Annual Report - Max F. Perutz Laboratories (MFPL)

Transcription

MFPLAnnRep2010-General_FV:Layout 1 16.05.11 21:46 Seite 1
M A X
F .
P E R U T Z
L A B O R A T O R I E S
Annual Report
The Max F. Perutz Laboratories are a joint venture of
H I S T O R Y
„In science, truth always wins.“
To honour an extraordinary teacher
and scientist, the Max F. Perutz
Laboratories were named after Max
Ferdinand Perutz, who, together with
John C. Kendrew, was awarded the
1962 Nobel Prize in Chemistry for
his studies on the structure of
globular proteins.
Max Perutz was born in 1914 in Vienna to a family
of textile manufacturers who made their fortune
during the industrial revolution in the 19th century,
through the introduction of mechanical spinning
and weaving. He attended the Theresianum, where
a perceptive teacher awakened his interest in
chemistry.
In 1932 he entered the University of Vienna, but
because of the poor prospects for a scientific career
MAX F. PERUTZ
in Austria he decided in 1936 to move to the
Cavendish Laboratory in Cambridge. After Hitler´s
invasion of Austria, the family business was expropriated, his parents became refugees and his natural
choice was to continue his career in Cambridge.
Perutz and his co-workers managed to solve the
structure of haemoglobin in 1959. The work was
published in Nature in February 1960, and Perutz
was awarded the Nobel Prize in Chemistry in 1962
together with John Kendrew, who had solved the
structure of myoglobin.
In addition to his studies, Perutz pioneered the new
research field of Molecular Biology and was instrumental in founding the Laboratory of Molecular
Biology (LMB) in Cambridge, UK. He was also involved in establishing the European Molecular Biology Organization (EMBO) in Heidelberg, Germany.
Max F. Perutz died in February 2002 in Cambridge.
History of the Max F. Perutz Laboratories
1992/1993
5 departments of the University of Vienna move to the VBC, three new chairs were established
1994
Start of the international VBC PhD Program
1996
Max Perutz Library established
1998
The biotech company Intercell is founded as a spin-off by Alexander v. Gabain and colleagues from IMP
1999
New chair for Immunobiology established
2001
Dept. for Structural Biology moves to the VBC, new Chair for Structural Biology / NMR established,
new Chair for X-Ray Crystallography established
2004
Medical University of Vienna established
2005
Dept. for Chromosome Biology moves to the VBC
Max F. Perutz Laboratories GmbH established, Harald Hochreiter appointed as Administrative Director
Scientific Advisory Board established
2007
Graham Warren appointed as Scientific Director
2008
First Junior Group Leaders appointed
2009
Start of MFPL International PhD Program
2010
4 new groups started
Fabien Martins appointed as new Administrative Director
M A X
F .
P E R U T Z
Annual Report
2010
T A B L E
O F
C O N T E N T
Content
Message from the Rectors
5
Report of the Directorate
6
The Max F. Perutz Laboratories
8
Awards and Honours
9
Focus on Research
10
Research Groups
14
Facilities
78
Education
89
Scientific Exchange
94
Service and Support
96
Social Life
98
Publications
100
The Campus Vienna Biocenter
104
Contact, Imprint
105
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R E S E A R C H
Research Groups
Gustav Ammerer
Manuela Baccarini
Andreas Bachmair
Andrea Barta
Dieter Blaas
Udo Bläsi
Cécile Brocard
Alexander Dammermann
Thomas Decker
Kristina Djinović-Carugo
Gang Dong
Silke Dorner
Roland Foisner
Peter Fuchs
Juraj Gregan
Alexander von Gabain
Arndt von Haeseler
Andreas Hartig
Erwin Heberle-Bors
Marcela Hermann
Joachim Hermisson
Reinhold Hofbauer
N.-Erwin Ivessa
Michael Jantsch
Verena Jantsch
Franz Klein
Alwin Köhler
Gottfried Köhler
Robert Konrat
Pavel Kovarik
Fritz Kragler
Karl Kuchler
Wolfgang Löffelhardt
Josef Loidl
Zdravko Lorkovic
Sascha Martens
Irute Meskiene
Isabella Moll
Ernst Müllner
Johannes Nimpf
Egon Ogris
Brigitte Poppenberger
Rainer Prohaska
Friedrich Propst
Florian Raible
Johann Rothenender
Peter Schlögelhofer
Wolfgang Schneider
Renée Schroeder
Christoph Schüller
Joachim Seipelt
Christian Seiser
Tobias Sieberer
Tim Skern
Markus Teige
Kristin Tessmar-Raible
Christina Waldsich
Graham Warren
Georg Weitzer
Gerhard Wiche
Angela Witte
Fanz Wohlrab
Bojan Zagrovic
Signal transduction and transcriptional regulation in yeast
Deciphering the MAPK pathway in vivo
Protein modifiers in plants and retrotransposon biology
Post-transcriptional regulation of gene expression in plants
Early interactions of viruses with host cells
Post-transcriptional regulation in Bacteria and Archaea
Protein networks and intracellular membrane remodeling
Centriole Assembly and Function
Host responses and innate immunity to bacteria
Structural Biology of Cytoskeleton
Structural studies of ciliogenesis
The regulation of gene expression by small ncRNAs
Lamins in nuclear organization and human disease
Stress response in simple epithelia
Chromosome segregation during mitosis and meiosis
R & D Programs at Intercell AG, a spin-off of the MFPL and the IMP
Bioinformatics
Origin and biogenesis of peroxisomes
Plant developmental genetics and biotechnology
LDL-R gene family, apolipoproteins and lipid transfer
Theoretical Population Genetics
Consequences of carnitine deficiency and CSF-1 inhibition
Protein biogenesis and degradation from the ER
Impact of RNA-editing on coding and non coding substrate RNAs
Meiosis in Caenorhabditis elegans
Chromosome Structure and Meiotic Recombination
Gene Expression and Chromosome Dynamics
Biomolecular optical spectroscopy
Computational Biology and Biomolecular NMR Spectroscopy
Signaling and gene expression in inflammation
Intercellular transport of proteins and RNAs regulating cell-fate
Host-Pathogen Interactions & Mechanisms of Fungal Pathogenesis
Cyanophora paradoxa, the key to plastid evolution
Meiotic chromosome pairing and recombination
Regulatory roles of cyclophilins in cellular signaling
Molecular Mechanisms of Autophagy
Cell signaling control by MAPK phosphatases
Ribosome Heterogeneity in Bacteria
Signal Transduction and Hematopoiesis/Erythropoiesis
ApoER2 and VLDL Receptor
PP2A enzyme biogenesis and monoclonal antibodies
Regulation of plant steroid hormone homeostasis
Stomatin, membrane microdomains and neuroacanthocytosis
The neuronal cytoskeleton in axon guidance
Origin and Diversification of Hormone Systems
Cell cycle regulation and DNA damage response
Meiotic Recombination
Molecular Mechanisms of Dyslipidemias and Atherogenesis
RNA Aptamers and RNA Chaperones
Demands on transcription in response to environmental stress
Virus Host cell Interactions
Chromatin modifiers in development and disease
Signaling networks in plant shoot formation
Interactions between viruses and cells
Plant Signaling
Lunar periodicity and inner brain photoreceptors
Exploring RNA folding: from structure to function
Biogenesis of the Golgi apparatus
Somatic Stem Cells of the Heart
The cytoskeleton in signaling and disease
fCh1, model for gene regulation in haloalkaliphilic Archaea
Function of zona pellucida domain proteins
Computational Biophysics of Macromolecules
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G R O U P S
M E S S A G E
F R O M
T H E
R E C T O R S
We see with great pleasure that our joint venture,
the Max F. Perutz Laboratories, is steadily developing.
The MFPL was founded as a ‘testbed’ for new approaches in university
research and science management
and continues in its fifth year to be
a success story.
In 2010, the MFPL scientists garned many prestigious grants from national and international
organizations, including an Austrian-funded network project on RNA. We would especially like to
congratulate the MFPL on two HFSP Young Investigator Grants, a FWF START Prize and two ERC Starting Grants awarded to its young group leaders, a
tangible sign that our investment in the new generation of scientists at the MFPL is bearing fruit.
We congratulate Renée Schroeder on her appointment to the Austrian Research Council, which advises the Minister on Science Policy; and Manuela
Baccarini, elected corresponding member of the
Austrian National Academy of Sciences.
to scientific independence. The MFPL now house
several PhD training programs, jointly organized
by the University and Medical University, and has
expanded its educational infrastructure for undergraduate and PhD students.
Both universities are happy to see that Vision 2020
- a major strategic plan for the Campus Vienna
Biocenter – has now become reality. The Campus
Support Facility provides high-end technologies to
the campus, helping to enhance its reputation as
one of Europe’s scientific centres and increasing
its attractiveness to outstanding scientists
Last year saw the departure of the Administrative
Director, Harald Hochreiter, who played a crucial
role in establishing the MFPL and in promoting
their successful development over the last five
years. We thank him for his dedication and wish
him all the best in his future career. His successor,
Fabien Martins, started work in the summer of
2010 and will, we feel sure, support the MFPL
through another prosperous phase of development.
We value and support the continuing efforts of
the MFPL to advance their education and training
programs at all academic career levels. One example
is VIPS, a unique and innovative program that
helps support post-doctoral fellows on their way
We reassert our continuing support to this increasingly successful enterprise and wish the Max F.
Perutz Laboratories all the best in their constant
striving for excellence.
Georg Winckler
Rector University of Vienna
Wolfgang Schütz
Rector Medical University of Vienna
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Georg Winckler
Wolfgang Schütz
M F P L
R E P O R T
2 0 1 0
Report of the Directorate
Four new junior group leaders
started their groups in 2010.
The end of 2009 and the beginning of 2010 was
marked by welcoming four new junior group leaders: Sascha Martens, a biochemist working on
membrane-bending proteins involved in autophagy,
was a post-doc with Harvey McMahon at the LMB
in Cambridge; Alwin Köhler, who was a Senior Research Fellow with Ed Hurt at the BZH in Heidelberg
works on molecular pathways involved in gene gating at nuclear pores in yeast; Alex Dammermann,
previously a post-doc with Karen Oegema at the
Ludwig Institute for Cancer Research at UCSD, studies the maturation of centrosomes during ciliogenesis in C. elegans; and Bojan Zagrovic, who
was Head of Computational Biophysics at the Mediterranean Institute for Life Sciences, Split, Croatia,
uses computational molecular dynamics simulations to study protein interactions and folding. At
the beginning of 2010 the City of Vienna and the
WWTF published a call for “Vienna Research Groups
for Young Investigators”. Two of the three successful candidates will be based at the MFPL: Alipasha
Vaziri (a joint appointee with the IMP) is a research
fellow at the Howard Hughes Medical Institute at
Janelia Farm and will move to the MFPL in the
spring of 2011, where he will focus on innovative
imaging and optogenetic approaches to understand
bio-molecular functions. Claudine Kraft from the
ETH in Zürich will start in the autumn of 2011,
working on the molecular regulation of autophagy.
2010 was a very successful
year for national and
international awards.
Of the nine HFSP Young Investigator Grants awarded in 2010, two were granted to MFPL group leaders: Juro Gregan and Kristin Tessmar. An Austrian
START prize was awarded to Bojan Zagrovic and
ERC starting grants were awarded to both Sascha
Martens and Florian Raible. Among the senior
From left: Fabien Martins (Administrative Director), Manuela Baccarini (Vice-Dean for the University of Vienna),
Graham Warren (Scientific Director), Roland Foisner (Vice-Dean for the Medical University of Vienna)
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M F P L
MFPL faculty, Renée Schroeder was elected a
member of the Austrian Research Council, which
advises the government on science policy. Manuela
Baccarini was appointed as corresponding member
of the Austrian Academy of Sciences.
We are also very pleased that the FWF recently
approved a new Special Research Program “RNA
regulation of the transcriptome” led by Renée
Schroeder, encompassing 11 research groups from
the MFPL, CEMM, IMP, IMBA, GMI, the University
of Vienna and the Medical University of Vienna.
The Vienna International
PostDoctoral Program VIPS
has started.
In early 2010, MFPL launched “VIPS”, an initiative
funded by the City of Vienna and the Ministry of
Science. This program aims to advance the careers
of young investigators at the postdoctoral level. It
offers funds for up to 18 post-docs, selected by
competitive international calls, who will spend the
first 3-5 years as a post-doc in a research group of
their choice at the MFPL and can then apply for
their own funding, publish independently, and so
take the first steps towards scientific independence.
In the first calls, in March and September 2010,
five PostDocs were selected from 358 applications.
The first VIPS post-doc, Justyna Sawa-Makarska,
started in September 2010 in Sascha Martens’
group.
Young investigators at the pre-doctoral level are
recruited via the MFPL International PhD selection
or by the Campus VBC PhD Program. In 2010, the
MFPL held two heavily oversubscribed selections,
which resulted in the recruitment of 37 PhD students from all over the world to the MFPL. Several
of these students participate in thematically focussed Doctoral programs funded by the Austrian
Research Fund FWF providing state-of-the-art training in the areas of Cell Signaling, RNA biology,
and Structural Biology.
R E P O R T
The new Campus Science
Support Facility becomes
reality.
Under the heading “Vision 2020”, the Austrian Ministry for Science and Research and the City of
Vienna awarded the Campus Vienna Biocenter 52
million euros over the next 10 years to fund stateof-the-art scientific infrastructure ranging from
Deep Sequencing to Phytoculture. Andreas Tiran
has been appointed as the managing director of
the Campus Support Facility GmbH, which is scheduled to start early in 2011.
A change in the administrative
management.
2010 also saw a change in the administrative management at the MFPL. The Administrative Director
Harald Hochreiter, who had built up the administrative structure at the MFPL over the last 5 years,
left our Institute, moving on to further challenges,
in which we wish him all the best. His place as
Administrative Director has been taken by Fabien
Martins, who looks back on 17 years of work experience, mostly in industry, but also with a strong
link to the scientific world.
Finally, research work at the MFPL is complemented
by social activities that range from Happy Hours
to the Dragonboat Race. We have continued the
tradition of a Christmas Pantomime, now extended
to include all Institutes on campus, providing the
writers even more material for their scripts. Our
productivity also extends beyond the publishing of
high profile papers to the equally joyful welcoming
of new family members to some of our junior group
leaders.
Fabien Martins
Manuela Baccarini
Graham Warren
Roland Foisner
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2 0 1 0
I N T R O D U C T I O N
The Max F. Perutz Laboratories
Founded in 2005 as a joint venture
of the University of Vienna and the
Medical University of Vienna, the
Max F. Perutz Laboratories (MFPL)
provide an environment for excellent,
internationally recognized research
and education in the field of
Molecular Cell Biology.
MFPL in numbers
470 people from more than 30 nations
63 research groups
70% of personnel funded by grants
Training for 700 undergraduate students
150 PhD students
Over 130 scientific publications in internationally recognized journals
• 12.5 million euro grant money
•
•
•
•
•
•
In 2010, the MFPL hosted 63 independent research
groups, involving more than 470 people from over
30 nations. Research at the Max F. Perutz Laboratories is curiosity-driven and spans the field of Molecular and Cell Biology. Most groups investigate
basic research questions but a significant number
are also active in more applied fields of biology.
Education
The Max F. Perutz Laboratories have a strong focus
on the education and training of young researchers.
Members of the MFPL faculty teach undergraduate
courses in the Life Sciences and Medicine, supervise
diploma students and train PhD students and PostDocs taking their first steps in their scientific career.
Research Areas
• Infection Biology
• RNA Biology
• Cell Signaling
• Computational and Structural Biology
• Chromosome Biology
• Membranes and the Cytoskeleton
More about opportunities for PhDs and Post-Docs
on page 90.
Funding
The Max F. Perutz Laboratories are jointly funded
by the University of Vienna and the Medical University of Vienna. The two universities cover around
two-thirds of the overall MFPL budget, providing
space, scientific infrastructure and some of the
staff. Most of the scientific personnel (70%) and
the running costs are covered by third-party funding raised by the MFPL group leaders.
The total volume of third party funding in 2010 was
12.5 million euros. The main external sources of
funding were the Austrian Research Fund (FWF),
Austrian Ministries and the Vienna Science and
Technology Fund (WWTF).
Scientific Advisory Board
The Scientific Advisory Board SAB visits MFPL every
year to monitor the scientific performance and discuss future developments with the Directorate and
the Faculty.
We thank our SAB members:
Jean Beggs, University of Edinburgh
Cyrus Chothia, MRC LMB Cambridge
Jorge Galan, Yale University
David Livingston, Dana-Farber Cancer Institute
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A W A R D S
A N D
H O N O U R S
Major Awards
HFSP Young
Investigator Grants
The Young Investigator Grant is awarded for interdisciplinary and international collaborations among
teams of young scientists. Two of nine grants in
2010 have been awarded to project leaders from
the MFPL. Juraj Gregan investigates the molecular
architecture and mechanical properties of the kinetochore, a protein structure at the chromosomes,
that plays an important role during cell division,
and Kristin Tessmar-Raible works on the molecular
mechanisms of light-dependent rhythmic processes
in the marine environment.
Honours
Nina Gratz
L’Oréal Austria Award “For Women in Science”
Juraj Gregan
Manuela Baccarini
Elected corresponding member of the Austrian
Academy of Sciences
Renée Schroeder
Appointed member of the Austrian Research Council
FWF START prize
The START program of the Austrian Science Fund
(FWF) is the best endowed and most prestigious
long-term funding program for young researchers
in Austria. Bojan Zagrovic joined the MFPL in
early 2010 and studies protein interactions and
folding using computational molecular dynamics
simulations.
Sabine Lagger
Campus Vienna Biocenter PhD Award
Elected member of the Royal Swedish Academy
ERC Grants
Starting Independent Researcher Grants from the
European Research Council (ERC) aim to support upand-coming young researchers who are about to
establish or consolidate a research team in Europe.
The two awardees – Sascha Martens and Florian
Raible – started their research groups at the Max
F. Perutz Laboratories in 2009 and 2008, respectively. Florian Raible will study moonlight-dependent
hormones that orchestrate the reproductive periodicity of the bristle worm Platynereis dumerilii.
Sascha Martens will investigate the molecular
mechanisms of autophagy, an important cellular
process, that plays an essential role during starvation, pathogen defense and in the removal of protein
aggregates and damaged organelles.
Bojan Zagrovic
Sascha Martens and
Florian Raible
WWTF - Vienna Research
Groups for Young
Investigators
With grants from the funding program "Vienna Research Groups for Young Investigators" of the
Vienna Research and Technology Fund (WWTF),
two outstanding young researchers will start their
research groups at MFPL. Claudine Kraft will work
on the molecular regulation of autophagy. Alipasha
Vaziri is a joint appointment of the IMP and MFPL
and will focus on innovative imaging and optogenetic approaches to understand bio-molecular
functions.
Claudine Kraft
Alipasha Vaziri
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F O C U S
O N
R E S E A R C H
Quis custodiet ipsos custodies?
How cells keep their guards in check.
Matthias Farlik
Thomas Decker
When cells are attacked by bacteria they use all
means at their disposal to defend themselves. But
cellular defence systems can damage the cells themselves and so need to be kept tightly in check. Recent
studies of Thomas Decker and his group have shed
light on the process by which cells produce Nitric
oxide (NO) fighting Listeria infection and at the
same time uncovered a new mechanism for regulating gene transcription. The synthesis of the NO producing enzyme (iNOS) requires the interaction of
two different signaling pathways – a mechanism to
ensure, that toxic NO is only produced when really
needed. Using a clever genetic trick, Decker and his
colleagues could separate the two immunological
signals and investigate them independently. So they
could show, that each of the signals triggers the
formation of only one part of an initiation complex
and that both pathways must be active to form the
entire complex and thus to switch on the gene for
iNOS production. If only one pathway is activated,
the part-complex works as a sort of molecular
memory: if the missing information arrives in time,
the gene is switched on, if not, the initial signal is
“forgotten” after a while – an ingenious mechanism
to combine two independent pathways, even if the
different signals do not arrive at the same time.
Farlik M, Reutterer B, Schindler C, Greten F, Vogl C, Müller
M and Decker T (2010). Nonconventional Initiation Complex Assembly by STAT and NF-kB Transcription Factors
Regulates Nitric Oxide Synthase Expression. Immunity,
Vol.32, issue 7.
Macrophages (immune cells) infected by listeria cells (small black
spots). The right macrophage has already been killed.
Why reinvent the wheel?
Annelid and arthropod segments
have a common origin.
Juliane Zantke
Annelids and arthropods share a similar segmented
organization of the body whose evolutionary origin
had been actively debated based on morphological
evidence. In her recent work, Kristin Tessmar-Raible,
Katharina Schipany and Juliane Zantke from the
MFPL together with collaborators at the CNRS in
France investigated the Hedgehog signaling pathway, which is crucial for arthropod embryonic segment patterning. While this pathway had not been
shown to have a similar function outside arthropods, Tessmar and colleagues found that the ligand
Hedgehog, the receptor Patched, and the transcription factor Gli are all expressed in striped patterns
before the morphological appearance of segments
in the annelid Platynereis dumerilii. Treatments
with small molecules antagonistic to Hedgehog
signaling disrupt segment formation. Platynereis
Hedgehog is not necessary to establish early segment patterns but is required to maintain them.
The molecular similarity of segment patterning
functions of the Hedgehog pathway in an annelid
and in arthropods strongly suggest a common origin
of segmentation in protostomes.
Dray N, Tessmar-Raible K, Le Gouar M, Vibert L, Christodoulou F, Schipany K, Guillou A, Zantke J, Snyman H, Béhague J, Vervoort M, Arendt D, Balavoine G. (2010) Hedgehog
signaling regulates segment formation in the annelid
Platynereis. Science. 329(5989):339-42
Platynereis dumerilii, showing the typical annelid segmentation
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F O C U S
O N
R E S E A R C H
Sometimes, it’s not the usual suspects …
Suprising role of HDAC1 for the
genesis of tumors.
University of Vienna, they also found, that the same
is true for human teratomas: lower levels of HDAC1
correlate with higher malignancy of the tumors.
Histone deacetylases (HDAC) influence the development of cells and have a crucial role for the
genesis of tumors. HDAC inhibitors are therefore
promising anti-cancer reagents. As the specific
HDAC isoforms that mediate these effects are not
yet identified, Christian Seiser and his colleagues
wanted to investigate the specific role of HDAC1
as it was suspected to increase the malignancy of
tumors.
These findings reveal a novel role for HDAC1 in
the control of tumour proliferation and could therefore constitute a new approach for the development
of anti-cancer reagents concentrating on specific
enzymes instead of a whole group. Additionally,
the results of the study identified HDAC1 as a potential marker for benign teratomas.
Surprisingly, they found out, that the teratomas (a
special type of germ cell tumors) they studied were
more aggressive, when HDAC1 was suppressed.
Together with clinical researchers from the Medical
Lagger S, Meunier D, Mikula M, Brunmeir R, Schlederer M,
Artaker M, Pusch O, Egger G, Hagelkruys A, Mikulits W,
Weitzer G, Muellner E, Susani M, Kenner L and Seiser C
(2010). Crucial function of histone deacetylase 1 for
differentiation of teratomas in mice and humans. The
EMBO Journal doi:10.1038/emboj. 2010.264
Human patient teratomas reflect the murine phenotype. Two mature (left) and two immature (right) human paraffin
embedded tumor sections were stained with antibodies against HDAC1 and HDAC2 (red staining). Samples were
counterstained with haematoxilin (blue staining) and pictures in a 20x magnification were taken. Strikingly, the
amount of HDAC1 and HDAC2 positive cells correlates with the tumor differentiation grade. Therefore, HDAC1 is
mainly found in differentiated teratomas, whereas HDAC2 staining is preferentially detected in immature,
undifferentiated germ cell tumors.
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Christian Seiser
Sabine Lagger
F O C U S
O N
R E S E A R C H
Research Initiatives and Networks
Scientific collaboration is a major
element and inevitable prerequisite
for excellence in basic research.
enabling them to work on the frontiers of their
thematic areas. Currently, MFPL researchers participate in three SFBs, two of which are hosted by the
MFPL.
Our group leaders are therefore actively involved
in local, national and international research networks. One example are Special Research Programs
(SFBs) funded by the Austrian Science Fund FWF.
SFBs are peer-reviewed, highly interactive research
networks, established to foster long-term, interdisciplinary co-operation of local research groups
Further collaborative research project coordinated
by the MFPL is the Center for Optimized Structural
Studies (COSS), funded by the Austrian Research
Promotion Agency FFG as a “Laura Bassi Centre of
Excellence”. The Laura Bassi funding program promotes research networks led by women at the interface between science and industry.
Laura Bassi Centre
“COSS – Center for Optimized
Structural Studies”
SFB17 “Modulators of
RNA Fate and Function”
Proteins are the building blocks of life and can be
found in every cell. Being able to decode the
structure of proteins means a better understanding
of numerous processes in the body. Structure determination at atomic detail requires high levels of
protein quality and in large quantity. To investigate
proteins for instance via X-ray diffraction analysis
they need not only to be produced and purified but
also to be crystallized, thus requiring more time
and labor intensive experiments. The major aim of
COSS is to research innovative methods to produce
sufficient quantities of high-quality protein that
can be used for the crystallization process. In the
long term, COSS could offer a platform and methodologies to complement the recently planned campus-wide structural biology facility.
HEAD
Kristina Djinovic-Carugo
PROJECT PARTNERS
Peggy Stolt-Bergner (IMP), Jan Michael Peters
(IMP), Markus Hanner (Intercell)
Today, RNA can be considered as the most versatile regulatory factor
in cellular metabolism.
RNA molecules are involved in gene expression at all levels in proand eukaryotes, including chromatin remodelling, transcription,
RNA stability, translation and post-translational events. The
Hfq hexamer with RNA molecule
SFB17 funded by the
FWF started in 2001
to study how proteins govern RNA structure and
function, mediate the interaction between nucleic
acids, and how they catalyze RNA maturation and
turnover. SFB 17 has been completed at the beginning of 2011 after almost ten years of intensive
and successful research. Besides numerous scientific
findings, it has contributed substantially to establish
an internationally recognized hot spot for RNA research in Vienna and served as a primer for the
PhD program “RNA Biology”, established in 2007.
SPEAKERS
Udo Bläsi, Renée Schroeder (Deputy)
PARTICIPATING GROUPS
Chlorite dismutase crystal
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Andrea Barta, Denise Barlow (CEMM), Kristina
Djinovic-Carugo, Michael Jantsch, Robert Konrat,
Anton Wutz (IMP)
Associated members: Silke Dorner, Isabella Moll,
Christina Waldsich
F O C U S
SFB “Jak-Stat Signaling:
from Basics to Disease“
Jak-Stat signaling is used by a large number of
cell surface receptors, particularly cytokine receptors, to reprogram gene expression and to regulate
many biological responses in virtually all cell types
and organs. The general objective of the SFB is to
jointly investigate how Jaks and Stats regulate
immunity to infection, inflammation and cancer.
The unifying aim is to study these topics and the
links between them. This concept is supported by
similarities concerning mechanisms of acute inflammation and cancer progression, and the association of signal transduction originating from
infection and inflammation with tumorigenesis.
Contributions of Jaks and Stats to cell autonomous
mechanisms of tumorigenesis are examined and
connected to Jak-Stat contributions to cancer immune surveillance or the establishment of an
inflammatory environment promoting cancer
growth. These studies consider a role for hitherto
poorly understood interactions with Jak-Stat partner molecules and they will test potential functions
of non-canonical Stat activation. Furthermore, they
address mechanisms by which Stats regulate expression of their target genes.
O N
R E S E A R C H
SFB “Chromosome Dynamics
– Unraveling the function
of chromosomal domains”
Chromosomes are not just simply receptacles for
our body plan, they are highly dynamic structures,
which change their properties dramatically according to the necessities of cell cycle and reproduction.
The SFB “Chromosome Dynamics”, started in 2008,
aims to define chromosomal domains, such as the
kinetochore, chromosome axis, loop domains and
recombination hotspots on a molecular level.
Various aspects of chromosome biology are studied
by altogether seven groups from the MFPL and the
IMP. The kinetochore - microtubule attachment
and the biochemistry of cohesins, both key aspects
of segregation, are studied in meiosis and in mitosis
in budding and fission yeast, as well as in human
cells. In meiosis I, chromosome segregation is ensured by recombination. Recombination hotspots
are studied in budding yeast and Arabidopsis
thaliana. High-end technological platforms, such
as mass spectroscopy, micro arrays and next generation sequencing are used as discovery tools.
Meiotic chromosome missegregation is a leading
cause of miscarriages and Down syndrome and
most cancers are associated with aberrant chromosome numbers. Knowledge of segregation mechanisms is thus required to understand the etiology
of these problems.
SPEAKER
Franz Klein, Jan Michael Peters (Deputy; IMP)
PARTNERS
Gustav Ammerer, Juraj Gregan, Franz Klein, Karl
Mechtler (IMP), Jan Michael Peters (IMP), Peter
Schlögelhofer, Stefan Westermann (IMP)
Signal transduction by the interferon (IFN) receptors.
More than 60 (instead
of 46) mitotic chromosomes in a human
cancer cell (HeLa),
with some chromosomal domains labelled
in different colors.
SPEAKERS
Mathias Müller, VetMedUni Vienna;
Thomas Decker, MFPL (Deputy)
Thomas Decker, Robert Eferl (LBI CR),
Wolfgang Mikulits (MedUni Wien), Richard Morrigl
(LBI CR), Mathias Müller (VetMedUni Vienna),
Veronika Sexl (MedUni Wien)
13
MFPLAnnRep2010_Groups_Teil1:Layout 1 16.05.11 21:50 Seite 14
R E S E A R C H
G R O U P S
GUSTAV AMMERER
Signal transduction and transcriptional regulation in yeast
One of our major aims is to
understand the cogs and wheels of
phosphorylation-modulated signal
transduction machineries in the
yeast Saccharomyces cerevisiae.
Gustav Ammerer
TEAM
Aleksandra Jovanovic
Christina Friedmann
Syam Yelamanchi
Aurora Zuzuarregui
Jiri Veis
Wolfgang Reiter
In this field, important still unresolved questions
concern the dynamic interactions between different
signaling factors and their effectors- e.g. in what
cellular context they might happen, how they are
controlled by phosphorylation events and how these
interactions change during signaling events.
To approach these questions we have established
and optimized a novel enzyme based protein proximity assay. This assay is based on a mammalian
histone methyl-transferase and its highly specific
substrate, the N-terminal fragment of histone 3.
Apart from successfully characterizing known
protein interactions in well studied signal systems
such as the high osmolarity response, we have also
started to use this approach for validating protein
interactions that have been suggested by quantitative mass spectrometry and/or by genetic data.
A second project deals with the problem of how
cell cycle dependent signals coordinate the transcriptional regulation of genes. In this case we have
focused on the regulation of the main mitotic cyclin
gene CLB2 in yeast.
Signal system mediating osmotic stress in yeast: cartoon
of the important factors and proximity assay between the
membrane sensor and adaptor Sho1, tagged with a histoneH3K9 methyl transferase and the protein kinase Ste11,
tagged with an H3HA epitope. The Western assays show
the methylation pattern obtained in wildtype cells and different signaling mutants before (-) and after stress (+).
α-Me3K9 depicts signals with methylation specific antibody, α-HA provides controls for protein levels of Ste11
This gene is repressed in the G1-phase of the cell
cycle, it is de-repressed at the START of S-phase
and fully induced by a positive feedback mechanism
during G2-phase and mitosis. In addition
we have found that genotoxic and replication stress will suppress the activation
of CLB2 as well as additional genes that
exhibit similar G2/M specific expression
patterns. We have therefore addressed the
question of how phosphorylation events
affect stability and function of the important transcriptional regulators, and
how their specific modifications can be
correlated with changes in the underlying
chromatin structure and chromatin modification patterns.
Cartoon of the proposed regulatory factor recruitment and chromatin
structures found at the CLB2 locus during different cell cycle stages
and arrest conditions in yeast
SELECTED PUBLICATIONS
Kijanska M. et al. 2010. Activation of Atg1 kinase in autophagy by regulated phosphorylation. Autophagy 6: 1168-78
n Rumpf C. et al. 2010. Casein kinase 1 is required for efficient removal of Rec8 during meiosis I. Cell Cycle 9:2655-60
n Lempiainen H. et al. 2009. Sfp1 interaction with TORC1 and Mrs6 reveals feedback regulation on TOR signaling.
Mol Cell 33:704-16
14
R E S E A R C H
G R O U P S
MANUELA BACCARINI
Deciphering the MAPK pathway in vivo
The Ras/Raf/MEK/ERK pathway
represents the oldest paradigm of a
cytosolic signal transduction cascade,
and its constitutive activation as a
result of mutations is considered a
key event in the development of
several human malignancies and
developmental disorders.
The components of the pathway, particularly the
Raf kinases A-, B-, and C-Raf (Raf-1), are considered
attractive therapeutic targets, but surprisingly little
is known about their essential functions in the
context of the whole organism. The research goal
of the Baccarini lab is to define the essential
function of C-Raf, B-Raf and their target Mek-1 in
in vivo models of tissue development, remodeling,
and neoplasia.
In the past few years, we have shown that B-Raf is
a crucial activator of the Erk pathway in vivo. In
contrast, C-Raf is essential to promote survival
and migration independently of its enzymatic activity as a MEK kinase. Instead, C-Raf binds to and
negatively regulates the proapoptotic kinases Mst2 and
Ask1, as well as the Rho-dependent kinase Rok-alpha, which
controls cell shape, migration,
and the expression of the death
receptor Fas.
Recently, we could demonstrate
that oncogenic Ras promotes the
interaction between C-Raf and
Rok-alpha, and that the negative
regulation of Rok-alpha by
C-Raf is absolutely necessary for
both development and maintenance of Ras-induced epidermal
tumors. In the absence of C-Raf,
Rok-alpha hyperactivity induces
the rapid differentiation of these
tumors, which disappear never
to relapse. Mechanistically, Rokalpha inhibition is mediated by
the autoinhibitory domain of
C-Raf, which is structurally similar to the autoinhibitory domain of Rok-alpha. Upon activation,
both C-Raf and Rok-alpha are converted from a
"closed" conformation, in which their autoinhibitory
domains contact and inhibit the respective kinase
domains, into an "open" conformation, in which
the kinase domains can accept and phosphorylate
substrates. In this situation, the autoinhibitory domain of C-Raf, like an ill-fitting lego brick, contacts
the kinase domain of Rok-alpha, restraining its
activity.
By showing that their essential in vivo functions
are fundamentally different, these results have
changed the way we look at Raf kinases and have
opened new possibilities for molecule-targeted
therapy.
Recently, we have also discovered an unexpected
essential role of MEK1 in downregulating MEK2/ERK signaling. MEK1 mediates the regulation
of MEK2 in the context of a Mek1:Mek2 heterodimer negatively regulated by ERK-mediated
phosphorylation of Mek1. These data establish
Mek1 as the critical modulator of Mek/Erk signaling
in vivo and in vitro.
Before and after – C-Raf ablation results in the regression of Ras-induced epidermal tumors. Once C-Raf is ablated, its binding partner Rok-alpha is hyperactive and induces the differentiation of keratinocytes (K10, brown staining), and
the regression of the tumors (Hematoxylin/Eosin staining), which never relapse.
Niault T, Sobczak I, Meissl K, Weitsman G, Piazzolla D, Maurer G, Kern F, Ehrenreiter K, Hamerl M, Moarefi I, Leung T, Carugo
O, Ng T, and Baccarini M (2009). From autoinhibition to inhibition in trans: the Raf-1 regulatory domain inhibits
Rok-alpha kinase activity. J Cell Biol, 187, 335-342 n Ehrenreiter K, Kern F, Velamoor V, Meissl K, Galabova-Kovacs G,
Sibilia M, and Baccarini M (2009). Raf-1 addiction in Ras-induced carcinogenesis. Cancer Cell, 16, 149-160
n Catalanotti F, Reyes G, Jesenberger V, Galabova-Kovacs G, de Mato Simoes R, Carugo O, and Baccarini M (2009). A MEK1MEK2 heterodimer determines the strength and duration of the ERK signal. Nat Struct Mol Biol, 16, 294-303
15
Manuela Baccarini
TEAM
Clemens Bogner
Anna Lina Cavallo
Katarina Cingelova
Botond Cseh
Eszter Doma
Karin Ehrenreiter
Thomas Kögler
Elisabeth Froschauer
Matthias Hamerl
Ines Jeric
Veronika Jesenberger
Florian Kern
Barbara Maier
Gabriele Maurer
Matthias Parrini
Josipa Raguz
Christian Rupp
Bartosz Tarkowski
Andrea Varga
Reiner Wimmer
R E S E A R C H
G R O U P S
ANDREAS BACHMAIR
Protein modifiers in plants and retrotransposon biology
Andreas Bachmair
TEAM
Gudrun Böhmdorfer
Karolin Eifler
Maria Granishchikova
Prabhavathi Talloji
Konstantin Tomanov
Andrea Tramontano
Many proteins are modified after
their synthesis. We are interested in
how the small modifier proteins
ubiquitin and SUMO are linked to
substrate proteins in plants, and
how these processes change
substrate properties.
