fluorophores,
can easily be discriminated above the
high autofluorescence background of primary cells for fine positioning and are
stable for long times. We have optimized
the use of QDots in living systems to
avoid the traits of blinking and non-fluorescence and have developed new automated detection algorithms for highthroughput screening of single-molecules
in living systems.
Sheet illumination microscopy for improved, sensitive imaging in tissues and
organisms
Our developed versions of selective
plane illumination microscopy (SPIM) and
ultramicroscopy. Use light sheet illumination parallel to the focal image plane of a
microscopy objective, a concept introduced
by Siedentopf in 1903. Our setup enables penetration depths beyond 1 mm
inliving embryos with resolution comparable to confocal microscopy. Growing evidence points to the importance
of tracking low numbers of proteins
in tissues and living organisms. We
successfully applied our SPIM setup to
detect single proteins bound to single
nano-crystals.
Detection of single quantum dots in
model systems with sheet illumination
microscopy
Single molecule detection and tracking
sometimes provides the only possible
method to observe the interactions of low
numbers of biomolecules, inlcuding DNA,
receptors and signal-mediating proteins
in living systems. However, most existing
imaging methods do not enable both high
sensitivity and non-invasive imaging of
large specimens. We now report a new
setup for selective plane illumination microscopy (SPIM), which enables fast imaging and single molecule tracking with
the resolution of confocal microscopy and
optical penetration beyond 300 µm. We
determined instrumental figures of merit,
control values of fluorescence properties
of single nano-crystals in comparison to
both standard widefield configurations,
and also values of nanocrystals in multicellular “fruiting bodies” of Dictyostelium, an
excellent control as a model developmental system.
by either binding to preformed homodimer
complexes of BRI, for signaling in 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 signalling can
now be described by observing individual
receptors on the surface of biologically
relevant cells. Together with Petra Knaus
(Biochemistry, FU Berlin), we developed
methods for single BMP receptor tracking with Qdots and applied this to the
non-signaling truncation mutant of BRII,
called TC1.
Rotational diffusion of the α2A adrenergic receptor revealed by FlAsH labeling
in living cells
The fluorescein arsenical hairpin binder
(FlAsH) shows much promise for determining the relative orientations of protein
regions and structures even in living cells
Fig. 2:
SPIM and Ultramicroscopy. Diagram, AAV-DsRed
image of a mouse spinal cord section, and
transmission/QuantumDotFluorescence image of
a Drosophila larvae.
Fig. 4.
Anisotropy measurements with FlAsH labeling.
Anisotropy images of the α2A adrenergic receptor
with FlAsH “I3-D” loop position in the equatorial
region of the cell. The cell membrane regions
parallel to the excitation polarization can be
clearly observed. Anisotropy: excitation
polarization-black arrow, emission polarization
high - blue arrow, emission polarization low –
green arrow. Anisotropy scaling: 0 to 0.5. Scale
bar: 10 µm.
Extramural Funding
DFG GK 1048
DFG GK 1342
Selected Publications
Bone morphogenetic protein signalling
The BMP signalling system regulates growth
and differentiation and is important for tissue engineering. BMP and BMP receptors
are implicated in diseases such preliminary
pulimary hypertension, juvinile polyposis,
breast cancer, colon cancer, and other
forms of cancer. BMPs, part the of transforming growth factor (TGF-β) super family,
are regulated through two types of receptors, BRI and BRII. The BMP ligand binding
event controls signalling and is regulated
relation time also allowed for comparison
with the theoretical relationship between
translational and rotational diffusion –
originally proposed by Saffman and Delbrück – and revealed a discrepancy of a
factor between 10 and 100.
Fig. 3.
Tracking and internalization of ACP-BRII-TC1 in
living HEK-293 cells. A. Transmission (upper)
and total internal reflection fluorescence image
(lower) of the CoA-quantum dot ACP-BRII-TC1 on
HEK-293 cells (left) and with two traces shown
in red and green of a zoomed portion of the left
image (right).
and the plasma membrane. Together with
Carsten Hoffmann and Martin Lohse, we
characterized FlAsH’s photophysical properties by steady-state anisotropy and timeresolved single photon counting for further
applications with G-protein coupled receptors (GPCRs). We characterized the FlAsH
fluorophore orientation in the α2A adrenergic receptor, revealing rigid orientations of
FlAsH in the membrane plane for rotational
correlation times of approximately 50 ns in
living cells. To elucidate the fluorophoremembrane orientation and rotational correlation time, an anisotropy treatment was
developed. The rotational correlation times
were observed to increase by up to 16 ns
after agonist addition. The rotational cor-
Spille, J. H., Zürn, A., Hoffmann, C.,
Lohse, M. J., and Harms G. S. (2010).
Rotational diffusion of the �α2A adrenergic receptor revealed by FlAsH labeling
in living cells. Biophys J, 100(4), 113948.
Zelman-Femiak, M., Gromova, K., Wang,
K., Knaus, P, and Harms, G. S. (2010).
Covalent quantum dot receptor linkage
via the acyl carrier protein for singlemolecule tracking, internalization and
traffickin studies. Biotechniques, 49,
574-79.
Gliem, M., Heupel, W. M., Spindler, V.,
Harms, G. S., and Waschke, J. (2010).
Actin reorganization contributes to loss
of cell adhesion in pemphigus vulgaris.
Am J Physiol Cell Physiol, 299, C 60613.
Herkert, B., Dwertmann, A., Herold, S.,
Abed, M., Naud, J. F., Finkernagel, F.,
Harms, G. S., Orian, A., Wanzel, M., and
Eilers, M. (2010). The Arf tumor suppressor protein inhibits Miz1 to suppress cell adhesion and induce apotosis.
J Cell Bio, 188, 905-918.
51
Manfred Heckmann
E-mail: [email protected]
Phone: +49(0)931 31 82731
Fax:
+49(0)931 31 82741
http://www.rudolf-virchow-zentrum.de/forschung/heckmann.html
The birth of synaptic connections between nerve cells is an intriguing developmental period that paves the way
for the complex functions executed by nervous systems. If the intricate network between neurons is improperly
formed during embryogenesis or is subsequently injured, network malfunctions cause severe disability. How
synapses are established during embryogenesis and by which molecular means their highly specialized properties
are maintained throughout a lifetime is studied in our group.
Neurons are highly polarized cell types that
dedicate enormous energy to establishing
and maintaining specific structures and
molecular domains for information reception, processing, and sending within a
single cell. Failure to polarize nerve cells
and deliver molecules that operate neural
information processing and synaptic communication inevitably results in grave neu-
rological and psychiatric diseases in humans. The combination of high-resolution
microscopic imaging techniques, molecular
biology, and electrophysiology allows the
study of synaptic formation and function in
the absence of molecular components necessary for proper synapse performance, and
aids in understanding the basis of neuropsychiatric pathologies.
Nude Crashpilots
At presynaptic active zones (AZs), the frequently observed tethering of synaptic
vesicles to an electron-dense cytomatrix
represents a process of largely unknown
functional significance. We identified a hypomorphic allele, brpnude, lacking merely the
last 1% of the C-terminal amino acids (17
of 1740) of the active zone protein Bruchpilot. In brpnude, electron-dense bodies were
properly shaped, although entirely bare of
synaptic vesicles. While basal glutamate
release was unchanged, paired-pulse and
sustained stimulation provoked depression. Furthermore, rapid recovery following
sustained release was slower. Our results
causally link, with intramolecular precision,
the tethering of vesicles at the AZ cytomatrix with synaptic depression (Fig. 1).
Stiff person syndrome
Synaptic inhibition is a central factor in
the fine tuning of neuronal activity in the
central nervous system. Symptoms consistent with reduced inhibitions such as
stiffness, spasms, and anxiety occur in
paraneoplastic stiff person syndrome with
autoantibodies against the intracellular
synaptic protein amphiphysin. We showed
that intrathecal application of purified
Fig. 1:
Impaired vesicle tethering at AZs of a bruchpilot mutant (brpnude) lacking the last 17 C-terminal amino acids. (A) Chemical induction of premature stop
codons resulted in truncated versions of the BRP protein (BRP; dark blue; boxes indicate coiled-coil domains) to 99% (brpnude; gray), 70% (brp1.3; green),
and 50% in length (brp5.45; blue). Below, illustration of the BRP protein (blue) within the AZ (red: Ca2+ channels; gray: synaptic vesicle) and the corresponding positions of the truncation (arrows). (B) All three BRP mutant animals (brpnude, brp1.3; brp5.45) showed severely impaired survival rates and walking skills.
(C) In the brpnude allele, a single base mutation at the C-terminal position 1724 leads to a premature stop codon and a BRP protein (BRPnude) lacking the
last 17 of 1740 aa. (D) STED images of neuromuscular junctions stained with a C-terminal BRP antibody (green; BRPNc82) and simultaneous confocal images
of an N-terminal BRP antibody (magenta; BRPN-term). The distribution of both antibody signals appears unaltered at brpnude synapses (right) compared to
controls (left). Arrows and arrowheads indicate planar and vertical AZs, respectively. (E) Examples of conventionally embedded AZs of control and brpnude.
Note fewer tethered vesicles in brpnude. (F) Height and length of the platform of the electron-dense cytomatrix (T-bar) for control and brpnude. (G) The number
of docked vesicles per AZ section for control and brpnude (n = 22 and 25 AZs, respectively) and the number of vesicles found within three shells (see inset)
each of 50 nm thickness surrounding the AZ (n = 20 and 31 AZs, respectively). Scale bars: D, 1 µm; E, 200 nm.
52
Extramural Funding
DFG (SFB 581, TP B27), (He 2621/4-2),
(La 2861/1-1)
Fig. 2:
Presynaptic localization of intrathecally injected anti-amphiphysin antibodies. (A) Double immunofluorescence labeling of anti-human IgG and the presynaptic markers VGAT, showed punctate staining
around motor neurons and dendrites in the anterior horn of the spinal cord (scale bar: 20 µm).
Staining for VGLUT, clathrin and bassoon gave similar patterns. (B) Stimulation emission depletion
high-resolution microscopy from an area of interest (small square in the left panel) revealed almost
complete overlay of human IgG with VGAT, partial overlay with VGLUT and clathrin, and largely
adjacent staining with the active zone marker bassoon, indicating presynaptic enrichment of injected
anti-amphiphysin antibodies (scale bar: 500 nm). (C) The amount of colocalization was determined
using volumetric data and calculating Pearson’s correlation coefficient.
anti-amphiphysin immunoglobulin G antibodies induces stiff person syndrome-like
symptoms in rats, including stiffness and
muscle spasms. Using in vivo recordings
of Hoffmann reflexes and dorsal root
potentials, we identified reduced presynaptic GABAergic inhibition as an underlying
mechanism. Anti-amphiphysin immunoglobulin G was internalized into neurons by
an epitope-specific mechanism and colocalized in vivo with presynaptic vesicular
proteins, as shown by stimulation emission
depletion microscopy. Neurons from amphiphysin deficient mice that did not internalize the immunoglobulin provided additional
evidence of the specificity in antibody uptake. GABAergic synapses appeared more
vulnerable than glutamatergic synapses to
defective endo- cytosis induced by antiamphiphysin immunoglobulin G, as shown
by increased clustering of the endocytic
protein AP180 and by defective loading of
FM 1–43, a styryl dye used to label cell
membranes. Incubation of cultured neurons
with anti-amphiphysin immunoglobulin G
reduced basal, and stimulated release of
gamma-aminobutyric acid substantially
more than that of glutamate. By whole-cell
patch-clamp analysis of GABAergic inhibitory transmission in hippocampus granule
cells we showed a faster, activity-dependent decrease in the amplitude of evoked
inhibitory postsynaptic currents in brain
slices treated with antibodies against
amphiphysin. We suggest that these findings may explain the pathophysiology of
the core signs of stiff person syndrome
at the molecular level, and show that
autoantibodies can alter the function of
inhibitory synapses in vivo upon binding to
an intraneuronal key protein by disturbing
vesicular endocytosis.
Selected Publications
Hallermann, S.*, Kittel, R. J.*, Wichmann, C.*, Weyhersmüller, A., Fouquet,
W., Mertel, S., Owald, D., Eimer, S.,
Depner, H., Schwärzel, M., Sigrist, S. J.,
and Heckmann, M. (2010). Naked dense
bodies provoke depression. J Neurosci,
30, 14340-45.
Geis, C., Weishaupt, A., Hallermann, S.,
Grünewald, B., Wessig, C., Wultsch, T.,
Reif, A., Byts, N., Beck, M., Jablonka,
S., Boettger, M.K., Üçeyler, N., Fouquet,
W., Gerlach, M., Meinck, H.M., Sirén,
A.L., Sigrist, S.J., Toyka, K.V., Heckmann, M.*, and Sommer, C*. (2010).
Stiff person syndrome-associated autoantibodies to amphiphysin mediate reduced GABAergic inhibition. Brain, 133,
3166-80. (scientific commentary in
Brain, 133, 3164-65)
Hallermann, S., Heckmann, M., and
Kittel, R. J. (2010). Mechanisms of
short-term plasticity at neuromuscular
active zones of Drosophila. HFSP J, 4,
72-84.
Wagner, N., Weyhersmüller, A., Blauth,
A., Schuhmann, T., Heckmann, M.,
Krohne, G., and Samakovlis C. (2010).
The Drosophila LEM-domain protein
MAN1 antagonizes BMP signaling at the
neuromuscular junction and the wing
crossveins. Dev Biol, 339, 1-13.
53
Martin Lohse
E-mail: [email protected]
Phone: +49(0)931 201 48401
Fax:
+49(0)931 201 48411
http://www.rudolf-virchow-zentrum.de/forschung/bioimagingcenter/lohse.html
The cyclic nucleotides – cyclic AMP (cAMP) and cyclic GMP (cGMP) – are the most important intracellular messengers. They link stimulation of receptors at the cell surface to cellular responses. We investigate how receptors
at the cell surface become activated and how they trigger and regulate cAMP production. For these studies, we
generated a number of fluorescent sensors for receptors and their downstream signaling proteins as well as fluorescent sensors for cAMP and cGMP. These sensors are used to generate images of receptor-generated signals in
intact cells, resolved in space and in time. These new technologies give unprecedented views on receptor signaling. For example, we have observed that the exact localization of receptors within a cell determines how they
signal and how a cell responds to stimuli. This has important (patho)physiological implications, which range
from the control of thyroid hormone secretion to heart failure.
Fluorescent sensors for receptor signaling and second messengers
The strategy to create sensors for the
various steps of receptor signaling – from
receptor activation down to cyclic nucleotides – is based on a technique called
fluorescence resonance energy transfer
(FRET). FRET is the transfer of energy from
one fluorescent moiety to another one
in close vicinity. In our sensors, these
moieties are generally cyan (CFP) and yellow (YFP) fluorescent proteins, but we are
also experimenting with small dyes that
can be attached to defined small epitopes
in proteins.
