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DFG Research Center for Experimental Biomedicine,University of Würzburg

Annual Report

2008

Welcome to the 2008 Annual Report of the Rudolf Virchow Center, the DFG Research Center for Experimen-tal Biomedicine of the University of Würzburg. This year has been an important and exciting one, withmany activities in research, teaching and public outreach, and most importantly many exciting research discoveries described in numerous publications. When a small group of researchers of the University fi rst developed the concept of a Research Center for Experimental Biomedicine, the idea was to create a center that is not only committed to excellent research, but also with a special focus on young researchers and the general public. In 2001, this idea was made possible with the funding of the DFG, the German Research Foundation. The Rudolf Virchow Center has by now become a highly dynamic and productive research center. International visibility was achieved not only through scientifi c publications, but also throughthe organization of international symposia and conferences, in Würzburg and elsewhere, membership in scientifi c academies, organizations and boards, and many national and international awards.Our research focuses on target proteins – proteins that exert key regulatory functions in a cell and may

serve as targets for diagnostic or therapeutic purposes, notably for cardiovascular diseases and cancer. Strong interdisciplinary collaborations in the Center, in Würzburg and elsewhere enable us to analyze these proteins from different perspectives, ranging from their molecular structure and mechanisms to their role in (patho-)physiological states.

Today, the Rudolf Virchow Center houses thirteen research groups in four areas – the Junior Research Groups, the Core Center Groups, the Research Professors and the associated Bio-Imaging Center (funded by the State of Bavaria and the University). In addition, the RVZ Network funds collaborative projects with seven extramural groups.

The Rudolf Virchow Center intends to remain a young center, and this entails constant change. This was particularly true for 2008. All members of the fi rst generation of group leaders successfully moved into senior positions here or elsewhere in Germany and abroad. We are very proud about the success of this fi rst generation and look forward to future collaborations with our alumni.

New group leaders were recruited who study target proteins with a special emphasis on the cardiovascular system. In 2008, Emmy Noether-Fellow Asparouh Iliev (coming from the European Neuroscience Institute in Göttingen) joined the Center. Current, ongoing recruitments concern Antje Gohla from the University of Düsseldorf, previously at the Scripps Research Institute, and Alma Zernecke from the RWTH Aachen. Additionally, Martin Eilers, University of Würzburg, Physiological Chemistry II, joined the Center with a new project in the RVZ Network. And Manfred Heckmann, a long-standing collaboration partner of Stephan Sigrist, has come from Leipzig and continues the Bio-Imaging research project on synaptic receptors. We are confi dent that the new generation of group leaders will continue to make the Rudolf Virchow Center a place of excellence in biomedical research as well as in teaching.

The Rudolf Virchow Center continues to play an active role in the research-oriented BSc/MSc-program in Biomedicine as well as in the Graduate School of Life Sciences, both realized together with the facul-ties of Sciences and Medicine. These programs consistently attract highly talented students from Germanyand abroad.

Our Public Science Center communicates our research and raises interest in biomedical science and re-search. In particular, it reaches out to elementary and high-school students. This year has been also a very event- and successful year for the Public Science Center. There was a marked increase in the number of media reports, a new school project for high-school students, the Virchowlab, was begun and, as a high-light, a team of scientists and the Public Science Center won the fi rst “Wissenschaft Interaktiv” Award by Wissenschaft im Dialog and the Stifterverband der deutschen Wissenschaft.

I hope that you will enjoy reading our 2008 Annual Report.

Chairman Rudolf Virchow Center/DFG Research Center for Experimental Biomedicine, University of Würzburg

Fore

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rdProf. Dr. Martin Lohse

I hope that you will enjoy reading our 2008 Annual Report.I hope that you will enjoy reading our 2008 Annual Report.

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ContentsRudolf Virchow Center 4

Research Activities 16

Junior Research Groups 18Stefan Engelhardt 18Heike Hermanns 20Asparouh Iliev 22Stephan Kissler 24Antje Gohla 26Alma Zernecke 27

Core Center 28Gregory Harms 28Caroline Kisker 30Hermann Schindelin 32Albert Sickmann 34

Research Professors 36Peter Friedl 36Bernhard Nieswandt 38Michael P. Schön 40Utz Fischer 42Manfred Gessler 44Thomas Hünig 46Thomas D. Müller 48Manfred Schartl 50Walter Sebald 52Martin Eilers 54Roland Jahns 55

Bio-Imaging Center 56Martin Lohse 56Stephan Sigrist/ Manfred Heckmann 58

Teaching & Training 60Training Activities 62

Public Science Center 66

Appendix 68Executive Committees and Scientific Members 68Academic Members and Supporting Staff 69Visiting Scientists 72Teaching Committees andBoard of the University of Würzburg Graduate Schools 73Bachelor and Master theses of the Undergraduate Program in Biomedicine 74PhD theses at the Virchow Graduate Program 76Publications 2008 79

Imprint 87

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Rudolf Virchow Center An Overview