This novel regulatory circuitry has been tested in
the model plant Arabidopsis thaliana and shall then
be used in other plants, in particular barley. Insertion of Tto1 into a plant´s genome generates mutations for gene analysis and, in case of a crop
plant, may provide genetic variability for breeding
programs.
Covalent attachment of ubiquitin to substrate proteins is essential for many processes. Best known
is its role in protein degradation. Several ubiquitin
moieties, linked as a chain to the substrate protein,
can serve as a signal for rapid proteolytic destruction of the substrate.
The focus of our interest is ubiquitylation during
plant cell death. We are studying two types of ubiquitylation reactions. One of them is linked to reactive oxygen species (ROS) signaling, and plays a
role in the induction or execution of fast cell death
programs. The other one, called N-end rule pathway, is important for senescence, a slow cell death
process specific to the plant kingdom. We use both
in vitro enzyme reactions, and characterization of
plants with mutations in ubiquitylation enzymes,
to elucidate pathway characteristics.
Our second focus is retrotransposon biology. Retrotransposons are pieces of DNA that can replicate
more often than the normal genomic DNA. They
do so by reverse transcribing their mRNA, and by
inserting the ensuing DNA copy into the host genome, often at a random position. In a synthetic
biology project, we want to convert retrotransposon
Tto1 into a novel tool for plant investigation and
improvement. On the way to this goal, we are learning a lot about the life cycle of Tto1. Activity of
retrotransposons is usually induced by stress, and
therefore not easy to control. We have modified
Tto1 such that its transpositional activity can be
induced by the chemical substance estradiol.
The model plant Arabidopsis thaliana grown in 8 hr day
and 16 hr night cycles.
Holman TJ et al. (2009) The N-end rule pathway of targeted protein degradation promotes seed germination and
establishment through removal of ABA sensitivity in Arabidopsis. Proc Natl Acad Sci USA 106, 4549-4554.
n Böhmdorfer G et al. (2010) A synthetic biology approach allows inducible retrotransposition in whole plants. Syst
Synthet Biol 4, 133-138. n Hermkes R et al. (2011) Distinct roles for Arabidopsis SUMO protease ESD4 and its closest
homolog ELS1. Planta 233, 63-73.
16
R E S E A R C H
G R O U P S
ANDREA BARTA
Post-transcriptional regulation of gene expression in plants
What determines the complexity of
higher organisms? No correlation has
been found to DNA content and
gene number and therefore studies
in the field are now focusing on
post-transcriptional processes
and the impact of the dynamic
transcriptome on gene expression.
Alternative splicing is one of the posttranscriptional
events to expand the repertoire of proteins and it
has been exploited for various differentiation processes. In plants, the significance of alternative
splicing was long underestimated, but we and
others have shown that it greatly impacts on development and response to the environment.
As alternative splicing in Arabidopsis is not well
defined we are using RNAseq to define the rules
and targets of alternative splicing. SR (Ser/Arg)
proteins are important splicing factors and to date
we have isolated and partially characterized several
Arabidopsis SR proteins, which are important for
splice site selection and spliceosome assembly.
In addition, we have isolated several regulatory
proteins which seem to be essential to drive the
splicing process, like SRPK kinases, helicases and
cyclophilins. To elucidate their mechanisms of action some of the plant SR proteins and cyclophilins
are currently characterized in greater detail in terms
of their RNA targets, interacting proteins and their
impact on flowering and UV-stress response.
Interestingly, some of these factors seem to connect
splicing to transcription and are therefore currently
investigated in greater detail.
Furthermore, a project has been started to investigate the influence of chromatin and DNA modifications on alternative splicing in plants. In another
line of research, we are developing a genomic method to select for riboswitches (these are regulatory
RNA elements which can bind metabolites) in
plants, as they are implicated in regulating alternative splicing and gene expression.
Alternative splicing of pre-mRNA: A: One example of an
alternative splicing event where an exon ist either included or excluded resulting in two different mRNA transcripts with different properties and coding potential. The
exons are symbolized by rectangles, the lines denote introns. B: Roles of cis-acting sequences and trans-acting
factors in determining the splicing code for splice site selection. ESE/ESS denote exonic splicing enhancer/silencer
sequences and ISE/ISS are intronic splicing enhancers/silencer sequences. Transacting factors include the constitutive splicing factors U1 snRNP, U2 snRNP, U2AF65,
U2AF35 and positive (+) or negative (-) splicing regulatory
proteins. BP, indicates the branch point sequences, Pytract, the pyrimidine track preceding the 3´splice site.
Andrea Barta
TEAM
Olga Bannikova
Armin Fuchs
Janett Göhring
Jacek Jaroslaw
Maria Kalyna
Branislav Kusenda
Yamile Marquez-Ortiz
Monika Maronova
Franz Stark
Anela Tosevska
Franziska Werba
Nicola Wiskocil
A high resolution alternative splicing RT-PCR panel to measure relative mRNA isoform
level changes when nonsense-mediated decay (NMD) was impaired in the Arabidopsis
NMD factor mutants, upf1-5 and upf3-1, or after cycloheximide treatment. Many transcripts increased in abundance identifying them as NMD targets.
Barta, A., Kalyna, M., and Reddy, A. (2010) Implementing a rational and consistent nomenclature for SR proteins in
plants. Plant Cell. Sep;22(9):2926-9. Epub 2010 Sep 30. n Barta A, Kalyna M, Lorković ZJ. Plant SR proteins and their
functions. Curr Top Microbiol Immunol. 2008;326:83-102. n Gullerova, M., Barta, A., and Lorkovic, ZJ (2007) Rct1, a
Nuclear RNA Recognition Motif-Containing Cyclophilin, Regulates Phosphorylation of the RNA Polymerase II CTerminal Domain. Mol. Cell. Biol. 27, 3601-3611
17
R E S E A R C H
G R O U P S
DIETER BLAAS
Early interactions of viruses with host cells
To infect a host cell, viruses usually
recognize particular structures on
the cell surface. Following binding to
these ‘viral receptors’, the virons are
taken up into the cell along different
entry routes.
Dieter Blaas
TEAM
Ernst Kenndler, guest professor
Irene Gösler
Shushan Harutyunyan
Kristina Hunyadi
Heinrich Kowalski
Mohit Kumar
Xavier Subirats
Rohan Wakade
Most of these pathways converge in vesicular
structures, the endosomes. Depending on whether
the virus is covered with a lipid membrane (enveloped) or lacking such a membrane (naked), its genome is then being released into the cytoplasm by
different mechanisms. Enveloped viruses usually
fuse with cellular membranes, which results in the
nucleocapsid arriving in the cytosol. Non-enveloped
viruses either disrupt the endosomal membrane,
with subviral particles being transferred into the
cytoplasm, or the genomic nucleic acids are threaded through a pore and the remaining empty
capsids are further shuttled to lysosomes for degradation. For some viruses there is experimental
support for RNA transfer between endosomal and
cytoplasmic compartments which, however, has so
far not been demonstrated explicitly and the putative membrane pore has not been visualized.
Working with human rhinoviruses (HRVs) that lack
a lipid membrane and are the predominant cause
of common colds, we aim at identifying so far
unknown viral receptors, the different mechanisms
underlying viral uptake, the process of genome
release and the structural basis of the transfer of
the viral genome through lipid membranes.
We address these questions by using biochemical,
molecular biological, biophysical, and structural
biology techniques, such as selection and characterization of viral mutants, expression library
screening, fluorescence correlation spectroscopy,
capillary electrophoresis, and cryo-electron microscopy.
In the last few years we have identified heparan
sulphate as an additional receptor for some rhinovirus types and characterized the uptake pathway
by this proteoglycan and by the intercellular adhesion molecule 1, the receptor of about 90 different HRV types. We developed a liposomal in vitro
system mimicking the transfer of the viral RNA
through the endosomal membrane that is currently
being used for structural analysis. Within the frame
of several international collaborations, solving the
3D structure of subviral particles is underway.
Binding geometry of two different soluble receptor fragments attached to a human rhinovirus (from Querol-Audi, J., Konecsni,
T., Pous, J., Carugo, O., Fita, I., Verdaguer, N., and Blaas, D. (2009). Minor group human rhinovirus-receptor interactions:
geometry of multimodular attachment and basis of recognition. FEBS Lett 583, 235-240.
Khan, A.G. et al. (2010). Human rhinovirus 14 enters rhabdomyosarcoma cells expressing icam-1 by a clathrin-, caveolin-, and flotillin-independent pathway. J Virol 84, 3984-3992. n Weiss, V.U. et al. (2010). Liposomal leakage induced by virus-derived peptides, viral proteins, and entire virions: rapid analysis by chip electrophoresis. Anal Chem
82, 8146-8152. n Khan, A.G. et al. (2011). Entry of a heparan sulphate-binding HRV8 variant strictly depends on dynamin but not on clathrin, caveolin, and flotillin. Virology (doi:10.1016/j.virol.2010.12.042)
18
R E S E A R C H
G R O U P S
UDO BLÄSI
Post-transcriptional regulation in Bacteria and Archaea
Bacteria are constantly challenged by
changing environmental conditions.
They employ a number of posttranscriptional control mechanisms
including trans-acting proteins, small
regulatory RNAs (sRNAs) as well as
features inherent to mRNA structure,
which permit a fast adaptation to
new environments or to different
kinds of stress.
coupled. Expression of phrS requires the oxygenresponsive regulator ANR. Thus, PhrS is the first
bacterial sRNA that provides a regulatory link between oxygen availability and quorum sensing,
which may impact on oxygen-limited growth in
P. aeruginosa biofilms.
Other projects are directed towards a better understanding of post-transcriptional regulatory mechanisms in the model crenarchaeon Sulfolobus solfataricus (Sso) . These studies revealed the sequence
of events in archaeal translation initiation as well
as unprecedented function(s) of archaeal translation initiation factors. We have shown that the
g-subunit of translation initiation factor aIF2 exhibits – besides its requirement for initiator-tRNA
binding – an additional function with resemblance
to the eukaryotic cap-complex. It binds to the
5´-triphosphate end of mRNAs and counteracts
mRNA decay in Sso by a recently identified Sso
RNAse with 5´->3´directionality. In addition, ongoing studies concentrate on the elucidation of
the function of archaeal Sm proteins in RNA metabolism and molecular mechanisms underlying
non-coding RNA mediated regulation in Sso.
Udo Bläsi
TEAM
David Hasenöhrl
Hermann Hämmerle
Johannes Kassmannhuber
Salim Manoharadas
Tetjana Milojec
Armin Resch
Alessandra Romeo
Nicole Roschanski
Elisabeth Sonnleitner
Lukas Zeichen
X-ray structure of the moonlighting gamma-subunit of S.
solfataricus translation initiation factor aIF2. Nucleotide
binding sites are circled (Stolboushkina et al., 2008).
We are focusing on post-transcriptional control
mechanisms exerted by the global regulatory protein Hfq in conjunction with sRNAs in Bacteria,
with emphasis on the human pathogen Pseudomonas aeruginosa. These studies revealed novel
molecular modes of sRNA-mediated regulation as
well as Pseudomonas sRNAs contributing to pathogenicity. We have identified the P. aeruginosa
regulatory RNA PhrS as an activator of PqsR synthesis, a key quorum sensing regulator involved in
the production of the virulence factor pyocyanin.
Genetic studies revealed a novel mode of regulation
by a sRNA, whereby PhrS uses a base-pairing mechanism to activate a short upstream open reading
frame to which the pqsR gene is translationally
PhrS-mediated activation of pqsR translation. In the absence of the sRNA PhrS, the ribosome
binding sites (rbs) of pqsR (red) and uof (orange) are partially masked by secondary structure,
which impedes translation initiation at either rbs. Under aerobic conditions the post-transcriptional regulation of the uof-pqsR entity leads to low levels of the virulence factor pyocyanin (Pyo) (yellowish color of the culture). When oxygen becomes limiting the sRNA PhrS
(blue) is synthesized and base-pairs upstream of the rbs of uof (highlighted in green), leading
to structural rearrangements followed by a stimulation of uof translation and consequently
that of pqsR. PhrS-mediated activation of pqsR translation leads to increased levels of pyocyanin (blue color of the culture) (Sonnleitner et al., 2011).
Hasenöhrl D, Konrat R and Bläsi U (2011). Identification of an RNase J ortholog in Sulfolobus solfataricus: implications for 5'-to-3' directional decay and 5'-end protection of mRNA in Crenarchaeota. RNA 2011 17, 99-107. n
Hasenöhrl D, Lombo T, Kaberdin V, Londei P and Bläsi U (2008). Translation initiation factor a/eIF2(-gamma) counteracts
5' to 3' mRNA decay in the archaeon Sulfolobus solfataricus. Proc Natl Acad Sci U S A. 105, 2146-50. n Vecerek B,
Beich-Frandsen M, Resch A and Bläsi U (2010). Translational activation of rpoS mRNA by the non-coding RNA DsrA
and Hfq does not require ribosome binding. Nucleic Acids Res. 38, 1284-93.
19
R E S E A R C H
G R O U P S
CÉCILE BROCARD
Protein networks and intracellular membrane remodeling
Eukaryotic cells are organized
into specialized membranebound compartments or
organelles, each serving
crucial cellular functions.
Cécile Brocard
TEAM
Christine David
Thomas Heil
Mathias Hochgerner
Johannes Koch
Alexandra Lärnsack
Sophie Melchior
Several transport mechanisms are implicated to maintain the identity as well
as the integrated function of the different
Cartoon representing the biogenic process of peroxisome formation
cellular organelles thereby allowing for
survival of the organisms in environ ments prone to constant changes. Accordingly,
The question is what molecular interactions ororganellar dysfunction alters the cellular metaganize such equilibrium?
bolism and affects the fitness of the cell.
Several membrane proteins have been shown to
Dynamic alterations of membrane structure are inparticipate in the process of peroxisome proliferatrinsic to organelle morphogenesis and homeostation in yeast and mammalian cells. We employ sesis. Among the cellular organelles, peroxisomes are
veral approaches based on quantitative proteomics
versatile single membrane-bound organelles that
using metabolic labeling as well as live-cell imaging
enclose essential functions mainly involved with
to analyze the molecular networks involved in the
lipid metabolism and degradation of reactive oxyregulation of peroxisome number in the cell. The
gen species. While peroxisomes are dispensable for
results of our studies in yeast cells reveal that large
unicellular organism such as yeast cells, they are
membrane proteins complexes physically link peressential for the proper development of multiceloxisomes to other sub-cellular organelles at spelular organisms.
cialized contact sites. We currently investigate the
formation of these membrane contact sites and
Peroxisomal dysfunction can lead to severe pathotheir consequence on peroxisome proliferation.
logical disorders such as adrenoleukodystrophy or
the Zellweger syndrome that are typically lethal
In studies on mammalian cells, using the membrane
diseases. Peroxisomes continuously adjust their
elongation factor PEX11 and photoactivatable GFP
shape, size, number and protein content according
appended with a peroxisomal targeting signal, we
to the metabolic requirements of the cell. Indeed,
recently illustrated the asymmetric inheritance of
molecular mechanisms exist that maintain the
peroxisomal matrix proteins in the process of pernumber and morphology of peroxisomes in the cell
oxisome proliferation a mechanism that might lead
through a delicate balance between biogenesis,
to rejuvenation of the peroxisome pool in the cell.
degradation and inheritance during cell division.
Whether the selective degradation of peroxisomes
via pexophagy is specifically targeted to “old” organelles is an attractive
question that we intend to
investigate in details.
Images represent confocal microscopy pictures. (B) and (C) represent single Z-layers of
the insert indicated in (A)
Koch J., Pranjic K., Huber A., Ellinger A., Hartig A., Kragler F. and Brocard C*. (2010) PEX11-family members are membrane
elongation factors that coordinate peroxisome proliferation and maintenance. Journal of Cell Science 123 (19) 33893340. n Zipor G., Haim L., Gelin-Licht R., Gadir N., Brocard C. and Gerst J.E.* (2009) Localization of mRNAs coding for
peroxisomal proteins in the yeast, Saccharomyces cerevisiae. Proc. Natl. Acad. Sci. U.S.A.;106 (47), 19848-19853. n
Brocard C.* and Hartig A. (2006) The Peroxisomal Targeting Signal 1: Is it really a simple tripeptide? Biochim. Biophys.
Acta Mol. Cell Res. 24 (23). 12(1763), 1563-1573.
20
R E S E A R C H
G R O U P S
ALEXANDER DAMMERMANN
Centriole Assembly and Function
Centrioles are small cylindrical
organelles whose distinguishing
feature is an outer wall composed
of a nine-fold symmetric array of
stabilized microtubules.
Centrioles perform two distinct functions in eukaryotic cells: 1) they recruit pericentriolar material
to form centrosomes that organize the microtubule
cytoskeleton and position the mitotic spindle, and
2) they template cilia, cellular projections that perform a variety of critical sensory and motile functions. Centrosome and cilia abnormalities have been
linked to aneuploidy and tumorigenesis as well as
developmental disorders including ciliopathies and
microcephaly. Despite their importance to human
physiology and pathology, centrioles have remained
poorly understood at the molecular level, largely
due to the technical challenges posed by the small
size of this organelle.
In our lab we are using a combination of biochemical, cell biological and genetic approaches in
the nematode C. elegans to investigate the fundamental and conserved molecular mechanisms underlying centriole assembly and function.
In previous work we have taken advantage of the
availability of data from genome-wide RNAi-based
screens to define the molecular requirements for
centriole assembly. The six-protein molecular pathway we identified has since been found to be conserved from ciliates to vertebrates, and is thought
to form the core of the centriole assembly machinery in all eukaryotes. We further identified the
hydrolethalus syndrome protein HYLS-1 as a core
centriolar protein that is incorporated into centrioles during their assembly to confer on them
the ability to initiate cilia. The single amino acid
missense mutation associated with hydrolethalus
syndrome impairs HYLS-1 function in ciliogenesis,
identifying this disorder as a severe (perinatal lethal)
ciliopathy.
Current research builds on this foundation, seeking
to answer three main questions: 1) How do centrioles assemble, in particular what are the specific
mechanistic contributions of each of the six proteins in the centriole assembly pathway; 2) how
do centrioles recruit pericentriolar material to form
centrosomes and what is the molecular nature of
this material; and 3) how do centrioles form cilia,
focusing on the events immediately downstream
of HYLS-1.
(A) Centriole assembly pathway as delineated in C. elegans. (B) C. elegans early embryo, stained for SAS-4 (centrioles, yellow), γ-tubulin (pericentriolar material, blue), Aurora-A (peripheral pericentriolar material and astral microtubules, red)
and microtubules (black). (C) Depletion of HYLS-1 in Xenopus embryo results in failure of cilia assembly (acetylated tubulin,
green). Basal bodies (γ-tubulin, blue) are disorganized.
Dammermann A, Muller-Reichert T, Pelletier L, Habermann B, Desai A, Oegema K (2004). Centriole assembly requires
both centriolar and pericentriolar material proteins. Dev Cell. 7:815-29. n Dammermann A, Maddox PS, Desai A, Oegema K (2008). SAS-4 is recruited to a dynamic structure in newly forming centrioles that is stabilized by the
gamma-tubulin-mediated addition of centriolar microtubules. J Cell Biol 180: 771-85. n Dammermann A, Pemble H,
Mitchell BJ, McLeod I, Yates JR, Kintner C, Desai A, Oegema K (2009). The hydrolethalus syndrome protein HYLS-1 links
core centriole structure to cilia formation. Genes Dev 23: 2046-59.
21
Alexander Dammermann
TEAM
Gabriela Cabral
Jesus Fernandez Rodriguez
Clementine Schouteden
R E S E A R C H
G R O U P S
THOMAS DECKER
Host responses and innate immunity to bacteria
A large number of pathogenic
microbes must be recognized by
the immune system and defense
mechanisms must be alerted.
Thomas Decker
TEAM
Matthias Farlik
Pia Gamradt
Amanda Jamieson
Elisabeth Kernbauer
Orest Kuzyk
Andrea Majoros
Birgit Rapp
Isabella Rauch
Sandra Westermayer
Sebastian Wienerroither
Fotima Touraeva
The first line of defense is set by the innate immune
system which rapidly limits antimicrobial and colonization and spread. To increase protection cells
participating in the innate response initiate an
adaptive immune response. Protection and immunoregulation by the innate immune system requires
that a microbe is detected and physical contact is
translated into altered gene expression of the infected cell. Antimicrobial gene products provide
protective effector mechanisms. Moreover, secreted
cytokines fulfill the task of communicating between
cells involved in the antimicrobial response to maximize the common antimicrobial effort. One important group of cytokines is formed by the interferons (IFN), subdivided into three distinct classes
(IFN-I, II, III). Collectively IFN play an indispensable
role in the immune system as humans or animals
with partial or complete losses of responses to IFN
are highly immunecompromised. To reprogram gene
expression in target cells, IFN employ Jak-Stat signal transduction: after binding to cell surface receptors, receptor- associated Jak tyrosine kinases
phosphorylate Stat transcription factors which
translocate to the nucleus to stimulate gene expression.
Our research aims at understanding how the synthesis of IFN-I is regulated when cells or animals
are infected with intracellular bacteria and how
the Stats activated upon IFN-I secretion communicate with additional bacteria-derived signals in
the process of activating antimicrobial genes. To
this end we infect normal cells and mice and compare them with infected mice that cannot synthesize IFN-I or that cannot respond to them. In addition, mice with reduced or absent responses to IFN
are used to study the impact of the cytokines in a
mouse model of acute intestinal inflammation. In
this situation we test the hypothesis that IFN contribute to inflammation, thus worsening the outcome. These efforts are coordinated with collaborators at the University of Vienna that determine
corresponding changes in the composition of the
intestinal microbiota.
Infections to viral pathogens such as influenza
virus are frequently followed by superinfection with
a bacterial pathogen and severe disease may result
from the bacterial rather than the original viral infection. In her project, Amanda Jamieson studies
mechanisms causing the immune response to influenza virus to alter and worsen the subsequent
infection by a bacterial pathogen. Her recent work
points towards an important role of influenza-induced glucocorticoids in suppressing the immune
response to a superinfecting bacterial pathogen.
Signaling in cells infected with Listeria monocytogenes. Bacteria are recognized by cell surface, endosomal and cytoplasmic
pattern recognition receptors that activate NFkB, MAPK and IRF pathways. IRFs stimulate transcription of type I interferon
(IFN-I) genes . IFN-I are produced and signal through Jak kinases and Stat transcripton factors. Together NFkB, MAPK –
activated transcription factors and Stats shape the gene expression signature of infected cells.
Stockinger, S., et al. (2009). Characterization of the interferon-producing cell in mice infected with Listeria monocytogenes. PLOS Pathogens, 5(3): e1000355. doi:10.1371/journal.ppat.1000355. n Jamieson, A.M. et al. (2010). Influenza
Virus-Induced Glucocorticoids Compromise Innate Host Defense against a Secondary Bacterial Infection. Cell Host
& Microbe, 7 (2), 103-14. n Farlik, M. et al. (2010). Stats and NFκB co-regulate gene expression in infected cells
through unconventional assembly of a transcription initiation complex. Immunity, 33:25-34.
22
R E S E A R C H
G R O U P S
KRISTINA DJINOVIC-CARUGO
Structural Biology of Cytoskeleton
We are interested in the molecular
mechanisms underlying the actinbased cytoskeleton of the striated
muscle.
The most striking feature of muscle proteins and
in particular of the specialised striated muscle sarcomere compartment Z-disk, is the high frequency
of multiple protein-protein interactions that form
part of a complex network. The aim of our research
is to generate detailed structural information on
the protein-protein interaction network in the Zdisk, starting from its important basic components
- alpha-actinin and filamin C - and moving towards
macromolecular complexes with adaptor and regulatory Z-disk proteins that are centred on them.
In collaboration with groups of the MFPL and the
Faculty of Life Sciences we are working on structural
studies of RNA chaperone Hfq and its interactions
with RNA (Blaesi), yeast magnesium channel Mrs2
(Schweyen), and trypasonomal cytoskeletal protein
MORN1 (Warren). In collaboration with M. Wagner
(Faculty for Life Sciences, Univ. Vienna) and C.
Obinger (University of Natural Resources and Life
Sciences, Vienna) we are studying the family of
bacterial chlorite dismutases.
Elucidation of chlorite dismutase reaction mechanism will be performed by analysis of the structures
of wild type, active site mutants and complexes
combined with biochemical studies with the aim
to elucidate the catalytic mechanism. In order to
study Hfq:RNA interactions at molecular level we
are employing a combination of solution scattering,
NMR and optical spectroscopy techniques.
Kristina Djinović-Carugo
Several of the components required for generation
of Z-disk complexes are already available in quantities and quality required for structural studies:
alpha-actinin, several segments of filamin C, myotilin and ZASP. We plan to proceed with generation
of binary and higher complexes and their biochemical, biophysical and structural characterisation
employing integrative structural biology approach
by combining high resolution studies (X-ray diffraction, NMR) with lower resolution approaches
that can either yield molecular envelopes (SAXS,
SANS, EM) or specific distance information (massspectrometry, NMR). These activities are complemented by the development of bioinformatics tools
for results and fine tuning of the protein constructs
to be structurally analyzed. New bioinformatics
strategies are being designed to extend our prediction capabilities.
Molecular surface and substrate entrance of Candidatus Nitrospira defluvii
chlorite dismutase (NdCld). (A) The position and accessibility of the heme
moiety in NdCld. The solvent accessible surface of one NdCld subunit is colored
according to its electrostatic potential (blue for positive, red for negative). The
semitransparent surface representations of other NdCld subunits forming the
NdCld pentamer are shown in grey, with iron shown as a red sphere. Hemes
are presented as green stick models. (B) Detailed view into the active site
chamber through the putative substrate entrance and product exit channel.
In order to overcome the major
bottlenecks in structural and
functional studies of proteins,
whoch are availability of milligram
amounts of active, chemically and
conformationally pure protein and
crystallization, an FFG funded Laura
Bassi Centre for Optimized Structural Studies (COSS) was established
as a joint venture of IMP, Intercell
and the MFPL with the goal to setup an efficient platform to combine
the recent advances in automated
expression screening of protein targets employing nested constructs
design and cell-free protein expression systems followed by biophysical characterization to find conditions best suited for structural and
functional studies.
Galkin VE, Orlova A, Salmazo A, Djinovic-Carugo K, Egelman EH (2010). Opening of tandem calponin homology domains
regulates their affinity for F-actin. Nat Struct Mol Biol. 17(5):614-6. n Sjöblom B, Polentarutti M, Djinovic-Carugo K
(2009). Structural study of X-ray induced activation of carbonic anhydrase. Proc Natl Acad Sci U S A. 106(26):10609-13.
n Djinovic-Carugo, K, Carugo O (2010). Structural portrait of filamin interaction mechanisms. Curr Protein Pept Sci
11(7): 639-50.
23
TEAM
Mads Beich-Frandsen
Oliviero Carugo
Eirini Gkougkoulia
Irina Grishkovskaya
(from Oct. 2010)
Kira Gysel
Muhammad Bashir Khan
Sviatlana Kirylava
Julius Kostan
Suresh Kumar
Anita Lehner
Jana Neuhold
Claudia Schreiner
Adekunle Onipe
Nikos Pinotsis
Euripedes de Almeida Ribeiro
Ulrich Salzer
Manivannan Sethurajan
Kresimir Sikic
Björn Sjöblom (till Sept. 2010)
Jaegeun Song
Christoph Szimoniuk
R E S E A R C H
G R O U P S
GANG DONG
Structural studies of ciliogenesis
Gang Dong
TEAM
Violet Feng
Clara Pleban
Renping Qiao
Ekaterina Shimanovskaya
Keni Vidilaseris
Eukaryotic cilia and flagella are
specialized organelles that are highly
conserved from protists to mammals.
These organelles consist of the membrane-sheathed axoneme, which is
an extension of the mother centriole,
and at least 360 associated proteins.
Cilia have attracted much attention in recent years
because of their role in the transduction of extracellular signals and their association with an expanding number of human disorders. Such disorders
include respiratory distress syndrome, male sterility,
polycystic kidney disease, retinal degeneration, and
Bardet-Biedl syndrome.
Cilium assembly is initiated by the docking and fusion of mother centriole to the apical membrane
of the cell. Cilia are assembled and maintained
through intraflagellar transport (IFT). This process
is carried out by two distinct protein complexes,
IFT complex A and B, which contain at least six
and sixteen subunits, respectively. These complexes
transport ciliary cargos within cilia and flagella by
interactions with the microtubule-associated motor
proteins kinesin-II and dynein.
Whereas the complexes IFT A/B have been studied
for some years, little is known about their architecture and assembly. The lack of high-resolution
structural information on these complexes has now
become a limiting step in gaining an understanding
of their function at the molecular level. We are
currently developing protocols for producing soluble
and stable protein complexes by reconstituting in
vitro or co-expressing all components in bacterial
or baculovirus-insect cell expression system. Our
goal is to elucidate at the atomic level the assembly
mechanisms of the protein complexes for cargo
transport to and within the cilium. We mainly use
X-ray crystallography to visualize these proteins
and their complexes. Other biophysical techniques
such as dynamic light scattering, differential scanning calorimetry, and analytical ultracentrifugation
will as well be employed to study the architecture
and assembly of the protein complexes. Large assemblies will also be examined by cryo-electron
microscopy. Our structural studies will be complemented by site-directed mutagenesis and in
vitro/vivo experiments to test our mechanistic hypotheses. The available new structures will enhance
our understanding of how these complexes function
and provide hints as to how their malfunction leads
to human diseases.
Intraflagellar transport and domain prediction for all known components of the IFT complexes
Dong G, Wearsch PA, Peaper DR, Cresswell P, and Reinisch KM (2009) Insights into MHC class I peptide loading from
the structure of the tapasin/ERp57 heterodimer. Immunity, 30: 21-32. Featured article. n Dong G, Medkova M, Novick
P, and Reinisch KM (2007) A catalytic coiled-coil: structural insights into the activation of the Rab GTPase Sec4p by
Sec2p. Mol. Cell, 25, 455-462. n Dong G, Hutagalung AH, Fu C, Novick PJ, and Reinisch KM (2005) Structures of Exo70p
and the Exo84p C-terminal domains reveal a common motif. Nat. Struct. Mol. Biol., 12, 1094-1100.
24
R E S E A R C H
G R O U P S
SILKE DORNER
The regulation of gene expression by small ncRNAs
Post-transcriptional processes such
as mRNA splicing, mRNA degradation,
mRNA surveillance, RNA editing,
translational repression and RNAmediated gene silencing play crucial
roles in the regulation of eukaryotic
gene expression.
logous mRNAs for degradation. A genome-wide
screen for components of the siRNA pathway identified several novel candidate genes in Drosophila
cultured cells. Thus, the main focus of this project
is the biochemical characterization of these novel
candidate genes. This will allow the identification
of their role in the siRNA pathway.
miRNA-mediated gene silencing
Silke Dorner
In the past decade the finding of small non-coding
RNAs has entirely revolutionized the way we think
about the regulation of gene expression. The major
focus of our research is the RNA-mediated gene
silencing by siRNAs (small interfering RNAs) and
miRNAs (micro RNAs) in Drosophila.
MicroRNAs (miRNAs) are an abundant class of
small non-coding RNAs (about 22 nt) that are
found in a variety of eukaryotic organisms. Over
the past decade these small RNAs emerged as crucial factors of gene regulation and play an essential
role in developmental and physiological processes.
Generally, animal miRNAs base pair imperfectly to
the 3’ untranslated region (3’ UTR) of target mRNAs
and have been well established as key regulators
of gene expression at the translational level. More
recently evidence accumulated that miRNAs can
also accelerate mRNA degradation of some of their
targets. It is important to note that miRNA-mediated decay of mRNA does not occur through an
endonucleolytic cleavage as in siRNA-mediated
gene silencing. However, miRNAs accelerate the
mRNA turnover by recruitment of the general
mRNA decay machinery. The underlying mechanisms by which miRNAs regulate gene expression
are still quite controversial. The aim of this project
is to develop kinetic tools to study the mechanism
by which miRNAs affect
the stability and translatability of their target
mRNA. Thus, we will investigate which particular
step of mRNA degradation
or translation is influenced
by miRNAs.
TEAM
siRNA mediated gene silencing or RNA
interference (RNAi)
RNA interference is thought to be a mechanism
used to defend viruses and other transposable elements. Once RNAi was discovered it quickly revolutionized reverse-genetic approaches in various
systems and thus has become a broadly powerful
tool for the analysis of gene function.
Post-transcriptional silencing by RNAi is initiated
by double-stranded RNAs (dsRNAs) that are processed into short interfering RNAs (siRNAs). siRNAs
get incorporated into the RNA-induced silencing
complex (RISC) which ultimately targets homo-
We established and inducible expression system for Drosophila cell culture that allows
the measurement of mRNA turnover rates. Left: Northern blot analysis of mRNA levels
after a transcriptional pulse. Right: Quantitative analysis of mRNA decay based on the
Northern blot experiments shown.
Jäger E and Dorner S. (2010). The decapping activator HPat a novel factor co-purifying with GW182 from Drosophila
cells. RNA Biol. 7(3), 381-5. n Dorner S, Lum L, Kim M, Paro R, Beachy PA, Green R. (2006). A genomewide screen for
components of the RNAi pathway in Drosophila cultured cells. Proc Natl Acad Sci U S A. 103, 11880-5. n Dorner S.,
Brunelle J.L., Sharma D., Green, R.(2006). The hybrid state of tRNA binding is an authentic translation elongation
intermediate. Nat. Struc. Mol. Biol. 13, 234-41.
25
Sanja Antic
Nadia Brillante
Yaprak Dönmez
Elisabeth Jäger
Eleonora Adami
R E S E A R C H
G R O U P S
ROLAND FOISNER
Lamins in nuclear organization and human disease
Lamins are nuclear intermediate
filament proteins in metazoan cells
that form the nuclear lamina, a
scaffold structure at the nuclear
envelope providing mechanical
stability for the nucleus.
retinoblastoma (pRb) and regulates the pRb/E2F
signaling pathway during cell proliferation and differentiation of tissue progenitor cells. LAP2α knockout mice lack lamins in the nuclear interior and
show impaired pRb activity, leading to hyperproliferation of early progenitor cells and hyperplasia in
regenerating tissues, such as epidermis (Fig1), intestine, skeletal muscle and the hematopoietic system.
Roland Foisner
TEAM
Andreas Brachner
Mirta Boban
Juliane Braun
Thomas Dechat
Kevin Gesson
Ursula Pilat
Sandra Vidak
Nikola Woisetschläger
Livija Zlopasa
The view of a mostly mechanical role of lamins,
however, has been challenged in the past years,
since mutations in lamins and lamin binding proteins
were found to cause a variety of human diseases
ranging from muscular dystrophy and lipodystrophy
to accelerated aging syndromes. There is increasing
evidence that lamins and associated proteins are
also involved in the control of gene expression and
signaling pathways that may contribute to the
disease pathologies, but the molecular details are
still unknown. We have been studying lamin-binding proteins and their role in lamin assembly and
lamin functions. Among the best-characterized lamin-binding proteins are the LEM (LAP-EmerinMAN1) domain protein family members. They associate with chromatin via their LEM domains and
are involved in higher order chromatin organization.
While most LEM proteins are integral membrane proteins of the
inner nuclear membrane and thus localize
to the nuclear envelope,
we studied unique
members of this protein family localizing
throughout the nucleus: Lamina-Associated Polypeptide 2α
(LAP2α) and Ankyrinand LEM domain-containing protein 1 (Ankle1).