FRET can cause an acceptor (for example
YFP) to emit light when a nearby donor (for
example CFP) is excited. FRET is very sensitive to changes in the distance between
the two fluorescent moieties: even small
increases in the distance lead to a large
loss in FRET. Figure 1 shows an example, a
sensor for cAMP. This sensor is built with
three elements: two fluorescent moieties
(CFP and YFP), which flank a binding
domain for cAMP. When cAMP binds to this
domain, it causes a movement of CFP away
from YFP (dotted red arrow), and this
results in a loss of FRET. Such changes in
FRET can be monitored by measuring the
signal intensities of the CFP- and YFP-emissions (and their ratio). They permit recording and imaging in real time, where and
when signals change within an intact cell.
54
Fig. 1:
Principle of a FRET-sensor. This sensor for cAMP comprises a cAMP-binding domain (grey) fused to cyan
(CFP) and yellow fluorescent proteins (YFP). Binding of cAMP moves CFP away from YFP and reduces
FRET; therefore, cyan emission increases and yellow emission decreases.
Mechanisms of receptor activation and
signaling
Activation of receptors is triggered by
binding of agonists and a subsequent conformational change. The exact nature of
this conformational change is still unclear.
Our studies with fluorescently labeled
receptors address the kinetics of these
changes in intact cells and the responses
to different types of ligands. We observed
that they occur with high speed (usually
30-80 ms). Fast switching can also be exerted by allosteric ligands; these are ligands that bind to additional binding sites
in receptors and appear to induce distinct
specific conformations of receptors. This
can decrease or increase the effects of regular, orthosteric ligands. The complex and
rapid interactions that different types of
ligands exert on receptors offer new strategies to alter receptor function, and may
provide new venues drug treatment.
A different type of allosteric mechanisms
has become a more recent research focus in
our group: the fact that some receptors
may form dimers, and that within such a
dimer one receptor may alter the function
of the other receptor. These dimers may result in complex interactions between drugs
acting at different receptors and may have
profound implications for drug therapy.
Fig. 2:
Orthosteric and allosteric effects on receptors.
The m2-muscarinic acetylcholine receptor
(m2AChR) can be activated via its “conventional”
orthosteric site (e.g. by acetylcholine) and can
be inhibited via a second, allosteric site (e.g. by
gallamine). FRET-recording of a fluorescently
labeled receptor show both the activation and
the inhibition.
The importance of receptor localization
for receptor signaling: a role in heart
failure
It is generally assumed that receptors are
evenly distributed over the entire surface
of a cell, and that they exert their classical
signaling function only from the cell surface. Recent studies in our group indicate
that both assumptions may be wrong.
The first set of studies investigated betaadrenergic receptors in cardiac myocytes,
the muscle cells of the heart. These cells
have two closely related receptors for the
“stress hormones” adrenaline and noradrenaline, which are called beta1- and
beta2-receptors. Both receptors cause an
increase in cAMP production in the cells,
but we observed earlier that the cAMP-signals produced by beta1-receptors extend
over the entire cell, while the cAMP-signals
triggered by beta2-receptors are locally
confined. In a collaboration with the labs
of J. Gorelik and Y. Korchev at Imperial
College in London, we have been able to
use very local stimulations, delivered
through the tiny tip of the pipette of a
scanning ion conductance microscope. This
permits stimulation at two very distinct
sites in the cells: directly at the cell surface, or in the t-tubules – long invaginations that emanate from the cell surface.
We observed that beta2-receptors cause
cAMP-signals only upon stimulation at the
t-tubules – suggesting that they are exclusively localized at these tubules. In contrasts, beta1-responses are found all over
the cell surface. The selective localization
of the beta2-receptors appears to be the
reason for their distinct, local character of
the cAMP-responses. Interestingly, both
receptors increase cardiac contractions;
however long-term stimulation of the
beta1-receptors causes growth and ulti-
mately death of cardiac myocytes, whereas
stimulation of beta2-receptors does not. We
postulate that this is due to the distinct
localization of these receptors, and that
this may provide a way of increasing cardiac contractility without damaging the
heart.
A similar, but distinct role of receptor
localization within a cell has become
evident from studies of thyroid follicles.
Here, receptors for the thyroid stimulating hormone (TSH) produce different
signals, whether they are at the cell surface or when they move together with
the TSH into the cell interior. In the latter
case, they produce long-lasting cAMP
signals that seem to regulate thyroid hormone secretion.
Extramural Funding
DFG-SFB487, TPA1
DFG-SFB688, TPB6
Leducq-Foundation, Transatlantic Network
of Excellence CAERUS
EMBO Fellowship to Veronika Hlavackova
ERC Advanced Investigator Grant, TOPAS
Selected Publications
Calebiro, D., Nikolaev, V.O., Persani, L.,
and Lohse, M. J. (2010). Signaling by
internalized G-protein-coupled receptors. Trends Pharmacol Sci, 31, 22128.
Hoffmann, C., Gaietta, G., Zürn, A., Adams, S. R., Terillon, S., Ellisman, M. H.,
Tsien, R. Y., and Lohse, M. J. (2010).
Fluorescent labelling of tetracysteinetagged proteins in intact cells. Nature
Protoc, 5, 1666-77.
Lohse, M. J. (2010). Dimerization in
GPCR mobility and signaling. Curr Opin
Pharmacol, 10, 53-58.
Maier-Peuschel, M., Frölich, N., Dees,
C., Hommers, L. G., Hoffmann, C., Nikolaev, V. O., and Lohse, M. J. (2010). A
FRET-based M2 muscarinic receptor sensor reveals rapid kinetics of allosteric
modulation. J Biol Chem, 285, 87938800.
Fig. 3:
Specific localization of receptors. A cardiac myocyte (blue cell) has many invaginations called ttubules (grey). Scanning ion conductance microscopy (SICM) reveals a pattern of t-tubular openings
(top, right). Delivery of a highly localized β2-adrenergic receptor stimulus through the SICM pipette
causes a cAMP-signal (recorded by FRET) from the t-tubules but not from the surface crest. This
indicates that these receptors are highly localized.
Nikolaev, V. O., Moshkov, A., Lyon, A. R.,
Miragoli, M., Novak, P., Paur, H., Lohse,
M. J., Korchev, Y. E., Harding, S. E., and
Gorelik, J. (2010). Beta2-Adrenergic
receptor redistribution in heart failure
changes cAMP compartmentation.
Science, 327, 1653-57.
55
Early Independence Program
Ingrid Tessmer
E-mail: [email protected]
Phone: +49(0)931 31 80425
Fax:
+49(0)931 31 87320
http://www.rudolf-virchow-zentrum.de/forschung/tessmer.html
Since January 2008, the Rudolf Virchow Center has run the early independence postdoctoral program to give excellent postdocs the opportunity to work on their own project.
We use the single molecule technique of atomic force microscopy (AFM) in combination with other biophysical
and biochemical techniques to study protein-DNA complexes involved in DNA repair. AFM uses a fine probe to
mechanically scan biological samples, producing topographical images with resolutions in the order of a few
nanometers, allowing us to directly visualize individual biological molecules such as proteins and their interactions. The high resolution of AFM imaging combined with its applicability in liquid environments (i.e. under
physiological conditions) renders this technique a complementary link between X-ray crystallography and functional in vitro and in vivo analyses of biological systems using optical microscopy approaches with resolutions of
typically a few hundred nanometers.
DNA repair is vitally important for the
maintenance and stability of our genetic
material. Different DNA repair mechanisms
often employ highly specialized approaches
to find and recognize their particular target sites within DNA. Disrupted damage
recognition and subsequently unrepaired
damage and errors in the DNA can cause
mutations in the transcribed proteins,
which in turn can lead to cancer and other
severe medical disorders. Understanding
damage recognition mechanisms and the
functional consequences of the protein
mutations involved is therefore of fundamental importance.
Our major interest is understanding different approaches to DNA damage recognition in various DNA repair systems. For
instance, alkylation damage in DNA, which
is not only introduced by endogenous
factors, but also exploited in chemotherapeutical treatment, is repaired by the pro-
Fig. 1:
AFM image of AGT multi-protein
complexes crosslinked to DNA fragments.
56
tein alkyl-guanine-transferase, AGT. In collaboration with Prof. Fried at the University
of Kentucky, we use AFM imaging of AGTDNA complexes to help us understand DNA
damage search and recognition strategies
employed by AGT (Figure 1).
The interplay between different types of
proteins provides enhanced levels of control and is ubiquitously employed in different DNA repair mechanisms. However, the
presence of multiple, different protein molecules complicates structural and functional studies of a protein complex. To approach this important analytical problem,
we are developing techniques for selective labeling of specific protein molecules
in heteromeric multi-protein complexes
detected in AFM images. One approach
is conjugation of a quantum dot to individual protein molecules to provide distinct topographical markers of the labeled
protein molecules in AFM images. [Wang
and Tessmer et al. 2008] We recently establis-hed the combinatory technique of FIONA-AFM, which overlays fluorescence and
AFM images of the same sample area with
high (nm) accuracy. In the resulting FIONA-AFM hybrid images, we can identify
fluorescently labeled molecules in the AFM
topography images. A first biological test
sample for FIONA-AFM was the prokaryotic
DNA damage recognition complex of UvrA
and UvrB bound to UV-damaged DNA. UvrB
molecules in the sample were conjugated
to quantum dots, and can subsequently be
identified by the topographical as well as
the fluorescent quantum dot signals (Figure 2). We are currently striving to further
optimize and apply FIONA-AFM to studies
of multi-protein complexes involved in
DNA repair.
Fig. 2:
3D FIONA-AFM image (top view in inset)
of the DNA repair protein UvrB bound
to UV-damage in DNA. The protein is
conjugated to a fluorescent quantum dot.
Fluorescence signals (red/yellow color)
are overlaid with AFM topography at nm
accuracy.
Selected Publications
Tsai, H. H., Huang, C. H., Tessmer, I.,
Erie, D. A., and Chen, C. W. (2011).
Linear Streptomices plasmids form
superhelical circles through interactions
between their terminal proteins. Nucleic Acids Res, 39(6), 2165-74.
Fronczek, D.N., Quammen, C., Wang, H.,
Kisker, C., Superfine, R., Taylor, R., Erie,
D.A., and Tessmer, I. (2011). High accuracy FIONA-AFM imaging. Ultramicroscopy, 111(5), 350-55.
Outlook
Katrin Heinze
E-mail: [email protected]
Phone: +49(0)931 201 48717
http://www.rudolf-virchow-zentrum.de/forschung/heinze.html
Discoveries in bioscience are frequently stimulated by the invention of new scientific tools. We have focused on
pushing fluorescence techniques beyond their usual limits of spatial and temporal resolution by combining highresolution concepts of fluorescence microscopy with tricks from material sciences. Suitable for live cell applications this low-invasive approach offers a fascinating prospect of observing biomolecules in their native environment and understanding how they act in concert.
Our research focuses on the measurement
and manipulation of inter- and intramolecular dynamics in a cellular setting. One
of the most fascinating tools we are working on is based on nanostructured materials that permit fast surface imaging with
lateral and possibly axial super-resolution.
The respective assembly or device is called
a superlens.
A superlens is a ‘planar lens’ consisting of
a metamaterial substrate (i.e. a sub-wavelength scale metal-dielectric structure, see
Figure), whose counterintuitive interactions with light permit among other things
imaging beyond the diffraction limit. Metamaterials are artificial materials engineered
to have effective properties that can not
be found in nature and gain their properties from structure rather than composition. One key feature of certain nanometerthin metal-based structures is that they
exhibit negative refractive properties. This
makes them a unique imaging tool with
great potential for applications where both
spatial and temporal resolution is crucial.
Furthermore, their interactions with molecules and emitters can be tuned systematically and lead to improved and novel biosensing applications.
However, the use of previous superlens
designs in bio-imaging was limited by the
required fabrication accuracies of the structures, as well as impractical and tedious
readout techniques. We refined a technique
for fabricating superlens structures with
unprecedented metal/dielectric interface
smoothness, which is critical for obtaining
optimal imaging results. Furthermore, we
confirmed the biocompatibility of these
structures in live cell experiments.
Recently we proposed a means of reading
out super-resolution information from a set
of simultaneous far-field measurements,
employing a spatial resolution that is
nearly an order of magnitude better than
that achieved using previous far-field
readout techniques. This new technique
relies on the use of a superlens design that
exploits the finite frequency bandwidth of
a fluorophore’s emission. We have shown,
in theory [Elsayad & Heinze 2010] as well
as in experiment [Elsayad et. al. 2010],
that a metal-dielectric stacked metamaterial structure can be designed to amplify
different discrete spatial Fourier components of an incident field at different optical frequencies. In other words, different
kinds of image details become color-coded,
thus spectrally distinguished, and can be
selectively detected. A final image or
movie containing all the different fine
details can be reconstructed later, allowing
us to study nanoscale dynamics that have
not been accessible before.
Extramural Funding
FWF (P23002-N24)
Selected Publications
Elsayad, K., and Heinze, K.G. (2010).
Multifrequency parallelized near-field
optical imaging with anistropic metaldielectric stacks. Physical Review A,
81(5).
Elsayad, K., Urich, A., Unterrainer, K.
and Heinze, K.G. (2010). Fast near-field
imaging of spectrally broad sources using layered metallic structures. Proc
SPIE, 7757, 73M.
Fig. 1:
Graphic of a layered metal-dielectric stack that can exhibit negative refractive properties and thus be
used for super-resolution surface imaging.
Elsayad, K. and Heinze, K.G. (2010).
Temperature dependence of the nearfield superlensing effect for single metal
layers and metal-dielectric films. Proc
SPIE, 7757, 73L.
57
Teaching & Training
Carmen Dengel
BSc/ MSc Program Biomedicine
Manfred Schartl
Graduate Training
Heike Hermanns
Stephan Kissler
Public Science Center
Kristina Kessler
58
59
Teaching & Training
Undergraduate and Graduate Programs
Coordinator: Bw. (VWA) Carmen Dengel
E-mail: [email protected]
Phone: +49(0)931 31 80378
Fax:
+49(0)931 31 83255
http://www.rudolf-virchow-zentrum.de/ausbildung/ausbildung.html
The Rudolf Virchow Center is not only dedicated to excellent research but also actively involved in numerous
educational programs for both undergraduate and graduate students. The Center has developed, and is hosting,
the undergraduate BSc/MSc program in Biomedicine and the Virchow Graduate Program, with the latter being
part of the Graduate School of Life Sciences (GSLS) of the University of Würzburg.
By combining both research and training, the Rudolf Virchow Center has adopted the Humboldt tradition, where
both are inseparable. The Center‘s training programs are fully integrated into University-wide programs and
have notably been the seed for restructuring other undergraduate and graduate programs at the University of
Würzburg. The Center thus provides a stimulating and nurturing environment for researchers and students alike.
The Rudolf Virchow Center strives to achieve excellence as much in its undergraduate and graduate training
programs as in its research program. Both training programs specifically target future researchers who will work
at the interface between life sciences and medicine. The undergraduate program in Biomedicine was initiated
in 2001, and seven classes have already graduated with a Bachelor of Science degree. Five classes have also
successfully completed the Master‘s course. The positions held by class alumni are testaments to the excellent
training they received, preparing them for successful careers.