Structurally, the Rudolf Virchow Center comprises seven areas:

Junior Research Groups: five-year groups with a tenure-track option, which are housed together to create a space of maximum freedom and dynamics,

Core Center: with long-term groups addressing basic mechanisms of protein structure and function with advanced technologies (structural biology, molecular microscopy, mass spectrometry and proteomics),

Research Professorships: offering established investigators the support and freedom to concentrate on a 5-year high-risk project; this area includes the RVZ Network, which offers 2-year support for collaborative projects within the Rudolf Virchow Center, or with other researchers in Würzburg,

Bio-Imaging Center: a more recent addition of four research groups in molecular biological imaging, funded by the State of Bavaria and the University,

Undergraduate Program in Biomedicine: a research oriented BSc/MSc- program with a focus on research training, and laboraory work.

A Graduate School of Biomedicine: developed from established interdisciplinary programs in various faculties and integrated into the Graduate School of Life Sciences.

Public Science Center: promoting the dialog with the public through press and media work, through several award-winning projects for children and high-school students, and public scientific debates.

An international Scientific Advisory Board: monitors the scientific progress and advises in recruiting Group Leaders and in awarding Research Professorships and RVZ Network projects.

Framework

The Rudolf Virchow Center – DFG Research Center for Experimental Biomedicine was approved by the Deutsche Forschungs-gemeinschaft in 2001 and established in January 2002. Its mission was the creation of a new center for target protein research at the interface between the medical and natural sciences.

The Rudolf Virchow Center follows two primary missions. Structurally, it aims to be a place that selects and recruits talented people at various stages of their careers ranging from pre-university to established investigators. It offers them maximum freedom and support for a generally limit-ed time to pursue training and research in experimental biomedicine, and it thereby creates a unique research institution un-der a common roof in a university setting. Scientifically, the Rudolf Virchow Center investigates “target proteins” – proteins that regulate key cellular functions and whose study may advance our understand-ing of the etiology, diagnosis or treatment of diseases. These proteins are investi-gated at various levels of complexity, rang-ing from their molecular structure to their (dys)function in animal models and human disease.

The Center aims to attract and recruit excellent scientists and to provide them with a maximum degree of freedom, but for a pre-defined time (5-year appointment, with tenure track option). These groups are recruited to either of two areas, “Junior Research Groups“ or “Research Professor-ships”, which allow junior or established researchers, respectively, to work on high-risk projects. The “Core Center” with long-term groups develops and utilizes new and expensive technologies in the areas of molecular microscopy, proteomics and structural biology. During the last years, the “Research Professorships” were com-plemented by the “RVZ Network”, in which collaborative projects are supported for two-year periods. More recently, another structural area was added, the “Bio-Imag-ing Center”: research professorships paid for by the State of Bavaria and the Univer-sity of Würzburg, which comprises groups that utilize advanced optical methods to analyze protein assemblies.

Fig. 1:Structure of the Rudolf Virchow Center.

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Fig.2:Academic Members oft the Rudolf Virchow Center at the annual Retreat 2008 in Rothenburg ob der Tauber.

Meanwhile, these structures have grown and evolved into a dynamic and fruitful environment for cutting edge research in the Center itself, as well as in national and international collaborations. The scientific success of all Junior Research Groups has propelled them into independent careers: all of them have developed internationally visible research projects and all of them have been offered professorial positions quite early during their 5-year period at the Rudolf Virchow Center. All members of the first generation successfully moved into senior positions here or elsewhere in Ger-many and abroad. Thus, while the overall structure remained largely constant during the last funding period, there have been major changes in its research groups.

One of our first Junior Group Leaders, Bernhard Nieswandt, who had been ap-pointed Research Professor in 2007, is now the new chair in Vascular Medicine, a position newly created by the Medical Faculty and the Rudolf Virchow Center. Gregory Harms will move to a Bio-Imaging position to build up a Technology Platform Microscopy in 2009.

New group leaders were recruited who study target proteins with a special em-phasis on the cardiovascular system. Stephan Kissler joined the center from the MIT in 2007, Heike Hermanns transferred from the RWTH in Aachen in 2007, and Asparouh Iliev joined the center in 2008 as an Emmy Noether-Fellow, coming from the European Neuroscience Institute in

Göttingen. Current, ongoing recruitments concern Antje Gohla from the University of Düsseldorf, previously at the Scripps Re-search Institute, with a research program on Rho-dependent signaling and the role of phosphatases, and Alma Zernecke from the RWTH Aachen, who studies the effects of chemokine receptors and their signaling in atherosclerosis.

Martin Eilers, University of Marburg, was recruited as a new chairman of Physi-ological Chemistry and joined the Center with a new RVZ Network project. And Manfred Heckmann from Leipzig became chairman of Physiology and continues the research program of his long-standing collaboration partner Stephan Sigrist on synaptic receptors.