Fig2: Immunofluorescence microscopy of Ankle1 expressed in HeLa before and after treatment with the
nuclear export-inhibiting drug Leptomycin (Ankle1,
green; DNA, blue). DNA damage is shown by γH2A.X
staining. Arrows, transfected cells; bar, 10µm.
In a complex with nucleoplasmic lamins,
LAP2α binds to the cell
cycle regulator protein
Fig1: Microscopic images of paw epidermis of LAP2α+/+
and LAP2α-/- mice, stained with haematoxylin and eosin
(H & E) or immunolabelled for keratin 5 (K5, proliferating
cells) and keratin 10 (K10, differentiated cells). Bar,50 μm.
Note the thickening of the “proliferating” layer.
We propose that lamin-LAP2α complexes regulate
the activity of adult stem cells and early progenitors
in tissue homeostasis and regeneration and that
an impairment of these functions in lamin-linked
diseases may contribute to the tissue pathologies.
Ankle1 is a LEM domain protein, which is predominantly expressed in hematopoietic cells and tissues and shuttles between the cytoplasm and the
nucleus. We have shown that Ankle1 contains a
C-terminal GIY-YIG domain first described in homing endonucleuases and possesses nuclease activity in vitro. Ectopically expressed Ankle1 in the
nucleus causes DNA damage. We propose that Ankle1 is a new component involved in DNA repair
and in DNA rearrangements during B-cell development.
Naetar N, Korbei B, Kozlov S, Kerenyi MA, Dorner D, Kral R, Gotic R, Fuchs P, Cohen TV, Bittner R, Stewart CL, Foisner R
(2008). Loss of nucleoplasmic LAP2alpha-lamin A complexes causes erythroid and epidermal progenitor hyperproliferation. Nat Cell Biol, 10, 1341-48. n Gotic I, Schmidt WM, Biadasiewicz K, Leschnik M, Spilka R, Braun J, Stewart CL,
Foisner R (2010). Loss of LAP2alpha Delays Satellite Cell Differentiation and Affects Postnatal Fiber Type Determination. Stem Cells, 28, 480-88. n Wilson KL, Foisner R (2010). Lamin-binding proteins. Cold Spring Harb Perspect Biol,
2(4):a000554.
26
R E S E A R C H
G R O U P S
PETER FUCHS
Stress response in simple epithelia
A major role of the keratin intermediate filaments in simple epithelia
is to protect cells from mechanical
and non-mechanical stresses.
There is increasing evidence for the involvement
of keratin-associated proteins with the modulation
of these functions. One of these proteins is epiplakin, a member of the plakin protein family. Compared to the other protein family members epiplakin
has an unusual structure comprising solely 16 (mouse)
or 13 (human) plakin repeat domains. Its expression
is restricted to epithelial tissues (Figure 1) and it
binds to intermediate filaments, mainly to keratins,
which are the only binding partners identified so
far. Epiplakin-deficient mice generated in our laboratory are viable and show no obvious phenotype.
These findings are in clear contrast to other proteins
belonging to the plakin protein family like plectin,
desmoplakin, and BPAG1, which play an important
role in mechanically strengthening the skin as
shown by phenotypes of knock-out mice.
Subsequent experiments using primary keratinocytes from epiplakin-deficient mice showed that
the biological role of epiplakin seems to be different
from these plakins and to be connected with cellular stress response (Figure 2) rather than with
maintenance and regulation of cytoskeletal architecture. This protective function appears to be more
prominent in simple epithelial tissues as shown by
the knock-down of epiplakin in HeLa cells which
led to the disruption of intermediate filament networks, contrasting the situation in keratinocytes.
Peter Fuchs
TEAM
Sandra Szabo
Karl Wögenstein
Fig.1: Immunolocalization of epiplakin in various mouse
tissues. Frozen sections prepared from tissues of adult
mice, as indicated, were processed for immunolabeling
using anti-epiplakin antibodies
However, a comprehensive analysis of the in vivo
function of epiplakin in simple epithelia using defined animal models is still missing to date. In order
to further elucidate the biological function of epiplakin in simple epithelia, we are performing a
combination of experiments using mouse injury
models and experiments based on
cell culture, biochemistry and video
microscopy. In the mouse we use
several stress models for simple epithelia in different organ systems
which are complemented by experiments with primary cells. In addition we use biochemical and cell
culture based methods to investiFig.2: Colocalization and subcellular co-distribution of epiplakin with keratin
gate epiplakin interaction with
aggregates after okadaic acid (OA)-induced filament disruption in wild-type
simple epithelial keratins in more
keratinocytes. Primary mouse keratinocytes, treated with OA for 2, 4 and 6
detail and to reveal epiplakin
hours were immunolabeled using epiplakin (red) and pan-keratin (green)
functions in keratin network recoantibodies.
very after stress.
Fuchs P et al. (2009). Targeted inactivation of a developmentally regulated neural plectin isoform (plectin 1c) in
mice leads to reduced motor nerve conduction velocity. J Biol Chem. Sep 25;284(39):26502-9. n Spazierer D et al.
(2008). Stress-induced recruitment of epiplakin to keratin networks increases their resistance to hyperphosphorylation-induced disruption. J Cell Sci. Mar 15; 121(Pt 6): 25-833. n Spazierer D et al. (2006). Epiplakin is dispensable
for skin barrier function and for integrity of keratin network cytoarchitecture in simple and stratified epithelia.
Mol Cell Biol. Jan; 26(2): 559-68.
27
R E S E A R C H
G R O U P S
JURO GREGAN
Chromosome segregation during mitosis and meiosis.
How does the cell ensure that during
cell division each daughter cell inherits
one copy of every chromosome?
Juro Gregan
Meiosis is a specialized cell division which produces
haploid gametes from diploid cells, how is this reduction of chromosome number achieved? We want
to understand how cells accurately segregate their
chromosomes during mitosis and meiosis.
TEAM
Shazia Ahmad
Lubos Cipak
Andrej Dudas
Ines Kovacikova
Silvia Polakova
Mirka Pozgajova
Cornelia Rumpf
Lijuan Zhang
It is important to understand this process because
defects in chromosome segregation (missegregation) during mitosis result in cells with abnormal
number of chromosomes. Such cells are hallmarks
of cancer. Moreover, defects during meiosis cause
miscarriages, infertility and genetic diseases such
as Down’s Syndrome.
Chromosome segregation during meiosis.
The reduction of chromosome number during meiosis is achieved by two successive rounds of chromosome segregation, called meiosis I and meiosis
II. While meiosis II is similar to mitosis in that sister
kinetochores are bi-oriented and segregate to
opposite poles, recombined homologous chromosomes segregate during the first meiotic division.
Formation of chiasmata, mono-orientation of sister
kinetochores and protection of centromeric cohesion
are three major features of meiosis I chromosomes
which ensure the reductional nature of chromosome segregation. In our studies we use the fission
yeast S. pombe, which is an excellent model organism amenable to both genetic and cell biology
techniques, to identify new proteins required for
proper segregation of chromosomes during meiosis.
In order to decipher molecular functions of identified proteins, we combine biochemical and cell
biology techniques. To test the possible functional
conservation of identified proteins, we plan to analyze the function of the respective homologs in
mammalian cells.
Chromosome segregation during mitosis.
Accurate chromosome segregation in mitosis depends on the establishment of correct (amphitelic)
kinetochore orientation. Merotelic kinetochore
orientation is an error which occurs when a single
kinetochore is attached to microtubules emanating
from opposite spindle poles. Recent studies showing
that merotelic kinetochore attachment represents
a major mechanism of aneuploidy in mitotic cells
and is the primary mechanism of chromosomal instability in cancer cells underline the importance
of studying merotely. We focus on fission yeast
proteins required to prevent and correct merotelic
attachments in order to understand how cells ensure high fidelity of chromosome segregation.
Pcs1/Mde4 complex is a putative clamp which ensures proper microtubule-kinetochore attachment.
Gregan, J. , Polakova, S., Zhang, L., Tolic-Norrelykke, I. and Cimini, D. (2011). Merotelic kinetochore attachment: causes
and effects. Trends in Cell Biology (in press) n Rumpf, C, Cipak, L., Dudas, A., Benko, A., Pozgajova, M., Riedel, C., Ammerer,
G., Mechtler, K., Gregan, J. (2010). Casein kinase 1 is required for efficient removal of Rec8 during meiosis I. Cell
Cycle, 9(13):2655-60. n Gregan, J., Riedel, C.G., Pidoux, A.L., Katou, Y., Rumpf, C., Schleiffer, A., Kearsey, S.E., Shirahige,
K., Allshire, R.C., Nasmyth, K. (2007). The kinetochore proteins Pcs1 and Mde4 and heterochromatin are required to
prevent merotelic orientation. Current Biology, 17(14): 1190-1201.
28
R E S E A R C H
G R O U P S
ALEXANDER VON GABAIN
R & D Programs at Intercell AG, a spin off of the MFPL and the IMP
driven Biotech Company takes advantage of this
trend and devotes its R&D programs to the development of novel infectious disease vaccines. The
company has worldwide launched a novel prophylactic vaccine against Japanese encephalitis virus
which is based on an attenuated, inactivated, cell
culture produced and highly purified vaccine antigen. In the clinical pipeline are protein subunit
vaccines against bacterial pathogens, such as
Mycobacterium tuberculosis, causing TB, and Pseudomas aeruginosa, Clostridium difficile, both cauHowever, development and launch of novel vaccines
sing hospital acquired infections, but also a therahas not seen a turn around before the late 1980ies.
peutic vaccine against the Hepatitis C virus. The
Dramatic progress made in the scientific fields of
Intercell team works also on the development of
immunology, molecular biology, genomics and hostanti-infective monoclonal antibodies that are deparasite interaction, but also in the arena of novel
rived from humans exposed to the pathogens. The
manufacturing technologies has facilitated the
vaccine development is supported by technology
development of novel vaccines. Intercell, a spin off
platforms that help to dissect the protective human
of the Campus Vienna Biocenter and researchimmune response, to identify vaccine antigens,
structures mediating protection
against the pathogen, and to design vaccine adjuvants, substances inducing and facilitating the
proper type of immunity in the
vaccinated subjects. Many of Intercell’s vaccine technologies and
R & D projects are partnered with
pharmaceutical industries, including Merck & co, sanofi and Novartis. Additionally, the company
is actively collaborating with
many academic and public institutions, including the Centre of
disease Control, CDC, Max Plank
Institutes, Karolinska Institute
and MFPL. Vaccine development
at Intercell is largely financed by
private investments and revenues. However, the company is
also grateful for the generous
support of Austrian, Viennese and
Vaccine development at Intercell is aiming to reduce the formulation to a miniUS funding agencies, but also inmal number of antigens and to an adjuvant. Consequently an adaptive and prodebted to PATH and AERAS fountective immune response is induced in the vaccinated subjects. Our antigens are
dations largely carried by the Meidentified by using the immune system of pathogen-exposed individuals as read
linda and Bill Gates Foundation.
out. We are optimizing our adjuvants by analysing their effect on the innate imFor more information:
mune system. Our needle-free delivery system is delivering antigens and adjuwww.intercell.com
Vaccination is arguably the most
successful medical intervention
which has become during the last
century a mandatory part of most
countries’ health care programs and
shown to be an effective instrument
in the control of infectious diseases
worldwide.
vant to the first layer of immune defence where macrophages are prevalent.
Senn BM et al. (2011). Monoclonal antibodies targeting different cell wall antigens of group B streptococcus mediate
protection in both Fc-dependent and independent manner. Vaccine April 2011; in press n Aichinger MC et al. (2011).
Adjuvating the adjuvant: facilitated delivery of an immunomodulatory oligonucleotide to TLR9 by a cationic antimicrobial peptide in dendritic cells. Vaccine, 29: 426-36. n Fritzer A et al. (2010). Novel conserved group A streptococcal proteins identified by the antigenome technology as vaccine candidates for a non-M protein-based vaccine.
Infect Immun. 78: 4051-67.
29
Professor at the MFPL, Foreign
Adjunct Professor at the Karolinska
Institute in Stockholm, Chairman
elect of the European Institute of
Innovation and Technology, EIT, and
Strategic advisor and Chairman of
the SAB at Intercell.
R E S E A R C H
G R O U P S
ARNDT VON HAESELER
Bioinformatics
The Center for Integrative Bioinformatics Vienna (CIBIV, www.cibiv.at)
serves as a central facility to coordinate the Bioinformatics activities at
the MFPL and the University of
Veterinary Medicine Vienna.
Arndt von Haeseler
TEAM
Quang Minh Bui
Ricardo de Matos Simoes
Huy Quang Dinh
Ingo Ebersberger
Rikard Erlandsson
Mareike Fischer
Wolfgang Fischl
Stefan Ganscha
Tanja Gesell
Tina Köstler
Anne Kupczok
Tobias Neumann
Minh Anh Thi Nguyen
Tung Lam Nguyen
Jovana Nolic
Mikhail Okun
Natasa Peric
Phuong Minh Pham
Philipp Rescheneder
Heiko Schmidt
Peter Schmitzberger
Fritz Sedlazeck
Andrea Setzer
Sascha Strauss
Stefanie Tauber
Moreover, it is involved in providing infrastructure
and bioinformatics expertise for the various research groups at MFPL and on campus. Thus, it is
involved in several collaborations with experimenters. Besides this data analysis part,
the CIBIV pursues its own research
agenda. The group’s main effort is to
understand the evolutionary processes
that have shaped the genomes of contemporary species. To this end, the CIBIV applies methods from statistics,
computer sciences, and mathematics
to detect the traces ancient evolutionary events have left in modern genomes. The CIBIV is involved in several
international projects, like the Deep
Metazoan Phylogeny project, where it
coordinates the Bioinformatics aspects
(www.deep-phylogeny.org).
To this end we have developed a graphic card
based optimal local alignment tool, which maps
millions of reads to a reference genome in a few
seconds. The mapping of reads to a reference genome is the first, and possibly crucial step for any
further analysis. To understand the performance
of different mapping strategies we suggest a new
evaluation tool that allows a graphical view of the
mapping accuracy (see Figure). The development
of efficient algorithms and further statistical tools
to analyse the data takes place in close collaborations with many research groups at the MFPL and
on campus.
More recently we have expanded our
research interests to address mathematically and computationally tractable problems that may help to assist
in conservation decisions. We have employed the integer linear programming
paradigm to explore conservation scenarios in the presence of external constraints. Here, we explore biodiversity
conservation questions regarding area
selection using a phylogenetic diversity
measure and apply it to a large data
set of 735 plant genera from the Cape
of South Africa. This work is still ongoing.
Finally, we have started to develop
tools to efficiently analyse deep sequencing data that pose a new challenge to bioinformatics.
Results of selected mapping programs for different simulations S1, S2,
S3 .For each data set the runtime is depicted in the middle of the rectangles. Here we measured the Wall clock time assuming a desktop PC.
K. Meusemann, B.M. von Reumont, S. Simon, F. Roeding, S. Strauss, P. Kück, I. Ebersberger, M. Walzl, G. Pass, S. Breuers, V.
Achter, A. von Haeseler, T. Burmester, H. Hadrys, W.W. Wägele and B. Misof (2010) A phylogenomic approach to resolve
the arthropod tree of life. Mol. Biol. Evol., 27, 2451-2464. n T. Köstler, A. von Haeseler, and I. Ebersberger (2010) FACT:
Functional annotation transfer between proteins with similar feature architecture. BMC Bioinform., 11, 417. n A.
Kupczok, H.A. Schmidt, and A. von Haeseler (2010) Accuracy of phylogeny reconstruction methods combining overlapping gene data sets. Algorithms Mol. Biol., 5, 37.
30
R E S E A R C H
G R O U P S
ANDREAS HARTIG
Origin and biogenesis of peroxisomes
Eukaryotic cells contain intracellular
membrane-surrounded compartments (organelles) to separate
metabolic pathways.
This spatial separation ensures optimal flux of metabolic intermediates and increases the efficiency
of the metabolism. Peroxisomes are highly versatile
organelles and essential for life. They participate
in many metabolic processes, most notably the degradation of fatty acids and the glyoxylate cycle.
Synthesis of organelles and their degradation has
to be tightly regulated in agreement with the metabolic status of the cell.
Accordingly, peroxisomes need to be maintained
in sufficient number to ensure metabolic homeostasis. A network of interacting proteins guarantees
the biogenesis of functional peroxisomes, the transport of peroxisomal matrix proteins across the organellar membrane, and the control of size, shape
and number of these compartments. Dispensable
peroxisomes are degraded in a process called pexophagy. Employing yeast as model system we aim
to elucidate the molecular mechanisms leading to
new peroxisomes either through proliferation of
already existing ones or via a de novo biogenesis
pathway through fission from the ER. Currently,
our main interest is focused on the mechanism of
the de novo biogenesis initiated at the ER.
Proteins exclusively involved in the biogenesis of
peroxisomes are called peroxins (Pex-proteins).
Among these the Pex11 protein is a membrane
elongation factor, and in yeast, we showed that
this protein acts only on already existing peroxisomes leading to proliferation. Two distantly related
yeast proteins, Pex25p and Pex27p, play similar roles at the peroxisomal membrane and, in addition,
participate in the de novo biogenesis. The Pex3
protein is the only peroxin demonstrated to accumulate under certain conditions at the ER and later
be transferred to peroxisomes. Distinct vesicles
emanating from the ER may slowly mature into
peroxisomes or may fuse with each other or already
existing peroxisomes to form mature organelles.
The priming event at the ER, the proteins involved
and the molecular mechanism are so far unknown,
and will be the focus of our future work.
Yeast cells expressing a fluorescent peroxisomal protein. Wild type cells (left) accumulate the protein in peroxisomes, in
mutant cells (right) lacking peroxisomes the fluorescent protein remains cytosolic.
Neuberger G, Maurer-Stroh S, Eisenhaber B, Hartig A, and Eisenhaber F (2003). Prediction of peroxisomal targeting
signal 1 containing proteins from amino acid sequence. J Mol Biol 328, 581-592. n Kunze M, Pracharoenwattana I,
Smith SM, and Hartig A (2006). A central role for the peroxisomal membrane in glyoxylate cycle function. Biochim
Biophys Acta Mol Cell Res 1763, 1441-1452. n Koch J, Pranjic K, Huber A, Ellinger A, Hartig A, Kragler F, and Brocard C
(2010). PEX11 family members are membrane elongation factors that coordinate peroxisome proliferation and maintenance. J Cell Science 123, 3389-3400.
31
Andreas Hartig
TEAM
Gisela Dechat
Anja Huber
R E S E A R C H
G R O U P S
ERWIN HEBERLE-BORS
Plant developmental genetics and biotechnology
Erwin Heberle-Bors
A method has been developed by
metabolic engineering of glutamine
for the creation of reversible malesterility in plants to be used for F1hybrid breeding.
In collaboration with Alisher Touraev’s group at
MFPL a gene called DCN1 has been characterized
in tobacco that regulates developmental phase
transitions, including totipotency, and that is involved in the neddylation of cullins, a component
of ubiquitin E3 ligases.
In collaboration with Fritz Kragler’s and Markus
Teige’s group at MFPL a MAP kinase, AtMPK10,
and a MAP kinase kinase, AtMKK2, have been identified that control flowering time, leaf size and
leaf vein formation by interacting with polar auxin
transport inhibitors.
Together with Roberto Nitsch in Joseph Penninger’s
lab we are investigating the role of mammalian
DCN1 by reverse genetics. Progress has been made
in using microspore embryogenesis for gene targeting via homologous recombination.
Expression of the MAP kinase
AtMPK10 in leaves of transgenic
Arabidopsis thaliana plants (blue,
center) coincides with auxin maxima (arrows) as reported by expression of teh auxin-response
gene DR5-GUS.
The image shows the leaf development schematically.
Ribarits A.,et al. 2007. Combination of reversible male sterility and doubled haploid production by dominant-negative
inhibition of cytoplasmic glutamine synthetase in developing anthers and pollen, Plant Biotech. J., 5: 483-494.
n Ribarits A. et al. 2007. Two tobacco proline dehydrogenases are differentially regulated and play a role in early
plant development. Planta 225: 1313-1324.
32
R E S E A R C H
G R O U P S
MARCELA HERMANN
LDL-R gene family, apolipoproteins and lipid transfer
Our studies focus on the biology of
the growing chicken oocyte and the
developing chicken embryo.
Specifically, we are interested in unraveling molecular mechanisms involved in the transport of VLDL
from the egg yolk to the embryo proper. In this
context, the roles of the LDL receptor gene family
members, apolipoproteins and lipid transfer proteins, are studied. The developing avian embryo
constitutes an excellent system for the study of lipid and lipoprotein transport phenomena. The yolk
is the major source of nutrients for the developing
embryo, but molecular details of the delivery mechanisms are largely unknown. During the vitellogenic phase of oocyte growth in the chicken, the
yolk accumulates via uptake from the circulation
of precursor proteins, serves as the sole source of
lipid, carbohydrate, and protein . Only 350 mg of
the 5-6 g of lipid in the yolk are mobilized during
the first two weeks of embryogenesis; the major
portion is transported during the final week. Such
uptake, to a large part, occurs via the yolk sac,
which utilizes the yolk lipoprotein components,
following their degradation or modification, for resynthesis of lipoproteins which are subsequently
secreted and delivered to the embryo through the
embryonic circulatory system.
The chick yolk sac is characterized by an outer
layer of loosely associated mesenchymal tissue
containing fetal blood islands and an inner single
layer of endodermal cells which line the lumen of
the yolk sac cavity. The yolk-sac derived lipoproteins, mainly VLDL contain much higher proportions
of cholesteryl esters than yolk VLDL and harbor the
intact form of apoB-100 rather than proteolytic
fragments thereof. Furthermore, they lack apoVLDLII, which is synthesized by laying hens and is present
in yolk VLDL. These findings suggest that processing
of yolk components inside the yolk sac proceeds in
controlled fashion, initially involving degradation
of their constituents.
early atherogenic events. Modifed LDL activates
endothelial cells to attract and bind monocytes,
and consecutively foam cells are formed, leading
to the appearance of the fatty streak lesion. Various
diseases such as diabetes, chronic renal insufficiency and obesity come along with elevated levels
of blood cholesterol and different modified LDL.
We are interested to identify compounds (synthetic,
natural) with the potential to act as catalysts or
inhibitors of the atherogenic modification of LDL.
Marcela Hermann
TEAM
Eva-Theres Gensberger
Domink Habrina
Patricia Mamesa
Clara Manns
Julia Plieschnig
Désirée Šubik
Hepato-oocyte-embryo axis for yolk transport and utilization. During oogenesis in the chicken, the yolk precursors (e.g., vitellogenin and VLDL) are synthesized by the
maternal liver under stringent hormonal control (E2) and
taken up into the oocyte via receptor-mediated endocytosis (LRs). After ovulation and fertilization, a major feature of development is the formation of a series of
extraembryonic structures including the amnion, chorion,
allantois and yolk sac membranes (modified from
http://chickscope.beckman.uiuc.edu/). Inset: The yolk sac
is a layer of tissue growing over the surface of the yolk
containing area vasculosa with blood vessels (bv), endothelial cells (EC), and an inner single layer of endodermal
epithelial cells (EEC) with endocytic LRs and basement
membrane (bm). A major role of the yolk sac is the uptake
of nutrients from the yolk, their degradation and/or modification for re-synthesis and secretion into the embryonic circulation.
We also focus on the roll of LDL modification in
atherogenesis. The onset of atherosclerosis is a
complex process, but there is now some evidence
that the modification of LDL may play a key role in
Marcela Hermann et al. (2000). Lipoprotein receptors in extraembryonic tissues of the chicken. J. Biol. Chem 275:
16837-16844 n Marcela Hermann et al. Regulation by Estrogen of Synthesis and Secretion of Apolipoprotein A-I
in the Chicken Hepatoma Cell Line, LMH-2A. Biochim. Biophys. Acta 1641: 25-33 n Sabine M. Schreier et al. (2011).
S-Carbamoylation impairs the oxidant scavenging activity of cysteine: its possible impact on increased LDL modification in uraemia. Biochimie 93(4): 772-7
33
R E S E A R C H
G R O U P S
JOACHIM HERMISSON
Theoretical Population Genetics
The work of the Mathematics and
Biosciences Group (MaBS) is on
theoretical population genetics
and evolutionary ecology.
Joachim Hermisson
TEAM
Claudia Bank
Gregory Ewing
Ines Hellmann
Christian Huber
MaBS members at Mathematics
Department:
Michael Kopp
Sebastian Matuszewski
Agnes Rettelbach
Claus Rüffler
Hannes Svardahl
Hildegard Uecker
Evolution is the unifying theory of the biological
sciences, and our aim is to design advanced mathematical methods and models that account for
the biological complexity involved in most evolutionary processes. Complexity arises on all levels
of biological organization: molecular, organismal,
and ecological. The key issues of evolutionary research, such as adaptation and speciation, are usually addressed in special sub-disciplines for each
of these levels, i.e. molecular population genetics,
quantitative genetics, and evolutionary ecology.
We work on all three fields with the special goal
to create an integrative approach, using a combination of different models, concepts, and methods.
Methods include analytical work (stochastic processes, differential equations), extensive computer
simulations, and statistical data analysis.
Molecular approaches
The availability of DNA polymorphism data on a genome-wide
scale (“population genomics”) is
arguably the most significant development in evolutionary research today. In this context, the
characterization of the adaptive
process on the level of the molecular genotype is a primary research focus in our group. Our
aim is to extend the population
genetic theory of molecular adaptation to a broader range of biological scenarios. Quantities of interest are fixation probabilities
and fixation times and the expected footprint of selection on
linked neutral variation (so-called
selective sweeps).
Phenotypic approaches
It is widely appreciated (and ever
better understood) that the genetic basis of most quantitative
traits consists of complex gene networks. However,
when and how gene interactions (epistasis) affect
evolutionary processes is far less clear. In a series
of articles, we have studied the evolutionary role
of epistasis in equilibrium and non-equilibrium systems. A special research focus is on the effects on
genetic variation and the adaptive process (epistatis
and evolvability) and on the evolution of the genotype-phenotype map (robustness, canalization,
and modularity).
Ecologically motivated approaches
The vast majority of population genetic models
work under the assumption of a constant fitness
landscape. Since fitness depends on variable environments, this is an idealization. Natural fitness
landscapes will change over space and time. And
because an important aspect of an individual's environment is the composition of phenotypes in its
own population, fitness will also depend on allele
frequencies. The aim of this third line of our research is to combine genetic models with ecological
factors. Recent studies have focused on conditions
for speciation in spatially structured populations
with gene-flow (parapatric speciation).
Does adaptive evolution typically proceed in many small steps or fewer larger
ones? This classical evolutionary question for the “genetic basis of adaptation”
has previously been addressed in theoretical models that do not account for
the mode of environmental change that causes the selection pressure. Kopp
and Hermisson (2009) demonstrate that this ecological information indeed
plays a crucial rule: If the environment changes slowly relative to the adaptive
potential of a population (small γ), the step sizes α will typically be small. In
contrast, large steps are expected for fast changes, when the speed of adaptation is only limited by the mutation rate.
Hermisson J. and Pfaffelhuber P. (2008). The pattern of genetic hitchhiking under recurrent mutation. Electronic
Journal of Probability 2008; 13:2069-2106. n Kopp M. and Hermisson J. 2009. The genetic basis of phenotypic adaptation II: The distribution of adaptive substitutions in the moving optimum model. Genetics 183:1453-1476.
n Ewing G and Hermisson J. MSMS: a coalescent simulation program including recombination, demographic structure
and selection at a single locus. Bioinformatics 2010; 26: 2064-2065.
34
R E S E A R C H
G R O U P S
REINHOLD HOFBAUER
Consequences of carnitine deficiency and CSF-1 inhibition
Although both research areas have a
completely different biological background, signaling processes are very
important for the transcriptional
activation of genes taking place under carnitine deprivation and CSF-1
inhibition.
The first research task is dealing with the effects
L-carnitine as a nutrigenomical metabolite exerts
upon gene expression. We study carnitine deficiency, itself defining a very critical clinical condition, followed by carnitine supplementation in an
artificial model system in human liver and fibroblast
cells. This cell culture model defines sharp metabolic condition comparable to a patient situation,
a precondition to study changes on mRNA expression levels. These promoter specific processes triggered by L-carnitine will be analyzed by a variety
of molecular techniques, including chip screen analysis, real time RT-PCR, reporter gene and (super)
band shift assays. We already have identified genes
directly involved in the transcriptional regulation
of the “L-carnitine effect”, thus being able to
approach clinical pathologies of hyperlipidemia,
insulin resistance and type 2 diabetes mellitus,
which are often very closely related.
We primarily want to reveal so called “candidate
or susceptibility” genes, which are associated with
these diseases and have an increased sensitivity to
diet (= main goal of nutrigenomics). The results of
this research will provide better insight in metabolic
aspects of pathologies and their regulation as well
as mitochondrial function.
The second research project is tracing the effects
associated with inhibition of the macrophage colony-stimulating factor (CSF-1), which plays a key
role in a wide variety of biologic processes. It primarily acts on cells of the mononuclear phagocyte
lineage by controlling the differentiation, proliferation and survival of precursor cells as well as the
activation of mature macrophages. As the latter
are present in many tissues, CSF-1 also has a role
in the pathogenesis of several disorders including
cancers, because it regulates the production of
MMPs and the uPA gene, which are heavily involved
in tissue remodeling and tumor invasion.
In view of the key role of CSF-1 in
tumor progression, we have investigated whether inhibition of CSF1 expression can serve as a valuable tool to fight tumor growth
and decrease the risk of metastasis. Microarray analyses have revealed very promising candidate
genes that are re- or induced during CSF-1 inhibition. In theory
their inhibition should enhance the
inhibitory effect of CSF-1 specific
antibodies or RNAi. Additional preclinical animal studies with additional inhibitory agents (monoclonal antibodies, RNAi) delineated
from chip screen candidates are
the next experimental aims.
The pivotal role of L-carnitine for the mitochondrial lipid metabolism
Blake SM et al. (2008) Thrombospondon-1 binds to ApoER2 and VLDL receptor and functions in postnatal neuronal
migration. EMBO J 27(22):3069-80. n Godarova A et al. (2005) L-Carnitine regulates mRNA expression levels of the
carnitine acyltransferases CPT I, CPT II and CRAT. Chem. Monthly 136, 1349-1363. n Hofbauer R et al. (2005) Chronic
hemodialysis and pregnancy- L-carnitine supplementation to human sera in vitro restoring normal expression levels
of carnitine acyltransferases. Chem. Monthly 136. 1509-1521.
35
Reinhold Hofbauer
TEAM
Marion Gamsjäger
Klemens Kienesberger
R E S E A R C H
G R O U P S
N. ERWIN IVESSA
Protein biogenesis and degradation from the ER
characterization of a quality control
system that operates in the endoplasmic reticulum (ER) to ensure
that only properly folded proteins
will be released.
N. Erwin Ivessa
TEAM
Johanna Levy
Karina Zöttl
Misfolded polypeptides are retro-translocated from
the ER to the cytosol, where they become polyubiquitinated and destructed by proteasomes. ERassociated degradation (ERAD) is of relevance for
a variety of genetically inherited, neurodegenerative, and virally transmitted diseases with protein
folding defects.
We have previously shown that a truncated form
of ribophorin I, a model glycoprotein for ERAD, is
degraded by the ubiquitin/proteasome system. The
role of N-linked glycans in ERAD was pinpointed
as temporary retention devices in the ER. Thus, interaction of N-glycosylated substrates with the
calnexin cycle prolongs their half lives.
Furthermore, the requirement of N-linked glycan
trimming for ERAD was shown, and from studies
with mutant cell lines with defects in N-glycan
assembly the activities of one or more ER α1,2mannosidases could be implicated in ERAD.
Interaction partners of ERAD substrate proteins in
these mutant cell lines will be determined in immunoprecipitation experiments with cell lysates
from cells grown in the presence of proteasome
and/or glycan processing inhibitors. Positive candidates will be identified using antibodies to known
ER-associated proteins, and/or by mass spectroscopy and then further characterized. Another
aspect of this project deals with the precise intracellular localization of the ERAD pathway of glycoproteins by indirect immunofluorescence and confocal laser scanning microscopy using appropriate
marker proteins.
The role of MTP und PDI in the assembly and secretion of atherogenic lipoprotein particles
Microsomal triglyceride transfer protein (MTP) is a
lipid transfer protein required for the assembly and
secretion of very low density lipoproteins (VLDL). Active MTP is a heterodimer containing a 97 kDa catalytic subunit and a 58 kDa subunit
identified as protein disulfide isomerase (PDI). The MTP complex catalyzes the loading of apolipoprotein B
(apoB) with lipids and/or the translocation of apoB into the lumen of
the endoplasmic reticulum (ER). In
avians, the synthesis of VLDL is inducible by estrogen. We are studying
the effect of estrogen treatment on
MTP activity and on the regulation
of VLDL secretion that is also determined by lipid availability and apoB
degradation. In this context, the consequence of altered intracellular MTP
activity on VLDL assembly and seModel for endoplasmic reticulum-associated degradation (ERAD): Enzymes,
cretion is being analyzed. Another
lectins and molecular chaperones work as folding factors on nascent
aspect of the project is concerned
(glyco)proteins in the lumen of the ER. After retrotranslocation of ERAD
with the mechanism of retention of
substrate proteins through a proteinaceous channel from the ER to the cythe MTP complex in the ER.
tosol, their degradation occurs via the ubiquitin proteasome pathway.
Kitzmüller C, Caprini A, Moore SE, Frénoy JP, Schwaiger E, Kellermann O, Ivessa NE, Ermonval M (2003). Processing of Nlinked glycans during endoplasmic-reticulum-associated degradation of a short-lived variant of ribophorin I. Biochem J. 376(3), 687-96. n Hermann M, Foisner R, Schneider WJ, Ivessa NE (2003). Regulation by estrogen of synthesis and
secretion of apolipoprotein A-I in the chicken hepatoma cell line, LMH-2A. Biochim Biophys Acta 1641(1), 25-33.
36
R E S E A R C H
G R O U P S
MICHAEL F. JANTSCH
Impact of RNA-editing on coding and non-coding substrate RNAs
RNA-editing by adenosine deaminases
acting on RNA (ADARs) leads to the
post-transcriptional conversion of
adenosines to inosines.
As inosines are interpreted as guanosines by most
cellular processes this type of editing can affect
the coding potential of an RNA, its folding, stablility,
or localization. ADAR mediated editing is widespread in metazoa and affects thousands of transcripts in the human transcriptome. Together with
alternative splicing, RNA-editing therefore leads
to massive diversification of the proteome.
This is exemplified best by the fact that both RNAediting, and alternative splicing are most abundant
in the mammalian brain. Consistent with its important function in modulating the transcriptome,
RNA editing is essential for normal life and development in many organisms. Our research is focused
on topics related to this type of RNA editing and
aims at understanding the biochemical, cellular,
and organismic consequences of A to I conversion.
Model of dsRNA-dependent nucleo-cytoplasmic shuttling
of ADAR1. Cytoplasmic ADAR1 is bound by TRN 1 and imported to the nucleus where TRN 1 is released by RanGTP.
Within the nucleus Exp-5 can interact with one or several
dsRBDs in ADAR1 in a RanGTP dependent manner leading
to nuclear export. This interaction can be stimulated by
dsRNA. After nuclear export RanGTP hydrolysis destabilizes
the complex in the cytoplasm. However, interaction and
thus re-import of RNA-bound ADAR1 is prevented by precluding an interaction of the third dsRBD with TRN 1 in the
presence of dsRNA. Potentially, substrate RNAs can be exported with ADAR1 from the nucleus to the cytoplasm.
Editing in protein coding mRNAs
A handful of highly conserved protein coding targets for A to I editing are known today. To understand the impact of editing on these RNAs and
their encoded proteins we are generating transgenic
mice that are impaired in specific editing events.
Our studies show that lack of editing of the mRNAs
encoding the actin crosslinking protein filamin A
leads to behavioral defects disturbed neuronal outgrowth underscoring the importance of editing for
proper neuronal function.