The development of the Virchow Graduate Program reflects the Center‘s dedication to graduate training.
Together with several other DFG-funded graduate programs, the Virchow Graduate Program has become
the nucleus of large-scale reform in graduate training at the University of Würzburg. This reform culminated
in the founding of the Graduate School for Life Sciences, which was awarded funding by the national
“Exzellenzinitiative” in 2006.
Fig. 1:
Historical auditorium, the students attend a lecture.
60
Teaching Activities
BSc/MSc Program in Biomedicine
The undergraduate program in biomedicine is a small, research-oriented
program that enrols 25-30 students each year. Its main focus is researchbased training at the interface between life sciences and medicine. Members of the RVZ Network and Core Center carry a considerable part of the
teaching load and also provide opportunities and supervision for many
theses.
Manfred Schartl
(Chairman of the Examination Committee)
Theodor Boveri Institute, Physiological Chemistry I
Bachelor`s Program (BSc – 6 semesters)
Admission is based on grades in the final high school examination. The three-year BSc curriculum combines elements of undergraduate programs in Natural Sciences (mathematics, physics, chemistry, biochemistry, molecular biology, cell biology)
with key modules in preclinical medicine (anatomy, physiology,
microbiology, immunology, virology, pharmacology, toxicology,
pathology). Many of the modules have been specifically developed for this course, while others were adapted from the
curricula of biology and medicine.
The curriculum has a strong focus on practical laboratory work
in order to prepare students for research. The topics are weighted to reflect direct relevance to state-of-the-art biomedical research. It also includes modules on scientific regulatory matters
to legally qualify students for chemical, radioactive and genetic
engineering work, as well as for animal experimentation.
To facilitate international exchange, the curriculum complies
with the European Credit Transfer System (ECTS). Credit points
(EP) and corresponding grades are collected during the
course and included in the final grade. Each module includes an
examination, which can be a written or practical test, a presentation of research results, or a piece of scientific writing. This
curriculum structure allows rapid progression. A thesis, written
in English and based on the student‘s own laboratory work,
is publicly defended in a final examination that concludes
the course.
Master`s Program (MSc – 3 semesters)
Admission to the MSc program is based on either a Bachelor`s
degree in biomedicine from the University of Würzburg or an
equivalent degree from another university.
The three-semester MSc curriculum allows for 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 (viruses, E. coli, Candida, S. cerevisiae,
Drosophila, zebrafish, mouse/rat).
The students then carry out two six-week rotations in laboratories of their choice, with the possibility of also spending time
in institutions not involved in the Biomedicine program, and
even abroad. Accompanying lectures cover molecular pathology,
biomaterials, neurobiology and cardiovascular biology.
The final part of the course is dedicated to a nine month
research project. The MSc program is concluded with a public
defense of the student‘s MSc thesis, again written in English and
based on the student‘s research. The MSc qualification can lead
directly into doctoral training and the thesis can be credited
towards a PhD degree.
Management of the programs
Two committees that include members of the Faculties of Biology and Medicine, as well as two coordinators, Carmen Dengel
and Michaela Reuter from the Rudolf Virchow Center, share the
responsibility for organizing the BSc/MSc program and taking
care of student affairs.
The examination committee, chaired by Prof. M. Schartl, supervises the organization of examinations and decides on admissions, transfers and accreditation of courses taken at other
universities or research institutions. The study committee,
chaired by Prof. M. Gessler, is responsible for the study program
and supervises the quality and content of teaching.
Results
The BSc curriculum started in 2001, with a new class starting
every winter term. It pioneered the implementation of the Bachelor/Master`s system in the University and can be regarded as
very successful. The number of applications has remained high,
with over 600 applications each year. To date, we have had 320
BSc students and 104 MSc students, of which 253 and 78 were
female, respectively.
Overall, the performance of BSc and MSc students has been
excellent so far. Two key features of these structured training
programs may be responsible for their popularity and success:
our students first acquire a particular ability to address research
problems, then design and present a relevant research project.
Second, more than half of the students take the opportunity to
spend study-time abroad. Most BSc graduates decided to continue their studies with the MSc program.
So far, 68 of our MSc graduates went on to get their PhD, 26
of these chose research topics in Würzburg, 1 in industry and 8
abroad, including Singapore, USA, Great Britain, and New Zealand. 33 of the former biomedicine students are pursuing doctoral work at different universities in Germany.
61
Graduate Training
Since its inception, the Rudolf Virchow Center has aimed to offer a structured doctoral training program of the
highest quality. The program is largely based on earlier experiences with doctoral training at the University of
Würzburg, notably in the context of several DFG-funded Research Training Groups (Graduiertenkollegs). Another
model for the Center‘s own program was the MD/PhD program initiated by the Faculties of Biology and Medicine
in 1996/97 as the first such program in Germany. These programs, after training several generations of basic and
clinical scientists, have shown the effectiveness and success of a more structured training concept. Accordingly,
the Rudolf Virchow Center is runs its own Graduate Program (see page 62).
Most notably, the Rudolf Virchow Center successfully catalyzed the introduction of structured graduate training
in the context of a Graduate School as the standard model throughout the whole University by proposing key
elements and helping to build the necessary structures.
Key elements of training in the Graduate Schools
The traditional single supervisor (“Doktorvater“) is
replaced by a three-person committee.
A panel of training activities is offered, from which
an individual program is tailored to each graduate
student.
Graduate students actively participate in the program
by giving and organizing courses and symposia.
A set of requirements has to be met to warrant a
common quality standard.
Mentoring System
Each student has an individual supervisory committee, which
meets with the doctoral student at regular intervals to monitor
progress and adjust the research and training activities. Additionally, the graduate students report on the status of their
project within the research groups and programs, exchanging
ideas and obtaining feedback within their peer group.
Training Activities
The training activities of at least 150 hours per year consist of
laboratory seminars, journal clubs, program seminars, methods
courses, and transferable skills workshops, as well as retreats
and international conferences.
Common Graduation Commission
The participating faculties form a common Graduation Commission within the Graduate School. The Commission is responsible
for conferring all doctoral degrees within the Graduate School.
This enforces common standards across disciplines and fosters
interdisciplinary cooperation in graduate training.
62
The Graduate School of Life Sciences (GSLS) is the culmination
of Rudolf Virchow Center initiative dating back to 2001/2, designed to implement structured doctoral training on a larger
scale. The GSLS was founded in 2006 and was awarded funding
by the “Exzellenzinitiative” that same year. The concept of
Graduate Schools bringing together broad fields of research
has since been extended to the entire University. Three more
Graduate Schools have started in the Humanities, Science and
Technology and Law, and Economics and Society. All of them
operate independently with respect to their research and training activities. Still, they are part of a single central institution,
the “University of Würzburg Graduate Schools” (UWGS). The
UWGS serves as a holding and monitors basic rules and standards, in addition to delivering general services. Martin Lohse,
speaker of the Rudolf Virchow Center, was elected director of
the UWGS in 2008.
The Graduate School of Life Sciences is jointly supported by
the faculties of Medicine, Biology, Chemistry & Pharmacy,
Physics & Astronomy as well as the Philosophical Faculty II.
All doctoral students enroll in the study program “Life Sciences”.
A Common Graduation Commission of all participating faculties awards doctoral students with the degree Dr. rer. nat.
or Ph.D.
The School has been growing rapidly since 2006. It started
with about 60 doctoral students and 28 founding members.
Now the numbers are close to 280 and 170, respectively,
and still rising.
University of Würzburg Graduate Schools
Graduate School of
Life Sciences
Graduate School of
Science & Technology
Graduate School of
Humanities
Graduate School of
Law, Economics &
Society
Section
Biomedicine
Section
Infection &
Immunity
Section
Integrative
Biology
Section
Neuroscience
Section
MD/PhD
Virchow
Graduate
Program
SFB 487
Regulatory
Membrane
Proteins
SFB 688
Cellular Interactions in the
Cardiovascular
System
TR 17
Ras-dependent
Cancer
GK 1048 Organ
Develop.
Fig. 1:
Structure of the University of Würzburg Graduate Schools.
The Graduate School comprises four scientific sections and an
MD/PhD-program, reflecting the research foci in the Life Sciences at our University. The section “Biomedicine” was initiated
by the RVZ and embodies the nucleus of the GSLS. This section
is still the largest with a total of 120 doctoral students. 32 of
the doctoral students in this section belong to the Virchow
Graduate Program, documenting the pivotal role of this
institution in its section. Other doctoral students working
at the RVZ have joined other sections, such as Infection and
Immunity, or Neuroscience, according to the main focus of
their research work.
A special fellowship program of the GSLS is the core element
of funding by the “Exzellenzinitiative”. Almost 1000 standardized
written applications have been evaluated so far in a staged process involving interviews, with more than 150 conducted by the
admission board - either in Würzburg, or using video conferencing, including from abroad. 67 fellows were recruited in total.
They originate from 19 different countries, 60% of the fellows
coming from abroad. The fellowships are portable and the
fellows can freely choose a laboratory and project among the
member laboratories of the GSLS. The RVZ proved to be exceptionally competitive in attracting fellowship recipients:
7 fellows (18.9%) are working at the Rudolf Virchow Center,
although the Center represents only 8% of all the eligible
laboratories within the GSLS.
63
Graduate Training
Program Section Biomedicine
The Section Biomedicine provides a structured training program in biomedicine for all graduate students. The Section not only serves as an interdisciplinary link between the different graduate programs but also between
scientific research and practical experience, such as graduate students have
the possibility to work in international teams with scientists from various
research areas.
Heike Hermanns
(Chairperson Section Biomedicine)
Rudolf Virchow Center
Training activities
In addition to training activities offered by each individual
program and its research groups, a number of activities were
organized for all graduate students in biomedicine and the
Life Sciences. Training activities and events in 2010, organized
by Carmen Dengel, coordinator at the Rudolf Virchow Center,
included:
Graduate Schools Day
In July the University of Würzburg Graduate Schools offered a
Graduate Schools Day to inform all interested GSLS doctoral students and project leaders about their programs and regulations.
Graduate School Study Trip
In October the Graduate School Study Trip brought interested students to Brussels, another European power center. The
trip gave insights into the work of the EU commission and EU
parliament. Since the Graduate School of Life Sciences brings
together students from both the EU, and also many non-EU
countries, study trips like this one give them a deeper understanding of social, economical and cultural differences between
different EU countries and their strategies for working together.
Graduate student-organized activities
Workshops “Writing for publication/Becoming a Better
Academic Writer” (5x)
Two-day workshops were held by a professional science
writer to provide tools to organize, structure and write
research papers.
Workshops “Giving academic talks/Effective Scientific
Presentation” (3x)
Two-day workshops provided opportunities to learn and to
test strategies for effective and concise oral presentations.
Workshops “Poster design/presentation” (3x)
One-day workshops focused on the key elements for
effective poster design.
Furthermore, final year students were offered special training
in writing cover letters or curriculum vitae and introduced to
job interviews. To foster academic research and development,
courses were offered in patent law and technologies in health
care for life sciences. Future principal investigator skills were
addressed in courses on project management, negotiation and
team building skills.
To improve and facilitate integration into the international
research environment special emphasis was given to language
courses in English and German.
Meetings and events
In addition to many international scientific conferences and
meetings that the graduate students attended, a number of
events were organized specifically for the graduate students.
Highlights were:
64
Since 2005, the graduate students from the Section Biomedicine, together with students from the MD/PhD program, have
organized a yearly international symposium with high-profile
speakers from around the world. Remarkably, the students are
responsible for all scientific and administrative aspects, such as
the selection and invitation of international speakers, raising
sponsorship funds from companies, and organizing the day‘s
event. The 2010 theme “Chiasma – on the crossroads of research” united 10 reknowned international speakers, 10 students selected from the collaborative research programs in
Würzburg and more than 90 participants in their discussion on
future directions of life science research.
For the first time, the 2010 international students symposium
also included a writing contest. PhD students who produced
outstanding pieces of scientific writing which communicated
a scientific topic in a creative way to non-scientists were
awarded.
Mentoring program
With an orientation meeting in March 2010 the GSLS initiated
a new program called “mentoring life sciences” which is promoted by the German Research Foundation (DFG). This program
addresses gender equality standards and aims at increasing
the proportion of women in life sciences, particularly at the
professorial level. Therefore, female students are supported by
experienced mentors and offered special training workshops.
So far, 15 participants took part in four workshops addressing
rhetoric as well as time, and self management.
Graduate Training
Virchow Graduate Program
The Virchow Graduate Program is the Rudolf Virchow Center‘s own graduate
program and is part of the Biomedicine Section of the Graduate School for
Life Sciences. The concept of this program is to bring together all the graduate students working in the Center, regardless of their source of funding and
external affiliations, to generate a collegial peer group within the Rudolf
Virchow Center. One of the primary goals is to promote interactions and cooperations at the graduate student level.
Stephan Kissler
(Coordinator of the Virchow Graduate Program)
Rudolf Virchow Center
In 2010, the Virchow Graduate Program comprised 55 students,
of whom 7 successfully completed their thesis in the course of
that year. A majority of projects aim to identify molecular mechanisms of disease and/or attempt to develop tools to monitor,
inhibit, or even abrogate pathologic cell behavior. The experimental strategies range from the analysis of single molecules to
complex in vivo disease models.
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:
Advanced molecular imaging of molecules and cells in
vitro and in vivo, including biosensors, single molecule and
multiphoton microscopy, as well as optical whole-mouse
imaging.
Cutting edge approaches to determine the structures of
biological macromolecules at the atomic level, together
with biophysical and biochemical methods to analyze their
functions.
The generation and analysis of transgenic and knockout
mouse models of disease, including modulation of gene
expression by RNA interference in vivo.
Events
Annual Retreat
This year’s annual Graduate retreat, held in conjunction with the
annual Rudolf Virchow Center retreat, took place at Kloster
Schöntal from September 29th to October 1st. Every graduate
student presented his or her work during the student symposium
or at the evening poster session. For the fourth year running, all
participants were encouraged to vote for their favorite talk and
poster. Gunnar Knobloch from the Gohla laboratory won the best
talk award, and Timo Vögtle from the Nieswandt group took the
prize for best poster. Overall, this year’s retreat proved very
successful, and displayed once again the impressive breadth
and quality of graduate students research.
Student activities
In addition to organizing their own seminar series, for presentation of ongoing work at the Center, students of the Virchow
Graduate Program also co-organized the 5th International GSLS
Symposium in October 2010. This symposium brought a remarkable number of renowned speakers to the Center, where the
event took place. During this symposium, students had the
opportunity to present their work both in oral presentations
and during a well-attended poster session, and to discuss their
research with the invited speakers.
The Virchow Graduate Program is fully integrated into the wider
biomedical research community in Würzburg. Exchange on a scientific as well as technological level with other laboratories, not
only in Würzburg but also nationally and internationally, is
highly encouraged and actively promoted. Furthermore, the program facilitates individualized hands-on training by organizing
practical training units, tutorials and small workshops. Our aim
is to provide training that truly prepares young scientists for a
career in biomedical research. Laboratory work is complemented
by opportunities to learn about relevant aspects of clinical
medicine and by seminars where students are taught to critically
review the scientific literature, organise their work-schedules efficiently, and communicate their science most effectively.
65
Public Science Center
Kristina Kessler
Since February 2010 the Public Science Center ist managed by Kristina Kessler.
E-mail: [email protected]
Phone: +49(0)0931 31 80895
Fax:
+49(0)0931 31 87283
http://www.rudolf-virchow-zentrum.de/public
The overall goal of the Public Science Center is to increase national and international visibility of the Rudolf
Virchow Center. It communicates openly with politicians and the scientific community about the newest research
discoveries, along with training the next generation of scientists. Next to discussions about the latest discoveries, it stimulates debates about ethical questions in biomedical research. In accordance with the overall concept
of the Rudolf Virchow Center, to win the brightest minds in science for research early on, young people are an
important target group for the work done at the Public Science Center. The most important forms of communication are therefore media releases, the annual report, and organizing public events such as school projects, science days, and opportunities for discussion.
Making science comprehensible
Press Officer relations employee of the
DFG, was present at the stand. Up to 3,000
visitors were at the hall during peak hours.
The informative material from the Rudolf
Virchow Center was well accepted and
stimulated active exchange at the stand.
Another public magnet was the exhibition
“MenschMikrobe”. The exhibition was displayed in the lobby of the Center for five
weeks, supervised and organized by the
Public Science Center. Over 5,300 visitors
came to get a living glimpse into current
knowledge about bacteria, viruses, and
parasites. Exceptionally large was the response from schools in the region. Approximately 150 classes used the free tours as
a colorful and refreshing addition to their
biology class. Würzburg was the first stop
of the traveling exhibition in southern
Germany. The reason for the project,
which is funded by the German Research
Foundation and the Robert Koch Institute,
was the 100th anniversary of the death
of Robert Koch.
Fig. 1:
The large model of an artery that visitors could
enter was a highlight at the Ecumenical Church
Congress in Munich in June 2010. The center
used the exhibit to make contact with visitors
and discuss current research.
A goal of the Public Science Center is to
bring science closer to the public. Thus,
the department took advantage of the opportunity for exchange at the Ecumenical
Church Congress 2010 in Munich. For three
days scientists and colleagues from the
Public Science Center presented a stand in
the hall “Dialog with Science”. The team
brought along two current research projects visualized by a large model of an
artery and a 3D projection of a protein.
There, visitors could interactively learn
about current discoveries and work at the
Center. Even Dr. Eva-Maria Streier, Chief
66
Fig. 2:
An engaged team of biology, medicine, and biomedicine students stood by during the exhibition to
answer questions from visitors and during the guided tours.
Attracting young people to science
Whoever does cutting-edge research must
also invest in the upcoming generation.
With numerous school projects the Center
wants to awaken interest in biomedical research early on. The programs are designed
for different age groups and build on one
another. Children between the ages of 8
and 19 can visit courses at the Rudolf
Virchow Center. Over the years, a complete
program could be established, from primary
school up to „Abitur“ (A-Level).
The project “Rudis Forschercamp” (Rudi´s
Reasearch Camp) was started in 2004. Once
a week, for four consecutive weeks, 8 to 12
Fig. 3:
Jana Kühlwein was the 500th child at “Rudis
Forschercamp” (research camp). Therefore she
was presented with a special research certificate
along with a laboratory kit “Genetics”. This will
enable her to continue her studies outside the
course
year-old “Young Scientists” are introduced
to four different areas of the natural
sciences. The camp focuses on the children
doing their own experiments. On the
last day parents are invited, giving the
young scientists the opportunity to present
everything they have learned. Hence, the
program brings science much closer to children and adults alike. Interest in the program is strong. This year, “Rudis Forschercamp” welcomed the 500th child.
Virchowlab
In September 2008, the Public Science
Center started the “Virchowlab”, a project
for pupils between the ages of 13 and 16.
Entire school classes can spend a whole
day at the Center. The contents were developed in close cooperation with teachers
and are compatible with the Bavarian cur-
riculum. During the course, pupils continue to work on topics they have already
learned in class in a practical manner, and
in doing so deepen their understanding.
The goal is to ignite and intensify interest
in biomedical research. The program has
received a great response. Approximately
1100 pupils have visited the Virchowlab
since the start. Currently, 11 schools are
participating. Due to its own laboratories,
the Public Science Center can flexibly
respond to the demand.
The “School Break Researcher”, offered
for the first time, involved experiments
similar to those from the “Virchowlab”. The
program was geared towards individual
students and not classes. The course was
a complete success. Within a short period
of time both courses were fully booked.
Numerous young people are already on
the waiting list for 2011.
The program “Gemeinsam Forschen” has
also newly started (Doing Research
Together). In 2010 the Public Science
Center, in cooperation with four schools
from the region, organized for the first
time a W-Seminar for the final years of high
school. Within the framework of the project,
the Center intensively accompanies around
60 students during their last two years at
school. The students gain valuable insights
into scientific work. One of the strengths of
the project is its close links to science,
since every seminar can count on the
valuable advice of a research group leader.
Together with Katja Weichbrodt from the
Public Science Center and other teachers,
our scientists focused on four separate
topics. The classes work on the theory
of these topics in school. In the summer
of 2011 the students will have the opportunity to visit their respective research
groups as a guest and put their theory
into practice.
Media Relations
The development and fostering of an extensive network of journalists is an important task at the Public Science Center. We
see journalists as our most important partner. Compared to our direct public campaigns, our press releases can reach a
broader audience. News about the Rudolf
Virchow Center is prepared and sent to
a network of journalists via press releases
and also distributed through science information services.
Fig. 4:
Making complicated science understandable for
the public is a challenge that the Public Science
Center must meet. A television production can
achieve just that in an exciting way.
Politicians and the scientific community
Politicians again visited the Center in
2010, including the Bavarian Parliament´s
working group for Universities, Research,
and Culture. The politicians visited the
departments of structural biology and
vascular medicine, along with the “Schülerlabor” (students laboratory). This gave
them a glimpse of the high scientific level
of work that goes on here. The significance
of the Center for Würzburg was also emphasized during the visit. Since its founding,
the Rudolf Virchow Center has become the
heart of biomedical research in Würzburg.
Highlighting this important role to the
representatives of Parliament was the main
goal of the visit.
Since 2006 the Public Science Center has
published an annual report with a circulation of around 1,500 copies. This report
is sent to politicians and decision-makers,
as well as employees, colleagues, research
centers, and scientists all over the world.
Selected Media Reports
„Fehlendes Protein schützt vor Herzinfarkt“, dpa, January 5, 2010
„750.000 US-Dollar für Würzburger Diabetes-Forschung“, Ärztezeitung, April
15, 2010
„Blutverdünner durch Wanze“, NDR
Fernsehen, March 31, 2010
„Gesicht des Tages: 500. Kind bei Rudis
Forschercamp“, Mainpost, August 3,
2010
„Schatten der Vergangenheit“, Bayerische Staatszeitung, August 6, 2010
„Mikroben und das Erbe Robert Kochs“,
Volksblatt, November 2, 2010
„CSU-Arbeitskreis zu Besuch“, TV Touring, November 18, 2010
67
Appendix
Executive Committees and Scientific Members
Chairman:
Vice-Chairs:
Members:
Prof. Dr. Martin Lohse, Bio-Imaging Center/ Institute of Pharmacology and Toxicology
Prof. Dr. Caroline Kisker, Rudolf Virchow Center
Prof. Dr. Manfred Schartl, Theodor-Boveri-Institute, Physiological Chemistry I
Prof. Dr. Georg Ertl, Department of Medicine I
Prof. Dr. Antje Gohla, Rudolf Virchow Center/ Institute of Pharmacology and Toxicology
Prof. Dr. Bernhard Nieswandt, Rudolf Virchow Center/ University Hospital
Prof. Dr. Michael Sendtner, Institute of Clinical Neurobiology
Scientific Advisory Board
Chairman:
Members:
Prof. Dr. Fritz Melchers, Basel Institute of Immunology/ MPI for Infection Biology, Berlin
Prof. Dr. Ueli Aebi, Biocenter, University of Basel, Switzerland
Prof. Dr. Volkmar Braun, University of Tübingen
Prof. Dr. Sabine Werner, ETH Zürich, Switzerland
Prof. Dr. Heiner Westphal, NICHD, Bethesda, MD, USA
Prof. Dr. Alfred Wittinghofer, MPI for Molecular Physiology, Dortmund
Prof. Dr. Claes Wollheim, University of Geneva, Switzerland
I. Funded Members
Prof. Dr. Roland Benz, Rudolf Virchow Center
Dr. Shashi Bhushan, Rudolf Virchow Center
Prof. Dr. Martin Eilers, Theodor-Boveri-Institute, Physiological Chemistry II
Prof. Dr. Utz Fischer, Theodor-Boveri-Institute, Biochemistry
Prof. Dr. Manfred Gessler, Theodor-Boveri-Institute, Developmental Biochemistry
Prof. Dr. Antje Gohla, Rudolf Virchow Center/ Institute of Pharmacology
Dr. Gregory Harms, Bio-Imaging Center
Prof. Dr. Manfred Heckmann, Bio-Imaging Center/ Institute of Physiology II
Prof. Dr. Martin Heisenberg, Rudolf Virchow Center
PD Dr. Heike Hermanns, Rudolf Virchow Center
Dr. Asparouh Iliev, Emmy Noether Fellow, Rudolf Virchow Center/ Institute of Pharmacology
Prof. Dr. Caroline Kisker, Rudolf Virchow Center
Dr. Stephan Kissler, Rudolf Virchow Center
Prof. Dr. Martin Lohse, Bio-Imaging Center/ Institute of Pharmacology
Prof. Dr. Thomas Müller, Julius-von-Sachs-Institute, Botany I
Prof. Dr. Bernhard Nieswandt, Rudolf Virchow Center/ University Hospital
Prof. Dr. Hermann Schindelin, Rudolf Virchow Center
PD Dr. Alma Zernecke, Heisenberg Fellow, Rudolf Virchow Center
II. Non-Funded Members
Prof. Dr. Gerhard Bringmann, Institute of Organic Chemistry I
Prof. Dr. Georg Ertl, Department of Medicine I
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 and Leopoldina, Halle
Prof. Dr. Bert Hölldobler, Theodor-Boveri-Institute, Zoology II
Prof. Dr. Thomas Hünig, Institute of Virology and Immunobiology
Prof. Dr. Roland Jahns, Institute of Pharmacology and Toxicology/ University Hospital
Prof. Dr. Peter Jakob, Institute of Physics, Biophysics
Prof. Dr. Hermann Koepsell, Institute of Anatomy I
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. Manfred Schartl, Theodor-Boveri-Institute, Physiological Chemistry I
Prof. Dr. Walter Sebald, Theodor-Boveri-Institute, Physiological Chemistry II
Prof. Dr. Michael Sendtner, Institute of Clinical Neurobiology
Prof. Dr. Klaus V. Toyka, Clinic of Neurobiology
Prof. Dr. Ulrich Walter, Institute of Clinical Biochemistry and Pathobiochemistry
68
Academic Members and Supporting Staff
Junior Research Groups
Group Shashi Bhushan
Group Asparouh Iliev
Group leader:
Dr. Shashi Bhushan
Group leader:
Dr. Asparouh Iliev (Emmy Noether Fellow)
Graduate students:
Rekha Rajasheskar
Kushal Sejwal
Rajkumar Singh
Graduate Students:
Christina Förtsch
Sabrina Hupp
Carolin Wippel
Technician:
Christian Kraft
Technician:
Alexandra Bohl
Group Heike Hermanns
Group Stephan Kissler
Group leader:
PD Dr. Heike Hermanns
Group leader:
Dr. Stephan Kissler
Postdoc:
Dr. Christine Mais
Graduate Students:
Kay Gerold
Julie Joseph
Lili Probst (med.)
Peilin Zheng
Graduate Students:
Johannes Drechsler
Carmen Schäfer
Sabine Walter
Diploma Student:
Sandra Spiegel
Technician:
Daniela Kraemer
Technicians:
Nicole Hain
Katharina Herrmann
Animal care taker:
Heike Rudolf
Group Alma Zernecke
Group leader:
PD Dr. Alma Zernecke (Heisenberg Fellow)
Postdoc:
Dr. Helga Manthey
Graduate Students:
Martin Busch
Sweena Chaudhari
Maik Drechsler
Miriam Koch
Technician:
Theresa Moritz
Melanie Schott
Bachelor Students:
Hendrik Beckert
Caroline Fecher
Karolina Scholtyschik
Core Center
Group Caroline Kisker
Group leader:
Prof. Dr. Caroline Kisker
Postdoc:
Dr. Jochen Kuper
Scientific staff:
Dr. Bernhard Fröhlich
Graduate Students:
Uwe Dietzel
Maria Hirschbeck
Sylvia Luckner
Shambhavi Mishra
Florian Rohleder
Heide Marie Roth
Johannes Schiebel
Dominik Schmitt
Stefanie Wolski
Diploma Students:
Verena Grundler
Claudia Hofmann
Felix Mattern
Christin Schäfer
Johannes Schiebel
Bachelor Student:
Marie-Christine Weller
Technicians:
Gudrun Michels
Dr. Antje Schäfer
Graduate Students:
Carolyn Delto
Gunnar Knobloch
Xaver Kober
Hans Maric
Bodo Sander
Daniel Völler
Group Hermann Schindelin
Diploma Students:
Constantin Braun
Kristina Haslinger
Kristina Keller
Group leader:
Prof. Dr. Hermann Schindelin
Technicians:
Nicole Bader
Postdocs:
Dr. Petra Hänzelmann
Dr. Daniela Schneeberger
69
Research Professors
Group Utz Fischer
Group leader:
Prof. Dr. Utz Fischer
Postdoc:
Dr. Martin Vielreicher
Graduate Students:
Florian Amelingmeier
Katrin Schäffler
Georg Stoll
Anu Tyagi
Group Antje Gohla
Group leader:
Prof. Dr. Antje Gohla
Postdoc:
Dr. Elisabeth Jeanclos
Graduate Students:
Gunnar Knobloch
Ambrish Saxena
Annegrit Seifried
Technicians:
Kerstin Hadamek
Angelika Keller
Anna-Karina Lamprecht
Beate Vogt
Diploma Student:
Melanie Radenz
Lab Manager:
Andreas Wittner
Bachelor Student:
Katharina Haneke
Technicians:
Stefanie Hartmann
Sylvia Hengst
Juliana Goldmann
Birgit Midloch
Jonas Müller
Group Bernhard Nieswandt
Group leader:
Prof. Dr. Bernhard Nieswandt
Postdocs:
Dr. Attila Braun
Dr. Lidija Chakarova
Dr. Margitta Elvers
Dr. Dr. Katharina Remer
Secretary:
Sandra Niklasch
Graduate Students:
Markus Bender
Shuchi Gupta
Ina Hagedorn
Sebastian Hofmann
Frauke May
Martina Morowski
Irina Pleines
David Stegner
Johannes Steinweg (med.)