Repetitive elements as modulators of gene
expression
Massive editing can be found in highly structured
3’ ends of mRNAs. These editing sites are localized
in basepaired regions formed between inverted repetitive elements of the SINE family. Analysis of
these untranslated regions in reporter gene assays
demonstrates that inverted SINES lead to a dramatic reduction in gene expression. Interestingly,
despite being a target for A to I editing, this phenomenon is not depending on editing activity but
is triggered by the double stranded structure formed
in the 3’ UTR. Deciphering the mechanisms by
which these 3’ ends control gene expression is
another research goal of our group.
Editing of repetitive elements in mRNAs. Repetitive elements (such as Alu elements) can basepair if inserted in
opposite orientation. The basepaired regions are recognized by ADAR enzymes and can thus provide a substrate for
RNA-editing.
Schoft, V., Schopoff, S., and Jantsch, M.F. (2007) Regulation of splicing by RNA editing. Nucleic Acids Res. 35: 37233732 n Fritz, J., Strehblow, A., Taschner, A., and Jantsch, MF. (2009) A double stranded RNAbinding domain in the
RNA-editing enzyme ADAR1 serves as an RNA-sensitive nucleo-cytoplasmic shuttling signal. Mol. Cell. Biol
MCB.01519-08 n Tian, N., Yang, Y., Sachsenmaier, N., Muggenhumer, D., Bi, J., Waldsich, C., Jantsch, M.F., and Jin, Y.
(2011) A structural determinant required for RNA editing. Nucleic Acids Research 2011; doi: 10.1093/nar/gkr144
37
Michael F. Jantsch
TEAM
Silpi Banerjee
Catarina Carrao
Cornelia Handl
Johann Schmuttermeier
Maja Stulic
Mansoureh Tajaddod
Aamira Tariq
Cornelia Vesely
R E S E A R C H
G R O U P S
VERENA JANTSCH
Meiosis in Caenorhabditis elegans
Meiosis is the specialized cell division
that generates haploid germ cells, a
requirement to compensate for
doubling of the chromosomal
content after fertilization.
Verena Jantsch
TEAM
Antoine Baudrimont
Anahita Daryabeigi
Marlene Jagut
Thomas Machacek
Christian Pflügl
Christina Wegrostek
Alexander Woglar
Meiosis also ensures genetic diversity by recombination. Defects in this process lead to unfaithful
chromosome segregation and are a major cause
for miscarriages and birth defects. For successful
recombination in meiotic prophase, homologous
chromosomes have to recognize each other, pair,
and finally closely associate, mediated by the synaptonemal complex, a well conserved, tripartite
proteinacious structure. Interestingly, few genes
and factors involved in the meiotic pairing process
have been identified today.
From our screen for mutants defective in meiotic
prophase we succeeded in cloning the novel pairing
gene, him-19. Most interestingly, with increasing
age, mutant him-19 hermaphrodites display an aggravation of phenotypes. Also, in feminized him19 worms defects are more serious while in male
him-19 worms meiosis is only mildly affected. In
older mutant animals presynaptic alignment and
pairing are reduced and synapsis is discontinuous
and non-homologous. Him-19 seems to be engaged
in multiple early meiotic events. Metastructural
analysis of the protein found similarities to an RNA
helicase and we want to address whether the gene
is involved in meiotic gene expression in aged worms.
Research in my lab is therefore directed towards
the identification of genes and processes essential
in meiotic prophase. Special emphasis is given the
study of the mechanisms of recognition and pairing
of homologous chromosomes.
Excellent forward and reverse genetics and easy
cytological observation of all meiotic stages make
the nematode Caenorhabditis elegans an excellent
model system for our studies. In forward genetic
screens we have isolated numerous novel meiotic
mutants that provide(d) us insight into prophase I
events.
In meiotic prophase I chromosomes are moved by
cytoplasmic forces transferred to the nucleus via
the SUN/KASH protein module (components of the
outer and inner nuclear envelope that connect
chromosomes to cytoplasmatic microtubules). Abrogation of chromosome movement, as we demonstrated with the sun-1(jf18) allele, leads to
precocious synapsis involving non-homologous
chromosomes. We study the nature of chromosome
movement and its regulation. Concomitant with
chromosome movement when the SUN-1 protein
redistributes into aggregates at chromosome ends,
the inner nuclear envelope protein SUN-1 is reversibly modified by multiple kinases. We try to understand the contribution of the modifications to
faithful chromosome segregation.
In early C. elegans meiosis one end of each chromosome
attaches to the nuclear envelope via meiosis-specific protein complexes (filled blue, yellow and orange circles). Cytoplasmic tubulin (pink bars) provide the driving forces
that move chromosomes (blue and brown lines) vigorously
along the surface of the inner nuclear envelope. Cytoplasmic driving forces are transmitted to the nucleus via SUNKASH protein complexes (green and magenta ellipses).
Concomitantly the synaptonemal complex forms between
homologous chromosomes (pink ladder like lines).
Baudrimont, A. et al. (2010). Leptotene/Zygotene Chromosome Movement Via the SUN/KASH Protein Bridge in Caenorhabditis elegans. PLoS Genet 6, e1001219. n n Penkner, A.M. et al. (2009). Meiotic chromosome homology
search involves modifications of the nuclear envelope protein Matefin/SUN-1. Cell 139, 920-933. n Penkner, A. et
al. (2007). The nuclear envelope protein Matefin/SUN-1 is required for homologous pairing in C. elegans meiosis.
Dev Cell 12, 873-885.
38
R E S E A R C H
G R O U P S
FRANZ KLEIN
Chromosome Structure and Meiotic Recombination
Chromosomes contain the individual
blue-prints of living organisms stored
as nucleotide sequences in their DNA.
During generation of gametes for sexual reproduction (meiosis), maternal and paternal chromosomes exchange fragments by recombination following the repair of meiotic DNA-breaks (DSBs),
ensuring that each gamete receives a unique mix
of parental properties. Chromosomes consist of
DNA and a large and variable set of proteins, which
is responsible for their dynamic behavior. A set of
structural chromosome elements is currently being
identified as playing key roles in many diverse processes. Such elements are sites where sister chromatids are held together by cohesin, which promote
loop and axis differentiation of chromosomes.
Meiotic recombination is embedded in this chromosomal landscape, as only loop sequences receive
DSBs and recombine, while axis-sequences don’t.
Our recent work has focused on the interplay between chromosome structure and recombination.
Two important recent results are described below:
1) Initiation of recombination occurs on DNAloops, which are tethered to axis-sites at the
time of break formation.
It had been known that recombination complexes
were often found in association with the chromo-
some axis, but it remained unclear how this is
achieved. We discovered that a number of proteins
essential for recombination, but whose contribution
to recombination was unclear for two decades,
mediate this tethering, together with axis components. We also obtained high resolution interactionmaps of these proteins along the chromosomes
and begin to understand, why recombination occurs
in some chromosomal regions rather than in others.
(submitted)
Franz Klein
TEAM
2) Sumoylation of the yeast SUMO E2 converts
it into a functional E3 and is essential for chromosome synapsis.
Synapsis is the alignment of chromosome axes during meiotic prophase at 100nm distance, important
to control CO distributions. The described result
was obtained in collaboration with Andrea Pichler
(Max Planck Institute, Freiburg). Andrea found that
upon Sumoylation of its Lysine153, Ubc9 loses its
E2-activity, but acquires “E3”-like competence in
stimulating unsumoylated Ubc9 to form free
SUMO-chains in vitro. In vivo ubc9-K153,157R
shows no detectable defect in vegetative cells, but
we discovered that it is completely devoid of synapsis in meiosis. Ubc9 is thus the first protein
whose sumoylation is important for synapsis and
teaches us how synapsis is established and regulated. In addition our observation is strong support
for the SUMO-relay hypothesis, proposing that free
SUMO-chains are sandwiched
between axis component Red1
and transversal filament Zip1 –
both SUMO binding proteins
(submitted).
The graph shows the interaction profile of a protein essential for initiating recombination (Rec114) with chromosome 3 as measured by chromatin IP and
micro-arrays. The binding positions coincide with cohesin binding sites and are
unchanged when DSBs can’t form in a catalytic Spo11 mutant (blue, red). Cdk
coordinates DSB formation with DNA replication and indeed, in the absence of
S-phase cyclins Clb5, Clb6 Rec114 is not recruited to the chromosome axis (black).
Penkner, A. M., Prinz, S., Ferscha, S., and Klein, F. (2005). Mnd2, an Essential Antagonist of the Anaphase-promoting
Complex during Meiotic Prophase. Cell 120, 1-13. n Jordan, P. W., F. Klein, and D. Leach (2007). Novel Roles for Selected
Genes in Meiotic DNA Processing. PLoS Genet 3(12): 2368-2380 n Mendoza-Parra, M., A., S. Panizza and F. Klein,
(2009). Analysis of Protein–DNA Interactions During Meiosis by Quantitative Chromatin Immunoprecipitation
(qChIP). Scott Keeney (ed.), Meiosis, Volume 1, Molecular and Genetic Methods, vol. 557, Humana Press
39
Lingzhi Huang
Jean Mbogning
Silvia Panizza
Feng Peng
Martin Xaver
Susanne Zich
R E S E A R C H
G R O U P S
ALWIN KÖHLER
Gene Expression and Chromosome Dynamics
My group is broadly interested in
genome organization and the mechanisms of gene expression.
Alwin Köhler
TEAM
Stephan Hütter
Ana Krolo
Noémi Mészáros
Maren Schneider
We focus on two areas: First, we explore the role
of nuclear pore complexes (NPCs) in genome regulation. Interphase chromosomes are not randomly
spread throughout the nucleus but are fairly well
organized, with different gene loci found in different regions of the nucleus. At the same time chromatin can undergo extensive motion. In fact, some
inducible genes dramatically change nuclear positions depending on whether they are active or not.
A fascinating new line of research suggests that
activated genes can become hooked to nuclear pores – large transport channels, which protrude into
the nuclear interior with a basket-like structure.
According to this view, NPCs serve as anchors for
the gene expression machineries and play a role in
tuning gene activities. We would like to understand
which factors mediate chromatin-NPC interactions,
how these links are formed and broken and how
they contribute mechanistically to transcription,
RNA processing and export. Ultimately, our goal is
to unravel basic principles of how nuclear architecture determines cellular function.
NPCs and proteins of the inner nuclear membrane partition the genome into areas of silent (yellow) and active
chromatin (green). Gene-NPC interactions require various
adaptors including the SAGA histone acetyltransferase
(HAT) (Köhler & Hurt, Mol Cell, 2010)
Second, we investigate how ubiquitin signaling
controls gene expression. While ubiquitin is wellknown for tagging proteins for destruction by the
proteasome, its role in regulating chromatin is far
less understood. We are particularly interested in
the enzymatic toolkit for histone ubiquitination
(ligases & deubiquitinases). When appended to histones ubiquitin can function as a reversible molecular switch to regulate transcription, gene silencing and DNA repair. Recently, we have determined
the structure of a histone deubiquitinase together
with our collaborators and uncovered its sophisticated activation mechanism. Intriguingly, the Ubp8
deubiquitinase forms a protein
module with three co-factors,
which act in concert to assemble
the module, shape the catalytic
center and recognize the substrate. The deubiquitinase module
is part of SAGA, a multifunctional
transcription co-activator. Our
studies serve as a paradigm to
explain how a deubiquitinase is
switched on at the right time and
place inside the cell. In addition,
we aim to discover novel ubiquitin functions related to RNA and
chromatin biology.
Multi-step activation and structure of a Ubiquitin Pac-Man
(Köhler et al., Cell, 2010)
Köhler A*, Zimmerman E, Schneider M, Hurt E, & Zheng N* (2010). Structural basis for assembly and activation of the
hetero-tetrameric SAGA histone H2B deubiquitinase module. Cell. 14;141(4):606-17. *corresponding authors n Köhler
A & Hurt E (2010). Gene regulation by nucleoporins and links to cancer. Mol Cell. 9;38(1):6-15. n Köhler A, Schneider
M, Cabal GG, Nehrbass U, Hurt E (2008). Yeast Ataxin-7 links histone deubiquitination with gene gating and mRNA
export. Nature Cell Biol, 10(6):707-15.
40
R E S E A R C H
G R O U P S
GOTTFRIED KÖHLER
Biomolecular optical spectroscopy
Biophysical characterisation of
biomolecules and of their interactions in solution as well as on
a live cell level represents the main
object of our research.
Methods include fluorescence and time resolved
techniques performed over a wide range of time
resolution. Studies by optical spectroscopy are complemented by biocalorimetry (DSC).
Among others, these methods are applied on studies
of ligand-receptor interactions relevant for hormone regulation and of the mechanisms of endocytosis and transport in single living cells. These
measurements provide the basis for mathematical
modelling of complex dynamic behaviour in biosystems, implemented in close cooperation with
other research groups.
Gottried Köhler
TEAM
Erwin Gaubitzer
Gottfried Grabner
Martin Knapp
Christoph Miksch
Karin Müller
Martin Puchinger
Julia Schindelar
Arthur Sedivy
Aamir Shazad
Quantitative studies on molecular dynamics on a
single molecule level are performed using advanced
fluorescence correlation techniques.
Consecutive threading of cyclodextrin macrocycles on green fluorescent coumarin dye was studied using fluorescence
correlation spectroscopy. Inclusion complexes of up to three macrocycles could be resolved. This construct becomes an
ideal water-soluble stain for lipoid structures in live cell imaging.
Ortner A., Wernig K., Kaisler R., Edetsberger M., Hajos F., Köhler G., Mosgoeller W. and Zimmer A. (2010) VPAC receptor
mediated tumour cell targeting by protamine based nanoparticles. J. Drug Targeting, 18 (2010) 457-467 n Shahzad
A., Knapp M., Lang I. and Köhler G. (2010) Interleukin 8 (IL-8) - a universal biomarker? International Archives of Medicine 3:11 (2010) n Smetana W., Balluch B., Atassi I., Kügler P., Gaubitzer E., Edetsberger M. and Köhler G. (2010) A Ceramic
Microfluidic Device for Monitoring Complex Biochemical reactive Systems, Biomedical Engineering Systems and Technologies, Communications in Computer and Information Science, Vol. 52 (2), pp. 110-132 (2010).
41
R E S E A R C H
G R O U P S
ROBERT KONRAT
Computational Biology and Biomolecular NMR Spectroscopy
The sequencing of the human genome has provided a ‘parts list’ of
the human inventory comprising
potential therapeutic targets for the
pharmaceutical and biotechnology
industry.
Finally, as much of protein function is predicated
on dynamics, we are developing novel methodological approaches that combine biochemistry, bioorganic chemistry and NMR spectroscopy to unravel
the microscopic details of functionally important
protein plasticity.
Robert Konrat
TEAM
Renate Auer
Sven Brüschweiler
Leonhard Geist
Morkos Henen
Gönül Kisilzavas
Matthias Hötzinger
Karin Kloiber
Karin Ledolter
Gerald Platzer
Thomas Schwarz
In order to cope with this huge number of targets
we introduced a new theoretical conception of
protein structural biology (meta-structure) that
can be used for protein sequence-to-function annotation and drug design. A hallmark of our research is the integrative application of this novel
conception and sophisticated NMR spectroscopy
directed towards a better understanding of fundamental biological processes.
The figure serves as an overview of currently pursued research topics in the group. A central structural biology topic in
the group is the structural analysis of the oncogenic transcription factor myc and its differentially regulated target genes.
(Top) We have used NMR spectroscopy to analyse the C-terminal (DNA-binding and dimerisation) domain of myc in the
individual stages of transcription. Additionally our structural analysis of myc target genes provided a first glimpse on
myc’s cell transforming potential. (Bottom) NMR spectroscopy is a unique tool to identify and analyse high-energy states
of proteins.
Coudevylle N, Geist L, Hötzinger M, Hartl M, Kontaxis G, Bister K, Konrat R (2010) The v-myc-induced Q83 lipocalin is
a siderocalin. J.Biol.Chem. 285, 41646-52. n Konrat R (2010) The meandering of disordered proteins in conformational
space, Structure , 18, 416-19. n Auer R, Neudecker P, Muhandiram DR, Lundstrom P, Hansen DF, Konrat R, Kay LE, (2010)
Measuring the signs of 1H(alpha) chemical shift différences between ground and excited protein states by off-resonance spin-lock R1ρ NMR spectroscopy, J.Am.Chem.Soc. 131, 10832-33.
42
R E S E A R C H
G R O U P S
PAVEL KOVARIK
Signaling and gene expression in inflammation
The innate immune system dynamically
responds to infecting pathogens by
initiation of protective host responses
and by a rapid termination of these
responses once the infection agent
is no longer present.
Inefficient or uncontrolled responses may result in
infectious or inflammatory diseases. We investigate
both the basic principles of balanced innate immune responses as well as aspects relevant for immune disorders. The molecular mechanisms that
allow a robust yet temporally precisely restricted
inflammatory reaction are studied in our laboratory
at the level of transcription, mRNA stability and
pathogen recognition.
Turning on/off and resetting inflammatory gene
transcription
The Stat transcription factors play a central role in
the immune system. An open question is the molecular mechanism that determines how often one
activated Stat molecule can initiate transcription
before becoming inactivated. Our recent studies
revealed that phosphorylation at serine 727, an
important modification of Stat1, is restricted only
to promoter-bound Stat1 molecules.
This suggests that the still not well understood
S727 kinase is a chromatin-associated enzyme involved in the feedback control of the transcription
cycle at the targeted gene. We are currently characterizing the kinases and their role in the Statdependent transcription cycle.
Control of immune homeostasis by
mRNA stability
A considerable proportion of the genes induced
during the acute phase of inflammation are strongly
regulated at the level of mRNA stability. Many proinflammatory mRNAs contain in their 3´ untranslated regions cis-acting AU-rich regulatory elements (AREs) that are targeted by RNA-stabilizing
and -destabilizing proteins. The RNA-destabilizing
protein tristetraprolin (TTP) plays a fundamental
role in the attenuation of inflammation. Our newest
findings revealed that TTP is one of the effector
molecules of the anti-inflammatory cytokine IL10. We are currently studying the role of TTP in inflammatory diseases using animals with conditional
ablation of the TTP gene.
Responses of innate immune cells
to Streptococcus pyogenes
S. pyogenes is a Gram-positive human pathogen causing mild (e.g.
tonsillitis) as well as severe (e.g. toxic
shock) diseases. It is still not known
how this bacterium is recognized by
the innate immune system. We have
recently shown that, surprisingly, S.
pyogenes is recognized by a receptor
that is distinct from any so far described receptors for bacterial pathogens. The identification of the receptor for S. pyogenes and the
elucidation of inflammatory signaling
cascades in the host cells are currently the major goals of the project.
Scheme of interferon induction in macrophages and dendritic cells infected
with Streptococcus pyogenes
Schaljo B et al. (2009). Tristetraprolin is required for full anti-inflammatory response of murine macrophages to IL10. J Immunol 183(2), 1197-206. n Sadzak I et al. (2008). Recruitment of Stat1 to chromatin is required for interferon-induced serine phosphorylation of Stat1 transactivation domain. Proc Natl Acad Sci U S A 105(26), 8944-9.
n Gratz N et al. (2008). Group A streptococcus activates type I interferon production and MyD88-dependent signaling
without involvement of TLR2, TLR4, and TLR9. J Biol Chem 283(29), 19879-87.
43
Pavel Kovarik
TEAM
Joanna Bancerek
Florian Ebner
Nina Gratz
Marton Janos
Franz Kratochvill
Ivana Mikulic
Vitaly Sedlyarov
R E S E A R C H
G R O U P S
FRIEDRICH KRAGLER
Intercellular transport of proteins and RNAs regulating cell-fate
We are far from having a comprehensive understanding of the signalling
systems organisms use to coordinate
tissue growth and cell differentiation.
Fritz Kragler
TEAM
Daniela Fichtenbauer
Gregor Kollwig
Martin Kragl
Kornelija Pranjic
Nikola Winter
Fritz Kragler moved to the
Max Planck Institute of
Molecular Plant Physiology
in Golm, Germany at the
beginning of 2011.
Our general goal is to shed light on a unique class
of signal molecules constituted by non-cellautonomous proteins and RNAs transported from
cell to cell and over long distances in plants. Distinct classes of cell-fate deciding transcription
factors and RNA molecules are recognized by receptors and actively transported from cell to cell
via intercellular channels named plasmodesmata.
Our long-term objective is to find answers to two
basic questions: Why are specific transcription
factors and RNA molecules transported between
tissues?, and: How are these macromolecules recognized by the plasmodesmal transport system?
Currently we focus on long-distance signals in form
of phloem delivered small RNA molecules and on
two recently identified proteins interacting with
non-cell-autonomous and cell-fate deciding
homeodomain proteins.
We have shown that the intercellular transported
homeodomain proteins KNOTTED1 (KN1) and
SHOOT MERISTEMLESS (STM) interact with MPB2C,
a microtubule-associated protein. This interaction
prevents intercellular transport and alters RNA-
binding of KN1/STM (Winter et al., Plant Cell, 2007).
In addition, we identified a novel protein KNB36,
interacting with both, KN1/STM and MPB2C, as a
potential factor triggering degradation of MPB2C
- KN1/STM complexes.
In collaboration with Hans-Hermann Gerdes, Bergen, Norway, we found evidence that plant homeodomain (HD) proteins such as STM and animal HD
proteins such as ENGRAILED and ANTENNAPEDIA
proteins are delivered via tunnelling nanotubes
(TNTs) to adjacent cells. Complementing this approach we are currently testing whether animal
homeodomain proteins are transferred via plasmodesmata in plants. The gained data will be
used to write a proposal to scrutinize the
potential function in regulating cell fate
by selective intercellular delivery of HD
protein in animal systems.
To tackle the long-distance RNA and protein transport system of plants (Zhang et
al., Plant Phys. 2009, Kragler 2010) we tested a number of RNA and protein fusion
constructs produced in leaf tissue for their
potential to enter meiotic tissues. We were
able to shown that specific RNA molecules
are systemically delivered via the phloem
stream to sporogenic tissue and interfere
with meiosis.
Zhang S, Sun L, Kragler F. The phloem-delivered RNA pool contains small noncoding RNAs and interferes with translation. Plant Physiol. 2009, 150: 378-387 n Bouyer D, Geier F, Kragler F, Schnittger A, Pesch M, Wester K, Balkunde R,
Timmer J, Fleck C, Hülskamp M. Two-dimensional patterning by a trapping/depletion mechanism: the role of TTG1
and GL3 in Arabidopsis trichome formation. PLoS Biol. 2008, 6:e141. n Winter N, Kollwig G, Zhang S, Kragler F. MPB2C,
a microtubule-associated protein, regulates non-cell-autonomy of the homeodomain protein KNOTTED1. Plant Cell.
2007, 19:3001-18.
44
R E S E A R C H
G R O U P S
KARL KUCHLER
Host-Pathogen Interactions & Mechanisms of Fungal Pathogenesis
We study the molecular mechanisms
of fungal pathogenicity and fundamental problems in infection biology,
using a combination of molecular, as
well as genome-wide and systems
biology approaches.
First, we use reverse genetics approaches to identify
virulence and antifungal drug resistance genes in
the most prevalent human fungal pathogens such
as Candida glabrata and C. albicans. For instance,
we have generated a genome-scale gene deletion
collection of C. glabrata currently comprising some
700 single gene deletions. Further, we decipher the
role of histone modification genes in morphogenetic switching, cell fate determination and virulence. We would like to define the genetic networks
and signaling pathways facilitating immune evasion
and driving invasion of host cells. We also investigate the genomic and genetic adaptations occurring in pathogen genomes during host niche or organ colonization and systemic dissemination.
Finally, we aim to identify genes and components
that counteract organ colonization and systemic
dissemination in the mammalian host.
Finally, we study structure-function relationships
of fungal ABC multidrug transporters, and we pursue systems biology approaches to answer how the
molecular cross-talk of stress response signaling
pathways impact cellular growth control and ion
homeostasis in simple model organisms such as
baker’s yeast.
Our work is supported by grants from the Christian
Doppler Research Society, the 7th European framework programme, the Austrian Science Foundation FWF, the transnational ERA-Net Pathogenomics scheme, a SysMO project through the
Austrian GenAU research programme, by the Austrian Academic Exchange Service OeAD and by the
Austrian Research Promotion Agency FFG.
On the host side, we are studying the mechanisms
of anti-Candida response in vitro using primary
innate immune cells, as well as mouse infection
models, to define the contributions of the host
immunity to pathogenesis. Along this line, we delineate the interplay of adaptive and innate immunity in the immune surveillance against fungal
pathogens. We particularly focus on the role of
type I interferons and their signaling pathways,
since they appear to be linked to virulence and
dissemination in host tissues and organs.
Candida albicans cells forming colonies of markedly different phenotypes on agar plates due to distinct chromatin modifications, which modulate transcriptional regulatory networks controlling morphogenesis.
Hnisz, D et al. (2010). The Set3/Hos2 histone deacetylase complex attenuates cAMP/PKA signaling to regulate morphogenesis and virulence of Candida albicans. PLoS Pathogens 6: e1000889. doi:10.1371 n Bourgeois, C et al. (2010).
Fungal attacks on mammalian hosts: pathogen elimination requires sensing and tasting. Current Opin. Microbiol.
13: 1-8 n Bourgeois, C et al. (2011). A type I-interferon response to Candida spp involves phagosomal TLR signaling
mediating an novel first wave IFNß release in innate immune cells. J. Immunol. 186: 3104-3112
45
Karl Kuchler
TEAM
Christelle Bourgeois
Ingrid Frohner (until 8/2010)
Walter Glaser (until 9/2010)
Christa Gregori
Kwang-Soo Hildering (until 7/2010)
Denes Hnisz
Fabian Istel
Regina Klaus
Cornelia Klein
Nathalie Landstetter (until 10/2010)
Iwona Lesiak-Markowicz (until
9/2010)
Olivia Majer
Christina Rashkova
Tobias Schwarzmüller
Eva Stappler
Lanay Tierney
Michael Tscherner
Martin Valachovic
Philipp Wittmann
Yangyang Xu
Florian Zwolanek
R E S E A R C H
G R O U P S
WOLFGANG LÖFFELHARDT
Cyanophora paradoxa, the key to plastid evolution
Phototrophic eukaryotes (algae)
originated about 1.2 billion years
ago through the so-called primary
endosymbiotic event: a heterotrophic
protist engulfed a cyanobacterium
without digesting it.
Wolfgang Löffelhardt
(retired since 2008)
COLLABORATORS
Juraj Krajc̀´ovic̀´
(Comenius-Univ. Bratislava)
Jürgen M. Steiner
(Univ. Halle)
Instead, the host cell made use of the photosynthesis products generated by the endosymbiont
and, in a lengthy and very complicated process,
prevented its escape through inducing gene transfer
from the genome of the former free-living cyanobacterium to the nucleus. In parallell, a selective
protein import apparatus was developed at the endosymbiont envelope and many but not all of the
exported genes were “reimported” as proteins.
C. paradoxa merits the status of a ”living fossil”
since the plastids (“muroplasts”, formerly named
“cyanelles”) are stabilized by a peptidoglycan wall
between the two envelope membranes – a situation
resembling the envelope of cyanobacteria and unique (i.e., restricted to Glaucocystophyte algae)
among eukaryotes (see figure).
Phylogenetic analysis of 143 concatenated nuclear genes for cytosolic proteins from
34 species. Glaucocystophytes, rhodophytes and chlorophytes/streptophytes group together to the exclusion of all other eukaryotes → monophyly of the kingdom Plantae.
The majority of researchers in the field assume a
single primary endosymbiotic event, i. e. monophyly
of the kingdom Plantae and we contributed phylogenetic analyses of muroplast and nuclear genes.
Immuno-EM of a dividing muroplast. Primary antibodies directed against peptidoglycan from E. coli. Gold particles
mainly decorate the envelope and the newly formed septum.
CB, Rubisco-containing central body (putative carboxysome).
Insert: The biflagellated protist harboring two muroplasts.
Other acivities in the past years dealt with the targeting sequences and the translocons involved in
protein import into muroplasts. For comparison,
proteins targeted into the secondary plastids of
Euglena gracilis were inspected. Furthermore, the
presence of a carbon-concentrating mechanism
and, potentially, of carboxysomes in muroplasts of
C. paradoxa was demonstrated (collaboration with
Hans J. Bohnert, Tucson and Hideya Fukuzawa,
Kyoto). Carboxysomes are Rubisco-microcompartments known from cyanobacteria but not from algae. The carboxysomal CCM leads to an enrichment
of bicarbonate in the cytosol by a factor of several
thousands. The presence of a carboxysome in muroplasts would explain the maintenance of the
peptidoglycan wall - as a stress-bearing layer.
Now, we participate in the Cyanophora genome
project (head: Debashish Bhattacharya, Rutgers
Univ.). At present, the draft genome (around 70Mb)
is subject to analysis and we concentrate on genes
for peptidoglycan synthesis, Sec, Tat, Toc and Tic
translocons, phycobilisome subunits, and potential
components other than Rubisco and Rubisco activase (the latter was recently identified by us) of
the putative carboxysomes.
Vesteg M et al. (2010) A possible role for short introns in the acquisition of stroma-targeting peptides in the flagellate
Euglena gracilis. DNA Res, 17(4), 223-31. n Löffelhardt W (2010) Low CO2 stress: Glaucocystophytes may have found
a unique solution. In: (Seckbach J and Grube M, eds.) Symbiosis and Stress: Joint Ventures in Biology, Cellular Origin,
Life in Extreme Habitats and Astrobiology 17, pp. 83-94, Springer Science+Business Media B.V., Dordrecht. n Steiner
JM and Löffelhardt W (2011) The Photosynthetic Apparatus of the Living Fossil, Cyanophora paradoxa. In: (Peschek
GA et al, eds) Bioenergetic Processes of Cyanobacteria, pp. 71-87, Springer Science+Business Media B.V., Dordrecht.
46
R E S E A R C H
G R O U P S
JOSEF LOIDL
Meiotic chromosome pairing and recombination
Meiosis originated at the dawn of
eukaryotic evolution as an integral
part of the sexual reproduction cycle.
Cells of sexually reproducing eukaryotes normally
contain two equal (homologous) sets of chromosomes, one contributed by the father, the other by
the mother during the fusion of gametes and the
formation of a zygote.
In particular we are investigating meiotic recombination in the fission yeast and in the ciliate
Tetrahymena, which have both lost their SC. Its
absence may have led to a similar non-canonical
processing of meiotic recombination intermediates,
which constitutes only a minor recombination
pathway in the majority of eukaryotes.
Moreover, the fission yeast and Tetrahymena have
in common unusual changes in shape and move-
Josef Loidl
TEAM
When eggs or sperm are produced, they must be endowed
with a single set of chromosomes. Therefore, germ progenitor cells undergo a reductional division, meiosis.
During meiosis, homologous
chromosomes of paternal and
maternal origin juxtapose and
become connected by a proFormation and repair of meiotic DNA double-strand breaks (DSBs) in Tetrahymena.
tein structure, the synaptoneDSBs create DNA fragments migrating on a pulsed-field gel whereas intact chromal complex (SC). They then
mosomes don´t enter the gel. Cytologically, DSBs are detected as fragmented chroexchange parts and segregate
mosomes. In the wild type, DSBs transiently appear during a meiotic timecourse and
to different daughter nuclei.
finally are repaired to resolve as 5 bivalents. In a repair-defective mutant, DSBs acWe are studying various
cumulate and cause chromosome fragmentation.
aspects of meiotic chromosome organisation and behaviour in evolutionarily divergent organisms such
ments of meiotic nuclei, which promote homoloas yeasts and ciliates to learn which adaptations
gous chromosome pairing and may have evolved
and amendments have occurred during the evoluto compensate for the lack of SCs.
tion of extant meiosis.
Ultimately, our studies will help
to understand the origin and
function of conserved meiotic
features such as the SCs, the
chromosomal bouquet, and the
regulation of meiotic recombination.
Ag-stained meiotic nuclei from an animal, fission yeast and Tetrahymena (not at
the same scale). While animals (like plants and most fungi) possess an SC, fission
yeast displays only rudimentary axes, and Tetrahymena completely lacks related
structures. They have evolved alternative strategies to pair chromosomes, process
meiotic DSBs and promote interhomolog crossing over. Note that Tetrahymena
meiotic nuclei are crescent-shaped.
Latypov V et al. (2010) Roles of Hop1 and Mek1 in meiotic chromosome pairing and recombination partner choice
in Schizosaccharomyces pombe. Mol Cell Biol 30, 1570-1581. n Lukaszewicz A et al. (2010) MRE11 and COM1/SAE2
are required for double-strand break repair and efficient chromosome pairing during meiosis of the protist Tetrahymena. Chromosoma 119, 505-518. n Spirek M et al. (2010) SUMOylation is required for normal development of linear elements and wild-type meiotic recombination in Schizosaccharomyces pombe. Chromosoma 119, 59-72.
47
Anna Estreicher
Rachel Howard-Till
Agnieszka Lukaszewicz
R E S E A R C H
G R O U P S
ZDRAVKO LORKOVIC
Regulatory roles of cyclophilins in cellular signaling
Zdravko Lorkovic
TEAM
Hana Kautmanova
Zdravko Lorkovic moved to the
Gregor Mendel Institute of
Molecular Plant Biology (GMI)
at the beginning of 2011.
Cyclophilins are ubiquitous proteins
which possess peptidyl-prolyl cistrans isomerase (PPIase) activity
(i.e. they catalyze cis to trans isomerisation of peptide bonds preceding
proline) and are implicated in
virtually all cellular processes.
Cis to trans isomerization of Pro imide peptide
bond by PPIases influences phosphorylation states
of their target proteins and consequently cellular
signalling pathways.
By using Schizosaccharomyces pombe we are studying pathways and regulatory roles of an essential
cyclophilin Rct1 in RNA polymerase II transcription
and its coupling with pre-mRNA processing as well
as in cell cycle and chromosome segregation.
Ser or Thr that precede Pro are major phosphorylation motifs in cells. These sites are phosphorylated
by a large family of Pro directed kinases, which include CDK, ERK, SAPK/JNK, p38, GSK3 and PLK kinases.
The largest subunit of RNAP II contains a C-terminal domain (CTD) consisting of YSPTSPS hepapeptide repeats. Phosphorylation of CTD at Ser5 by Mcs6 kinease is required for transcription initiation whereas posphorylation at Ser2 by Lsk1 and
Cdk9 (also active on Ser5; broken arrow) is prerequisite for transcript elongation. Rct1 negatively regulates Cdk9 activity in
vivo and in vitro, thereby regulating transcriptional activity of RNAP II. For a new round of transcription, CTD has to be dephosphorylated by CTD phosphatases Fcp1 an Ssu72.
Lorković ZJ 2009. Role of plant RNA binding proteins in development, stress responses and genome organisation.
Trends Plant Sci. 14, 229-236. n Gao Z., H.-L. Liu, L. Daxinger, O. Pontes, X. He, W. Qian, H. Lin, M. Xie, Z.J. Lorković, S.
Zhang, D. Miki, X. Zhan, D. Pontier, T. Lagrange, H. Jin, A.J. Matzke, M. Matzke, C.S. Pikaard, J.-K. Zhu (2010) An RNA
polymerase II- and AGO4-associated protein acts in RNA-directed DNA methylation. Nature 465, 106-109. n Kanno.
T., E. Bucher, L. Daxinger, B. Huettel, D.P. Kreil, F. Breinig, M. Lind, M. Schmitt, S.A. Simon, G. Sai Ranjan, M.C. Meyers, Z.J.
Lorković, A.J.M. Matzke, M. Matzke (2010) RNA-directed DNA methylation and plant development require an IWR1type transcription factor. EMBO Rep. 11, 65-71.
48
R E S E A R C H
G R O U P S
SASCHA MARTENS
Molecular Mechanisms of Autophagy
Autophagy is an evolutionarily conserved and important process during
which our cells digest or cannibalize
small parts of themselves.
Autophagy plays an essential role during starvation,
the defense against pathogenic microorganisms,
the removal of protein aggregates and the degradation of damaged organelles. Misregulated or defective autophagy can result in neurodegeneration
and premature aging and is thus highly relevant to
a plethora of human diseases.
Although many genes that are important for autophagy have been identified we have only a very
limited understanding of how this important and
fascinating process is regulated and executed. Thus,
the challenge now is to assign functions to these
genes in order to gain a better understanding of
the mechanisms that orchestrate autophagy.