Ina Thielmann
Timo Vögtle
Senior Professors
Group Roland Benz
Group Martin Heisenberg
Group leader:
Prof. Dr. Roland Benz
Group leader:
Prof. Dr. Martin Heisenberg
Graduate Students:
Ivan Barcena-Uribarri
Christoph Beitzinger
Angelika Kronhardt
Research Assistant:
Reinhardt Wolf
Technicians:
Elke Maier
Diploma Students:
Kerstin Duscha
Maike Eberhardt
Bachelor Students:
Iraida Gil
Eulalia Sans
70
Graduate Students:
Preeti Sareen
Narendra Solanki
Zhenghong Yang
Technician:
Juliane Clessen
Diploma Students:
Sebastian König
Franziska Toepfer
Animal care taker:
Mario Müller
Laboratory care taker:
Alexander Fink
Diploma Students:
Wenchun Chen
Ronmy Rivera Galdos
Master Students:
Marianne Frings
Friederike Mühlpfordt
Bachelor Students:
Johanna Andersson
Melanie Hüttenrauch
Iris Mair
RVZ Network
Group Martin Eilers
Group Roland Jahns
Group Thomas Müller
Group leader:
Prof. Dr. Martin Eilers
Group leaders:
Prof. Dr. Roland Jahns
Dr. Valerie Boivin
Group leader:
Prof. Dr. Thomas D. Müller
Postdoc:
Dr. Elmar Wolf
Postdoc:
Dr. Vladimir Kocoski
Graduate Student:
Stefan Saremba
Group Manfred Gessler
Group leader:
Prof. Dr. Manfred Gessler
Graduate Students:
Traudel Schmidt
Jenny Wegert
Graduate Students:
Sonja Hartmann
Elisabeth Klinger (med.)
Priyadarshini Panjwani
Mathias Sättele (med.)
Angela Schlipp
Yuxiang Ye
Technicians:
Christin Bitterer
Katja Graf
Tanja Röder
Julia Ullrich
Christina Zechmeister
Bio-Imaging Center
Microscopy Technology Group
Group Manfred Heckmann
Group Martin Lohse
Group leader:
Dr. Gregory Harms
Group leader:
Prof. Dr. Manfred Heckmann
Group leader:
Prof. Dr. Martin Lohse
Graduate Students:
Jörg Blachutzik
Qiang Gan
Kun Wang
Monika Zelman-Femiak
Postdocs:
Dr. Tobias Langenhan
Dr. Nicole Wagner
Postdocs:
Dr. Viacheslav Nikolaev
Dr. Davide Calebiro (Humboldt Fellow)
Dr. Veronika Hlavackova (EMBO Fellow)
Dr. Silvia Volpe
Technicians:
Mike Friedrich
Markus Hirschberg
Graduate Students:
Martin Pauli
Patrick Stock
Technicians:
Christian Geiger
Uta Maas
Graduate Students:
Katharina Deiß
Nadine Frölich
Susanne Nuber
Technicians:
Monika Frank
Sonja Kachler
Bianca Klüpfel
71
Early Independence Program
Group Ingrid Tessmer
Central Technologies
Transgene Technology Group
Teaching and Training
Administration
Group leader:
Dr. Bettina Holtmann
Coordinator:
Carmen Dengel
Administrative Director:
Prof. Dr. Karl-Norbert Klotz
Postdoc:
Dr. Dr. Katharina Remer
Assistant:
Michaela Reuter
Administration Manager:
Bianca Klotz
Technician:
Daniela Östreich
Animal care taker:
Azer Achmedov
Valentina Zemskova
Public Science Center
Administrative Assistants:
Gerhard Antlitz
Eva Bernhardt
Tanja Böhm
Hilke Gehret
Maria Weidner
Group leaders:
Kristina Kessler
Assistant:
Katja Weichbrodt
System Administrator:
Christian Weinberger
Facility Technicians:
Daniel Göbel
Sebastian Hämmerling
72
Teaching Committees (Faculties Biology and Medicine)
BSc/MSc Study Committee
Chairman:
Members:
Prof. Dr. Manfred Gessler, Theodor-Boveri-Institute, Developmental Biochemistry
Dr. Ursula Rdest, Theodor-Boveri-Institute, Microbiology
Prof. Dr. Wolfgang Rößler, Theodor-Boveri-Institute, Zoology II
BSc/MSc Examination Committee
Chairman:
Members:
Prof. Dr. Manfred Schartl, Theodor-Boveri-Institute, Physiological Chemistry I
Prof. Dr. Jürgen Kreft, Theodor-Boveri-Institute, Microbiology
Prof. Dr. Thomas Müller, Theodor-Boveri-Institute, Botany I
Prof. Dr. Georg Nagel, Theodor-Boveri-Institute, Botany I
Prof. Dr. Hermann Schindelin, Rudolf Virchow Center
Prof. Dr. Helga Stopper, Institute of Pharmacology and Toxicology
University of Würzburg Graduate Schools
Board of Directors
Director:
Members:
Prof. Dr. Martin Lohse, Institute of Pharmacology and Toxicology, Rudolf Virchow Center
Prof. Dr. Brigitte Burrichter, Institute of Modern Languages and Linguistics, Romance Languages and Literature II
Prof. Dr. Bernhard Heininger, New Testament Exegesis
Prof. Dr. Caroline Kisker, Rudolf Virchow Center
Prof. Dr. Heidrun Moll, Institute of Molecular Infection Biology
Prof. Dr. Werner Porod, Theoretical Physics II
Prof. Dr. Wolfgang Schneider, Psychology IV
Graduate School of Life Sciences (since 09/2006)
Dean:
Vice Deans:
Prof. Dr. Caroline Kisker, Rudolf Virchow Center
Prof. Dr. Martin Lohse, Institute of Pharmacology and Toxicology
Prof. Dr. Heidrun Moll, Institute of Molecular Infection Biology
Section Biomedicine (Graduate School of Life Sciences)
Speakers:
PD Dr. Heike Hermanns, Rudolf Virchow Center
Prof. Dr. Helga Stopper, Institute of Pharmacology and Toxicology
73
Undergraduate program in Biomedicine
The following theses are written in the context of the RVZ
Undergraduate Program in Biomedicine:
Bachelor´s theses 2010 (Biomedicine)
“Untersuchungen zur Hemmung autophager Prozesse in
Leishmania major durch Cysteincathepsininhibitoren”
Baum, Ulrike
“Darstellung von DCM Genen über long-range PCR“
Bode, Elisabeth
“Wirkstoffscreening ausgewählter Proteaseinhibitoren an
den Blutstadien und Sexualstadien des Malariaerregers
Plasmodium falciparum“
Leubner, Monika
“Functional charakterization of dmrt1bY in Medaka“
Engel, Mareen
“Untersuchung zur Rolle von Rho-GTPasen bei Thrombozytenfunktion und –bildung in doppelt defizienten
Mäusen“
Mair, Iris
“Dendritische Zellen in der Regulation von T-Zellfunktionen“
Fecher, Caroline
“Klonierung einer pluripotenten murinen ES-Zelllinie
zum reversiblen Gen-Knockdown“
Neumann, Björn
“Influence of Resveratrol on Genomic Stability“
Fesenfeld, Michaela
“Ligand induzierte beta-Arrestin Intraktion mit dem
humanen M1 ACh Rezeptor“
Rikeit, Paul
“Imaging of the dynamic interaction between cell
membrane and stabilizing microtubules after a challenge
with the Streptococcus pneumoniae toxin pneumolysin”
Fries, Maximilian Werner
“Molekulare Charakterisierung einer Proteinkinase A
(PKA) aus Echinococcus multilocularis“
Schilling, Maximilian Thomas
“Role of the LINC/DREAM complex in gene repression
and activation during the cell cycle”
Günster, Regina Agnes
“Untersuchung der Permeabilität verschiedener Connexine für cAMP und cGMP“
Schmidt, Nadine
“Funktionelle Charakterisierung von AUM”
Haneke, Katharina
“MicoRNAs in der Regulation von Funktionen dendritischer Zellen“
Scholtyschik, Karolina Alexandra
“Studien zur Blutplättchenfunktion in genetisch
veränderten Mäusen“
Hüttenrauch, Melanie
“Untersuchung der Funktion von dmrt1a in Medaka –
Fischen“
Klughammer, Johanna
“NFATc Transcription Factors in Lymphocyte Function”
Kober, Christina
“Untersuchung zur Stabilität des antiapoptotischen
Protein A1/Bfl1“
Kozareva, Desislava Asenova
“Toxizität und Mutagenität biliärer Metaboliten des
Hepatokanzerogens Furan“
Kratz, Anne-Sophie
74
“Optimierung der Transduktionsrate von foamyviralen
Vektoren“
Stratmann, Anna Theresa
“Vorbereitende Untersuchungen zur Gentherapie des
Morbus Fabry“
Stritt, Simon Tobias
“Modulation der Strahlenempfindlichkeit maligner Zellen
unterschiedlicher Entitäten mittels neuartiger Inhibitoren des Hitzeschockproteins und m-TOR“
Wack, Linda-Jacqueline Maria
“Funktionelle Charakterisierung der Nukleotid-ExzisionsReparatur“
Weller, Marie-Christine
Master´s theses in 2010 (Biomedicine)
“Molekulare Eigenschaften des Endothels bei der Up- und
Downstream - Regulation des Transkriptionsfaktors
Runx1”
Baumann, Claudia
“Funktionsanalyse von humanen Plättchen nach Phytoprostan-Exposition“
Damm, Anna Maria
“Dissecting new parasitophorous vacular resident
proteins that may specifically interact with PbUIS4 and
PbUIS3 in developing liver stage parasites“
Fraschka, Sabine Anne-Kristin
”Studies on molecular mechanisms of thrombus formation in a model of laser induced endothelial injury in
mice”
Frings, Marianne
“Charakterisierung neuartiger Antagonisten der HIV-Vif/
Apobec3G Wechselwirkung“
Otto, Mirjam
”Effects of Early-Life Stress on Behavior of Mice Lacking
the M1 Muscarinic Acetylcholine Receptor Subtype”
Popp, Sandy
”Effects of prenatal stress on epigenetic programming in
serotonin transporter knock out mice”
Schraut, Karla-Gerlinde
”Untersuchung der neuronalen Funktionen der Kinase
S6KII in Drosophila melanogaster”
Strecker, Katrin
”Phosphorylation of microneme proteins in the malaria
parasite”
Thiessen, Anja
“Der Einfluss von Kalziumkanälen-Agonisten auf das
Differenzierungsverhalten von Smn-defizienten und TrkBdefekten Motoneuronen“
Godzik, Katharina
“Einfluss von Transport- und Adaptorproteinen auf die
APP-Prozessierung in primären Neuronen“
Hellrung, Anke
“Novel Roles for KATP channels in the endothelium”
Herget, Sabine Stephanie Jutta
“Cytoskeletal functions and differential migratory
behaviour of myeloid cell subsets”
Imle, Andrea
”Analyzing the loss of pluripotency in mouse embryonic
stem cells”
Jakob, Burkhard Helmar
”Molecular studies on BAR domain proteins in murine
hematopoietic cells”
Mühlpfordt, Friederike
“Cellular source of early lnterleukin-10 in the murine
model of infection with Leishmania major“
Nahrendorf, Wiebke
75
PhD theses of the Virchow Graduate Program (2010)
“Analysis of G protein-coupled receptor activation by
optical methods”
Ahles, Andrea
“Studies on platelet activation mechanisms in genetically modified mouse lines”
Hofmann, Sebastian
“Structure, development and plasticity of mammalian
synapses”
Andlauer, Till
“Role of sub-cellular and sub-compartmental distribution
of regulatory GEFs and GAPs in mediating activation of
small GTPases by pneumolysin”
Hupp, Sabrina
“Porins in the genus Borrelia”
Barcena-Uribarri, Ivan
“MicroRNAs in the regulation of dendritic cell functions
in atherosclerosis”
Busch, Martin
“Role of HIF-1alpha in the immune-regulation of
atherosclerosis”
Chaudhari, Sweena
“Architecture of gephyrin”
Delto, Carolyn
“X-ray crystallographic studies on Rhodesain and SARSPLpro, two papain-like proteases in complex with new
inhibitors”
Dietzel, Uwe
“Impact of Interleukin-6-like cytokines on cardiovascular
diseases”
Drechsler, Johannes
“Pore formation and small GTPase activation by pneumolysin”
Förtsch, Christina
“In vivo visualization of Smad signaling by dynamic high
resolution microscopy“
Gan, Qiang
“Translational control in cardiomyocytes”
Ganesan, Jayavarshni
“Role and function of the human susceptibility gene
KIAA0350 in the NOD mouse model of type 1 diabetes”
Gerold, Kay
“Studies on cytoskeletal regulation of platelet spreading,
granule release and coagulant activity”
Gupta, Shuchi
“In vitro and in vivo analysis of thrombus formation
under flow conditions”
Hagedorn, Ina
“Structure based drug design on enzymes of the bacterial
fatty acid synthesis pathway II”
Hirschbeck, Maria
76
“Signaling mechanisms in cardiac failure”
Jentzsch, Claudia
“Evaluating the therapeutic potential of Men1 modulation in the NOD model of type 1 diabetes”
Joseph, Julie
“Biochemical and structural basis of Chronophin
activation”
Knobloch, Gunnar
“Structural and functional characterization of protein
disulfide isomerise”
Kober, Xaver
“Platelet - T cell interactions in atherosclerosis”
Koch, Miriam
“Anchoring of GABA(A) receptors”
Maric, Hans
“Signaling in platelets via ITAM-coupled receptors”
May, Frauke
“Structure based drug design on essential proteins from
Mycobacterium tuberculosis”
Mishra, Shambhavi
“Studies on thrombus formation under flow conditions”
Morowski, Martina
“Molecular mechanism of antibiotic mediated inhibition
of translating bacterial ribosome”
Raj, Kumar
“Structural analysis of a eukaryotic DNA repair mechanism”
Rohleder, Florian
“Nucleotide excision repair: From recognition to incision
of damaged DNA”
Roth, Heide-Marie
“Profiling the gephyrin-neuroligin2 interaction”
Sander, Bodo
PhD theses of the Virchow Graduate Program (2010)
“Role of the novel human tyrosine phosphatase AUM for
cell adhesion”
Saxena, Ambrish
“Influence of Ca2+ on the signal transduction of proinflammatory cytokines”
Schäfer, Carmen
“Structure based drug design on enzymes of the fatty
acid biosynthesis”
Schiebel, Johannes
“Evaluation of activating antibodies by means of
fluorescence resonance energy transfer microscopy”
Schlipp, Angela
“Effects of pneumolysin on dendritic spine function and
synapse formation via small GTPases”
Wippel, Carolin
“Structural and functional characterization of Nucleotide-Excision-Repair proteins”
Wolski, Stefanie
“Tracking and signaling of Interleukin receptors”
Zelman-Femiak, Monika
“Role of the susceptibility gene Ptpn22 in the selection
and function of T cell in the NOD model of type 1
diabetes”
Zheng, Peilin
“Towards a crystal structure of the human TFIIH complex”
Schmitt, Dominik
Concluded in 2010:
“Role of chronophin for glioma cell migration and
invasion”
Schulze, Markus
“Collective cancer cell invasion along blood vessels“
Alexander, Stephanie
“Biochemical and structural characterization of AUM, a
novel aspartate-based tyrosine phosphatase”
Seifried, Annegrit
„Studies on patelet cytoskeletal dynamics and receptor
regulation in genetically modified mice”
Bender, Markus
“Structural investigations of various functional states of
the ribosomes”
Sejwal, Kushal
“Function of the stromal interaction molecule 2 (STIM2)
in hemostasis and thrombosis“
Berna Erro, Alejandro
“Generation and characterization of mice deficient in
gi24”
Stegner, David
“Identification and characterization of AUM, a novel
human tyrosine phosphatase”
Duraphe, Prashant
“Studies on the role of phospholipase D in platelet
biology isoforms”
Thielmann, Ina
“The role of miRNAs in cardiac disease”
Gross, Carina
“The role of Like-Sm (LSm) proteins in the replication
cycle of positive strand RNA viruses”
Tyagi, Anu
“Store-operated calcium entry in immune cell activation
and signalling”
Vögtle, Timo
“The role of the Rho GTPases Rac1 and Cdc42 for platelet
function and formation”
Pleines, Irina
“Characterisation of posttranslational modifications of
guanylyl cyclase A (GC-A)”
Schröter, Juliane
“Structural and functional studies of the ubiquitin
activating enzyme”
Völler, Daniel
“Structural and functional analysis of the oncostatin M
receptor”
Walter, Sabine
77
Publications
Junior Research Groups
Group Shashi Bhushan
Group Asparouh Iliev
Armache, J. P., Jarasch, A., Anger, A. M.,
Villa, E., Becker, T., Bhushan, S., Jossinet,
F., Habeck, M., Dindar, G., Franckenberg,
S., Marquez, V., Mielke, T., Thomm, M.,
Berninghausen, O., Beatrix, B., Söding, J.,
Westhof, E., Wilson, D.N., and Beckmann,
R. (2010). Cryo-EM structure and rRNA
model of a translating eukaryotic 80S
ribosome at 5.5 Å resolution. PNAS, 107,
19748-53.