Autophagy is induced by an upstream signal such
as starvation, the detection of pathogenic microorganisms in the cytosol or by damaged mitochondria.
This signal triggers the most enigmatic and fascinating step of autophagy, the de novo formation
of autophagosomes. Initially a small double membrane bound structure is formed, which grows and
adopts the shape of a cup. This cup-shaped
structure eventually fuses at its rims to form a
double membrane bound organelle enclosing a part
Sascha Martens
TEAM
Scheme showing the generation of autophagosomes. Initially a small double membrane-bound structure called isolation membrane is formed. This structure expands to adopt
a cup-like shape thereby gradually enclosing cytoplasmic
cargo. This structure fuses at its rims giving rise to the mature autophagosome. Subsequently, autophagosomes fuse
with lysosomes. Within these so-called autolysosomes the
inner membrane and the cargo are degraded.
of the cell’s cytoplasm. The autophagosome then
fuses with components of the classical endosomal
system thereby maturing to an autolysosome within
which the content is degraded. The degraded content
can subsequently be used for the synthesis of factors
that are essential for the survival of the cell.
We employ biochemistry, light- and electron microscopy to investigate these mechanisms. We are
particularly interested in the mechanisms that
sculpt cellular membranes
into autophagosomes. Our
ultimate goal is to reconstitute crucial steps of
autophagosome formation
in vitro and to translate our
findings back to in vivo
models.
(A) A picture taken by confocal microscopy showing giant unilamellar vesicles (GUVs).
The membrane of the GUVs was labelled by incorporation of a fluorescent lipid.
(B) A picture showing human cells which express green and red labelled proteins that
are targeted to autophagosomes.
Our findings will give important insights into the
generation of membrane
curvature, the formation
of specialized membrane
domains and organelle
formation in general.
Groffen, A.J.*, Martens, S.*, Arazola, R.D., Cornelisse, L.N., Lozovaya, N., de Jong, A.P.H., Goriounova, N.A., Habets, R.L.P.,
Takai, Y., Borst, J.G., et al. (2010). Doc2b Is a High-Affinity Ca2+ Sensor for Spontaneous Neurotransmitter Release.
Science 327, 1614-1618. * first and corresponding authors n McMahon, H.T., Kozlov, M.M., and Martens, S. (2010). Membrane Curvature in Synaptic Vesicle Fusion and Beyond. Cell 140, 601-605. n Martens, S., Kozlov, M.M., and McMahon,
H. (2007). How synaptotagmin promotes membrane fusion. Science 316, 1205-1208.
49
Julia Romanov
Justyna Sawa-Makarska
Marta Walczak
Bettina Wurzer
R E S E A R C H
G R O U P S
IRUTE MESKIENE
Cell signaling control by MAPK phosphatases
Signals induced by environmental
stress or during development have
to be transmitted inside the cell to
generate appropriate responses.
Irute Meskiene
TEAM
Zahra Ayatollahi
Justyna Boniecka
Alois Schweighofer
Verena Unterwurzacher
In response to extracellular signals proteins are
phosphorylated by reversible protein phosphorylation mechanism as a major principle of intracellular
signaling, where protein phosphatases act as important regulators. The model plant Arabidopsis
provides excellent possibilities to investigate regulation of stress-induced or developmental signalings on cellular and on the whole plant level.
activation is induced in a minutes after exposure
to stress, such as wounding of pathogen elicitors,
but also during stomata development. Ser/Thr specific protein phosphatases from PP2C family
AP2C1-4 dephosphorylate the phospho-Thr of the
MAPK activation loop and thus inhibit their kinase
activity. Inactivation of the MAPKs leads to the
signal transduction „swich off“ ensuring its transient nature. Interestingly, stress and developmental
signaling pathways are using the very same components, such as MPK3 and MPK6, to mediate signals to different responses, raising the question
how the specificity is attained in these pathways.
Our experiments performed on Arabidopsis plants
demonstrated that AP2C1-4 gene expressions are
stress-induced and tissue-specific, suggesting that
AP2C protein phosphatases may coordinate inactivation of MPK3/MPK6/MPK4 in tissue/cell-specific
manner or under stress conditions.
We study Arabidopsis protein phosphatases of
PP2C-type (AP2Cs) in regulation of signaling that
is mediated by MAPKs (mitogen-activated protein
kinases). We have demonstrated that AP2Cs regulate activity of stress-induced
MAPKs, which mediate pathogen
and abiotic stress pathways as
well as control of stomata development. Stomata are cells on
plant epidermal surfaces that are
specialized to regulate gas and
water exchange with environment. Stomata developmental
pathway involves MAPK signaling
cascades and control by a PP2Ctype MAPK phosphatase AP2C3.
We have shown that AP2C3
MAPK phosphatase modulates
cell cycle and stomata differenA - AP2C1 modulates plant immunity to infection by fungus Botrytis.
tiation, while AP2C1 MAPK phosB - AP2C3 affects epidermal cell fate inducing differentiation of multiple
phatase controls plant immunity
stomata.
and stress hormone production.
Our recent findings reveal that
gene expression profiles, different plant hormones
Studying protein phosphatase single and double
and plant phenotypes are strongly affected by these
mutant plants we have also succeeded to identify
MAPK phosphatases. Currently, we are studying
specific gene expression profiles induced by a spehow MAPK phosphatases channel signaling pathcific MAPK pathway. We suggest that AP2C protein
ways towards specific responses under stress conphosphatases may help to channel MAPK-mediated
ditions and during cellular differentiation in stosignals to specific targets. Our results are linking
mata developmental pathway.
MAPK signaling to transcriptional reprogramming
of plant cells and demonstrate importance of proArabidopsis MAPKs, such as MPK3, MPK4 and
tein phosphatases in regulation of plant growth,
MPK6 are activated by phosphorylation of the condevelopment and survival under stress conditions.
served Thr and Tyr in their activation loop. This
Umbrasaite et al. (2010). MAPK phosphatase AP2C3 induces ectopic proliferation of epidermal cells leading to stomata development in Arabidopsis. PLoS ONE 5, e15357. n Schweighofer et al. (2007). The PP2Ctype Phosphatase
AP2C1 Which Negatively Regulates MPK4 and MPK6, Modulates Innate Immunity, JasmonicAcid and Ethylene
Levels in Arabidopsis. Plant Cell 19:221-24. n Michniewicz et al. (2007). Antagonistic Regulation of PIN Phosphorylation by PP2A and PINOID Directs Auxin Flux. Cell 130, 1044-1056.
50
R E S E A R C H
G R O U P S
ISABELLA MOLL
Ribosome Heterogeneity in Bacteria
One of the most intricate and
fundamental processes of life is
the translation of the genetic
code into proteins.
Decoding mRNA-based information into the corresponding sequence of amino acids is performed
by a complex ribonucleoprotein particle, the ribosome. Although the structure of the ribosome is
solved at atomic resolution and the process of translation has been extensively studied, little is known
about the modulation of protein synthesis mediated
by the generation of heterogeneous ribosomes in
bacteria. Our main aim is to decipher molecular
mechanisms that lead to selective synthesis of distinct classes of mRNA during stress adaptation. In
particular, we focus on adverse conditions bacteria
encounter upon infection of their host.
Recently, we have shown that treatment of E. coli
with the aminoglycoside antibiotic kasugamycin
results in the formation of ribosomes depleted for
several essential proteins of the small subunit including the functionally important proteins S1 and
S12. However, these 61S ribosomes are proficient
in translation of leaderless mRNAs, which directly
start with a 5´-terminal AUG and thus lack kingdom-specific ribosome recruitment signals (Kaberdina et al., 2009). These results provided the first
evidence for the formation of a subpopulation of
functionally distinct ribosomes under adverse conditions.
During these studies we observed the resumed synthesis of specific proteins upon prolonged antibiotic
treatment. We identified some of these proteins
as chaperones, stress proteins, ribosomal proteins
and ribosome modifying enzymes. Surprisingly, we
discovered that the respective
mRNAs became leaderless in the
presence of the antibiotic, which
apparently allows translation by
protein-depleted ribosomes.
Further investigations revealed
that diverse stress conditions likewise result in the formation and
selective translation of leaderless
mRNAs. Currently, we are investigating the underlying molecular
mechanisms and our recent results hint towards a novel stress
response mechanism, which is
based on the formation of heterogeneous ribosomes that provide
a means for selective synthesis of
The antibiotic Kasugamycin (Ksg) leads to the formation of protein-depleted
a sub-class of proteins.
61S ribosomes functional in translation of leaderless mRNAs: (A) The 30S subunit seen from the subunit interface. The helices affected by binding of Ksg are
highlighted. The r-proteins S2, S6, S12, S18 and S21 are absent in 61S particles.
(B) The 30S subunit shown from the solvent side. H26 is located on the platform
interacting with proteins S18 and S1 (not shown). (C) Working model for the
formation of the 61S particle: (1) 70S ribosomes form an initiation complex exclusively with leaderless mRNA in the presence of Ksg (2) Ksg changes the 70S
conformation, in particular that of the loop-forming helices h2, h27, h24, h28,
and h26 (indicated in A) (3) disruption of some of these helices (h2, h26, and
h27) triggers the release of r-proteins directly or indirectly attached to this
loop. (Kaberdina et al., 2009)
Based on these studies another
focus of our work concerns the
interaction of protein S1 with the
ribosome. In particular, we concentrate on the interaction surface between proteins S1 and S2,
which could serve as antimicrobial drug target, as for Gramnegative bacteria binding of S1
to the ribosome is essential.
Kaberdina AC, Szaflarski, W, Nierhaus, KH and Moll, I (2009) An unexpected type of ribosomes induced by kasugamycin: A look into ancestral times of protein synthesis? Mol. Cell, 33 (2), 227-36.
51
Isabella Moll
TEAM
Olga Ballauri
Konstantin Byrgazov
Niklas Janisch
Martina Sauert
Hannes Temmel
Oliver Vesper
R E S E A R C H
G R O U P S
ERNST W. MÜLLNER
Signal Transduction and Hematopoiesis / Erythropoiesis
Hematopoiesis starts from stem-cells
in the bone marrow which gradually
differentiate towards the well-known
specific lineages of blood cells.
Ernst W. Müllner
TEAM
Matthias Artaker
Katrin Fischhuber
Manfred Schifrer
The process ends with late-stage committed progenitors undergoing terminal maturation. Our
group focused on molecules critically involved in
reaching the balance between sustained proliferation versus terminal differentiation of progenitors,
with particular emphasis on erythropoiesis.
As systems, we used fetal liver- or bone marrowderived mouse erythroblasts as well as myeloid progenitors from various genetically modified mouse
strains. Moreover, we employed cells from cord- or
peripheral blood of healthy or diseased human donors. These cell types could be kept immature in
culture for extended periods and terminally differentiated in a highly synchronous manner.
These cellular tools were mainly used to study
1 – Signalling pathways emanating from extracellular ligands like growth factors (stem cell factor,
Wnt, erythropoietin) or steroid hormones (thyroid
hormone T3, glucocorticoid, androgen)
(Stat5DN/DN), impeding full analyses. Most complete
Stat5-/- embryos developed to term but suffered
from severe microcytic anemia resulting in perinatal
lethality. We could attribute this phenotype to multiple defects in erythropoiesis, mainly a massive
increase in apoptosis in the fetal liver.
ad 2 – We also uncovered an additional unexpected
defect on cellular iron metabolism due to Stat5
deficiency, involving a significant down regulation
of TfR-1 expression in vivo, resulting in massively
reduced iron uptake. In particular, levels of the
erythroid TfR-1 regulator IRP-2 were strongly decreased. IRP-2 itself could be shown to be a direct
target gene of Stat5 (see Figure).
ad 3 – Experiments with e.g. erythroblasts and fibroblasts synchronized in the cell cycle by centrifugal elutriation provided evidence that vertebrate
cells respond to cell size alterations (induced
through different growth factor signaling or DNA
synthesis inhibitors) by compensatory shortening
of the subsequent G1 phase. This suggests an active
size-threshold mechanism in G1 to re-adjust cellcycle duration in the next cycle, ensuring maintenance of a proper balance between growth and
proliferation rates.
2 – Cell-type specific features in
the regulation of iron metabolism during erythropoiesis and
3 – Cell size control. This topic
was originally triggered by the
decrease in cell volume accompanying erythroid maturation.
ad 1 – Erythropoiesis depends on
signaling through the EpoRJak2-Stat5 axis, regulating proliferation, differentiation and
survival. While Jak2 and EpoR
deficiency are embryonic lethal,
the exact role of Stat5 had remained puzzling, since the original Stat5-ko mice had carried
a hypomorphic Stat5 allele
Reduced iron uptake into Stat5-/- erythroblasts. Cells display decreased Irp-2 expression (left; Western blot and quantification) due to functional Stat5 binding
sites in the Irp-2 promoter (right; promoter architecture and reporter assay) which
in turn down-modulates levels of TfR-1 protein and thus iron import.
Kerenyi MA et al. (2008). Stat5 regulates cellular iron uptake of erythroid cells via IRP-2 and TfR-1. Blood 112, 387888. ”Doc-Award 2009” from the University of Vienna; “Promotion Award of the City of Vienna 2009” (Förderungspreis der Stadt Wien); featured by the Austrian daily newspaper "Die Presse" n Kerenyi MA and Müllner EW (2009).
Muscle iron in stress erythropoiesis? BLOOD 113, 6507-8. n Friedbichler K et al. (2010). Stat5a serine 725 and 779
phosphorylation is a prerequisite for hematopoietic transformation. BLOOD 116, 1548-58. ”Doc-Award 2011” from
the University of Vienna
52
R E S E A R C H
G R O U P S
JOHANNES NIMPF
ApoER2 and VLDL Receptor
We study the biology of LDL receptor
related proteins (VLDL receptor and
ApoER2), a group of cell surface
receptors which mediate transport
of macromolecules across cell
membranes and play important
roles in signal transduction.
The biological systems we are working with are
the chicken oocyte and the mammalian brain. These
two systems reflect the functional dichotomy of
the receptors which function in endocytosis (follicles) and signal transduction (brain development).
The best characterized function of VLDLR in follicles
of egg laying species is endocytosis of yolk precursors into the growing oocyte.
These yolk precursors (VLDL and Vitellogenin) are
synthesized in the liver and rapidly taken up by
the growing oocyte. Recently we have started to
elucidate cell signalling functions of VLDLR and
ApoER2 in granulosa cells which support the maturation of oocytes within the follicle. In respect
to brain development both receptors act as Reelin-signal transducers. The
Reelin signal orchestrates the
correct positioning of newly
generated neurons within laminated structures of the
brain. In the development of
the olfactory system in rodents the structure of olfactory bulb depends on neurons generated throughout life
in the subventricular zone.
Model of the intracellular fates of ApoER2 and VLDLR upon Reelin stimulation. Upon
binding of Reelin, both ApoER2 and VLDLR mediate phosphorylation of Dab1 (1).
VLDLR internalizes Reelin rapidly via Clathrin-mediated endocytosis (2) and is separated from Reelin in the compartment of uncoupling of receptor and ligand (3). VLDLR
then recycles back to the plasma membrane (4) while Reelin is delivered to the lysosome for degradation (5). ApoER2 internalizes Reelin via the same pathway although
the receptor originally resides in lipid rafts and endocytoses its ligand with a much
slower rate. In contrast to VLDLR, ApoER2 is not recycled but ends up in the lysosome
together with Reelin (6). As an additional feedback mechanism, Reelin stimulation
induces secretase-mediated cleavage of ApoER2, thereby generating a soluble extracellular fragment (8). This fragment can, together with another N-terminal fragment produced from an ApoER2 isoform by furin cleavage (9), inhibit the Reelin signal
by sequestering free Reelin in the cell’s surrounding. The function of the soluble intracellular domain of ApoER2 is not well understood yet.
These neurons migrate via the
rostral migratory stream towards the olfactory bulb. This
migration also depends on the
presence of ApoER2 and
VLDLR but seems to be independent on Reelin. To this end
we have characterized thrombospondin-1 as novel ligand
for ApoEr2 and VLDLR present
in the rostral migratory
stream.
Currently we are focusing on
the search for other alternative ligands for the receptors
which are involved in orchestrating the migration of neuroblasts and/or which are involved in maturation of the
follicle.
Duit S, Mayer H, Blake S.M, Schneider W.J, Nimpf J (2010). Differential functions of ApoER2 and Very low density lipoprotein receptor in reelin signaling depend on differential sorting of the receptors. J.Biol.Chem. 285, 4896-4908
n Blake M.S, Strasser V, Andrade N, Duit S, Horbauer R, Schneider W.J, Nimpf J (2008). Thrombospondin-1 binds to ApoER2
and VLDL receptor and functions in postnatal neuronal migration. EMBO J. 27, 3069-80 n Andrade N, Komnenovic V,
Blak S, Jossin Y, Howell B, Goffinet A, Schneider W.J, Nimpf J (2007). ApoER2/VLDL receptor and Dab1 in the rostral migratory stream function in postnatal migration independently of Reelin. Proc. Natl. Acad. Sci. 104, 8508-8513
53
Johannes Nimpf
TEAM
Christine Ehresheim
Christian Leeb
R E S E A R C H
G R O U P S
EGON OGRIS
PP2A enzyme biogenesis and monoclonal antibodies
Cells employ the
phosphorylation/dephosphorylation
of proteins to regulate protein
function.
Egon Ogris
TEAM
Hibbah Auf
Bhumika Bhatt
Ingrid Frohner
Sonja Kuderer
Thomas Kupka
Ingrid Mudrak
Stefan Schüchner
Claudia Stanzel
Gerald Zwinger
The enzymes catalyzing these reactions, the protein
kinases and phosphatases, are thus important regulators of almost all aspects of life. One of the
major phosphatase enzymes in the cell is protein
phosphatase 2A (PP2A), a known tumor suppressor
and target of cancer causing viruses. PP2A is a family of protein serine/threonine phosphatases with
prototypical multisubunit architecture, in which a
catalytic subunit achieves substrate specificity
through the interaction with regulatory subunits.
The PP2A family consists of over 70 different holoenzymes that possess probably many hundred
substrates in a cell. How holoenzyme assembly is
regulated and what the substrates of different holoenzymes are, is largely unknown. A pathological
decrease of PP2A activity has been linked to the
development of human diseases such as cancer or
Alzheimer. Thus, our major research goals are to
understand the molecular mechanisms of PP2A regulation and to identify the substrates and processes regulated by PP2A.
Our study of PP2A biogenesis in yeast led to a model, in which the generation of the active enzyme
is tightly coupled to the assembly of substratespecific holoenzymes (Hombauer et al., 2007 PLoS
Biology).
Model of PP2A biogenesis in yeast: PP2A phosphatase activator, RRD/PTPA, scaffolding A subunit,
regulatory B subunit, catalytic C subunit, PP2A
methyltransferase PPM1
This process is under surveillance of the PP2A methylesterase, PPE1, which
seems to control the correct
order of the PP2A biogenesis cascade. How PPE1
exerts its surveillance function and how PP2A biogenesis is regulated and by
which signaling pathways,
is currently investigated in
the lab.
PP2A activity is decreased in Alzheimer brain tissue
suggesting a potential causal role for PP2A in Alzheimer pathogenesis (Sontag et al., 2010). We are
investigating in collaboration with Estelle Sontag
(University of Newcastle, Australia) whether dysfunction of PP2A biogenesis might be involved in
Alzheimer disease development.
PP2A substrate identification is difficult with the
currently available methods due to the transient
enzyme-substrate interaction during catalysis. We
have adapted a novel two-hybrid system for the
detection of transient PP2A interactions and are
using this method for PP2A substrate validation
and identification.
Immunofluorescence of laminopathy nucleus with a general
anti Lamin A/C antibody (red) and the point-mutant specific
anti R453W antibody (green). Fluorescence intensities of the
lamin A/C stainings are shown below the respective images.
The second more business-oriented focus of the
lab is the generation of monoclonal antibodies
against human disease-linked point-mutant proteins. We show that antibodies with such exquisite
specificity represent a new type of research tools
for the analysis of disease mechanism (Roblek et
al.,2010 PloS ONE). In 2009 our lab established the
MFPL Monoclonal Antibody Facility and since then
generated custom-tailored monoclonal antibodies
for internal and external customers (please see report of Monoclonal Antibody Facility).
Roblek, M., Schuchner, S., Huber, V., Ollram, K., Vlcek-Vesely, S., Foisner, R., Wehnert, M., and Ogris, E. (2010). Monoclonal
antibodies specific for disease-associated point-mutants: lamin A/C R453W and R482W. PLoS One 5, e10604. n
Sontag, J.M., Nunbhakdi-Craig, V., Mitterhuber, M., Ogris, E., and Sontag, E. (2010). Regulation of protein phosphatase
2A methylation by LCMT1 and PME-1 plays a critical role in differentiation of neuroblastoma cells. J Neurochem
115, 1455-1465 n Hombauer, H., Weismann, D., Mudrak, I., Stanzel, C., Fellner, T., Lackner, D.H., and Ogris, E. (2007). Generation of active protein phosphatase 2A is coupled to holoenzyme assembly. PLoS Biol 5, e155.
54
R E S E A R C H
G R O U P S
BRIGITTE POPPENBERGER
Regulation of plant steroid hormone homeostasis
Plants have evolved sophisticated
mechanisms which enable them to
adapt rapidly to the constant
changes in their environment.
Plant hormones, a group of organic substances
that function as signalling molecules and influence
physiological processes at low concentrations, play
a crucial role in regulating the growth and development of plants.
One class of plant hormones are the brassinosteroids (BRs). BRs are polyhydroxylated sterol derivatives, structurally similar to cholesterol-derived
animal steroid hormones and ecdysteroids from in-
sects. The BRs function in cell elongation, cell division and differentiation and have been particularly studied in relation to processes such as germination, development in the light and dark,
senescence, vascular development and abiotic and
biotic stress responses. Like their animal steroid
counterparts BRs are highly bioactive substances;
to maintain balanced BR levels - a homeostasis is a prerequisite for normal growth.
We are investigating the mechanisms that regulate
BR cellular homeostasis in Arabidopsis thaliana.
On the one hand we aim to elucidate the contribution of catabolic inactivation (glucosylation) to
the regulation of BR bioactivity and in this context
are analyzing the role of the UDP-glycosyltransferases UGT73C5 and UGT73C6 in regulating BR activity. On the other
hand we study factors that control
BR biosynthesis. Recently we have
identified the novel basic helixloop-helix transcription factor
CESTA (CES), that acts as an essential positive regulator of BR biosynthetic gene expression and other BR
responses. CES directly binds to the
promoters of BR biosynthesis genes.
Also we have evidence that CES is
phosphorylated by a GSK3 shaggylike kinase, BIN2, that act as negative regulators of BR signaling and
are currently investigating the physiological significance of this phophorylation event in vivo.
(A) BR cellular homeostasis is believed to be regulated either by adapting BR
biosynthesis (via a feedback regulatory loop) and/or by initiating BR inactivation events. (B) Defects in BR homeostasis have severe effects on plant development. Adult plants of Arabidopsis thaliana that either over-accumulate
BRs (in the ces-D mutant) or are deficient in BRs (due to increased BR glucosylation) are shown.
In addition we are also very interested in understanding which signalling pathways cross-talk with
those controlling BR homeostasis.
In particular we are investigating
the influence of external stimuli
such as light and cold on BR responses.
Husar S, Berthiller F, Fujioka S, Rozhon W, Khan M, Kalaivanan F, Elias L, Higgings GS, Li Y, Schuhmacher R, Krska R, Seto H,
Vaistij FE, Bowles D and Poppenberger B (2011). Overexpression of UGT73C6 alters brassinosteroid glucoside formation
in Arabidopsis thaliana. BMC Plant Biol. 11: 51. n Poppenberger B, Rozhon W, Khan M, Husar S, Adam G, Luschnig C, Fujioka
S and Sieberer T (2011) CESTA a positive regulator of brassinosteroid biosynthesis. EMBO J., 30 (6), 1149-61. n Poppenberger B, Fujioka S, Sueno K, George GL, Vaistij FE, Seto H, Hiranuma S, Takastuto S, Adam G, Yoshida S and Bowles D (2005)
The UGT73C5 of Arabidopsis thaliana glucosylates brassinosteroids. Proc.Nat.Acad.Sci. USA., 102 (42), 15253-15258.
55
Brigitte Poppenberger
TEAM
Sigrid Husar
Florian Kalaivanan
Mamoona Khan
Renata Milcevicova
Elisabeth Piehslinger
Theresa Ringwald
Rozhon Wilfried
R E S E A R C H
G R O U P S
RAINER PROHASKA
Stomatin, membrane microdomains and neuroacanthocytosis
Rainer Prohaska
TEAM
Stefanie Rungaldier
Claudia Roos
Membrane microdomains or "lipid
rafts", lateral complexes of distinct
proteins and lipids, are implicated in
a variety of functions like signal
transduction, vesicle trafficking,
sorting of certain proteins and
lipids, and cell polarity.
cholesterol in stomatin-GLUT1 complex formation.
For our investigations we are using biochemical,
molecular and cell biological methods, complemented by collaborative studies together with partners from the LIMES Institute, Univ. Bonn, the Institute of Biophysics, JKU Linz, and the IGMM, Univ.
Montpellier.
European Multidisciplinary Initiative on Neuroacanthocytosis (EMINA)
We investigate the functions of oligomeric membrane proteins that are typical lipid raft markers,
the stomatins and flotillins, particularly in the context of cholesterol trafficking, membrane scaffolding and vesiculation. In a second project, we study
red blood cell membrane microdomains in the context of cholesterol availability and associated morphological changes, as presented in the form of
acanthocytes in the disease neuroacanthocytosis.
An ERA-NET/E-RARE network coordinated by Prof.
Adrian Danek (LMU, Munich) comprises six partner
organizations in Germany, Austria, the Netherlands,
France, and Turkey, to take a significant step forward in both basic research and applied clinical
research into the neuroacanthocytosis (NA) syndromes. These syndromes are a group of rare neurological illnesses, which affect mostly young
adults. NA is associated with neurodegeneration
Stomatin-specific microdomains
in the brain, similar to Huntington’s Disease, but
can be differentiated by the presence of spiky eryStomatin is an oligomeric, cholesterol-binding, lipid
throcytes (acanthocytes) in the blood. Several canraft-associated, integral membrane protein that is
didate genes for various NA forms have been idenlocalized to the cytoplasmic side of the plasma
tified: VPS13A (vacuolar protein sorting 13A) for
membrane and late endosomes of many cell types.
chorea-acanthocytosis (ChAc), XK (Kell system) for
We are studying the structure and function of stoMcLeod Syndrome (MLS), JPH3 (junctophilin 3) for
matin focusing on oligomerization, cholesterolHuntington’s Disease-Like 2 (HDL2), and PANK2
binding, lipid raft-association, and interactions with
(pantothenate kinase 2) for Pantothenate Kinaseother membrane proteins, such as the glucose
Associated Neurodegeneration (PKAN). Our group
transporter GLUT1. Moreover, we study the role of
participates in EMINA basic research by focusing
on the identification of
acanthocyte membrane
domains that differ in
composition from the normal, discocyte membrane.
We hypothesize that the
VPS13A defect will affect
vesicle sorting and autophagy in late stage erythropoiesis and thus lead
to changes during reticulocyte membrane reorganiStomatin is localized to recycling and late endosomes. Wild type stomatin-GFP (green)
zation. Similar sorting delocalizes, like endogenous stomatin, to the plasma membrane and perinuclear vesicles.
fects may affect autophagy
Co-localization with (A) the recycling endosome marker Rab11 (red) and (B) the late
in neurons leading to acendosomal /lysosomal marker NPC1 (red), respectively, verifies this statement. Nuclei
cumulation of protein agwere stained with DAPI (blue).
gregates and cell death.
Mairhofer M, Steiner M, Salzer U and Prohaska R (2009). Stomatin-like protein-1 interacts with stomatin and is targeted to late endosomes. J Biol Chem, 284(42), 29218-29. n Montel-Hagen A, Kinet S, Manel N, Mongellaz C, Prohaska
R, Battini JL, Delaunay J, Sitbon M and Taylor N (2008). Erythrocyte Glut1 triggers dehydroascorbic acid uptake in
mammals unable to synthesize vitamin C. Cell, 132(6), 1039-48. n Umlauf E, Mairhofer M and Prohaska R (2006). Characterization of the stomatin domain involved in homo-oligomerization and lipid raft association. J Biol Chem,
281(33), 23349-56.
56
R E S E A R C H
G R O U P S
FRITZ PROPST
The neuronal cytoskeleton in axon guidance
Axon extension, axon branching, and
axon retraction are major morpho logical changes that neurons have
to execute to accomplish correct
wiring of the nervous system
during development and during
regeneration after injury.
These transformations are guided by extracellular
signals which ultimately need to be translated into
the rearrangement of the neuronal cytoskeleton.
We study signaling mechanisms and posttranslational modifications of microtubule-associated proteins and other components of the cytoskeleton
that regulate the orchestrated reorganization of
microtubules and actin in response to extracellular
signals. Our approach combines gene ablation in
the mouse with cell biological and molecular analyses in cultured neurons and other primary cells.
One focus of our research is the role of microtubule-associated proteins of the MAP1 family. In a
recent study we found that MAP1B is necessary
for nitric oxide signaling to the cytoskeleton in
axon retraction. Post-translational modification of
MAP1B by S-nitrosylation changes its interaction
with microtubules. Thus, we showed that MAP1B
is a component of a pathway that links calcium
influx and activation of neuronal nitric oxide synthase to reconfiguration of axonal microtubules
and might contribute to the physiological and pathological effects of nitric oxide in the brain. We
have since expanded our investigation towards the
role of S-nitrosylation of tubulin in neuronal morphogenesis. Another current topic is the role of
MAP1B in repulsive axon guidance in the brain.
We have obtained evidence that MAP1B is essential
for signal transduction of several unrelated repulsive axon guidance cues.
We have also analyzed the functional properties
of other MAP1 proteins and found that the light
chains of these proteins determine to some extent
their functional characteristics. Moreover, we characterized a novel member of the MAP1 family,
which we termed MAP1S. MAP1S is expressed not
only in the brain, but also in a wide range of other
tissues and represents the non-neuronal counterpart of MAP1A and MAP1B. We have generated
MAP1S deficient mice and are exploring the role
of this protein in cell division, cell migration, and
tumorigenesis.
MAP1B is essential for nitric oxide-induced axon retraction. a) Cultured neurons from adult wild-type mice
(dorsal root ganglion) were treated with the nitric oxide
donor SNAP, fixed, stained for tubulin and analyzed by
confocal fluorescence microscopy. Cellular morphology
was scored as unchanged (left) or retracted (right). b) Cultured neurons from adult MAP1B+/+ or MAP1B-/- mice
were treated with SNAP for 1 h and processed as above.
Microtubule configuration was classified as unchanged
(compared to untreated cells) or displaying retraction hallmarks (sinusoidal bends along the axon, a trailing remnant, and a retraction bulb). MAP1B-/- neurons displayed
a severely reduced capacity to respond to SNAP by axon
retraction.
Stroissnigg H*, Tranc̀´íková A*, Descovich L, Fuhrmann J, Kutschera W, Kostan J, Meixner A, Nothias F and Propst F (2007).
S-nitrosylation of microtubule-associated protein 1B mediates nitric oxide induced axon retraction. Nat Cell Biol,
9, 1035-45. *Equal contribution n Noiges R, Eichinger R, Kutschera W, Fischer I, Németh Z, Wiche G and Propst F (2002).
MAP1A and MAP1B: light chains determine distinct functional properties. J Neurosci, 22, 2106-14. n Orbán-Németh
Z, Simader H, Badurek S, Trančiková A and Propst F ( 2005). Microtubule-associated protein 1S, a short and ubiquitously
expressed member of the microtubule-associated protein 1 family. J Biol Chem, 280, 2257-65.
57
Fritz Propst
TEAM
Michael Ebner
Anton Kamnev
Waltraud Kutschera
Rajeshwari Meli
Zsuzsanna Orban-Nemeth
R E S E A R C H
G R O U P S
FLORIAN RAIBLE
Origin and Diversification of Hormone Systems
We are interested in the evolution of
hormone systems. Central to our
work is the exploration of a novel
invertebrate model system,
Platynereis dumerilii.
Florian Raible
TEAM
Benjamin Backfisch
Mingliu Du
Stefan Hammer
Claudia Lohs
Fiorella Schischlik
These experiments are supported by an ERC starting
grant (HOR.MOON) we have recently attracted, and
provide us with first molecular insight into the
enigmatic hormone machinery underlying lunar reproductive periodicity.
Our past work has shown that this marine worm
exhibits a unique combination of ancestral-type
genomic characteristics not found in insect and
nematode model species. Moreover, we have identified numerous components of ancestral-type
hormone pathways in Platynereis. Therefore,
Platynereis is highly interesting for comparison
with the vertebrate hormone system, and for our
understanding of marine life.
The hormonal control of reproduction
and regeneration
What could be the function of ancestral-type hormones in Platynereis? One of the systems that we
aim to dissect is the hormonal machinery orchestrating reproduction and regeneration. Platynereis
is an excellent object for this analysis, as it has
been a central model for the link between chronobiology and reproduction. Our molecular analyses
have identified a spectrum of conserved hormones
in Platynereis. Thanks to the establishment of new
molecular tools, we are now able to systematically
assess the impact of these candidates on the development and maturation of the animals.
Hormonal orchestration of regeneration and reproduction by the medial Platynereis
brain. (A) Classical transplantation studies revealed that immature Platynereis
heads are the source for an endocrine brain hormone inhibiting maturation and
supporting regeneration. (B) Implantations of small brain fragments (blue) map
the source of the brain hormone to the medial Platynereis brain. The new molecular
tools established in the lab allow us for the first time to identify and study the cellular circuits and hormonal cues responsible for these functions.
Ancestral-type hormones in a simple invertebrate. Individual hormone-producing cells are visualized (green color)
in an adult Platynereis brain. Whereas cell bodies (“mc”)
localize to the medial brain, neuronal projections (np) project into the region of the infracerebral gland, an annelid
neurohemal organ.
Exploring a new marine model system
Over the past years, Platynereis has emerged as a
very promising “next-generation” model system.
We have pioneered transgenic technology in
Platynereis that allows us to mark and interrogate
cell types with unprecedented precision. Moreover,
we explore transcriptomic technology and functional assays to dissect the logic of hormone regulation and action in the animal. Finally, we make use
of the remarkable transparency of Platynereis to
observe cells and molecules in action.
These approaches provide entry points into the fascinating biology of a new marine model species.
Besides the action of hormones, we are actively
investigating the evolution of gene-regulatory logic
and the orchestration of cellular processes involved
in the sculpting of bristles. Our vision is to firmly
establish Platynereis as a reference species for marine biology.
Tessmar-Raible K, Raible F, Arboleda E (2011). Another place, another timer: Marine species and the rhythms of life.
Bioessays. 33(3):165-172. n Christodoulou F, Raible F, Tomer R, Simakov O, Trachana K, Klaus S, Snyman H, Hannon GJ,
Bork P, Arendt D. (2010). Ancient animal microRNAs and the evolution of tissue identity. Nature. 463(7284):10841088. n Raible F, Tessmar-Raible K, Osoegawa K, Wincker P, Jubin C, Balavoine G, Ferrier D, Benes V, de Jong P, Weissenbach
J, Bork P, Arendt D. (2005). Vertebrate-type intron-rich genes in the marine annelid Platynereis dumerilii. Science.
310(5752):1325-1326.
58
R E S E A R C H
G R O U P S
HANS ROTHENEDER
Cell cycle regulation and DNA damage response
My laboratory is focused on the
mechanisms controlling growth
and cell cycle of the mammalian
cell. They respond to perturbations
like replication errors or DNA
damage by inducing cell cycle
arrest, senescence, or apoptosis.
Dysfunction of these mechanisms often results in
the malignant transformation of a cell and the development of cancer. E2F is a family of transcription
factors that integrate cell-cycle progression with
transcription through cyclical interactions with important cell cycle regulators. We have recently
identified and characterized a protein that we
called EAPP (E2F Associated PhosphoProtein). EAPP
interacts with E2F1-3, comprising the activator
group of E2F proteins, and modulates E2F-dependent transcription. Tumour cells often overexpress
EAPP, indicating that it confers a selective advantage to these cells. EAPP levels increase upon DNA
damage and higher EAPP levels seem to protect
cells from apoptosis. This protection can also be
achieved by ectopic expression of EAPP and correlates with an increased number of cells in G1 phase
and an upregulation of p21. Increased p21 inhibits
cyclin/cdk activity which is required for cell cycle
progression, but has also interferes with apoptosis.