Förtsch, C., Hupp, S., Ma, J., Mitchell, T.
J., Maier, E., Benz R., and Iliev, A. I.
(2011). Changes in astrocyte shape
induced by sublytic concentrations of the
cholesterol-dependent cytolysin pneumolysin still require pore-forming capacity.
Toxins, 3(1), 43-62.
Armache, J. P., Jarasch, A., Anger, A. M.,
Villa, E., Becker, T., Bhushan, S., Jossinet,
F., Habeck, M., Dindar, G., Franckenberg,
S., Marquez, V., Mielke, T., Thomm, M.,
Berninghausen, O., Beatrix, B., Söding, J.,
Westhof, E., Wilson, D. N., and Beckmann,
R. (2010). Localization of eukaryotespecific ribosomal proteins: in a 5.5-Å
cryo-EM map of the 80S eucaryotic
ribosome. PNAS, 107, 19754-59.
Bhushan, S., Gartmann, M., Halic, M.,
Armache, J. P., Jarasch, A., Mielke, T.,
Berninghausen, O., Wilson, D.N., and
Beckmann, R. (2010). Alpha-helical
nascent polypeptide chains visualized
within distinct regions of the ribosomal
exit tunnel. Nat Struct Mol Biol, 17,
313-17.
Bhushan, S., Mayer, H., Mielke, T.,
Berninghausen, O., Sattler, M., Wilson,
D.N., and Beckmann, R. (2010). Structural
basis for translational stalling by human
cytomegalovirus and fungal arginine
attenuator peptide. Mol Cell, 40, 138-46.
Bhushan, S., Hoffman, T., Seidelt, B.,
Frauenfeld, J., Mielke, T., Berninghausen,
O., Wilson, D. N., and Beckmann, R. (2011).
secM-stalled ribosomes adopt an altered
geometry at the peptidyl transferase
center. PLOS Biology, 9, e10000581.
Group Heike Hermanns
Radtke, S., Wüller, S., Yang, X. P., Lippok,
B. E., Mütze, B., Mais, C., Schmitz-Van de
Leur, H., Bode, J. G., Gaestel, M.,
Heinrich, P.C., Behrmann, I., Schaper, F.*
and Hermanns, H. M.* (2010). Crossregulation of cytokine signalling: proinflammatory cytokines restrict IL-6
signalling through receptor internalisation
and degradation. J Cell Sci, 123, 947-59.
(*equal contribution).
78
Group Stephan Kissler
Acharya, M., Mukhopadhyay, S., Païdassi,
H., Jamil, T., Chow, C., Kissler, S., Stuart,
L. M., Hynes, R. O., and Lacy-Hulbert, A.
(2010). αv Integrin expression by DCs is
required for Th17 cell differentiation and
development of experimental autoimmune
encephalomyelitis in mice. J Clin Invest,
120, 4445-52.
Group Alma Zernecke
Goossens, P., Gijbels, M. J., Zernecke, A.,
Eijgelaar, W., Vergouwe, M. N., van der
Made, I., Vanderlocht, J., Beckers, L.,
Buurman, W. A., Daemen, M. J., Kalinke,
U., Weber, C., Lutgens, E., and de Winther,
M. P. (2010). Myeloid type I interferon
signaling promotes atherosclerosis by
stimulating macrophage recruitment to
Lesions. Cell Metab, 12, 142-53.
Hristov, M., Gümbel, D., Lutgens, E.,
Zernecke, A., and Weber, C. (2010).
Soluble CD40 ligand impairs the function
of peripheral blood angiogenic outgrowth
cells and increases neointimal formation
after arterial injury. Circulation, 121,
315-24.
Kraemer, S., Lue, H., Zernecke A.,
Kapurniotu, A., Andreetto, E., Frank, R.,
Lennartz, B., Weber, C., and Bernhagen, J.
(2011). MIF-chemokine receptor interactions in atherogenesis are dependent on
an N-loop-based 2-site binding mechanism. FASEB J, 25(3), 894-906.
Liehn, E. A., Piccinini, A. M., Koenen,
R. R., Soehnlein, O., Adage, T., Fatu,
R., Curaj, A., Popescu, A., Zernecke, A.,
Kungl, A. J., and Weber, C. (2010). A new
monocyte chemotactic protein-1/chemokine CC motif ligand-2 competitor limiting
neointima formation and myocardial
ischemia/reperfusion injury in mice. J Am
Coll Cardiol, 56, 1847-57.
Lievens, D.*, Zernecke, A.*, Seijkens, T.,
Soehnlein, O., Beckers, L., Munnix, I.,
Wijnands, E., Goossens, P., van Kruchten,
R., Thevissen, L., Boon, L., Flavell, R. A.,
Noelle, R. J., Gerdes, N., Biessen, E. A.,
Daemen, M. J., Heemskerk, J. W., Weber,
C., and Lutgens, E. (2010). Platelet CD40L
mediates thrombotic and inflammatory
processes in atherosclerosis. Blood, 116,
4317-27. (*equal contribution).
Lutgens, E., Lievens, D., Beckers, L.,
Wijnands, E., Soehnlein, O., Zernecke, A.,
Seijkens, T., Engel, D., Cleutjens, J.,
Keller, A. M., Naik, S. H., Boon, L.,
Oufella, H. A., Mallat, Z., Ahonen, C. L.,
Noelle, R. J., de Winther, M.P., Daemen,
M. J., Biessen, E. A., and Weber, C. (2010).
Deficient CD40-TRAF6 signaling in
leukocytes prevents atherosclerosis by
skewing the immune response toward an
antiinflammatory profile. J Exp Med, 207,
391-404.
Schreinemachers, M. C., Doorschodt, B.
M., Florquin, S., van den Bergh Weerman,
M. A., Zernecke, A., Idu, M. M., Tolba, R.
H., and van Gulik, T. M. (2010). Pulsatile
perfusion preservation of warm ischaemiadamaged experimentalkidney grafts. Br J
Surg, 97, 349-58.
Shagdarsuren, E., Bidzhekov, K., Mause,
S. F., Simsekyilmaz, S., Polakowski, T.,
Hawlisch, H., Gessner, J. E., Zernecke, A.,
and Weber, C. (2010). C5a receptor
targeting in neointima formation after
arterial injury in atherosclerosis-prone
mice. Circulation, 122, 1026-36.
Weber, C., Schober, A., and Zernecke, A.
(2010). MicroRNAs in arterial remodelling,
inflammation and atherosclerosis. Curr
Drug Targets, 11, 950-56.
Zernecke, A., and Weber, C. (2010)
Chemokines in the vascular inflammatory
response of atherosclerosis. Cardiovasc
Res, 86, 192-201.
Zimmermann, H. W., Seidler, S., Nattermann, J., Gassler, N., Hellerbrand, C.,
Zernecke, A., Tischendorf, J. J., Luedde,
T., Weiskirchen, R., Trautwein, C., and
Tacke, F. (2010). Functional contribution of elevated circulating and hepatic
non-classical CD14CD16 monocytes to
inflammation and human liver fibrosis.
PLoS One, 5, e11049.
Core Center
Group Caroline Kisker
Group Hermann Schindelin
Basu, A., Broyde, S., Iwai, S., and Kisker,
C. (2010). DNA damage, mutagenesis, and
DNA repair. J Nucleic Acids, 2010 182894.
Völler, D., and Schindelin, H. (2010). And
yet it moves: active site remodeling in the
SUMO E1. Structure, 18, 419-21.
Breuning, A., Degel, B., Schulz, F.,
Buchold, C., Stempka, M., Machon, U.,
Heppner, S., Gelhaus, C., Leippe, M., Leyh,
M., Kisker, C., Rath, J., Stich, A., Gut, J.,
Rosenthal, P. J., Schmuck, C., and
Schirmeister, T. (2010). Michael acceptor
based antiplasmodial and antitrypanosomal cysteine protease inhibitors with
unusual amino acids. J Med Chem, 53,
1951-63.
Hänzelmann, P., Stingele, J., Hofmann, K.,
Schindelin, H., and Raasi, S. (2010). The
yeast E4 ubiquitin ligase Ufd2 interacts
with the ubiquitin-like domains of Rad23
and Dsk2 via a novel and distinct
ubiquitin-like binding domain. J Biol
Chem, 285, 20390-98.
Luckner, S. R., Liu, N., am Ende, C. W.,
Tonge, P. J., and Kisker, C. (2010). A slow,
tight binding inhibitor of InhA, the enoylacyl carrier protein reductase from
Mycobacterium tuberculosis. J Biol Chem,
285, 14330-37.
Machutta, C. A., Bommineni, G. R.,
Luckner, S. R., Kapilashrami, K., Ruzsicska,
B., Simmerling, C., Kisker, C., and Tonge,
P. J. (2010). Slow onset inhibition of
bacterial beta-ketoacyl-acyl carrier protein
synthases by thiolactomycin. J Biol
Chem, 285, 6161-69.
Qiu, J. A., Wilson, H. L., Pushie, M. J.,
Kisker, C., George, G. N., and Rajagopalan,
K. V. (2010). The structures of the C185S
and C185A mutants of sulfite oxidase
reveal rearrangement of the active site.
Biochemistry, 49, 3989-4000.
Schlereth, K., Beinoraviciute-Kellner, R.,
Zeitlinger, M. K., Bretz, A.C., Sauer, M.,
Charles, J. P., Vogiatzi, F., Leich, E.,
Samans, B., Eilers, M., Kisker, C., Rosenwald, A., and Stiewe, T. (2010). DNA
binding cooperativity of p53 modulates
the decision between cell-cycle arrest and
apoptosis. Mol Cell, 38, 356-68.
Wolski, S. C., Kuper, J., and Kisker, C.
(2010). The XPD helicase: XPanDing
archaeal XPD structures to get a grip
on human DNA repair. Biol Chem, 391,
761-65.
79
Research Professors
Group Utz Fischer
Group Antje Gohla
Chari, A., and Fischer, U. (2010). Cellular
strategies for the assembly of molecular
machines. Trends Biochem Sci, 35(12),
676-83.
Stan, A., Pielarski, K.N., Brigadski, T.,
Wittenmayer, N., Fedorchenko, O., Gohla,
A., Lessmann, V., Dresbach, T., and
Gottmann, K. (2010). Essential cooperation of N-cadherin and neuroligin-1 in the
transsynaptic control of vesicle accumulation. Proc Natl Acad Sci USA, 107,
11116-21.
Galao, R.P., Chari, A., Alves-Rodriggues,
I., Labao, D., Mas, A., Kambach, C.,
Fischer, U., and Diez, J. (2010). LSm1-7
complexes bind to specific sites in viral
RNA genomes and regulate their translation and replication. RNA ,16, 817-27.
Grimm, C., Chari, A., Reuter, K., and
Fischer, U. (2010). A crystallization screen
based on alternative polymeric precipitants. Acta crystallographica, Section D,
ACTA CRYSTALLOGR D, 66 (Pt 6), 685-97.
Martin, G., Ostareck-Lederer, A., Chari, A.,
Neuenkirchen, N., Dettwiler, S., Blank, D.,
Ruegsegger, U., Fischer, U., and Keller, W.
(2010). Arginine methylation in subunits
of mammalian pre-mRNA cleavage factor I.
RNA, 16, 1646-59.
Schäffler, K., Schulz, K., Hirmer, A.,
Wiesner, J., Grimm, M., Sickmann, A., and
Fischer, U. (2010). A stimulatory role for
the La-related protein 4B in translation.
RNA, 16, 1488-99.
Linder, B., Dill, H., Hirmer, A., Brocher, J.,
Lee, G. P., Mathavan, S., Bolz, H. J.,
Winkler, C., Laggerbauer, B., and Fischer,
U. (2011). Systemic splicing factor
deficiency causes tissue-specific defects: a
zebrafisch model for retinitis pigmentosa.
Hum Mol Genet, 20(2), 368-77.
Guderian, G., Peter, C., Wiesner, J.,
Sickmann, A., Schulze-Osthoff, K., Fischer,
U., and Grimmler, M. (2011). RioK1, a new
interactor of protein arginine methyltransferase 5 (PRMT5), competes with plCln for
binding and modulates PRMT5 complex
composition and substrate specificity.
J Biol Chem, 286(3), 1976-86.
80
Bender, M., Eckly, A., Hartwig, J.H.,
Elvers, M., Pleines, I., Gupta, S., Krohne,
G., Jeanclos, E., Gohla, A., Gurniak, C.,
Gachet, C., Witke, W., and Nieswandt, B.