The RNAi-mediated knock down of p21 reduces
the anti-apoptotic activities of overexpressed EAPP.
This suggests that p21 at least in part mediates
this activity of EAPP.
EAPP stimulates p21 expression by binding to its
promoter and seems to be required for the assembly
of the transcription initiation. The knock down of
EAPP facilitates apoptosis and goes along with reduced p21.
Our findings suggest that EAPP is indispensable
for the survival of a cell. The required amount of
EAPP seems to depend on the environmental conditions. Preliminary evidence suggests that the role
of EAPP in transcription is not limited to the p21
promoter. Active promoters are occupied by multiple types of complexes and EAPP seems to be an
important component of at least some of them.
Lowering EAPP levels influences the expression of
some of the genes examined including important
cell-cycle regulators. To examine which genes are
influenced by EAPP and to scrutinize the biochemical
details of its activity will be focus of future.
A model showing three different scenarios with elevated, normal and reduced levels of EAPP
Andorfer, P., and Rotheneder, H (2011). EAPP: Gatekeeper at the crossroad of apoptosis and p21-mediated cell-cycle
arrest. Oncogene (Jan. 24. epub ahead of print). n Schwarzmayr, L., Andorfer, P., Novy, M., and Rotheneder, H. (2008).
Regulation of the E2F-associated phosphoprotein promoter by GC-box binding proteins. Int J Biochem Cell Biol 40,
2845-2853. n Novy, M., Pohn, R., Andorfer, P., Novy-Weiland, T., Galos, B., Schwarzmayr, L., and Rotheneder, H. (2005).
EAPP, a Novel E2F Binding Protein That Modulates E2F-dependent Transcription. Mol Biol Cell 16, 2181-2190.
59
Hans Rotheneder
TEAM
Peter Andorfer
Nazanin Najafi
R E S E A R C H
G R O U P S
PETER SCHLÖGELHOFER
Meiotic Recombination
We focus our research on meiotic
recombination, mainly working with
the model plant Arabidopsis thaliana
and to some extend with the yeast
Saccharomyces cerevisiae.
Peter Schlögelhofer
TEAM
Bernd Edlinger
Mona von Harder
Manuel Hofer
Fritz Hunger
Michael Janisiw
Edith Kolleger
Marie-Therese Kurzbauer
Clemens Uanschou
Our research efforts are well embedded in the Department of Chromosome Biology with five other
groups performing meiosis research in various organisms. Meiosis is a specialised, two-step cell
division that ensures the reduction of the genome
prior to the formation of generative cells. During
meiosis, homologous centromeres are segregated
during the first, and sister centromers during the
second division. As there is no intervening DNA
replication between the two meiotic divisions,
each of the final division products contains only
half of the initial DNA content. For a given diploid
organism the developing generative cells are then
haploid. It is important to note, that during meiosis, genetic information between maternal and
paternal chromosomes is mutually exchanged,
leading to novel combinations of genetic traits in
the following generation.
Two genetically diverse generative cells fuse during
the process of fertilization, re-establish the speciesspecific original genome content and constitute
an individual with a unique genetic set-up.
Novel combinations between parts of paternal and
maternal chromosomes are generated through the
process of homologous recombination (HR). A prerequisite for HR are DNA double strand breaks
(DSBs), generated by a protein complex with the
conserved protein SPO11 being its catalytically
active subunit. DSBs are formed at non-random
sites throughout the genome, known as hot spots
of meiotic recombination.
We are interested in 1) cis and trans acting factors
that mediate meiotic DSB formation, 2) mechanisms of meiotic DSB processing, 3) the biochemical
details of subsequent DSB repair and 4) the coordination of all these events. We use a broad range
of techniques (molecular biology, cytology, biochemistry and genetics) and take advantage of the
on-site facilities (Bio-optics, deep-sequencing,
mass-spectrometry, bioinformatics).
Figure 1: The panel shows a preparation of meiotic chromosomes isolated from meiocytes of a mutant Arabidopsis
plant. The depicted stage of meiosis is called “pachytene”
with all five chromosome pairs in close alignment, stabilized by a protein complex known as the “synaptonemal
complex” (SC). To visualise the DNA and associated proteins a specific DNA dye (DAPI) and antibodies (coupled to
fluorescent molecules) specifically detecting certain meiotic proteins have been applied. The DNA is stained in blue,
a protein of the SC is stained in green, and a DNA repair
protein is stained in red.
Edlinger B, Schlögelhofer P (2011). Have a break: determinants of meiotic DNA double strand break (DSB) formation
and processing in plants. J Exp Bot. 2011 Mar;62(5):1545-63. n Kurzbauer MT, Schlögelhofer P (2011). Retinoblastoma
protein goes green: the role of RBR in Arabidopsis meiosis. EMBO J. 2011 Feb 16;30(4):631-3. n Uanschou C, Siwiec
T, Pedrosa-Harand A, Kerzendorfer C, Sanchez-Moran E, Novatchkova M, Akimcheva S, Woglar A, Klein F, Schlögelhofer P
(2007). A novel plant gene essential for meiosis is related to the human CtIP and the yeast COM1/SAE2 gene. EMBO
J. 2007 Dec 12;26(24):5061-70.
60
R E S E A R C H
G R O U P S
WOLFGANG SCHNEIDER
Molecular Mechanisms of Dyslipidemias and Atherogenesis
The work of my group focuses on
receptor-mediated endocytic
processes.
In detail, we investigate molecular genetic, cell
biological, and biochemical details of (i) the low
density lipoprotein (LDL) receptor gene family in
oocyte growth and embryo development, and of
the receptor LR11 in smooth muscle cell biology,
(ii) avian lipases and transfer proteins (i.e., the lipolytic proteome) of the granulosa cells surrounding the chicken oocyte as well as of the extraembryonic yolk sac, which mediate yolk lipid
deposition and subsequent utilization by the embryo, respectively, (iii) the molecular genetic basis
for human atherosclerosis caused by single-gene
mutations that reduce or abolish receptor-mediated
transport of lipoproteins and/or cholesterol, and
(iv) the role of the recently discovered apolipoprotein, apo-AV, in the etiology of human pathological
hypertriglyceridemia.
In regards to (i), we have elucidated
the role of the LDLR family member
LR11 in Ang-II stimulated vascular
smooth muscle cell migration. A circulating soluble form of LR11, sLR11,
is a novel marker of carotid IMT (intima-media thickness), and targeted
disruption of the LR11 gene greatly
reduces intimal thickening of arteries
through attenuation of AngII-induced migration of SMCs (Ref. 1).
In the projects (ii) we have shown,
for the first time in any system, that
hepatic arylacetamide deacetylase
(AADA), like the key lipase ATGL, is
upregulated by fasting, and that its
affinity for insoluble carboxylester
substrates is compatible with an in
vivo function similar to that of ATGL.
Unknown heretofore, hepatic expression
of chicken AADA is estrogen-responsive, and is induced to the same degree as the stimulation of
hepatic VLDL-production by estrogen. These observations support the hypothesis that chicken
ATGL, PNPLA3, and AADA play roles in acylglycerol
metabolism related to the high rates of VLDL synthesis essential for reproduction (Ref. 2). We have
characterized patatin-like phospholipases, and revealed their unique tissue distribution patterns in
the laying hen.
Wolfgang Schneider
TEAM
In project (iv), we have achieved the first molecular
and functional characterization of a nonmammalian
ApoA-V, and have described novel mechanism for
modulation of triglyceride levels by ApoA-V proposed based on discovery that the apo binds to LRs.
Finally, we have established that a new chondroitin
sulfate-modified collagen forms a follicular membrane which to date has been assumed to be a
bona-fide basement membrane (Ref. 3).
VLDL particles in coated structures of oocytes. The electron micropraph
shows serum-derived lipoprotein particles (VLDL) in clathrin-coated pits
(c.p.) being internalized via invagination and pinching-off of coated vesicles
(c.v.) in a chicken oocyte. The receptor gene family involved has been extensively characterized in my group. Adapted from M.M. Perry and A.B. Gilbert, J. Cell Sci. 39:257, 1979.
Jiang M, Bujo H, Ohwaki K, Unoki H, Yamazaki H, Kanaki T, Shibasaki M, Azuma K, Harigaya K, Schneider WJ, Saito Y (2008).
Ang II-stimulated migration of vascular smooth muscle cells is dependent on LR11 in mice. J Clin Invest. 118(8):273346. n Riegler B, C Besenboeck, R Bauer, J Nimpf and WJ Schneider (2011). Enzymes involved in hepatic acylglycerol
metabolism in the chicken. Biochem. Biophys. Res. Commun., 406:257-61. n Schneider WJ (2009). Receptor-mediated
mechanisms in ovarian follicle and oocyte development. Gen. Comp. Endocrinol. 163: 18 – 23, 2009.
61
Raimund Bauer
Mary-Rose Espina
Yasmin Gravogl
Barbara Riegler
Tanja Strini
Fan Zhang (MCW Dipl. Student)
R E S E A R C H
G R O U P S
RENÉE SCHROEDER
RNA Aptamers and RNA Chaperones
Renée Schroeder
RNA is at the center of all steps of
gene expression. Cells can be defined
by their transcriptomes, not by
their genomes. We are interested
in discovering many regulatory
elements that are part of the RNA
regulon and in identifying their interacting partners and their targets.
human genomes. These experiments delivered thousands of genomic RNA aptamers that regulate gene
expression. We are currently analyzing the mode
of action of these aptamers. Another focus in our
laboratory deals with proteins that promote RNA
folding: RNA chaperones.
As model examples we are analyzing the mode of
action of the E. coli protein StpA and the HIV-1
Tat peptide. While StpA promotes RNA annealing
TEAM
Lucia Aronica
Ivana Bilusic
Johanna Bisich
Jennifer Boots
Martina Dötsch
Ece Ergir
Boris Fürtig
Thomas Gstrein
Megahn Lybecker
Katarzyba Matylla Kulinska
Nadia Tukhtubaeva
Adam Weiss
Robert Zimmermann
To achieve this goal we
adapted the classical SELEX
procedure to be used in
combination with genome
sequences and deep sequencing. Genomic systematic evolution of ligands
by exponential enrichment
(SELEX) allows the isolation
of protein binding RNAs independently of computational predictions and expression conditions.
The HIV-1 Tar RNA hairpin undergoes refolding during transcription of the HIV genome. The refolding is promoted by the tat peptide.v
We used genomic SELEX with an E. coli library to
isolate RNA aptamers against RNA polymerase and
the regulator protein Hfq. We further selected RNA
polymerase II binding aptamers from the yeast and
and strand exchange, HIV-1 Tat only promotes RNA
annealing. Using biochemical and biophysical methods (NMR) we study the structural dynamics of
both RNA and protein.
Overview of SELEX procedure. A genomic RNA library derived from DNA is submitted to several rounds of selection (b-e)
and amplification (f-g,a) until a pool enriched in desired sequences is obtained. Deep sequencing of enriched pools allows
the annotation of genomic aptamers to analysed genomes.
LORENZ, C. et al. (2010). Genomic SELEX for Hfq-binding RNAs identifies genomic aptamers predominantly in antisense transcripts. Nucleic Acids Res, 38, 3794-3808. n ZIMMERMANN, B. et al. (2010). Monitoring Genomic Sequences
During SELEX Using High-Throughput Sequencing: Neutral SELEX. PlosOne, 5, e9169. n DOESTSCH, M. et al. (2011).
The RNA annealing mechanism of the HIV-1 Tat peptide: conversion of the RNA into an annealing-competent
conformation. Nucleic Acids Res. Feb 4.
62
R E S E A R C H
G R O U P S
CHRISTOPH SCHÜLLER
Demands on transcription in response to environmental stress
The cellular DNA contains all the
information required to build and
maintain an organism. Changes in
the environment of a cell demand
changes in gene expression.
This is a complicated process involving several layers of gathering and mediating information and a
number of factors (both RNA and Protein) interacting directly or indirectly with DNA. Our interest
is to understand how signals reach the genomic
DNA and lead to controlled interpretation of the
stored information. How is expression of genes under environmental or stress control regulated? This
complex problem is solved by the cell by the concerted action of chromatin remodeling complexes,
RNA polymerase and transcription factors.
The simple unicellular eukaryote Saccharomyces
cerevisiae (also called Baker´s yeast) has been a
perfect organism to decipher many fundamental
principles of the interaction of these components.
In yeast, powerful systematic genetic and chemogenomic methods have been developed and being
used by us to follow the path of information from
the environment to the genome. An especially fascinating aspect is their rapid and dramatic response
demanding special adjustment of genen transcription.
We are also investigating the environmental response of the human fungal pathogen Candida
glabrata. This yeast-like organism is adapted to
the environment of a mammalian host which is
different in many aspects to the environment
S. cerevisiae is adapted to. However, C. glabrata
uses the same gene products and mechanisms to
survive inside the mammalian body.
We are investigating the mechanisms how these
fungal cells adapt to the activity of innate immune
cells respond to their attack and manage to survive
as successful commensals. We integrate a number
of methods to determine the strategy of these
simple cells to adapt to their environment.
Systematic high throughput analysis with yeast.
Left: Geneexpression Microarrays measure gene expression
of all 6200 yeast genes. Right: Microscopic analysis of
individual yeast cells. Background: Systematic phenotypic
analysis with 4700 yeast gene deletion strains.
Klopf E et al. (2009). Cooperation between the INO80 complex and histone chaperones determines adaptation of
stress gene transcription in the yeast S. cerevisiae. Mol Cell Biol. Sep;29(18):4994-5007. n Roetzer A et al. ( 2010).
Autophagy supports Candida glabrata survival during phagocytosis. Cell. Microbiol. Feb;12(2):199-216. n Hosiner D
et al. (2009). Arsenic toxicity to Saccharomyces cerevisiae is a consequence of inhibition of the TORC1 kinase combined with a chronic stress response. Mol Biol Cell. Feb;20(3):1048-57.
63
Christoph Schüller
TEAM
Christine Grasschopf
Dagmar Hosiner
Zejlka Jandric
Eva Klopf
Gerhard Niederacher
R E S E A R C H
G R O U P S
JOACHIM SEIPELT
Virus Host cell Interactions
mechanisms of interactions between
viruses and host cells. Our main
interest is the biology of Human
Rhinovirus, this virus is the
causative agent of common cold.
Joachim Seipelt
Joachim Seipelt is on university
leave since July 2010 and works
at the biotech company AVIR
Green Hills Biotechnology AG.
AVIR is developing a novel
intranasal influenza vaccine
(deltaFLU).
Its relatives within the picornavirus genus are important pathogens in humans such as poliovirus or
coxsackievirus and in animals, e.g. foot and mouth
disease virus.
These small and simple viruses can – in a short timeframe – very efficiently subvert a complex eucaryotic cell into a virus producing machine. Cells try
to defend themselves against intruders, but at the
same time viruses have evolved complex strategies
to avoid cellular defense reactions. Analysis of this
interplay between host and virus can provide new
insights into both viral and cellular functions.
Here we would like to discriminate between cellmediated and virus mediated processes. We have
found significant differences in the induction of
apoptosis when analyzing different serotypes of
HRVs. These data hint on substantial biological diversity of these viruses.
Furthermore, we have identified a set of antiviral
compounds that show striking properties. We try
to identify the antiviral mechanism of these compounds in molecular detail.
Understanding the mode of action of these compounds might be used on a medium term scale to
find novel chemical molecules with improved antiviral activity against these viruses. In collaboration
with Karl Kuchler, MFPL, we have used yeast as a
model system to identify underlying mechanisms.
Within this theme we work on several topics: We
have in the past analyzed interactions of viruses
with the cytoskeleton. We have found that a viral
proteinase 2A cleaves cytokeratin 8 during virus
multiplication. Another area of interest is the onset
of cell-death (apoptosis) during virus infection.
HeLa cells (left) afer infection with human rhinovirus (right). Actin is shown in red, cytokeratin 8 in green.
Krenn BM, Gaudernak E, Holzer B, Lanke K, Van Kuppeveld FJ, Seipelt J. (2009). Antiviral Activity of Zinc Ionophores
Pyrithione and Hinokitiol against Picornaviral Infections. J. Virol. 83, (1), 58–64. n Krenn BM, Holzer B, Gaudernak E,
Triendl A, van Kuppeveld FJ, Seipelt J. (2005). Inhibition of polyprotein processing and RNA replication of human rhinovirus by pyrrolidine dithiocarbamate involves metal ions. J Virol. 79(22):13892-9. n Landstetter N, Glaser W, Gregori
C, Seipelt J, Kuchler K. (2010). Functional genomics of drug-induced ion homeostasis identifies a novel regulatory
crosstalk of iron and zinc regulons in yeast. OMICS.;14(6):651-63
64
R E S E A R C H
G R O U P S
CHRISTIAN SEISER
Chromatin modifiers in development and disease
DNA, the carrier of genetic information in our cells, is organized with
the help of histone proteins as
chromatin.
Histone modifications affect the chromatin
structure and thereby the accessibility of particular
genomic regions and are important for fundamental
biological processes such as transcription, replication and DNA repair. Our group is specifically interested in the role of histone deacetylases (HDACs)
in development and disease. HDAC inhibitor treatment of tumor cells leads to cell cycle arrest, differentiation or apoptosis. Therefore, HDACs are potential targets for anti-tumor drugs and several
HDAC inhibitors are currently tested in clinical trials. HDACs are considered as transcriptional corepressors and 18 members of the HDAC family
have been identified in mammalian cells.
We have originally identified mouse HDAC1 as
growth factor inducible in cytolytic T cells. HDAC1
gene disruption in mice leads to reduced proliferation and severe developmental problems resulting
in embryonic lethality of HDAC1 knockout mice.
One crucial function of HDAC1 in the context of
proliferation control is the repression of the CDK
inhibitor p21/WAF1 suggesting a potential role of
HDAC1 in tumorigenesis. Surprisingly, absence or
reduced expression of HDAC1 in murine or human
teratomas led to increased proliferation and reduced differentiation and was linked with a more
malignant phenotype. We are currently studying
the function of HDAC enzymes in different cell types and tissues by using conditional HDAC knockout
mice.
Christian Seiser
TEAM
Induced phosphorylation of histone H3 at serine 10 upon
stress stimulation of proliferating Swiss 3T3 fibroblasts.
Indirect immunofluorescence analysis using a histone
H3S10ph antibody; nuclear DNA was stained with DAPI
(Krahmer and Taubenschmid).
In a second project, we examine the role of histone
phosphorylation during the activation of mammalian genes by stress and growth factors. The presence of histone H3 phosphorylation marks at the
regulatory regions of several mammalian genes
correlates with the induced expression of dozens
of target genes in mammalian cells. We have recently shown that 14-3-3 zeta can act as reader
protein for S10- and S28-phosphorylated histone.
We have now identified the phosphatase PP2A as
chromatin associated transcriptional repressor, which removes the active chromatin
mark from specific target genes. In the near future, we
plan to analyze the genome
wide presence of H3 phosphorylation marks in mouse cells.
The chromatin modifier HDAC1 is a potential marker for benign teratomas. HDAC1
is expressed at high levels in differentiated benign human teratomas, while its closest homologue HDAC2 is expressed at low levels (left panel). Immature human teratomas show the reciprocal expression patterns for HDAC1 and HDAC2 (right
panel). Taken from Lagger et al. EMBO J. 2010.
Lagger S et al. (2010). Crucial Function of Histone Deacetylase 1 for Differentiation of Teratomas in Mice and Humans.
EMBO J. 29:3992-4007. n Grausenburger R*, Bilic I* et al. (2010). Conditional Deletion of Histone Deacetylase 1 in T
Cells Leads to Enhanced Airway Inflammation and Increased Th2 Cytokine Production. J Immunol. 185:3489-349. (*
equal contribution) n Simboeck E et al. (2010). A phosphorylation switch regulates the transcriptional activation of
cell cycle regulator p21 by histone deacetylase inhibitors. J Biol Chem. 285:41062-73.
65
Raphael Bitterer
Astrid Hagelkruys
Sabine Lagger
Mirjam Moser
Magdalena Rennmayr
Anna Sawicka
Mircea Winter
Gordin Zupkovitz
R E S E A R C H
G R O U P S
TOBIAS SIEBERER
Signaling networks in shoot organ formation
In contrast to animals, plants show
an indeterminate mode of development, where organ formation mainly
occurs post-embryonically and is
highly adaptive to the environment.
Tobias Sieberer
TEAM
Wenwen Huang
Christine Marizzi
Matthias Nagler
Delphine Pitorre
Olena Poretska
Karin Zwerger
The above ground organs of higher plants are generated through the activity of stem cell centres,
the so-called shoot meristems. To ensure correct
growth, the plant must tightly balance the ratio
between pluripotent stem cells and differentiating
cells, which are consumed by organ formation. This
requires constant intercellular communication
achieved by complex interactions of numerous signalling molecules. In addition to this short range
communication in the meristem plants also developed sophisticated long range signals to communicate between meristems to concert overall
growth. We are interested in the molecular nature
and function of these signals, which in some respect represent an analogous communication system to the neuronal network of animals.
Apart from gaining essential new insights in the
molecular control of plant growth the knowledge
should be applicable to improve the production of
food and renewable energy resources. Shoot system
architecture affects light harvesting potential, the
synchrony of flowering and seed set, and the number of flowers and seeds per plant. Thus, changes
in architectural characteristics have been, and continue to be central breeding targets in agriculture,
horticulture and forestry.
We pursue an highly interdisciplinary approach to
define novel pathways and mechanisms, which
control the growth rate and architecture of shoots.
Besides modern functional genomic tools to identify
and characterize genes of interest, we perform
phenotypic and reporter-based screens to find small
molecules affecting these processes. Those com-
Fig.1: Development of an interdisciplinary toolset to identify novel regulators of shoot organ formation.
pounds are then used to determine their molecular
targets in the plant by biochemical and genetic
means.
Currently we are focusing on the following projects:
1) the AMP1 pathway, which controls stem cell
pool size and leaf initiation rate. 2) the strigolactone pathway, which regulates the outgrowth
of lateral branches.
Fig. 2: Plants mutated in the AMP1
gene (B) form leaves quicker than
wild-type plants (A). We identified a
small molecule that can enhance the
leaf initiation rate in wild-type plants
(C: control plant, D: treated plant) to a
similar extend as the amp1 mutation.
(E) Plants lacking the hormone strigolactone generate more shoot branches
(right) than wild-type plants (left).
Poppenberger, B. et al. (2011). CESTA a positive regulator of brassinosteroid biosynthesis. EMBO J. 30: 1149–1161. n
Crawford, S. et al. (2010). Strigolactones enhance competition between shoot branches by dampening auxin transport.
Development 137:2905-13. n Anzola, J.M. et al. (2010). Putative Arabidopsis Transcriptional Adaptor Protein (PROPORZ1) is required to modulate histone acetylation in response to auxin. Proc Natl Acad Sci U S A 107:10308-13
66
R E S E A R C H
G R O U P S
TIM SKERN
Interactions between viruses and cells
Most viruses interfere with or
modulate host systems to ensure
successful replication. My group has
been looking at the interactions
between the leader proteinase of
foot-and-mouth disease virus and
the 2A proteinase of the common
cold virus and coxsackieviruses with
the cellular protein called eukaryotic
initiation factor 4G (eIF4G).
This protein is involved in recruiting capped cellular
mRNA to the ribosomes for protein synthesis. Cleavage of this molecule during replication of the
above mentioned viruses thus prevents capped cellular mRNA being translated. Viral protein synthesis
is unaffected as it initiates internally downstream
of the 5’ end of its RNA. We have determined the
molecular structures of three of these proteinases
and investigated the sites at which they interact
with eIF4G.
Tim Skern
TEAM
15N NMR signals of the wild-type leader proteinase and
the C-terminal P187A mutant
For the 2A proteinase, our recent work is concentrating on obtaining a crystal structure of the poliovirus enzyme. This may allow inhibitors to be
designed that can be used in the poliovirus eradication campaign of the WHO.
The leader proteinase is a relative of the plant cysteine proteinase papain. However, in contrast to
papain, the leader proteinase is very specific, with
only three target proteins identified at present.
Nevertheless, a consensus sequence representing
the cleavage site has been difficult to define as
the three known cleavage sites show considerable
differences (Santos et al, 2009). We have recently
started to use our knowledge of the cleavage sites
to develop specific inhibitors of the enzyme.
Recently, in collaboration with Christian Mandl and
Franz Heinz (Medical University of Vienna), we
have started to examine how the tick-borne encephalitis virus (TBEV) and West Nile virus (WNV)
proteins are synthesised in an infected cell. Specifically, we have replaced one of the viral proteinase
cleavage sites with a sequence from foot-andmouth disease virus that can split the polypeptide
chain without viral proteinase activity.
The foot-and-mouth disease leader proteinase
Both TBEV and WNV containing with this sequence
were viable. However, whereas the TBEV mutant
grew only in mammalian cells but not tick cells,
the WNV mutant was unstable in mammalian cells
but grew well in mosquito cells (Schrauf et al,
2009). This system reveals that different flaviviruses
have different requirements in their cognate host
cells and will be useful in elucidating the nature
of the differences, especially in studying the interactions of the viral proteins with structures found
in the host cells.
Santos, J, Gouvea, I.E, Judice, W.A.S., Izidoro, M.A.S, Alves, F. M., Melo, R.L., Juliano, M.A., Skern, T., and Juliano, L. (2009).
Hydrolytic Properties and Substrate Specificity of the Foot-and-Mouth Disease Leader Protease. Biochemistry 48,
7948-7958. n Schrauf, S., Mandl, Ch., Bell-Sakyi, L. and Skern, T. (2009). Extension of flaviviral protein C differentially
affects early 1 RNA synthesis and growth in mammalian and arthropod host cells. J. Virol. 83, 11201-11210. n
Schlick, P. and Skern, T. (2008). Investigating human immunodeficiency virus-1 proteinase specificity at positions P4
to P2 using a bacterial screening system. Anal. Biochem. 377, 162–169.
67
Martina Aumayr
Sofiya Fedosyuk
David Neubauer
Melanie Niemer
Toma Sara
Katharina Ruzicska
Ulrike Seifert
Jutta Steinberger
R E S E A R C H
G R O U P S
MARKUS TEIGE
Plant Signaling
We study how plants adapt and
acclimate to a changing environment
or to stress conditions. How are
environmental signals perceived
and further processed in plants,
and which processes are regulated?
Markus Teige
TEAM
Andrea Mair
Andrea Simeunovic
Simon Stael
Bernhard Wurzinger
Helga Waltenberger
To answer these questions, we investigate different
pathways, which are triggered by calcium-signals
and test different mutants for a physiological phenotype. We are particularly interested in the subcellular localization of the signaling molecules with
a special focus on chloroplast-related metabolism.
Therefore we performed directed proteomic studies
in order to identify new signaling components in
chloroplasts (Bayer et al. 2011) and studied effects
of protein modification in the subcellular targeting
of protein kinases (Stael et al. 2011).
Detailed functional studies of chloroplast-related
signaling are currently performed within the Marie-
Curie training network
(ITN) COSI (www.univie.ac.at/cosi).
The role of calcium signals is studied using
calcium-dependent
protein kinases (CDPKs)
as example. We identified a CDPK, which is
required for acclimation
to salt-stress (Mehlmer
et al. 2010).
This CDPK seems to regulate different cellular
Isolated Arabidopsis chlotargets at the vacuole,
roplasts on a Percoll grathe plasma membrane,
dient (upper part) and
in the cytosol, and also
three chloroplasts expresin the nucleus. The mosing a green-fluoresent
lecular mechanism beprotein (GFP) fusion. Red,
hind the observed
Chlorophyll-fluorescence;
growth phenotype ungreen, GFP signal.
der salt-stress could
therefore be manifold
from regulation of ion
channels or transporters at different membranes to metabolic reprogramming by phosphorylation of metabolic enzymes or transcriptional regulators. Such mechanisms
of metabolic reprogramming in response to different growth conditions are currently tested in the
framework of the ITN MERIT
(www.uu.nl/science/merit).
Here we analyze the regulation of a bZIP transcription factor by phosphorylation in collaboration
with eight European partner groups.
Phenotype of bZIP transcription factor overexpressor- and
knock-out plants under salt stress (200 mM NaCl).
Future directions of our work will be to untangle
how phosphorylation of these transcription factors
regulates the activity and thereby the expression
of downstream genes, and to study newly discovered factors in chloroplast signaling. A particular
focus in this aspect will be the decoding of calcium
signals in the chloroplast.
Bayer RG, Stael S, Csaszar E, Teige M (2011). Mining the soluble chloroplast proteome by affinity chromatography.
Proteomics, doi: 10.1002/pmic.201000495. n Stael S, Bayer RG, Mehlmer N, Teige M (2011). Protein N-acylation overrides differing targeting signals. FEBS Lett., 585(3), 517-22. n Mehlmer N, Wurzinger B, Stael S, Hofmann-Rodrigues
D, Csaszar E, Pfister B, Bayer R, Teige M (2010). The Ca(2+)-dependent protein kinase CPK3 is required for MAPK-independent salt-stress acclimation in Arabidopsis. Plant J., 63(3), 484–498.
68
R E S E A R C H
G R O U P S
KRISTIN TESSMAR-RAIBLE
Lunar periodicity and inner brain photoreceptors
Ever since the dawn of life, eco systems are governed by periodic
changes in light conditions that
act as reliable cues to synchronize
biotic processes.
Most, if not all organisms feed this light information into molecular clockworks that allow them to
anticipate rhythmic changes in their environment
and to synchronize specific biological events.
For decades, efforts to study the underlying molecular mechanisms have focused almost exclusively
on land model species and their most prominent
light-dark cycle: the circadian rhythm. A key finding
of this work was that in eukaryotes the underlying
common core mechanism consists of negative transcriptional/translational feedback loops formed by
a set of regulatory genes ('clock').
However, life evolved in the sea, and a rich body of
literature describes rhythmic phenomena in marine
organisms that are unparalleled on land.
These exceed circadian rhythms and include tidal,
lunar and semilunar rhythms. Yet, albeit important
and widespread, not a single molecule has clearly
been implicated in a moon-entrained clock, due to
the lack of a suitable molecular model species.
We aim to fill this gap by analyzing the molecules
that govern the lunar reproductive periodicity of
the marine bristle worm Platynereis dumerilii. Its
large sequence resources and molecular tools, along
with its ancestral-type nervous system make it an
ideal starting point not only to unravel the molecular principles of its circalunar and circadian
clocks, but also to place both clocks in an evolutionary context. Using these worms, we have
started to address the following three main questions:
• How are lunar and circadian clock
interconnected?
• What is the nature of the lunar clock in
Platynereis?
• What are the moon light sensors on the
cellular and molecular level?
Platynereis possesses multiple Opsin-based photoreceptor cells, some of which
are part of the eyes, while others are located in the medial region of its forebrain. We have started to especially focus
on the latter as candidate moon light receptors.
Lunar-controlled rhythms are widespread and of fundamental importance for marine organisms.
Simplified phylogeny of metazoan groups with representatives exhibiting moon-controlled rhythms. In most mentioned cases evidence
for a free-running lunar clock mechanism exist. All mentioned groups
represent marine species. Lunar-controlled rhythms have also been
described outside metazoans in green and brown algae (bottom).
Interestingly, such inner brain photoreceptor cells also exist in the forebrain
of vertebrates. In fact, we could show
that these cells belong to the most ancient cell types present in the vertebrate
brain. Yet, we know nothing about their
function. We have also started to solve
this riddle, using the zebrafish and medakafish. We approach the function of
these Opsins using biochemical analyses,
zinc-finger mediated mutagenesis, transgenesis, whole mount in situ hybridization, immunocytochemistry, as well as
electron microscopy.
Tessmar-Raible K, Raible F, Arboleda E. (2011). Another place, another timer: Marine species and the rhythms of life.
Bioessays. Mar;33(3):165-72. n Dray N*, Tessmar-Raible K,* Le Gouar M*, Vibert L, Christodoulou F, Schipany K, Guillou
A, Zantke J, Snyman H, Béhague J, Vervoort M, Arendt D, Balavoine G. (2010) Hedgehog signaling regulates segment
formation in the annelid Platynereis. Science 16;329(5989):339-42. * equal contribution. n K. Tessmar-Raible, Raible
F, Christodoulou F, Guy K, Rembold M, Hausen H, Arendt D. (2007), Conserved sensory-neurosecretory cell types in annelid and fish forebrain: insights into hypothalamus evolution. Cell 129, 1389.
69
TEAM
Enrique Arboleda
Vinoth Babu Veedin Rajan
Andrea Brezovich
Susanne Bloch
Ruth Fischer
Stefan Keplinger
Anna Pazos
Katharina Schipany
Juliane Zantke
R E S E A R C H
G R O U P S
CHRISTINA WALDSICH
Exploring RNA folding: from structure to function
RNAs regulate biology: In the past
years it has become increasingly evident that RNA is the driving force in
most cellular processes.
Christina Waldsich
TEAM
Samuel Flores
Andreas Liebeg
Nora Sachsenmaier
Eva Steiner
Michael Wildauer
Georgeta Zemora
Although these RNAs are highly diverse and fulfill
very different tasks, they share their strict dependence on acquiring a specific 3D architecture to
be functional. The process of folding describes how
RNA undergoes the transition from a disordered,
unfolded state to the native, functional conformation. Our research focuses on understanding this
most essential aspect of RNA function by investigating RNA structure and folding pathways. Specifically, we aim to provide novel insights into protein-facilitated RNA folding and how RNAs fold in
the living cell. Little is known about how RNA folds
in vivo and how they interact with their targets
despite RNA’s importance for cell viability. Therefore,
it is of fundamental importance to gain insights
into the forces driving RNA folding in vivo and to
establish the contribution and impact of the cellular
environment, in order to understand the basic mechanism of these RNA-dependent processes.
Catalytic RNAs, in particular group II introns, are
the best-suited model system to study RNA folding
in the living cell, as their structure and folding
pathways are well characterized in vitro and formation of the native conformation can be measured
as a function of catalysis.
Therefore, we investigate the intracellular folding
pathway of the Sc. ai5γ group II intron. Importantly,
Sc. ai5γ and other yeast mitochondrial introns depend on trans-acting protein factors for efficient
splicing in vivo. Consequently, we are interested in
exploring how these proteins shape folding of their
target RNAs. This allows us to derive principles governing in vivo RNA folding facilitated by proteins
and other cellular factors.
Aside from studying catalytic RNA, we are fascinated by Telomerase; an RNP that has received
considerable attention because of its significant
up-regulation in the majority of cancer cells and
its role in preventing chromosomal instability and
senescence as well as in inherited human disorders.
In spite of the high level of interest in the bio-medical importance of telomerase, telomerase RNA
and protein components have largely eluded
structural characterization.
In this regard, we are interested in exploring the
structure of telomerase RNA and in studying the
interplay of RNA folding and RNP assembly. Ultimately, deciphering the rules governing RNA folding
will advance our understanding of the basic mechanism of RNA-dependent processes, like selfsplicing and telomere addition, and the role of RNA
in disease.
The folding pathway of the Sc. ai5γ group II ribozyme. In the unfolded state only the secondary structure is formed, while
in case of the intermediate state Domain 1 (blue) compacts and forms tertiary structure thereby providing the scaffold
for docking of Domains 3 (green) and 5 (red), which completes folding to the native conformation (Pyle, Fedorova and
Waldsich, TiBS 2007).
Liebeg A, Mayer O and Waldsich C (2010). DEAD-box protein facilitated RNA folding in vivo. RNA Biol 7, 103-111. n
Zemora G and Waldsich C (2010). RNA folding in the living cell. RNA Biol 7, 8-15. n Liebeg A and Waldsich C (2009).
Probing RNA structure within living cells. Methods Enzymol. 468, 219-238.
70
R E S E A R C H
G R O U P S
GRAHAM WARREN
Biogenesis of the Golgi apparatus
During normal growth and division,
cells double in mass and divide into
two equally-sized daughters. All cell
constituents must be duplicated and
then segregated equally during
mitosis.