(2010). ADF/n-cofilin-dependent actin
turnover determines platelet formation
and sizing. Blood, 116, 1767-75.
von Holleben, M.*, Gohla, A.*, Janssen,
K. P., Iritani, B. M., and Beer-Hammer, S.
(2011). Immunoinhibitory adapter protein
Src homology domain 3 lymphocyte
protein 2 (SLy2) regulates actin dynamics
and B cell spreading. J Bio Chem, 286
(15), 13489-501. *contributed equally
Research Professors
Group Bernhard Nieswandt
Bender, M., Eckly, A., Hartwig, J. H.,
Elvers, M., Pleines, I., Gupta, S., Krohne,
G., Jeanclos, E., Gohla, A., Gurniak, C.,
Gachet, C., Witke, W., and Nieswandt B.
(2010). ADF/n-cofilin-dependent actin
turnover determines platelet formation
and sizing. Blood, 116(10), 1767-75.
Hagedorn, I., Schmidbauer, S., Pleines, I.,
Kleinschnitz, C., Kronthaler, U., Stoll, G.,
Dickneite, G., and Nieswandt, B. (2010).
Factor FXII inhibitor rHA-Infestin-4
abolishes arterial thrombous formation
without affecting bleeding. Circulation,
121(13), 1510-17.
Bender, M., Hofmann, S., Stegner, D.,
Chalaris, A., Bösl, M., Braun, A., Scheller,
J., Rose-John, S., and Nieswandt, B.
(2010). Differentially regulated GPVI
ectodomain shedding by multiple plateletexpressed proteinases. Blood, 116(17),
3347-55.
Hagedorn, I., Vögtle, T., and Nieswandt,
B. (2010). Arterial thrombus formation.
Novel mechanisms and targets Novel
mechanisms and targets. Hämostaseologie, 30(3), 127-35.
Bültmann, A., Li, Z., Wagner, S., Peluso,
M., Schönberger, T., Weis, C., Konrad, I.,
Stellos, K., Massberg, S., Nieswandt, B.,
Gawaz, M., Ungerer, M., and Münch, G.
(2010). Impact of glycoprotein VI and
platelet adhesion on atherosclerosis--a
possible role of fibronectin. J Mol Cell
Cardiol, 49(3), 532-42.
De Meyer, S. F., Schwarz, T., Deckmyn, H.,
Denis, C. V., Nieswandt, B., Stoll, G.,
Vanhoorelbeke, K., and Kleinschnitz, C.
(2010). Binding of von Willebrand factor
to collagen and glycoprotein Ibalpha, but
not to glycoprotein IIb/IIIa, contributes
to ischemic stroke in mice--brief report.
Arterioscler Thromb Vasc Biol, 30(10),
1949-51.
Elvers, M., Pozgaj, R., Pleines, I., May, F.,
Kuijpers, M. E., Heemskerk, J. W., Yu, P.,
and Nieswandt, B. (2010). Platelet
hyperreactivity and a prothrombotic
phenotype in mice with a gain-of-function
mutation in phospholipase Cγ2. J Thromb
Haemost, 8(6), 1353-63.
Elvers, M., Stegner, D., Hagedorn, I.,
Kleinschnitz, C., Braun, A., Kuijpers, M. E.,
Boesl, M., Chen, Q., Heemskerk, J. W.,
Stoll, G., Frohman, M. A. and Nieswandt,
B. (2010). Impaired alpha(IIb)beta(3)
integrin activation and shear-dependent
thrombus formation in mice lacking
phospholipase D1. Sci Signal, 3(103), ra1.
Gilio, K., van Kruchten, R., Braun, A.,
Berna-Erro, A., Feijge, M. A., Stegner, D.,
van der Meijden, P. E., Kuijpers, M. J.,
Varga-Szabo, D., Heemskerk, J.W., and
Nieswandt, B. (2010). Roles of platelet
STIM1 and Orai1 in glycoprotein VI- and
thrombin-dependent procoagulant activity
and thrombus formation. J Biol Chem,
285(31), 23629-38.
Kleinschnitz, C., Schwab, N., Kraft, P.,
Hagedorn, I., Dreykluft, A., Schwarz, T.,
Austinat, M., Nieswandt, B., Wiendl, H.,
and Stoll, G. (2010). Early detrimental Tcell effects in experimental cerebral
ischemia are neither related to adaptive
immunity nor thrombus formation. Blood,
115(18), 3835-42.
Nieswandt, B. and Stoll, G. (2010). The
smaller, the better: VWF in stroke. Blood,
115(8), 1477-78.
Stoll, G., Kleinschnitz, C., and Nieswandt,
B. (2010). The role of glycoprotein Ibalpha and von Willebrand factor interaction
in stroke development. Hämostaseologie,
30(3), 136-38.
Braun, A., Vögtle, T., Varga-Szabo, D., and
Nieswandt, B. (2011). STIM and Orai in
hemostasis and thrombosis. Front Biosci,
in press.
Nieswandt, B., and Stoll, G. (2011). Sugar
rush bleeds the brain. Nat Med, 17(2),
161-62.
Stegner, D., and Nieswandt, B. (2011).
Platelet receptor signaling in thrombus
formation. J Mol Med, 89(2), 109-21.
Pleines, I., Eckly, A., Elvers, M., Hagedorn, I., Eliautou, S., Bender, M., Wu, X.,
Lanza, F., Gachet, C., Brakebusch, C., and
Nieswandt, B. (2010). Multiple alterations of platelet functions dominated by
increased secretion in mice lacking Cdc42
in platelets. Blood, 115(16), 3364-73.
Petri, B., Broermann, A., Li, H., Khandoga,
A.G., Zarbock, A., Krombach, F., Goerge,
T., Schneider, S. W., Jones, C., Nieswandt,
B., Wild, M. K., and Vestweber, D. (2010).
von Willebrand factor promotes leukocyte
extravasation. Blood, 116(22), 4712-19.
Pham, M., Kleinschnitz, C., Helluy, X.,
Bartsch, A. J., Austinat, M., Behr, V. C.,
Renné, T., Nieswandt, B., Stoll, G., and
Bendszus, M. (2010). Enhanced cortical
reperfusion protects coagulation factor
XII-deficient mice from ischemic stroke as
revealed by high-field MRI. Neuroimage,
49(4), 2907-14.
Pleines, I., Eckly, A., Elvers, M., Hagedorn,
I., Eliautou, S., Bender, M., Wu, X., Lanza,
F., Gachet, C., Brakebusch, C., and
Nieswandt, B. (2010). Multiple alterations
of platelet functions dominated by
increased secretion in mice lacking Cdc42
in platelets. Blood, 115(16), 3364-73.
Stoll, G., Kleinschnitz, C., and Nieswandt,
B. (2010). Combating innate inflammation: a new paradigm for acute treatment of stroke? Ann N Y Acad Sci, 1207,
149-54.
81
Senior Professors
Group Roland Benz
Bárcena-Uribarri, I., Thein, M., Sacher, A.,
Bunikis, I., Bonde, M., Bergström, S., and
Benz, R. (2010). P66 porins are present in
both Lyme dis-ease and relapsing fever
spirochetes: a comparison of the biophysical prop-erties of P66 porins from six
Borrelia species. Biochim Biophys Acta,
1798(6), 1197-203.
Barth, E., Barceló, M. A., Kläckta, C., and
Benz, R. (2010). Reconstitution experiments and gene deletions reveal the
existence of two-component major cell
wall channels in the genus Corynebacterium. J Bacteriol, 192(3), 786-800.
Ebner, M., Stalph, P., Michel, M., and
Benz, R. (2010). Evolutionary parameter
optimization of a fuzzy controller which is
used to control a sewage treatment plant.
Water Sci Technol, 61(1), 53-66.
Fahrer, J., Kuban, J., Heine, K., Rupps, G.,
Kaiser, E., Felder, E., Benz, R., and Barth,
H. (2010). Selective and specific internalization of clostridial C3 ADP-ribosyltransferases into macrophages and monocytes.
Cell Microbiol, 12(2), 233-47.
Huc, E., Meniche, X., Benz, R., Bayan, N.,
Ghazi, A., Tropis, M., and Daffé, M. (2010).
O-mycoloylated proteins from Corynebacterium: an unprecedented post-translational modification in bacteria. J Biol
Chem, 285(29), 21908-12.
Knapp, O., Maier, E., Mkaddem, S. B.,
Benz, R., Bens, M., Chenal, A., Geny, B.,
Vandewalle, A., and Popoff, M. R. (2010).
Clostridium septicum alpha-toxin forms
pores and induces rapid cell necrosis.
Toxicon, 55(1), 61-72.
Ludwig, A., Völkerink, G., von Rhein, C.,
Bauer, S., Maier, E., Bergmann, B., Goebel,
W., and Benz, R. (2010). Mutations affecting export and activity of cytolysin A from
Escherichia coli. J Bacteriol, 192(15),
4001-11.
Polzien, L., Benz, R., and Rapp, U. R.
(2010). Can BAD pores be good? New
insights from examining BAD as a target
of RAF kinases. Adv Enzyme Regul, 50(1),
147-59.
82
Group Martin Heisenberg
Kläckta, C., Knörzer, P., Rieß, F., and
Benz, R. (2011). Hetero-oligomeric cell
wall channels (porins) of Nocardia
farcinica. Biochim Biophys Acta, 1808(6),
1601-10.
Polzien, L., Baljuls, A., Roth, H. M., Kuper,
J., Benz, R., Schweimer, K., Hekman, M.,
and Rapp, U. R. (2011). Pore-forming
activity of BAD is regulated by specific
phosphorylation and structural transitions
of the C-terminal part. Biochim Biophys
Acta, 1810(2), 162-69.
Pei, X.Y ., Hinchliffe, P., Symmons, M. F.,
Koronakis, E., Benz, R., Hughes, C., and
Koronakis V. (2011). Structures of sequential open states in a symmetrical opening
transition of the TolC exit duct. PNAS,
108(5), 2112-17.
Genisyuerek, S., Papatheodorou, P., Guttenberg, G., Schubert, R., Benz, R., and
Aktories, K. (2011). Structural Determinants for Membrane Insertion, Pore Formation and Translocation of Clostridium
difficile Toxin B. Mol Microbiol, Jan 14.
doi:10.1111/j.1365-2958.2011.07549.x.
[Epub ahead of print].
Heisenberg, M. (2010). Von Natur aus frei
– Die Organisation menschlichen und
tierischen Verhaltens ermöglicht Freiheit.
Theologie und Glaube, 100, 208–15.
Schmid, B., Schindelin, J., Cardona, A.,
Longair, M., and Heisenberg, M. (2010).
A high-level 3D visualization API for Java
and ImageJ. BMC Bioinformatics, 11,
274.
Yamaguchi, S., Desplan, C., and Heisenberg, M. (2010). Contribution of photoreceptor subtypes to spectral wavelength
preference in Drosophila. Proc Natl Acad
Sci, 107, 5634-39.
RVZ Network
Group Martin Eilers
Cannell, I. G., Kong, Y. W., Johnston, S.
J., Chen, M. L., Collins, H. M., Dobbyn, H.
C., Elia, A., Kress, T. R., Dickens, M.,
Clemens, M. J., Heery, D. M., Gaestel, M.,
Eilers, M., Willis, A. E., and Bushell, M.
(2010). p38 MAPK/MK2-mediated
induction of miR-34c following DNA
damage prevents Myc-dependent DNA
replication. PNAS, 107, 5375-80.
Singh, G., Singh, S. K., König, A.,
Reutlinger, K., Nye, M. D., Adhikary, T., Eilers, M., Gress, T. M., Fernandez-Zapico, M.
E., and Ellenrieder, V. (2010). Sequential
activation of NFAT and c-Myc transcription factors mediates the TGF-beta switch
from a suppressor to a promoter of cancer
cell proliferation. J Biol Chemistry, 285,
27241-50.
Jahns, R., Boivin, V., and Lohse, M. J.
(2010). Pathogenetical Relevance of
Autoantibodies in Dilated Cardiomyo-pathy. In: Inflammatory Cardiomyopathy –
DCMi – Pathogenesis and Therapy. Series
Progress in In-flammation Research. Parnham MJ Ed., Birkhäuser Verlag AG, Basel
(Switzerland); pp 159-72.
Iraci, N. Diolaiti, D., Papa, A., Porro, A.,
Valli, E., Gherardi, S., Herold, S., Eilers,
M., Haber, M., Norris, M., Bernardoni, R.,
Della Valle, G., and Perini, G. (2010). A
Sp1/Miz1/MycN repression complex
recruits HDAC1 at the TRKA and p75NTR
promoters and affects neuroblastoma
malignancy by inhibiting the cell response
to NGF. Cancer Res, Dec 1. [Epub ahead
of print].
Teutschbein, J., Haydn, J. M., Samans,
B., Krause, M., Eilers, M., Schartl, M., and
Meierjohann S. (2010). Gene expression
analysis after receptor tyrosine kinase
activation reveals new potential melanoma
proteins. BMC Cancer, 10, 386.
Group Thomas D. Müller
Herkert, B., Dwertmann, A., Herold, S.,
Naud, J. F.,Finkernagel, F., Harms, G. S.,
Wanzel, M., and Eilers, M. (2010). The Arf
tumor suppressor protein inhibits Miz1 to
suppress cell adhesion and induce
apoptosis. J Cell Biol, 188 (6), 905-18.
Herkert, B.,and Eilers, M. (2010). Transcriptional Repression: the dark side of
Myc. Genes and Cancer, 1, 580-86.
Kosan, C., Saba, I., Godmann, M., Herold,
S., Herkert, B., Eilers, M., and Möröy, T.
(2010). Transcription factor miz-1 is
required to regulate interleukin-7 receptor
signaling at early commitment stages of B
cell differentiation. Immunity, 33, 91728.
Müller, J., Samans, B., van Riggelen, J.,
Fagà, G., Peh, R., Wei, C. L., Müller, H.,
Amati, B., Felsher, D., and Eilers, M.
(2010). TGF beta-dependent gene
expression shows that senescence
correlates with abortive differentiation
along several lineages in Myc-induced
lymphomas. Cell Cycle, 9, 4622-26.
Popov, N., Schülein, C., Jaenicke, L. and
Eilers, M. (2010). Ubiquitylation of the
amino-terminus of Myc by SCF (beta-TrCP)
antagonizes SCF (Fbw7)-mediated
degradation. Nat Cell Biol, 12, 973-81.
Schlereth, K., Beinoraviciute-Kellner, R.,
Zeitlinger, M. K., Bretz, A.C., Sauer, M.,
Charles, J. P., Vogiatzi, F., Leich, E., Samans, B., Eilers, M., Kisker, C., Rosenwald,
A., and Stiewe, T. (2010). DNA binding cooperativity of p53 modulates the decision
between cell-cycle arrest and apoptosis.
Mol Cell, 14, 356-68.
vanRiggelen, J., Müller, J., Otto, T.,
Beuger, V., Samans, B., Yetil, A., Tao, J.,
Choi, P., Kosan, C., Möröy, T., Felsher, D.,
and Eilers, M. (2010). The interaction
between Myc and Miz1 is required to antagonize TGF beta- dependent atutocrine
signaling during lymphoma formation and
maintenance. Gene Dev, 24, 1281-94.
Group Manfred Gessler
Bielesz, B., Sirin, Y., Si, H., Niranjan, T.,
Gruenwald, A., Ahn, S., Kato, H., Pullman,
J., Gessler, M., Haase, V.H., and Suzstak,
K. (2010). Epithelial Notch signaling
regulates interstitial fibrosis development
in the kidneys of mice and humans. J Clin
Invest, 120, 4040-54.
Wiese, C., Heisig, J., and Gessler, M.
(2010). Hey bHLH factors in cardiovascular development. Pediatr Cardiol, 31,
363-70.
Group Roland Jahns
Jahns, R., Schlipp, A., Boivin, V., and Lohse,
M. J. (2010). Targeting receptor-antibodies
in immune-cardiomyopathy Semin. Thromb.
Hemost, 36, 212-18.
Deubner, N., Berliner, D., Schlipp, A.,
Gelbrich, G., Caforio, A. L. P., Felix, S. B.,
Fu, M., Katus, H., Angermann, C. E., Lohse,
M. J., Ertl, G., Störk, S., and Jahns, R.
(2010). Cardiac Beta1-Adrenoceptor Autoantibodies in Human Heart Disease: Rationale and Design of the Etiology, TitreCourse, and Survival (ETiCS) Study - on
behalf of the ETiCS-Study Group. Eur J
Heart Fail, 12, 753-62.
Dunkel, M., Müller, T., Hedrich, R., and
Geiger, D. (2010). K+ transport characteristics of the plasma membrane tandem-pore
channel TPK4 and pore chimeras with its
vacuolar homologs. FEBS Lett, 584(11),
2433-39.
Harth, S., Kotzsch, A., Hu, J., Sebald, W.,
and Mueller, T.D. (2010). A selection fit
mechanism in BMP receptor IA as a
possible source for BMP ligand-receptor
promiscuity. PLoS One, 5(9), e13049.
Harth, S., Kotzsch, A., Sebald, W., and
Mueller, T.D. (2010). Crystallization of
BMP receptor type IA bound to the
antibody Fab fragment AbD1556. Acta
Cryst F, 66(Pt 8), 964-68.
Krause, C., Korchynskyi, O., de Rooij, K.,
Weidauer, S.E., de Gorter, D.J., van
Bezooijen, R.L., Hatsell, S., Economides,
A.N., Mueller, T.D., Lowik, C.W., and Ten
Dijke, P. (2010). Distinct modes of
inhibition by sclerostin on bone morphogenetic protein and Wnt signaling
pathways. J Biol Chem, 285(53), 4161426.
Piters, E., Culha, C., Moester, M., Van
Bezooijen, R., Adriaensen, D., Mueller, T.,
Weidauer, S., Jennes, K., de Freitas, F.,
Loewik, C., Timmermans, J.P., Van Hul, W.,
and Papapoulos, S. (2010). First missense
mutation in the SOST gene causing
sclerosteosis by loss of sclerostin
function. Hum Mutat, 31(7), E1526-43.
Sebald, W., Nickel, J., Zhang, J.L., and
Mueller, T.D. (2010). Molecular basis of
cytokine signalling--theme and variations.
Febs J, 277(1), 106-18.
83
Bio-Imaging Center
Group Gregory Harms
Group Manfred Heckmann
Gliem, M., Heupel, W.M., Spindler, V.,
Harms, G.S., and Waschke, J. (2010) Actin
reorganization contributes to loss of cell
adhesion in pemphigus vulgaris. Am J
Physiol Cell Physiol, 299, C606-13.
Hallermann, S.*, Kittel, R. J.*, Wichmann,
C.*, Weyhersmüller, A., Fouquet, W.,
Mertel, S., Owald, D., Eimer, S., Depner,
H., Schwärzel, M., Sigrist, S. J., and
Heckmann, M. (2010) Naked dense bodies
provoke depression. J Neurosci, 30,
14340-45.
Herkert, B., Dwertmann, A., Herold, S.,
Abed, M., Naud, J. F., Finkernagel, F.,
Harms, G.S., Orian, A., Wanzel, M., and
Eilers, M. (2010) The Arf tumor suppressor
protein inhibits Miz1 to suppress cell
adhesion and induce apotosis. J Cell Bio,
188, 905-18.
Spille, J. H., Zürn, A., Hoffmann, C.,
Lohse, M. J., and Harms G. S. (2011)
Rotational diffusion of the α2a adrenergic
receptor revealed by FlAsH labeling in
living cells. Biophys J, 100(4), 1139-48.
Zelman-Femiak, M., Gromova, K., Wang,
K., Knaus, P, and Harms, G. S. (2010)
Covalent quantum dot receptor linkage via
the acyl carrier protein for single-molecule
tracking, internalization and trafficking
studies. Biotechniques, 49, 574-79.
84
Geis, C., Weishaupt, A., Hallermann, S.,
Grünewald, B., Wessig, C., Wultsch, T.,
Reif, A., Byts, N., Beck, M., Jablonka, S.,
Boettger, M. K., Üçeyler, N., Fouquet, W.,
Gerlach, M., Meinck, H. M., Sirén, A.L.,
Sigrist, S. J., Toyka, K. V., Heckmann, M.*,
and Sommer, C*. (2010) Stiff person
syndrome-associated autoantibodies to
amphiphysin mediate reduced GABAergic
inhibition. Brain, 133, 3166-80. (scientific commentary in Brain, 133, 3164-65).
Hallermann, S., Heckmann, M., and Kittel,
R. J. (2010) Mechanisms of short-term
plasticity at neuromuscular active zones of
Drosophila. HFSP J, 4, 72-84.
Wagner, N., Weyhersmüller, A., Blauth, A.,
Schuhmann, T., Heckmann, M., Krohne,
ans G., Samakovlis C. (2010) The Drosophila LEM-domain protein MAN1 antagonizes
BMP signaling at the neuromuscular
junction and the wing crossveins. Dev
Biol, 339, 1-13.
Bio-Imaging Center
Group Martin Lohse
Ambrosio, M., and Lohse, M. J. (2010).
Microscopy: GPCR dimers moving closer.
Nat Chem Biol, 6, 570-71.
Lohse, M. J. (2010). Dimerization in GPCR
mobility and signaling. Curr Opin Pharmacol, 10, 53-58.
Calebiro, D., Nikolaev, V.O., and Lohse,
M. J. (2010). Imaging of persistent cAMP
signaling by internalized G proteincoupled receptors. J Mol Endocrinol, 45,
1-8.
Lompré, A. M., Hajjar, R. J., Harding,
S. E., Kranias, E. G., Lohse, M. J., and
Marks, A. R. (2010). Ca2+ cycling and new
therapeutic approaches for heart failure.
Circulation, 121, 822-30.
Calebiro, D., Nikolaev, V.O., Persani, L.,
and Lohse, M. J. (2010). Signaling by
internalized G-protein-coupled receptors.
Trends Pharmacol Sci, 31, 221-28.
Maier-Peuschel, M., Frölich, N., Dees, C.,
Hommers, L. G., Hoffmann, C., Nikolaev,
V. O., and Lohse, M. J. (2010). A FRETbased M2 muscarinic receptor sensor
reveals rapid kinetics of allosteric modulation. J Biol Chem, 285, 8793-8800.
Hoffmann, C., Gaietta, G., Zürn, A.,
Adams, S.R., Terillon, S., Ellisman, M.H.,
Tsien, R.Y., and Lohse, M.J. (2010). Fluorescent labelling of tetracysteine-tagged
proteins in intact cells. Nature Protoc, 5,
1666-77.
Jacobs, S., Calebiro, D., Nikolaev, V.O.,
Lohse, M.J., and Schulz, S. (2010). Realtime monitoring of somatostatin receptorcAMP signaling in live pituitary. Endocrinology, 151, 4560-65.
Khelashvili, G., Dorff, K., Shan, J., Camacho-Artacho, M., Skrabanek, L., Vroling,
B., Bouvier, M., Devi, L. A., George, S. R.,
Javitch, J. A., Lohse, M. J., Milligan, G.,
Neubig, R. R., Palczewski, K., Parmentier,
M., Pin, J. P., Vriend, G., Campagne, F.,
and Filizola, M. (2010). GPCR-OKB: the G
protein coupled receptor oligomer knowledge base. Bioinformatics, 26, 1804-05.
Nikolaev, V. O., Moshkov, A., Lyon, A. R.,
Miragoli, M., Novak, P., Paur, H., Lohse,
M. J., Korchev, Y. E., Harding, S. E., and
Gorelik, J. (2010). Beta2-Adrenergic receptor redistribution in heart failure changes
in cAMP compartmentation. Science, 327,
1653-57.
Ziegler, N., Bätz, J., Zabel, U., Lohse,
M.J., and Hoffmann, C. (2010). FRETbased sensors for the human M1-, M3-,
and M5-acetylcholine receptors. Bioorgan
Med Chem, 19, 1048-54.
Ambrosio, M., Zürn, A., and Lohse, M.J.
(2011). Sensing G-protein-coupled receptor activation. Neuropharmacology,
60, 45-51.
Spille, J.H., Zürn, A., Hoffmann, C., Lohse,
M.J., and Harms, G.S. (2011). Rotational
diffusion of the α2a-adrenergic receptor
revealed by FlAsH labeling in living cells.
Biophys J, 100, 1139-48.
Werthmann, R. C., Lohse, M. J., and
Bünemann, M. (2011). Temporally resolved
cAMP monitoring in endothelial cells
uncovers a thrombin-induced [cAMP]
elevation mediated via the Ca²+-dependent
production of prostacyclin. J Physiol,
589(Pt1), 181-93.
Reiner, S., Ambrosio, M., Hoffmann, C.,
and Lohse, M. J. (2010). Differential signaling of the endogenous agonists at the
beta2-adrenergic receptor. J Biol Chem,
285, 36188-98.
von Hayn, K., Werthmann, R.C., Nikolaev,
V. O., Hommers, L. G., Lohse, M. J., and
Bünemann, M. (2010). Gq-mediated Ca2+
signals inhibit adenylyl cyclases 5/6
in vascular smooth muscle cells. Am J
Physiol Cell Physiol, 298, C324-32.
Klenk, C., Schulz, S., Calebiro, D., and
Lohse M. J. (2010). Agonist-regulated
cleavage of the extracellular domain of
parathyroid hormone receptor type 1. J
Biol Chem, 285, 8665-74.
Volpe, S., Thelen, S., Pertel, T., Lohse, M.
J., and Thelen, M. (2010). Polarization
of migrating monocytic cells is independent of PI 3-kinase activity. PLoS One, 5,
e10159.
Klenk, C., Vetter, T., Zürn, A., Vilardaga, J.
P., Friedman, P. A., Wang, B., and Lohse,
M. J. (2010). Formation of a ternary
complex among NHERF1, beta-arrestin,
and parathyroid hormone receptor. J Biol
Chem, 285, 30355-62.
Wachten, S., Masada, N., Ayling, L. J.,
Ciruela, A., Nikolaev, V. O., Lohse, M. J.,
and Cooper D. M. (2010). Distinct pools of
cAMP centre on different isoforms of adenylyl cyclase in pituitary-derived GH3B6
cells. J Cell Sci, 123, 95-106.
Lampert, K. P., Schmidt, C., Fischer, P.,
Volff, J. N., Hoffmann, C., Muck, J., Lohse,
M., Ryan, M. J., and Schartl, M. (2010).
Determination of onset of sexual maturation and mating behavior by melanocortin
receptor 4 polymorphisms. Curr Biol, 20,
1729-34.
Zürn, A., Klenk, C., Zabel, U., Reiner, S.,
Lohse, M.J., and Hoffmann, C. (2010).
Site-specific, orthogonal labeling of
proteins in intact cells with two small
biarsenical fluorophores. Bioconj Chem,
21, 853-59.
85
Early Independence Program
Ingrid Tessmer – Early Independence
Program
Tsai, H. H., Huang, C. H., Tessmer, I., Erie,
D. A., and Chen, C. W. (2011). Linear
Streptomices plasmids form superhelical
circles through interactions between their
terminal proteins. Nucleic Acids Res,
39(6), 2165-74.
Fronczek, D. N., Quammen, C., Wang, H.,
Kisker, C., Superfine, R., Taylor, R., Erie,
D. A., and Tessmer, I. (2011). High
accuracy FIONA-AFM hybrid imaging.
Ultramicroscopy, 111(5), 350-55.
86
The Annual Report 2010 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.
Editor:
Rudolf Virchow Center /DFG Research Center for Experimental Biomedicine of the University of Würzburg
Editors in chief:
Kristina Kessler
Imprin t
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Images:
Wolfgang Dürr (U1), Science-Images (from left): Caroline Kisker/ Bernhard Nieswandt/Shashi Bhushan,
Schmelz (U2), Archiv Pathologie Universität Würzburg (U4), Sascha Kreger (p.4), Kristina Kessler (p.5),
Thomas Martin Pieruschek (p.6), Thorsten Winter (p.7), Hermann Schindelin/Alma Zernecke (p.8),
Ingrid Tessmer/Roland Benz (p.9), Bernhard Nieswandt (p.10), Sonja Jülich-Abbas (p.11), Kristina Kessler/
Sascha Kreger/Andy Krapf (p.12), Thomas Martin Pieruschek (p.13), Sascha Kreger, Horst Pfrang (p.14),
Sascha Kreger (p.15), (from top) Alma Zernecke/Hermann Schindelin/Antje Gohla/ Martin Lohse (p.17),
Shashi Bhushan (p.18-19), Heike Hermanns (p.20-21), Asparouh Iliev (p.22-23), Stephan Kissler (p.24-25),
Alma Zernecke (p.26-27), Caroline Kisker (p.28-29), Hermann Schindelin (p.30-31), Utz Fischer (p.32-33),
Antje Gohla (p.34-35), Bernhard Nieswandt (p.36-37), Roland Benz (p.38-39), Martin Heisenberg (p.40-41),
Martin Eilers (p.42-43), Manfred Gessler (p.44-45), Roland Jahns (p.46-47), Thomas Müller (p.48-49),
Gregory Harms (p.50-51), Manfred Heckmann (p.52-53), Martin Lohse (p.54-55), Ingrid Tessmer (p.56),
Katrin Heinze (p.57), (from top) Stephan Schröder-Köhne/Kristina Kessler/ Sonja Jülich-Abbas/Ilja C. Hendel
für Wissenschaft im Dialog (p.59) Carmen Dengel, Waldemar Salesski (p.60), Manfred Schartl (p.61), Sascha
Kreger (p. 63), Heike Hermanns (p.64), Stephan Kissler (p.65) Waldemar Salesski, Kristina Kessler, Stephan
Rinke (p.66), Klaus Scheuplein, Kristina Kessler (p.67)