For some constituents, such as the chromosomes,
the underlying principles and the mechanistic details are relatively clear. For membrane-bound organelles, such as the Golgi, the principles and mechanism have been controversial. The primary aim
of our research work is to understand how the cell
creates another copy of the Golgi during the cell
cycle and partitions them equally between the two
daughter cells, thereby ensuring that this organelle
is propagated through successive generations.
tioning of the Golgi during mitosis in mammalian
cells and most studies suggest that the Golgi undergoes a dramatic conversion to thousands of
small vesicles that can then be stochastically distributed between daughter cells. This conversion is
triggered by mitotic kinases acting on structural
proteins such as GRASPs and golgins.
Golgi duplication has been more difficult to study
since most mammalian cells have several hundred
copies, subsumed into a ribbon-like structure next
to the centrosomes and often the nucleus. This
precludes facile observation of the duplication process. We have solved this problem by focusing on
organisms that have only a single Golgi that undergoes duplication during the cell cycle and partitioning during mitosis. Protozoan parasites are
the best model systems since many have had their
genomes sequenced and a variety of molecular
biological techniques are
available to manipulate protein levels.
Trypanosoma brucei is the
causative agent of sleeping
sickness in sub-Saharan
Africa, and provides a highlysimplified and organized secretory system that is ideal
Early in the cell cycle (left panel) the old Golgi (G; red) in T. brucei is located near to
for studying the process of
one lobe of a bi-lobe structure (green, closed arrowheads). Later in the cell cycle
Golgi biogenesis. The dupli(right panel) the new Golgi is found associated with the other lobe suggesting that
cation of the single Golgi can
the bi-lobe has a role to play in the duplication process. N=nucleus; K=kinetoplast
be observed using GFP-tag(mitDNA); open arrowheads=basal bodies.
ged Golgi proteins, and video
fluorescence
microscopy
The Golgi lies at the heart of the secretory pathway
shows that the old Golgi is involved in the conreceiving the entire output of newly-synthesized
struction of the new.
cargo proteins from the endoplasmic reticulum,
modifying any bound oligosaccharides, and then
Furthermore, both are located on a novel bilobe
sorting them to their final destinations. Typically
structure that appears to act as a template, detercomprising a stack of closely-apposed and flattened
mining both the size of the Golgi and its inhericisternae, the Golgi presents a complex architecture
tance. The composition and duplication of this bithat needs to be duplicated and partitioned every
lobe are presently under investigation as is the
cell cycle. Most studies have focused on the partimolecular mechanism that generates the new Golgi.
de Graffenried, C. L., Ho, H. H., and Warren, G. (2008). Polo-like kinase is required for Golgi and bilobe biogenesis in
Trypanosoma brucei. J Cell Biol 181, 431-438. n He, C. Y., Pypaert, M., and Warren, G. (2005). Golgi duplication in Trypanosoma brucei requires Centrin2. Science 310, 1196-1198. n Morriswood, B., He, C.Y., Sealey-Cardona, M., Yelinek,
J., Pypart, M., Warren, G. (2009). The bilobe structure of Trypanosoma brucei contains a MORN-repeat protein. Mol.
Biochem. Parasitol. 167(2): 95-103
71
Graham Warren
TEAM
Chris de Graffenried
Lars Demmel
Heather Esson
Katharina Havlicek
Andrea Hessenberger
Kyojiro Ikeda
Ana Lozano
Brooke Morriswood
Marco Sealey
Sevil Yavuz
R E S E A R C H
G R O U P S
GEORG WEITZER
Somatic Stem Cells of the Heart
In recent years, numerous groups
provided compelling evidence for
the existence of somatic stem cells
in the heart of different
mammalian species.
Georg Weitzer
TEAM
Phillip Heher
Teresa Gottschamel
Brigitte Gundacker
Maximillian Miksch
Jasmin Taubenschmid
Dania Walder
Stem cells and progenitor cells are supposed to
exist in niches, where they remain in an undifferentiated and quasi-dormant state until external
signals stimulate commitment and differentiation
to specific somatic cells which may contribute to
the repair or maintenance of homeostasis of an
organ. Mimicking a stem cell niche of the heart in
vitro, we succeeded in the isolation of somatic
stem cells from postnatal murine hearts and could
maintain these cells as monoclonal self-renewing
cells lines expressing Oct4, Sox2 and Nanog for
several years (see figure).
These cells obviously committed to the mesodermal
lineage and expressing early myocardial transcrip-
tion factors Brachyury, Nkx2.5, GATA4, and Isl1 exclusively differentiate to cardiomyocytes, smooth
muscle cells, and vascular endothelial cells and
thus were named cardiovascular progenitor cells
(CVPCs).
Cardiomyogenic progenitors further differentiate
to equal numbers of functional pacemakers, atrial
and ventricular cardiomyocytes with a near-adult
action potential. Stimulation of CVPCs with Activin
A and Retinoic Acid did not yield any cell types of
the endodermal and ectodermal lineage, respectively. Addition of BMP2 and SPARC promoted cardiomyogenesis and led to the upregulation of genes
for the mesoderm specific transcription factor Brachyury and the early myocardial transcription factor
Nkx2.5.
In our ongoing research, we try to reveal the molecular pathways which allow SPARC, BMP2 and
Nodal to activate Brachyury and Nkx2.5 expression
in CVPCs and how Brachyury, Nanog and Nkx2.5
interact on the transcriptional
level in undifferentiated and differentiating CVPCs. Our long term
scientific goal is to understand
early cardiomyogenesis and how
somatic stem cells may contribute to homeostasis of the heart.
Understanding the molecular
and cellular interplay regulating
stem cell self-renewal and differentiation may contribute to
future targeted therapies utilizing growth factors or small molecules for the temporal endogenous activation of the stem
cell pool.
Localisation of Oct4 protein during cell division of CVPCs. Immunofluorescence
microscopy of CVPCs with Oct4 antibodies (green), and DAPI (blue). Bar: 15 µm.
Arrows, top, Metaphase; middle, Anaphase, and bottom, Telophase. Asterisks,
Oct4 negative nucleus of a SNL76/7 feeder cell.
Lagger,S, Meunier,D, Mikula,M, Brunmeir,R, Schlederer,M, Artaker,M, Pusch, O, Egger,G, Hagelkruys,A, Mikulits,W, Weitzer,G,
Muellner,EW, Susani,M, Kenner, L, and Seiser, C (2010). Crucial function of histone deacetylase 1 for differentiation of
Teratomas in mice and humans. EMBO J 21, 2672-2681. n Hofner, M., Höllrigl, A., Puz, S., Stary, M., Weitzer, G. (2007).
Desmin stimulates differentiation of cardiomyocytes and upregulation of brachyury and nkx2.5. Differentiation 75,
605-615. n Stary, M., Pasteiner, W., Summer, A., Hrdina, A., Eger, A., and Weitzer, G. (2005). Parietal endoderm secreted
SPARC promotes early cardiomyogenesis in vitro. Experimental Cell Research, 310, 331-343.
72
R E S E A R C H
G R O U P S
GERHARD WICHE
The cytoskeleton in signaling and disease
The cytoskeleton provides the
structural basis for physical robustness, shape, movement, and intra cellular dynamics of eukaryotic cells.
In muscle cells, it forms the contractile apparatus,
confers structural support and positions organelles;
in neurons, it maintains the asymmetric cell shape
and polarity; and in epithelial cells, it plays a pivotal
role in maintaining cell and tissue integrity. We
are interested in cytoskeletal linker proteins (cytolinkers), a family of multi-modular, highly versatile
proteins of exceptional size, that by networking
and anchoring cytoskeletal filaments regulate cytoskeleton dynamics and architecture. We are studying the role of cytolinkers in normal development,
cellular stress response, and disease, combining
mouse genetics with cell and structural biology.
Several years ago we discovered plectin, a ubiquitous cytolinker that became the prototype of what
meanwhile is a whole family of similar proteins.
Plectin has key functions in shaping cell architecture, mechanical stabilization and polarization of
cells, positioning of organelles, signal transduction
and nerve conduction. Thus, loss or dysfunction of
plectin leads to diseases affecting a variety of cell
types and tissues. Plectin’s versatility is based on
an unusual diversity of isoforms differing in small
N-terminal sequences that determine the protein’s
localization. We have generated a panel of transgenic mouse lines, including full knockout (KO),
single isoform and conditional/tissue-restricted KO,
and knock-in lines. Serving us in analyzing isoform-specific functions and providing animal models for plectin-related human diseases, we use
these systems focusing on:
Myofibrillar myopathies.
We found myofiber integrity, including mitochondrial function, in skeletal muscle to be dependent
on the proper targeting of desmin intermediate
filament (IF) networks to strategic cellular sites via
distinct plectin isoforms. Plectin-unanchored desmin networks collapse and form protein aggregates
leading to dysfunctional myofibers. In addition,
unbalanced plectin levels cause diabetes. Other
topics are plectin-related heart dysfunctions and
failures in myofiber regeneration.
Plectin in epithelia (skin).
Severe skin blistering (EBS) is the hallmark of most
plectinopathies. The analysis of a knock-in mouse
line mimicking the dominant plectin mutation of
EBS-Ogna patients provided new insights into
hemidesmosome(HD)-stabilizing mechanisms and
revealed plectin-isoform-specific proteolysis as a
novel mechanism regulating HD-homeostasis.
Role of plectin in neural cells.
Having identified the major neuronal plectin-isoform as a microtubule regulator, we are assessing
its role in synaptic transmission, nerve conduction,
and glucose uptake.
Fig 2: Perinuclear cage-like vimentin intermediate filament core (red), visualized by confocal 3-D Z-stack projection. A specific isoform of plectin (P1f) stabilizes the
core structure and enables cells to become polarized.
(Blue) Microtubules.
Plectin-dependent signaling and stress response
in endothelial and fibroblast cells.
We found plectin scaffolds to antagonize oxidative
stress-mediated alterations of cell cytoarchitecture.
Present studies are focused on plectin’s role in
stress response of endothelial and fibroblast cells
(see figure).
Burgstaller G, Gregor M, Winter L, Wiche G. (2010). Keeping the vimentin network under control: cell-matrix adhesion-associated plectin 1f affects cell shape and polarity of fibroblasts. Mol Biol Cell 21(19), 3362-75. n Winter L,
Abrahamsberg C, Wiche G. (2008). Plectin isoform 1b mediates mitochondrion-intermediate filament network linkage
and controls organelle shape. J Cell Biol 181(6), 903-11. n Konieczny P, Fuchs P, Reipert S, Kunz WS, Zeöld A, Fischer I,
Paulin D, Schröder R, Wiche G. (2008). Myofiber integrity depends on desmin network targeting to Z-disks and
costameres via distinct plectin isoforms. J Cell Biol 181(19), 667-81.
73
Gerhard Wiche
TEAM
Irmgard Fischer
Peter Fuchs
Rocio Garcia de la Cruz Valencia
Karin Groß
Eva Mihailovska
Selma Osmanagic-Myers
Marianne Raith
Günther Rezniczek
Karina Scherrer
Ilona Staszewska
Gernot Walko
Lilli Winter
Karl Wögenstein
R E S E A R C H
G R O U P S
ANGELA WITTE
fCh1, model for gene regulation in haloalkaliphilic Archaea
The virus fCh1 was found by
spontaneous lysis of a culture of the
haloalkaliphilic, archaeon, Natrialba
magadii, an isolate from the soda
lake, Lake Magadii in Kenya.
Angela Witte
TEAM
Bea Alte
Daniel Kiesenhofer
Michael Reiter
Regina Selb
Tatjana Svoboda
Petra Till
This organism has an optimal growth at 3.5M NaCl
and at a pH of 9.5. The virus itself is used as a
model system to analyse gene expression in haloalkaliphilic organisms, facing with two extremes:
a high pH and high concentrations of salt. The sequence of fCh1, infecting the haloalkaliphilic archaeon Natrialba magadii, contains an open reading frame (int1) in the central part of its genome
that belongs to the l integrase family of site-specific recombinases. Sequence similarities to known
integrases include the highly conserved tetrad RH-R-Y. The flanking sequences of int1 contain several direct repeats of 30 bp in length (IR-L and
IR-R), which are orientated in an inverted direction.
The invertible region encodes two structural proteins (gp34 and gp36, encoded by ORF34 and
ORF36) expected to represent the viral tail fibre
proteins.
In vitro experiments
using purified protein
variants gp341 and
gp3452 (containing the
C-terminus of gp36) revealed exclusive binding of gp3452 but not
of gp341 to cells of the
cured strain Nab. magadii L13. This specific
binding could be inhibited by the addition of
a-D-galactose. a-DElectron micrograph of
galactose also signififCh1 particle negatively
cantly reduced the instained with uranylacetate.
fectivity of fCh1.
Binding experiments
employing distinct domains of gp341 and gp3452
indicated the C-terminus to be responsible for binding to the receptor on the cell surface of Nab.
magadii L13. This C-terminus contains a domain
with similarities to the super-family of ìgalactoselike bindingî proteins. In summary, the experiments
gave evidence that gp3452 represents the anti-receptor of fCh1 that binds to specific carbohydrate
ligands located on the cell surface of Nab. magadii.
Currently the work concentrates
on the identification and
function of repressor and activator molecules encoded by the
virus, gene regulation due to a
recombination event, identification of the receptor for the virus
on the cell surface of Nab. magadii and the transformation/
shuttle vector system developed
by the group. In addition the method is used to construct different mutants.
Schematically representation of the genome of fCh1
Klein R, Baranyi U, Rössler N, Greineder B, Scholz H and Witte A (2002). Natrialba magadii virus fCh1: first complete
nucleotide sequence and functional organization of a virus infecting a haloalkaliphilic archaeon. Mol Microbiol, 45,
851-863. n Rössler N, Klein R, Scholz H and Witte A (2004). Inversion within the haloalkaliphilic virus fCh1 DNA
results in differential expression of structural proteins. Mol Microbiol 52, 413-426. n Iro M, Klein R, Galos B, Baranyi
U, Rössler N. and Witte A (2007). The lysogenic region of virus fCh1: identification of a repressor-operator system
and determination of its activity in halophilic Archaea. Extremoph 11, 383-396
74
R E S E A R C H
G R O U P S
FRANZ WOHLRAB
Function of zona pellucida domain proteins
The first specific interaction between
sperm and egg occurs at an extra cellular matrix (ECM) called the zona
pellucida (zp). The zp does not only
provide specific receptors for incoming
sperm, but has other important
functions.
It can induce the acrosome reaction on the sperm
head, and by reacting to the release of cortical
granules from the oocyte, provides the major block
to polyspermy. It also serves as a protection for
the fertilized egg and early mammalian embryo
during its travel to the point of implantation. The
zp then has to break open in a tightly controlled
timed program to allow the embryo to hatch and
contact the maternal endometrium.
In addition, the number of ovarian zp proteins besides the three canonical zp components is steadily
increasing. Thus, in birds, we have up to now identified 8 follicular zp proteins. The function of these
factors is entirely unknown.
Another aim of this project is to delineate the roles
these proteins play during the lifecycle of the ovary
and in different organs. We have already shown
that one of the liver-derived zp proteins, ZPAY, is
not targeted to the ovary, but is associated in the
brain and the kidney with cells lining tubular
structures, such as cerebral smooth muscle cells,
and the proximal tubulus, respectively.
Studies in mice have shown that the zp consists of
only 3 glycoproteins which all share a 260 amino
acid domain called the zp domain. These proteins
are coordinately expressed by the oocyte at the
transition from the primordial to the primary
stage. In other animals, however, they are often
expressed by somatic tissues. In birds, we have
shown that the two main components of the zp,
ZP1 and ZPC, are made by the liver and granulosa
cells, respectively.
Similar extraoocytic expression of zp proteins is
now established in many animals including mammals. In these cases, the proteins have to travel to
their final destination and then polymerize to form
the growing zp. A major aim of this project is to
investigate how these factors are targeted to the
site of zp assembly. We have shown that purified
native ZPC will self-assemble to larger structures
if its concentration is sufficiently high and focus
our attention on optimal conditions for assembly
of the zp in vitro. Special interest is being paid to
the role of follicle-derived factors like GDF9,
BMP15, activin, perlecan, etc. in this process.
Avian liver tubuli are lined with the zona pellucida
protein ZPAY.
Bausek N, Ruckenbauer HH, Pfeifer S, Schneider WJ, and Wohlrab F (2004). Interaction of Sperm with Purified Native
Chicken ZP1 and ZPC Proteins. Biol Reprod 71, 684-690. n Stewart SS, Bausek B, Wohlrab F, Schneider WJ, Horrocks
AJ, and Wishart GJ (2004). Species Specificity in Avian Sperm: Perivitelline Interaction. Comp. Biochem. Physiol. Part
A. 137, 657-663.
75
Franz Wohlrab
R E S E A R C H
G R O U P S
BOJAN ZAGROVIC
Computational Biophysics of Macromolecules
Bojan Zagrovic
TEAM
Mario Hlevnjak
Antonija Kuzmanic
Christian Margreitter
(since 5/2011)
Bianca Mladek
Drazen Petrov
Matija Piskorec
Anton Polyansky
Rita Santos (until 7/2010)
Ruben Zubac (until 4/2011)
Biological function on the molecular
level is directly related to the 3-dimensional structure of biomolecules,
their dynamics and finally their
interactions, both with the
environment as well as with
other biomolecules.
Conceptually, the research interests of our laboratory revolve around exploring different faces of this
fundamental principle through the use of computational and theoretical methods in close collaboration with experimentalists. While experimental
approaches are making breathtaking steps forward,
currently the only way to probe biomolecular
structure and dynamics on a single molecule level
and with all-atom, femtosecond resolution is
through molecular dynamics computer simulations,
our primary workhorse.
Specifically, we are interested in intrinsically disordered proteins, which, despite their lack of stable
tertiary structure, carry out a multitude of amazing
functions in our cells. We strongly believe that
these molecules will teach us some completely new
principles of how biological systems function on
the molecular level.
Conformational selection and induced fit underlie specificity in non-covalent interactions of ubiquitin (Wlodarski
et al., PNAS, 106, 2009).
The intrinsically disordered regions in nudix hydrolaze of
the bacterium D. radiodurans may be an important component in the bacterium's adaptation to desiccation
(Awile et al. PLOS Comp. Bio, 6, 2010).
Also, we are interested in the role of dynamics and
entropy (in particular, conformational entropy) in
biomolecular processes in general. In many cases,
a change in conformational entropy of a protein
may be enough to alter its functional state, without
any associated conformational change on the level
of the average structure. We are developing new
methods both for calculating conformational entropy of biomolecules from computer simulations
and for measuring it in the experiment.
Dynamics also subtly affects our attempts to determine what biomolecules look like. Namely, biomolecular structures are static models derived from
structural experiments (e.g. X-ray or NMR) performed typically on cca. 1020 dynamic copies of the
same molecule. We are interested in using computer simulations to help interpret such time- and
ensemble-averaged experimental data and to analyze the impact of conformational averaging on
the properties of the derived structures.
Finally, all of biomolecular processes occur in
crowded, dynamic, constantly changing environments. We are interested in exploring how this affects basic biomolecular processes such as protein
folding, covalent protein modifications or proteinprotein interactions. In particular, we have a strong
interest in studying how binding partners find each
other in the crowded cell.
n Wlodarski T and Zagrovic B (2009). Conformational selection and induced fit mechanism underlie specificity in
non-covalent interactions with ubiquitin. Proc Nat Acad Sci USA, 106(46), 19346-19351. n Hlevnjak M, Zitkovic G
and Zagrovic B (2010). Hydrophilicity matching – a potential prerequisite for the formation of protein-protein complexes in the cell. PLoS ONE, 5(6): e11169. n Awile O, Krisko A, Sbalzarini IF & Zagrovic B (2010). Intrinsically disordered
regions may lower the hydration free energy in proteins: a case study of nudix hydrolase in the desiccation-resistant
bacterium D. radiodurans. PLOS Computational Biology, 6(7), e1000854.
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MFPLAnnRep2010-Facilities:Layout 1 16.05.11 21:54 Seite 78
F A C I L I T I E S
BioOptics - Light Microscopy
The Bio-Optics facility
feels dedicated to provide
state-of-the art light microscopy equipment to
MFPL researchers.
Besides professional training, the facility personnel
assist in experimental
planning, technical setup
and troubleshooting.
FACILITY HEAD
Josef Gotzmann
Current Equipment:
• three laser scanning confocal microscopes
• a live-imaging station
• a microdissection / laser ablation instrument
• an image-restoration epifluorescence microscope
• a dissection-/stereo-microscope
• a tissue-culture unit
• two image processing workstations
Single nerve fiber from mouse sciatic nerve stained with
antibodies to dystrophin-related protein 2 (DRP2, green)
and vimentin intermediate filaments (red). ©Gernot Walko
Our current equipment allows MFPL people to perform most standard biooptical techniques, ranging
from routine fluorescence microscopy to optical
sectioning, including the study of dynamic processes. Moreover, with the microdissection equipment
the integration of optical techniques into molecular
biological experimental setups is now available.
Professional software packages to restore, process
and analyze acquired images in a multi-dimensional
way complement the hardware-driven projects.
Our current equipment is rather frequently used
by the majority of MFPL groups. To date 288 MFPL
people (62%) from 46 labs (70%) are registered as
trained microscope users. This statistical data clearly reflect the need and integration of advanced
bioimaging techniques in all scientific research
fields covered by MFPL research groups. The usage
is still biased for routine confocal microscopy applications, although a clear tendency towards an
increased need for live-imaging instrumentation
becomes more and more obvious.
Activities in 2010:
• Training routines for users, including introductory
and advanced lectures, established
• Image processing unit, providing off-site licenses
for microscope’s software and a professional deconvolution and 3-/4-D image processing (“Huygens”) package, implemented
• successful launch of hands-on image processing
workshops for students and post-graduates (together with JP Koch, Brocard lab)
• Initiation of an annual application-based workshop for students of the DK “Molecular and cellular signaling”
• Installation of the new epifluorescence restoration microscope “personal Deltavision”
Current Activities:
• Assessment on available hardware and projecting
upgrades and replacements of existing micro scopes
• Involvement in establishment of the Campus
Support Facility section “IMAS”
• Preparation of a lecture on “Advanced Microscopy Techniques”
Beatrix Karl - the Austrian Minister for Science and
Research visiting the BioOptics Facility in October 2010
Schneider M, Lu W, Neumann S, Brachner A, Gotzmann J, Noegel AA, Karakesisoglou I. (2010). Molecular mechanisms of
centrosome and cytoskeleton anchorage at the nuclear envelope. Cell Mol Life Sci., DOI: 10.1007/s00018-010-0535-z
n Lu W, Gotzmann J, Sironi L, Jaeger VM, Schneider M, Lüke Y, Uhlén M, Szigyarto CA, Brachner A, Ellenberg J, Foisner R,
Noegel AA, Karakesisoglou I. (2008). Sun1 forms immobile macromolecular assemblies at the nuclear envelope. Biochim
Biophys Acta. 1783(12):2415-26. n Brachner A, Reipert S, Foisner R, Gotzmann J. (2005). LEM2 is a novel MAN1related inner nuclear membrane protein associated with A-type lamins. J Cell Sci.118(24):5797-810.
78
F A C I L I T I E S
BioOptics - Flow Cytometry
The facility runs three flow cytometers for the measurement of fluorescence, size and granularity of
cells or other particles in solution.
The outdated Partec PASIII will be eliminated in
may 2011. It uses an UV lamp for excitation and is
useful for DNA analysis
The current workhorse is a FACS Calibur machine
(laser lines 488 and 635). It is a multi-purpose
analytical instrument that can simultaneously analyze
two scatters and up to four fluorescences.
Thanks and farewell to
Edgar Wawra
Edgar retires in March
2011. About 25 years ago,
he was one of the first
scientists in Austria, who
realized the possibilities of
flow cytometry and established the first Partec at
Institute of Molecular Biology in 1090 Wien.
One of his main interests was the regulation of
DNA precursor synthesis and the ensuing mechanisms of cell cycle control. This was complemented
by the establishment of a facility dedicated to
fractionating and harvesting cell preparations synchronized in the cell cycle by centrifugal elutriation,
a long-time reference lab in Austria. Moreover he
developed and synthesized fluorescent DNA precursor analogs which were successfully used in
flow cytometry studies on various model systems
of tumor progression.
FACS Aria
The FACS Aria (laser lines 407, 488, 633) can analyze
on the basis of two scatters and up to nine fluorescences. Importantly, this aparatus offers highthroughput sorting with a maximum speed of
20 000 events per second, thus enabling preparative
work as well as cultivation of separated cell populations.
Flow cytometer: schematic construction – fluorescent cells – a typical result of analysis
79
SCIENTIFIC SUPERVISOR
Edgar Wawra (until Feb. 2011)
Thomas Decker (from Feb. 2011)
FACILITY HEAD
Thomas Sauer
F A C I L I T I E S
Electron Microscopy Facility
Electron microscopes are
essential tools for elucidation of the cellular
“nano-world”. The Electron
Microscopy Facility at the
MFPL comprises a transmission electron microscope JEOL 1210, built
1992, equipped with a 11
megapixel CCD camera for
routine use.
Siegfried Reipert
and disease, great efforts will be taken to establish
a follow-up FWF-project for systematic studies of
the so-called tubulohelical membrane arrays (see
figure) in the context of the cell-and ciliary cycle.
For our point of view, this approach could provide
a scientific backbone for future development of
the EM facility.
In addition, the facility offers an opportunity for surface visualization with
a tabletop scanning electron microscope. The robust
and easy to use Hitachi TM-1000 allows studies of
both frozen and critical point-dried samples.
Besides of the microscopes themselves, a comprehensive set of equipment for biological sample
preparation is available, which includes devices for
freezing at ambient and high-pressure, automated
tissue processing, and ultrathin sectioning of
resin-embedded or frozen samples.
The methods we apply range from routine preparations of cells and tissues for phenotyping of their
ultrastructure, cryopreparations, and immunoelectron microscopy. In particular we aim at rapid
immobilization and fixation of the living state by
microwave-accelerated chemical fixation, or
high-pressure freezing. The latter is followed by
low-temperature chemical fixation and embedding
in epoxy-or methacrylic resins. In 2010, progress
was made and applications were extended towards
common model systems of molecular biological
and genetic interest, such as yeast strains and tripanosomes. To cope with the increase in demand
of cryo-preparation, an additional automated
freeze-substitution unit (AFS2, LEICA Microsystems)
was purchased by Graham Warren.
Our continuous efforts for methodological development enable us to use state-of-the art preparation
in a flexible way for both service and research. The
latter resulted in the finding of a novel, singleorganelle like membrane array and in addressing
of cell biological questions from unexpected perspectives (Reipert et al., 2009, 2010). Considering
a possible impact of our observations for health
Spotlighting cellular “Nanotechnology”: The Tubulohelical
Membrane Array (TUHMA). A “giant” cellular lipid membrane array was discovered in the rat kangaroo epithelial
cell line PtK2 (Reipert et al., 2009). Transmission electron
microscopy of rapidly microwave-fixed, epoxy resinembedded cell monolayers reveals nanoperiodic, tubular
entities consisting of lipid- and proteinaceous components. Center, top, left: TUHMA surrounded by mitochondria. Center, top, right: TUHMA stripped of its lipid
membranes by treatment with detergent Triton X-100
(Bar, 200 nm). Continuous, helix-like threads are exposed.
Underneath: Fine structure of TUHMAs displaying characteristic zigzag patterns of threads confining core tubules,
ca. 80 nm in diameter. Series of confocal microscopic
sections on top and at the bottom of the panel: Polarized
orientation of TUHMAs (red) either in parallel (top) or perpendicular (bottom) to the cell nucleus (blue). Both
TUHMAs and nuclear pores are labeled by antibodies
against nuclear pore proteins. In contrast to annulate lamellae, TUHMAs show a single-organelle-like appearance.
Reipert S, Wesierska-Gadek J, Wienerroither S. (2010). Tubulohelical membrane arrays: From the initial observation to
the elucidation of nanophysical properties and cellular function. PMC Biophys. 3(1):13. n Reipert S, Kotisch H, Wysoudil B, Neumüller J.(2009). Tubulohelical membrane arrays: novel association of helical structures with intracellular
membranes. Cell Biol Int. 33(2):217-23.
80
F A C I L I T I E S
Fish & Marine Facilities
Fish Facility
The MFPL fish facility provides maintenance and
stock supply for the research groups working with
both zebrafish (Danio rerio) and medakafish
(Oryzias latipes). Both fish species have become
attractive models for the molecular analysis of
vertebrate development and physiology.
The current facility is designed to host around
25,000 fish in more than 580 aquariums of different
sizes. For chronobiological experiments and the
study of light-dependent processes, the facility also
provides special shelving systems that can be set
to individual light regimes and light of different
wavelengths.
The MFPL fish facility includes laboratory space
and equipment to perform high-throughput microinjections, transplantations or other micromanipulations.
The facility organization including breeding and
feeding routine is provided by a highly motivated
animal care team and supported by the lab members of the Tessmar and Raible groups.
Adult zebrafish (Danio rerio)
Marine Facility
The Marine Facility at the
Max F. Perutz Labs comprises four environmentally
controlled culture rooms
as well as a salt-water
preparation unit. The
facility serves to maintain
and propagate several
marine species.
One central organism is the
annelid worm Platynereis
dumerilii, a model species for evolutionary, developmental and chronobiological research. The facility
houses a unique collection of transgenic and highly
inbred natural strains. Due to their quality, the MFPL
inbred strains have become the major resource for
Platynereis genome and transcriptome sequencing.
Besides worms, the facility also houses eight strains
of marine midges (Clunio marinus), another chronobiological model species. The special equipment of
the facility includes fully light-controlled culture
racks and a state-of-the-art injection setup for
high-throughput microinjection.
Adult bristleworm (Platynereis dumerilii). Female (top)
and male (bottom) animals shortly before spawning.
Adult Medaka (Oryzias latipes)
Adult male of Clunio marinus
81
SCIENTIFIC SUPERVISORS
Florian Raible
Kristin Tessmar
HEAD - FISH FACILITY
Claudia Lohs
HEAD - MARINE FACILITY
Katharina Schipany
TEAM
Patrick Berthold
Alexandra Biach
Denise Vorauer
F A C I L I T I E S
Functional Genomics & Microarray Facility
The Functional Genomics &
Microarray Facility (FGMF)
supports MFPL scientists
and external customers in
their efforts to generate
and analyze data from
high-throughput functional genomics experiments,
including
phenotypic
screening or gene expression analysis using microarrays and qPCR.
Karl Kuchler
FACILITY HEAD
Walter Glaser
TECHNICIAN
Andriy Petryshyn
Services include programming and operation of robots, training and experimental planning support,
as well as processing and bioinformatics analysis
of microarray data. Users of the Max Perutz Labs
pay non-profit based service fees.
Microarray Unit
The services include RNA quality control using the
NanoDrop and Agilent 2100 Bioanalyzer; labeling,
hybridization, washing, staining and both basic
and advanced bioinformatic analysis of datasets.
The available equipment in the FGMF includes:
• Axon 4000B Scanner; NanoDrop2000c; Agilent
2100 Bioanalyzer
• Agilent 2-micron resolution high-throughput
scanner, which also allows for scanning of nonAgilent microarray platforms
• Affymetrix GCS3000 7G System, consisting of a
scanner, a fluidics station and the hybridization
oven.
Robotics Unit
The FGMF robotics equipment consists of two robots:
• The SINGER RoTor HDA is a pinning robot for
ultra-fast manipulation of high-density arrays
of microbial colonies (yeast, bacteria). This robot
uses pin-pads to transfer microbial colonies and
cells between plates.
• The Hamilton Star Line 8-channel liquid-handling
robot has an integrated fluorescence microplate
reader and a CO2 incubator with adjustable temperature, ranging from 4 - 50 °C. This high-end
robot is suitable for diverse large-scale processes,
including DNA/RNA/protein preparations, growth
monitoring, mutant screening, as well as HTS. Advanced features of this robot include a shaker, a
vacuum unit, a barcode reader, a light table, as
well as a camera for automated colony-picking.
Hamilton Star Line liquid handling robot
Real-Time PCR Unit
Our Real-Time PCR equipment consists of two Eppendorf Realplex4S Master Cyclers and one Roche
LightCycler 480. For multiplex PCR experiments,
the Mastercycler allows for simultaneous detection
of up to 4 flourescent dyes.
Agilent DNA Microarray Scanner
Eppendorf Realplex MasterCycler
82
F A C I L I T I E S
Histology Facility
The Histology Facility is located in room 4.518 on
the 4th floor of the VBC1 building. The facility is
equipped to produce high quality microscopic sections of frozen and paraffin embedded material.
Digital image capture and image analysis are available for bright field and stereomicroscopy. Introduction, basic training and updates sessions are
held periodically by Histocom specialists.
In addition, a practical course on basic histological
techniques such as tissue processing, immunohistochemistry, special stains and image analysis is
organized by the facility for PhD students of the
International PhD program 'Molecular Mechanisms
of Cell Signaling' at the MFPL.
Mouse liver – Staining of lipid deposits (Oil Red O Staining)
The Facility is supervised/
organized by the Baccarini
lab and is open to all MFPL
staff and students, and to
external Institutions on a
user pays basis.
Equipment:
• Tissue processor
• Paraffine-embedding
center
• Microtomes, Cryotome
• ASS-1 automated H&E staining center
• Shandon Sequenza, Workstation for immunohistochemistry
• Stereomicroscope Zeiss SteREO Discovery V.12,
bright field Microscope Zeiss “Axioimager”
• Image capture & analysis: Leica Camera DFC
320, Computer and Software for image analysis
Mouse cerebellum – Purkinje cells stained with an anticalbindin antibody
83
Manuela Baccarini
FACILITY HEAD
Christian Rupp
F A C I L I T I E S
Mass Spectrometry Facility
The Mass Spectrometry
Facility of the MFPL is currently run by two parttime post doc researchers
and three full-time technicians.
Gustav Ammerer
FACILITY HEADS
Dorothea Anrather
Edina Csaszar
TEAM
Sonja Frosch
Rainer Gith
Verena Unterwurzacher
We carried out sequence
identifications for over
350 samples in 2010, localized and characterized
different post translational
modifications in 150 samples and analyzed more than 40 cell lysates with
respect to selective identification and quantification of phosphorylated residues. Twenty four groups
within the MFPL as well as five external academic
and two industrial clients made use of our service.
The MS facility contributes substantially to the following major projects:
• qualitative and quantitative analysis of phosphorylation pattern of yeast proteins under certain conditions e.g. specific inhibition of the
MAP kinase HOG1 or mutation of the regulatory
subunits of the protein phosphatase PP2A,
• characterization of multi drug resistance proteins,
• quantitative characterization of the phosphoproteome in mouse brain during learning,
• localization of sumoylation on purified proteins.
The acquisition of our new generation high mass
accuracy instrument, the LTQ Orbitrap Velos, was
a milestone towards top quality mass spectral data.
We achieve more reliable identifications with this
mass spectrometer with lower false discovery rates,
higher sensitivity and can provide more accurate
quantification than with the first generation linear
ion trap.
Furthermore we participate in the beta testing of
software packages for data processing to keep our
tools up-to-date and get early access to the newest
developments.
The aim of our facility is not only the generation
of mass spectral raw data but we take active part
in the setup of the biological experiment. The strong
interaction between us and the sample owners is
a prerequisite for a successful analysis. We therefore invite the users to be trained in the basic requirements of the measurements. Data interpretation and its discussion with the users is a further
fundamental aspect of our work.
The quantitative analysis of the phosphoproteome
of Trypanosoma brucei is currently in the test phase,
and we are working on getting deeper into the
proteome of Saccharomyces pombe by pre-fractionation steps.
Our Thermo Scientific LTQ Orbitrap Velos coupled to the
Dionex nanoHPLC U3000
Frohner IE, Gregori C, Anrather D, Roitinger E, Schüller C, Ammerer G, Kuchler K. (2010) Weak organic acid stress triggers
hyperphosphorylation of the yeast zinc-finger transcription factor War1 and dampens stress adaptation. OMICS. 2010
Oct;14(5):575-86. Epub 2010 Aug 20. n Mehlmer N, Wurzinger B, Stael S, Hofmann-Rodrigues D, Csaszar E, Pfister B, Bayer
R, Teige M.(2010) The Ca(2+)-dependent protein kinase CPK3 is required for MAPK-independent salt-stress acclimation
in Arabidopsis. Plant J. 2010 August; 63(3): 484–498. Epub 2010 May 20 n Spirek M, Estreicher A, Csaszar E, Wells J,
McFarlane RJ, Watts FZ, Loidl J. (2010) SUMOylation is required for normal development of linear elements and wildtype meiotic recombination in Schizosaccharomyces pombe. Chromosoma. 2010 Feb;119(1):59-72. Epub 2009 Sep 12.
84
F A C I L I T I E S
Monoclonal Antibody Facility
Based on our decade-long expertise in the development of mouse monoclonal antibodies that has
led to unique antibodies such as the anti-methyl
protein phosphatase 2A, clone 2A10, or the antimyc tag, clone 4A6, the MFPL Monoclonal Antibody
Facility (MAF) was established in 2009 by Egon
Ogris and Stefan Schüchner as a service facility
offering in-house development of custom monoclonal antibodies. The aim of the MFPL MAF is to
produce novel high quality mouse monoclonal antibodies specific for any custom antigens, e.g. peptides, recombinant proteins, or post-translational
modifications. Such antibodies may not only be
applied in basic research, but may also serve in
clinical applications, e.g. as diagnostic markers.
Besides the realization of
custom projects, we have
acquired extensive expertise in the generation of
point-mutant/isoform
specific antibodies. In the
course of an FWF-funded
translational research project, we have succeeded in
the generation of the only
commercially available
antibody against human
progerin as well as antibodies specific for two other disease-associated
lamin A/C single point-mutants.
During the past year, the MFPL MAF was able to
attract internal as well as external customers, for
whom we could generate custom-tailored monoclonal antibodies against a variety of antigens, including fungal pathogens, bacterial membrane
transport proteins, signaling molecules and organelle constituents.
Our antibody collection also includes an antibody
specific for apolipoprotein E4, the high risk isoform
of apolipoprotein E associated with Alzheimer´s
disease, and modification-specific antibodies, e.g.
against methylated histone species.
Key to the isolation of antibodies with custom-designed properties is the close collaboration with
the customers during the project, which is one of
our foremost priorities.
In addition, funded by another FWF translational
research grant, we are aiming to refine the methods
for the generation of monoclonal antibodies and
to apply the improved method for the development
of yet more antibodies specific for disease-relevant
human proteins.
(a) Centrin 2 and 4 antibodies specifically detect the respective isoforms by Western blotting using
lysates of T.brucei expressing GFPtagged centrins.
(b) Immunofluorescence images of
T.brucei cells stained with the isoform specific antibodies reveal distinct subcellular localization of
centrins 2 and 4.
Roblek M, Schüchner S, Huber V, Ollram K, Vlcek-Vesely S, Foisner R, Wehnert M, Ogris E (2010). Monoclonal Antibodies
specific for disease-associated point-mutants: lamin A/C R453W and R482W. PLoS One, 5(5):e10604. n Capanni, C.,
Cenni, V., Haraguchi, T., Squarzoni, S., Schuchner, S., Ogris, E., Novelli, G., Maraldi, N.M., and Lattanzi, G. (2010). Lamin A
precursor induces barrier-to-autointegration factor nuclear localization. Cell Cycle, 9.
85
Egon Ogris
FACILITY HEAD
Stefan Schüchner
TEAM
Ingrid Mudrak
Marko Roblek
F A C I L I T I E S
Bio-NMR Facility
Robert Konrat
FACILITY HEAD
Georg Kontaxis
TECHNICIAN
Rebby Precilla
USER COMMITTEE
Kristina Djinovic-Carugo
Renée Schroeder
Tim Skern
The NMR (Nuclear Magnet
Resonance) facility of the
Max F. Perutz Laboratories
is based in building VBC5
in the Department of
Structural and Computational Biology of the University of Vienna. It currently houses a 500, two
600 and an 800MHz
spectrometer. In addition,
the unit also has IT facilities for structure calculations, molecular modeling and NMR data analysis.
Its mission is to apply state-of-the-art NMR spectroscopy it to solve biological problems and provide
the best possible NMR support and training to interested users and collaborators.
We offer a full range of NMR research services
from routine sample characterization to determination of solution-structures of biological macromolecules. Furthermore determination of biophysical
properties of proteins (e.g. diffusion measurement,
relaxation times, folding-/unfolding equilibria, ligand binding) is possible. The strength of NMR is
that in can go beyond structure and study dynamic
phenomena. Starting from known protein structures, or structures that have been determined in
cooperation by different methods (mainly X-ray
crystallography), NMR can be used to probe their
function and interactions.
NMR can also be applied in the complete absence
of tertiary structure to investigate intrinsically unstructured or disordered proteins (IUPs, IDPs), whose
role in biology is more and more appreciated. In
such cases NMR can detect residual structural elements and conformational preferences, which are
relevant for their biological roles and interactions.
Furthermore NMR is also routinely used to screen
various protein constructs to assess their likelihood
of crystallization.
NMR is applied in combination with other techniques in structural biology, especially X-ray crystallography, which is also available in house, but also
small angle X-ray scattering (SAXS) or electron
microscopy (EM) thus using a truly integrated approach to determination of biological structure and
function. We do this in collaboration with our colleagues from the Max F. Perutz Laboratories, the
Vienna Biocenter Campus and beyond.
Comparative NMR analysis of two variants of
the RNA binding protein Hfq: a truncated version HfqEc65 and wild-type full-length
HfqEc.containing a C-terminal extension (A)
Overlay of 15N-HSQC spectra of HfqEc65
(black) (and full-length HfqEc (red) demonstrating the unstructured nature of the C-terminus,
with multiple intense peaks occurring in the
7.5–8.5 ppm region of the 1H-dimension. (B)
Chemical 1H–15N shift differences plotted
against residue positions. (C) Ribbon representation of the proximal face and side of E. coli
Hfq (amino acids 6–65) hexamer. Chemical 1H–
15N shift differences between HfqEc65 and
full-length HfqEc are color-coded from blue
(zero) to red (largest shift) and mapped onto the
structure to illustrate the effect of the largely
unstructured C-terminus (data from BeichFrandsen et al. (2008))
Beich-Frandsen M, Vecerek B, Konarev PV, Sjöblom B, Kloiber K, Hämmerle H, Rajkowitsch L, Miles AJ, Kontaxis G, Wallace
BA, Svergun DI, Konrat R, Bläsi U, Djinovic-Carugo K. (2010), Structural insights into the dynamics and function
of the C-terminus of the E. coli RNA chaperone Hfq. Nucleic Acids Research (in print) n Coudevylle N, Geist L, Hötzinger M, Hartl M, Kontaxis G, Bister K, Konrat R. (2010), The v-myc-induced Q83 lipocalin is a siderocalin. J Biol Chem.
285(53), 41646-52. n Schedlbauer A, Auer R, Ledolter K, Tollinger M, Kloiber K, Lichtenecker R, Ruedisser S, Hommel U,
Schmid W, Konrat R, Kontaxis G. (2008), Direct methods and residue type specific isotope labeling in NMR structure
determination and model-driven sequential assignment. J Biomol NMR. 42(2), 111-27.
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F A C I L I T I E S
Plant Growth Facility
The plant growth facility has been established to
support educational and scientific needs of MFPL
scientists utilizing plants for research. The facility
encompasses greenhouses, controlled environment
growth rooms and growth incubators. A variety of
parameters including temperature, light and humidity can be adjusted to generate different growth
environments. Best control of parameters is possible
in incubators and in growth rooms, whereas greenhouses allow less strict control of conditions, but
offer more space. In total, more than 250 square
meters of space are available.
The facility has dedicated potting and service areas.
We have established a user fee system to contribute
to the running costs of the facility. Space in greenhouses, where conditions can be less well controlled, is cheaper than space in the walk-in growth
chambers or in incubators.
Expert personnel organizes the supply with consumables, and provides support in pest management, horticulture, and maintenance of the
facility. Users can choose between two types of
soil, to fit individual preferences.
Pest management occurs mainly through biological
measures. In particular, we use strains of the fungus
Trichoderma harzianum,
and of bacteria Bacillus
subtilis and Bacillus licheniformis as soil additives
to contain soil pathogens.
Regular administration of
nematodes prevents the
spread of fly larvae. Treatment with agrochemicals
is also possible in case of
plant diseases where biological measures are ineffective.
We have a set of mobile miniature temperature
and humidity probes to determine and record these
parameters on local spots in the plant growth area,
so that we routinely obtain information about
actual values and unexpected deviations from the
preset values.
One room contains laminar air flow boxes for seed
sterilization and plant tissue culture work. There is
space for growth of plant tissue culture and sterilized seedlings in two dedicated rooms. Currently,
these rooms deviate significantly from set temperature regimes, so that improvement of the cooling
system has been initiated.
Greenhouse (A), growth room (B), incubator (C) and tissue culture room (D) of the plant growth facility.
87
SCIENTIFIC SUPERVISORS
Andreas Bachmair
Andrea Barta
FACILITY HEAD
Marlene Zwettler
E D U C A T I O N
Promoting new generations of scientists
One of the MFPL’s biggest assets is
the strong focus on education and
training of young scientists. The
MFPL provide an exciting scientific
environment for diploma students,
PhDs and PostDocs in an outstanding
international community at the
Campus Vienna Biocenter.
Study Service Center
The Study Service Center provides information about
the study programs of the University of Vienna at
the MFPL, helps students and teachers with administrative procedures and organizes all teaching affairs
from the scheduling of lectures up to the awarding
degrees.
Located at the Campus Vienna Biocenter, the MFPL
enable students to participate in high quality research in an academic environment and to establish
connections with nearby companies and institutes.
Studies at the University of Vienna:
• Bachelor of Biology
• Masters of Molecular Sciences
• PhD programs
SSC TEAM
Studies at the Medical University of Vienna:
• Diploma in Human Medicine
• PhD in Medical Science
Renate Fauland (left)
Barbara Hamilton, Head (middle)
Angela Witte, Deputy Head (right)
Undergraduate Studies and Teaching
MFPL scientists participate in the undergraduate
curricula for students of the University of Vienna
and the Medical University of Vienna. In 2010, they
invested around 1200 hours to educate and inspire
new generations of scientists. Most of the practical
courses for the studies of molecular life sciences
take place on the premises of the MFPL. Over 460 m2
of teaching lab space was refurbished in the past
year to offer well equipped state-of-the-art training workspace for undergraduate students.
VBC Summer School
A number of our research groups participate in the
Vienna Biocenter Summer School, which offers
12 week courses for undergraduate students during
the summer months. Working side by side with our
scientists, the students can get insight into worldclass research and prepare for graduate studies in
molecular or cell biology.
www.vbcsummerschool.at
89
E D U C A T I O N
The Max Perutz International PhD Program
The MFPL are strongly committed
to provide interdisciplinary training
and research opportunities for PhD
students in a highly attractive and
inspiring research environment.
Our mission is to educate talented PhD students
to become excellent researchers with a competitive
professional profile, by fostering independence, inquisitive thinking and scientific rigor. MFPL are
currently home to 150 PhD students from more
than 30 countries who pursue their research in the
open, collaborative environment of the Campus
Vienna Biocenter. PhD students are recruited via a
structured selection and interview process usually
held twice a year. They have a primary affiliation
with one of the participating research groups, and
are enrolled as graduate students at the University
of Vienna or the Medical University of Vienna. All
MFPL PhD students are employed with a full-time
contract and a competitive salary conforming to
the guidelines of the Austrian Research Funds (FWF).
Additionally, the MFPL participate in the campuswide VBC PhD Program which includes our close
neighbors IMP, IMBA and GMI.
International Excellence
Doctoral Programs
The MFPL are proud to host three Doctoral Programs reviewed and funded by the Austrian Science
Fund (FWF). Each of these programs involves several
of our research groups and offers a specific curriculum fitting its scientific focus.
Molecular Mechanisms
of Cell Signaling
Cells manage to survive, proliferate, and differentiate in their environment by interpreting the signals
they receive from it and translating them into the
right output. If signaling goes awry, even only in
part of the cells, the whole organism is at risk.
The MFPL are home to a strong group of scientists
whose common long-term research goal is to
investigate and understand signal transduction mechanisms in a variety of cell-based and organismal
systems. The program offers structured, state-ofthe-art training in signal transduction and competitive PhD projects that combine biochemistry,
molecular biology, cell biology, and genetics to study
cell signaling in different model organisms.
PHD PROGRAM COORDINATOR
Alwin Köhler
SPEAKER
Manuela Baccarini
MFPL GRADUATE SCHOOL OFFICE
Gerlinde Aschauer
PROGRAM MANAGER
Elisabeth Froschauer
Thomas Decker, Roland Foisner, Pavel Kovarik,
Irute Meskiene, Egon Ogris, Friedrich Propst,
Christian Seiser, Graham Warren, Gerhard Wiche
Associated Groups: Claudine Kraft, Sascha
Martens, Florian Raible, Kristin Tessmar
B-Raf ablation perturbs ERK activation (brown staining)
in extraembryonic tissues but not in the embryo proper.
Superimposed: structure of a Mek1:Mek2 heterodimer
90
E D U C A T I O N
Structure and Interaction
of Biological Macromolecules
RNA Biology
Tim Skern
RNA biology is at the heart of
many exciting research areas
today. The consortium of this PhD
program unites researchers from
the MFPL, the Medical University
of Vienna, IMP, IMBA and CeMM,
to study main aspects of RNA processing (editing, splicing and folding), RNA localisation and degradation, RNA mediated translational
regulation (RNAi, microRNAs,
In silico analysis of RNA polymerase II binding elements.
small non-coding RNAs) and the
influence of small and long ncRNAs
on chromosomal function (DNA degradation, gene
silencing). Students have the additional advantage
of being integrated into the special research program on “Regulatory ncRNAs” and in two European
Networks of Excellence (EPIGENOME; EURASNET).
PROGRAM MANAGER
SPEAKER
The determination of a biological structure is the
starting point for understanding how macromolecules work and how they interact with their binding
partners. The doctoral program was created to examine the central themes of the thematic framework
in cooperation of scientists from MFPL, IMP and
IMBA. Projects within the doctoral program will
cover a comprehensive range of research areas introducing state-of-the-art techniques, methodology and theory to the PhD students. They are being
guided by a supervisor and a PhD committee, a
scheme that will ensure an intensive contact and
exchange of ideas between the students and the
faculty members.
SPEAKER
Ulrike Seifert
Dieter Blaas, Tim Clausen (IMP),
Kristina Djinovic Carugo, Robert Konrat,
Thomas Marlovits (IMBA), Jan-Michael Peters
(IMP), Peggy Stolt-Bergner (IMP)
Andrea Barta
PROGRAM MANAGER
Nicola Wiskocil
Denise Barlow (CeMM), Andrea Barta, Udo Bläsi,
Ivo Hofacker (Univ. Vienna), Michael Jantsch,
Michael Kiebler (Medical Univ. Vienna), Alwin
Köhler, Javier Martinez (IMBA), Kazufumi
Mochizuki (IMBA), Isabella Moll, Renée Schroeder
Associated Groups: Silke Dorner, Christina
Waldsich
PhD community
With more than 150 people from all over the
world, the PhD students form an active community at the MFPL. Their elected representatives
organize professional as well as social activities
and make their voices heard in MFPL’s decision
bodies.
PHD REPRESENTATIVES
Florian Kern (until Nov. 2010)
Mingliu Du (from Nov. 2010)
Sebastian Wienerroither (from Nov. 2010)
Interaction of a common cold virus pentamer (blue,
green and yellow) with a fragment of a cellular receptor,
the low density lipoprotein receptor (red).
91
E D U C A T I O N
VIPS – Vienna International Postdoctoral Program
VIPS is a pioneering program promoting outstanding young researchers
on their way to scientific independence. With support from the
Austrian government and the City
of Vienna, VIPS offers a three to
five year postdoctoral fellowship at
the MFPL, an individual research budget and travel money at free disposal.
The VIPS PostDocs will be encouraged and supported to develop their own research topics and to
apply for independent grants towards the end of
their fellowship. This will help them to establish
their first project that they will take with them
when they leave the MFPL to start their own research
groups as principal investigators.
VIPS PostDocs are selected through a highly competitive screening process in international calls.
Successful applicants are affiliated with one of
the eligible research groups and will also choose
an independent postdoctoral mentor from the faculty who will provide additional advice and career
support.
Career development for postgraduates
Besides the PostDoc positions, VIPS offers a wide
range of career development activities not only for
the VIPS PostDocs but for all postgraduates at MFPL:
•
•
•
•
•
mentoring program
individual coaching
grant writing support
communication trainings
family support and childcare.
The first VIPS PostDocs: Ana Catarina Ribero Carrao, Stephanie Bannister, Tobias Kaiser, Justyna Sawa-Makarska,
Bianca Mladek and Petronela Weisová with Program Manager Gabriele Permoser
92
E D U C A T I O N
First calls in 2010
VIPS Voices
In 2010, VIPS launched the first two calls for applicants, one in March and the second in September.
Out of over 350 applications answering the first
call, 6 outstanding young researchers were offered
PostDoc positions after a thorough selection in June
2010. Justyna Sawa-Makarska was the first of these
five successful candidates to take up pipettes and
put on gloves in October 2010. The other five PostDocs - Stephanie Bannister, Ana Catarina Ribero
Carrao, Tobias Kaiser, Bianca Mladek and Petronela
Weisová - will start at the beginning of 2011.
Justyna, why did you
apply for VIPS?
“The program itself is really
unusual and it gives additional opportunities that a regular PostDoc position doesn’t
offer. So obviously it was
really very tempting and if I
could combine it with staying in Vienna, this was
just the perfect situation.”
Justyna Sawa-Makarska originally comes from Poland
where she studied Biotechnology before she moved
to Vienna to do her PhD at the IMP.
VIPS SCIENTIFIC COORDINATOR
Renée Schroeder
PROGRAM MANAGER
Gabriele Permoser
PostDoc community
The PostDoc community at the MFPL comprises
over 90 young researchers. Their representatives
offer help and support and they organize joint
PostDoc activities.
POST DOC REPRESENTATIVES
Christelle Bourgeois & Jennifer Boots
(until November 2010)
Isabella Rauch & Heather Esson
(since November 2010)
Stephanie, how is it like
to move to Vienna from
Tasmania?
“I am really excited about living in a traditionally nonEnglish speaking country and
looking forward to learning
some German/Austrian!
Being from Australia, where the research community
is much smaller than in Europe or the UK, I feel it’s
also important for me to be exposed to a larger research community and have the opportunity to build
new networks for friendship, mentoring and collaboration.”
Stephanie Bannister comes from Tasmania in Australia. She did her PhD at the CSIRO’s Animal Health
Laboratory in Geelong, Australia.
Tobias, did you have any
preference for a certain
lab when you applied?
“Yes, I did. I was absolutely
determined to continue my
PhD work on lunar rhythms
by advancing to the molecular level. As far as I know, the
Tessmar group is the only group in Europe investigating the molecular biology of lunar rhythms.
Therefore, for me this was the place to go.”
Tobias Kaiser studied biology at the Universities of
Leipzig and Freiburg in Germany and at the ETH
Zürich. For his PhD, he joined the Max Planck Institute for Chemical Ecology in Jena.
93
VIPS is supported by
Federal Ministry of Science and Research
City of Vienna
S C I E N T I F I C
E X C H A N G E
The whole is greater than the sum of its parts
Research is a profession dependent
on collaboration and intellectual
exchange.
Participants of the PhD and PostDoc
retreat 2010
The MFPL PhD and Post Doc Retreat
At the Campus Vienna Biocenter, scientific exchange is not an exception, but an intrinsic quality
of the strong interconnection among all institutions,
basic research centres and companies alike. Regular
seminar series are organized by all institutions,
campus-wide as well as intra-institutional.
The weekly VBC Seminar Series, organized by the
IMP, serves as a platform for scientists from all
over the campus to exchange ideas and discuss
their research.
Apart from the regular seminar series, the MFPL students also organize annual retreats. In 2010, the PhD
and Post Doc Retreat was held in Krems. For the second
year, students were able to present their work in talks
and poster presentations to their colleagues and invited
guests. A total number of 55 PhDs and PostDocs participated in the retreat, which comprised eight talks
from MFPL scientific staff, two poster sessions for all
other students, two career-perspective talks and a
complementary social program with tours and excursions. The MFPL PhD and PostDoc retreat is organized
by students, for students and although the tradition is
only two years old, it is already a success story!
MFPL Regular seminars
The AKH Collaboration Day
The weekly MFPL Faculty Lunches serve as a platform for group leaders to present their work in a
chalk board talk to other faculty members and discuss new ideas and directions for their groups. The
bi-monthly MFPL Group Seminars offer the opportunity for PhDs, PostDocs and staff scientists
to give a progress report on their individual projects
to the whole Campus Vienna Biocenter. This keeps
colleagues up to date concerning the research done
at the MFPL and also offers the chance to gather
different perspectives on their work. In addition to
these regular talks, there are also thematically
focused seminar series, like the Seminar Series
“Modern Concepts in Structural Biology”, which
started at the end of 2009 and already saw more
than 40 talks by international speakers.
In 2009, four collaborative projects between researchers at the MFPL and the Medical University
of Vienna received collaboration grants to support
initial work on research projects to prepare for a
joint grant application for long-term funding. This
fostering of collaboration between clinicians and
basic researchers proved a great success and led
to several grant applications.
94
Half a year later, the awardees convened to present
their work. Furthermore, the Junior Group Leaders
Alwin Köhler and Gang Dong from the MFPL as
well as four selected researchers from the Medical
University of Vienna gave talks on their current
work to spark new ideas for collaborations in the
future.
S C I E N C E
C O M M U N I C A T I O N S
Talking about Science
Science Communications is an important topic for the Max F. Perutz
Laboratories. Our goal is to explain
the research done at the institute
and convey its implications for the
understanding of diseases and of the
world around us not only to other
scientists, but also to the general
public.
We want to foster interest for the natural sciences
in upcoming generations of researchers and correct
the often wrong perception of the unworldly,
misanthrope and confused scientist.
'MFPL researchers spinning the wheel': Karl Kuchler (above), Renée Schroeder (lower left) and
Andrea Barta (lower right).
The “Wiener Forschungsfest 2010”
The “Wiener Forschungsfest” as an initiative of the
“Zentrum für Innovation and Technologie (ZIT)” offers
an annual platform for researchers to present and
communicate their work in a public setting. From
the 18th to the 20th of September 2010, the Prater,
a traditional Viennese attraction for tourists, was
transformed into a hub for scientists and the interested public. In the middle of it were Renée Schroeder, Andrea Barta and Karl Kuchler of the Max F.
Perutz Laboratories. They joined their colleagues
from various institutes on the Vienna Giant Ferris
Wheel, where they explained their research to visitors
while enjoying a gentle ride in the red wagons
high above the ground.
Site-visit of the Federal Minister for Science and
Research
The goal of Science Communication is to make the
research done at the MFPL visible to the public.
This also includes welcoming national and international delegations from other research centers,
official bodies and politicians to the institute. In
October 2010, the Max F. Perutz Laboratories had
the pleasure of welcoming the Austrian Federal
Minister for Science and Research Dr. Beatrix Karl.
During her two-hour stay, we were able to offer a
broad perspective on the activities at the MFPL by
organizing a visit to our scientific facilities, teaching
and research labs. We were happy to show that
public funds allocated to MFPL are put to good
use and to see that our ongoing contribution to
the scientific expertise and international prominence of Austria is valued not only by colleagues
in the field, but also by politicians.
Beatrix Karl, the Austrian Minister for Science
and Research visiting the MFPL
95
S E R V I C E
A N D
S U P P O R T
IT-Support and Lab Technicians
IT SUPPORT
Christian Bernhard
Markus Klocker
Günther Leitgeb
Harald Nierlich
TECHNICIANS/LAB ASSISTANTS
Wolfgang Binder
Barbara Bublava
Sharif Duale
Andrea Fellner
Romana Finsterberger
Isabel del Pino Gomez
Mirjana Iliev
Elisabeth Jursa
Monika Kastler
Werner König
Harald Nierlich
Ralica Nikolova
Birgit Rapp
Harald Rumpler
Matthias Scheinast
Silvia Tömö
Fotima Touraeva
Administration and Office Support
FLOOR OFFICES
Gerlinde Aschauer
Maria Bausback
Katharina Haberler
Erna Huber
Thomas Lenert
Angela Martins
Jovana Nolic
Natasa Peric
Karin Pfeiffer
Rita Stadler
Sabine Tschanter
Gabriele Waidringer
Helga Wieltschnig
Sabina Winter
MFPL ADMINISTRATION
Georg Bauer
Wolfgang Binder
Romana Bohnenstingl
Ulrike Deuerling
Aini Kyynäräinen-Rennert
Barbara Miksch
Gabriele Schaller
96
R E S E A R C H
F U N D I N G
MFPL want to thank the following institutions for
financial support of research projects:
Owners
University of Vienna
Medical University of Vienna
Funding organizations and programs
AICR UK – Association for International Cancer Research, United Kingdom
BMWF – Austrian Ministry of Science and Research
GEN-AU – Genome Research Austria
CDG – Christian Doppler Research Association
DEBRA Austria
City of Vienna
DFG – German Research Foundation
EU – European Union
FEBS – Federation of Biochemical Societies
FFG – Austrian Research Promotion Agency
FWF – Austrian Science Fund
Herzfelder Stiftung
HFSP – Human Frontier Science Program
Johanna Mahlke geb. Obermann-Stiftung zur Förderung der Krebsforschung an der Uni Wien
ÖAW – Austrian Academy of Sciences
Theodor Körner Fonds
Wings for Life Spinal Cord Research Foundation
WWTF – Vienna Science and Technology Fund
97
S O C I A L
L I F E
Science is hard work – but work is not everything!
The Max F. Perutz Laboratories aim
at offering researchers an environment to develop their full potential.
This means not only providing our
scientists with adequate lab space
and tools to realize their research
plans, but also making sure that they
have the opportunity to relax every
once in a while and recharge their
batteries.
The Campus Vienna Biocenter Amateur Dramatic
Club (VBC-ADC)
Strength through diversity at the MFPL does not
only refer to research, but also to the personal
interests of our scientists. In 2010, a group of theatre
aficionados under the lead of Brooke Morriswood,
a PostDoc in the lab of Graham Warren, started
performing theatre on campus. The inaugural
production of “Amadeus” by Peter Schaffer, was a
great success and was followed by an on campus
open-air production of “A Midsummer Night’s
Dream” by William Shakespeare.
Happy Hours
Every last Thursday from April to October, volunteering research groups organize the famous MFPL
Happy Hours – regular institute-wide get-togethers
with food and drinks sponsored by the MFPL. These
parties are a unique chance to break the daily routine and get to know colleagues on a different
level than at the bench or in a seminar. Owing to
the creativity of the organizing scientists, the range
of themes and topics has no limits and mirrors the
diversity of nations, interests and people at the institute. In 2010, the MFPL saw many great parties
with themes like ”Brazil”, “Show your Nation”,
“Chopstick Challenge”, “Pirates” and “Folk”, just to
name a few of them, which leaves everyone in
anticipation of what the next season in 2011 will
bring!
“A Midsummer Night's Dream”
Carnival Happy Hour
98
S O C I AL
In December, the whole campus celebrated Christmas and was entertained by the annual Christmas
Pantomime, in which the classic story of “Peter
Pan” was presented in a remake full of anecdotes
and gossip about the institutes and the campus.
The VBC-ADC is open to everyone who is interested
in playing theatre.
MFPL Sports
Mind over matter only goes so far. The Max Perutz
Labs therefore offer all researchers at the institute
a convenient and easy way to stay in shape. The
newly instated sports committee is responsible for
the planning and organization of institute-wide
sport events and practices.
The 2010 Christmas Pantomime “Peter Pan”
This includes a variety of regular trainings for Badminton, Volleyball, Basketball, Climbing, Running
and Soccer. Apart from that, the MFPL also organizes special sport events such as the MFPL Ski-trip
or the participation in the Viennese Dragonboat
Cup, the Vienna City Marathon, the Cancer Research Run and many more.
The MFPL Dragonboat
Climbing
Basketball tournament on Campus
99
LI FE
P U B L I C A T I O N S
Publications 2010
Adlassnig W; Pranjic K; Mayer E; Steinhauser G;
Hejjas F; Lichtscheidl I. (2010), The Abiotic Environment of Heliamphora nutans (Sarraceniaceae): Pedological and Microclimatic Observations on Roraima Tepui, Braz. Arch. Biol.
Technol.,Vol.53, No.2, 425-430
Al-Dubai H; Oberhofer G; Kerleta V; Hinterwirth
H; Strobl M; Gabor F. (2010), Cleavage of antibodies using dihydrolipoamide and anchoring of
antibody fragments on to biocompatibly coated
carriers, Mon. Chem., Vol.141, No.4, 485-490.
Anzola, Jeanette Moulinier; Sieberer, Tobias; Ortbauer, Martina; Butt, Haroon; Korbei, Barbara;
Weinhofer, Isabelle; Müllner, Almuth Elise; Luschnig, Christian (2010), Putative Arabidopsis
transcriptional adaptor protein (PROPORZ1) is
required to modulate histone acetylation in response to auxin., Proc. Natl. Acad. Sci. U. S. A.
PMID 20479223.
Assinger, Alice; Koller, Franz; Schmid, Werner;
Zellner, Maria; Babeluk, Rita; Koller, Elisabeth;
Volf, Ivo (2010), Specific binding of hypochlorite-oxidized HDL to platelet CD36 triggers proinflammatory and procoagulant effects., Atherosclerosis PMID 20684828.
Auer, Renate; Hansen, D Flemming; Neudecker,
Philipp; Korzhnev, Dmitry M; Muhandiram, D Ranjith; Konrat, Robert; Kay, Lewis E (2010), Measurement of signs of chemical shift differences
between ground and excited protein states: a
comparison between H(S/M)QC and R1rho methods., J. Biomol. NMR PMID 20033258.
Auer, Renate; Kloiber, Karin; Vavrinska, Andrea;
Geist, Leonhard; Coudevylle, Nicolas; Konrat, Robert (2010), Pharmacophore mapping via crossrelaxation during adiabatic fast passage., J. Am.
Chem. Soc. PMID 20078057.
Barnat, Monia; Enslen, Hervé; Propst, Friedrich;
Davis, Roger J; Soares, Sylvia; Nothias, Fatiha
(2010), Distinct roles of c-Jun N-terminal kinase
isoforms in neurite initiation and elongation
during axonal regeneration., J. Neurosci. PMID
20534829.
Barta, Andrea; Kalyna, Maria; Reddy, Anireddy S
N (2010), Implementing a rational and consistent
nomenclature for serine/arginine-rich protein
splicing factors (SR proteins) in plants., Plant
Cell PMID 20884799.
Barta, Andrea; Schümperli, Daniel (2010), Editorial
on alternative splicing and disease., RNA Biol.
PMID 21140604.
Batova, Monika; Klobucnikova, Vlasta; Oblasova,
Zuzana; Gregan, Juraj; Zahradnik, Pavol; Hapala,
Ivan; Subik, Julius; Schüller, Christoph (2010),
Chemogenomic and transcriptome analysis
identifies mode of action of the chemosensitizing agent CTBT (7-chlorotetrazolo[5,1c]benzo [1,2,4]triazine)., BMC Genomics PMID
20202201.
Baubec, Tuncay; Dinh, Huy Q; Pecinka, Ales; Rakic,
Branislava; Rozhon, Wilfried; Wohlrab, Bonnie;
von Haeseler, Arndt; Mittelsten Scheid, Ortrun
(2010), Cooperation of multiple chromatin modifications can generate unanticipated stability
of epigenetic States in Arabidopsis., Plant Cell
PMID 20097869.
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Baudrimont, Antoine; Penkner, Alexandra; Woglar,
Alexander; Machacek, Thomas; Wegrostek, Christina; Gloggnitzer, Jiradet; Fridkin, Alexandra; Klein,
Franz; Gruenbaum, Yosef; Pasierbek, Pawel;
Jantsch, Verena (2010), Leptotene/zygotene chromosome movement via the SUN/KASH protein
bridge in Caenorhabditis elegans., PLoS Genet.
PMID 21124819.
Boban, Mirta; Braun, Juliane; Foisner, Roland
(2010), Lamins: 'structure goes cycling'., Biochem. Soc. Trans. PMID 20074079.
Böhmdorfer, Gudrun; Tramontano, Andrea; Luxa,
Kerstin; Bachmair, Andreas (2010), A synthetic
biology approach allows inducible retrotransposition in whole plants., Syst Synth Biol PMID
20805932.
Bourgeois, Christelle; Majer, Olivia; Frohner, Ingrid
E; Tierney, Lanay; Kuchler, Karl (2010), Fungal attacks on mammalian hosts: pathogen elimination requires sensing and tasting., Curr. Opin.
Microbiol. PMID 20538507.
Brunmeir, Reinhard; Lagger, Sabine; Simboeck,
Elisabeth; Sawicka, Anna; Egger, Gerda; Hagelkruys, Astrid; Zhang, Yu; Matthias, Patrick; Miller,
Wolfgang J; Seiser, Christian (2010), Epigenetic
regulation of a murine retrotransposon by a
dual histone modification mark., PLoS Genet.
PMID 20442873.
Burgstaller, Gerald; Gregor, Martin; Winter, Lilli;
Wiche, Gerhard (2010), Keeping the Vimentin Network under Control: Cell-Matrix Adhesion-associated Plectin 1f Affects Cell Shape and Polarity
of Fibroblasts., Mol. Biol. Cell PMID 20702585.
Carugo, O. (2010), Structural similarity between
native proteins and chimera constructs obtained
by inverting the amino acid sequence., Acta
Chim. Slov., 57, 936–940.
Carugo, Oliviero (2010), Clustering criteria and
algorithms., Methods Mol Biol PMID 20221920.
Carugo, Oliviero (2010), Clustering tendency in
the protein fold space., Bioinformation PMID
20975898.
Carugo, Oliviero (2010), Proximity measures for
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103
A R O U N D
M F P L
Campus Vienna Biocenter
The Campus Vienna Biocenter is one
of the most outstanding and prominent life sciences hubs in Austria
with more than 1,400 scientists
working in an area of 67,000 m2.
To support the researchers, the Campus has its own
day-care centre for children starting from three
months of age. Tailored to the needs of our highly
committed international staff the day-care center
is run by English-speaking personnel and offers
flexible opening hours.
The Campus Vienna Biocenter was founded in 1992,
as offspring of the close relationships between private enterprise and the molecular life science labs
of the University of Vienna. The core was comprised
of eight university departments (which now build
the MFPL) and the Institute of Molecular Pathology
(IMP), a research institute established by Boehringer
Ingelheim.
Access to state of the art infrastructure has become a decisive element for cutting edge research.
The Campus Vienna Biocenter developed a vision
for communal use of infrastructure. In 2010, new
campus science services facilities (CSF GmbH) with
a comprehensive range of new technologies were
set up with substantial financial support from the
Austrian Ministry of Science and Research and the
City of Vienna.
Today, the Campus is also home to the Institute of
Molecular Biotechnology IMBA and the Gregor
Mendel Institute for Molecular Plant Biology GMI,
a University of Applied Sciences, to several biotechnology companies such as Intercell and Affiris,
and to the Vienna Open Lab.
4
3
1
2
1 MFPL Main Building
IMP - Institute of Molecular
2 Pathology
IMBA - Institute of Molecular
3 Biotechnology and GMI - Gregor Mendel
Institute of Molecular Plant Biology
4 Intercell
104
D I R E C T I O N S
Contact
Max F. Perutz Laboratories
Dr. Bohr-Gasse 9, 1030 Vienna, Austria
T +43 1 4277-24001
F +43 1 4277-9240
[email protected]
www.mfpl.ac.at
Imprint
Published by
Editors
Pictures
Design/Layout
Max F. Perutz Laboratories GmbH
Gabriele Schaller, Georg Bauer with contributions from MFPL researchers
MFPL staff and scientists, Point of View, Barbara Mair
Friedrich Vesely, MSc | www.indeco.cc
105
M A X
F .
P E R U T Z
Dr. Bohr-Gasse 9, 1030 Vienna, Austria
T +43 1 4277-24001
F +43 1 4277-9240
[email protected]
w w w.mf p l .a c.a t

Annual Report - Max F. Perutz Laboratories (MFPL)

Transcription

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