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Fig. 3:“Hereinspaziert – Biomedizinische Forschung XXL!” Model of an artery and the development of a thrombus presented by a team of scienctists and the Public Science Center at the Science Summer in Leipzig 2008.

The next generation of scientists

A key element of the Rudolf Virchow Center is the close intertwining of research with undergraduate and graduate training. This sets it apart from similarly equipped non-university institutes. The aim of the Rudolf Virchow Center is not only to add scientific and experimental elements to teaching, but also to teach and train the next generation of future scientists as undergraduate and graduate students.

Undergraduate and graduate training

A new research-oriented undergraduate BSc/MSc-program “Biomedicine“ was established together with the faculties of Sciences and Medicine and is coordinated by the Rudolf Virchow Center. This program is aimed at future scientists at the interface of science and medicine. Therefore, the students are integrated early on into the research environment of the Rudolf Virchow Center as well as other research in-stitutes. The program continues to attract large numbers of excellent students (900 applications per year), allowing us to be highly selective since there are on average ~30 applicants per slot. In 2008, the third group of students received their MSc diplo-mas (all of them with excellent grades), while the fifth group of BSc students com-pleted their studies. We are excited to see that most of them (85%) continue their careers in science.Structured graduate training helps to at-tract good graduate students and to offer them what they need for a career in sci-ence. To this end, the Rudolf Virchow Center initiated several years ago, to-gether with other graduate programs, a Graduate School of Biomedicine. In 2006, this program was integrated into the new, larger Graduate School of Life Sciences, which won support in the na-tional “Excellence Initiative“. Our gradu-ate program offers laboratory courses, soft skills and seminars that complement the students’ research projects. In 2009, a new group, from all over the world, will start their graduate work.

Attracting people to science

In line with the overall concept of the Rudolf Virchow Center to attract the brightest minds to science early on, we start with the very young. Our Public Science Center offers a broad spectrum of programs for young people from eight to eighteen years of age. Demand for participation in all projects is extreme-ly high, with nearly 800 young people taking part from the start of the first pro-ject in 2004. From simple experiments to scientifically ambitious projects in a real laboratory and in close contact to scien-tists – different demands can be met re-sponding to age and interests. Various awards, grants and collaborations mirror the overall success of these projects.

The Public Science Center is furthermore committed to promoting the dialog be-tween science and society. It plays an im-portant role in making the Rudolf Virchow Center but also biomedical research in

general visible to scientists and to the general public. This annual report is dis-tributed to research centers and scientists world-wide and is also available on our website.

47 press releases in 2008 resulted in more than 600 reports in print, radio, TV and online media, increasingly with national distribution. In 2008, the Rudolf Virchow Center provided insight into its research at several public events, such as the Science Day in September 2008 and the Science Summer 2008 in Leipzig. At Germany´s biggest science festival, a team of the Public Science Center and four young scientists presented their interactive exhibit “Herein-spaziert – Biomedizinische Forschung XXL!“ This exhibit received the first “Wissen- schaft Interactiv” Award by Wissenschaft im Dialog and the Stifterverband der deutschen Wissenschaft.

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Rudolf Virchow CenterResearch Program

In all its elements, the Rudolf Virchow Center has been guided by the successful principle of giving talented scientists the opportunity to realize their scien-tific dreams. Thus, while the general topics of research are defined by the overall theme, it is the primary goal of this Center to attract and to support the very best scientists.

Fig. 4:Target proteins investigated at the Rudolf Virchow Center.

The research pursued at the Rudolf Virchow Center deals with target proteins – proteins that exert key regulatory functions and are, therefore, good candidates for un-derstanding the etiology of diseases and

potentially also for their diagnosis and treatment. Major questions regarding these proteins address their folding and mobil-ity, the ways they are modified and how they are ultimately degraded. Recogni-tion between proteins and small ligands is essential to understand how the function of signaling proteins such as receptors can be regulated and how activation switches work. Similarly recognition between pro-teins and nucleic acids is fundamental for signaling to the nucleus and for the use and maintenance of genetic information. This research can, therefore, be grouped

into four Research Fields: (1) Protein Struc-ture and Function, (2) Proteins in Cellular Signaling, (3) Nucleic Acid Binding Pro-teins, and (4) Proteins in Cell-Cell Interac-tions and Motility. The main projects that are studied reflect the focus on cell surface proteins and their signaling proteins and on nucleic acid binding proteins. While each group studies its own set of proteins, as indicated in the figure, many projects are carried out in collaborations involv-ing two or more groups that provide either different technologies or complementary biomedical expertise.

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Rudolf Virchow Center

Fig. 5:Protein structures provide evolutionary insights. (A) Structure of the ubiquitin-activating enzyme (Lee and Schindelin, Cell, 2008) in complex with ubiquitin (yellow). Active and inactive adenylation domains are shown in magenta and cyan, respectively. (B) Structure of the heterotetrameric MoeB-MoaD complex (Lake et al., Nature, 2001) in an equivalent orientation with the MoeB monomers in magenta and cyan and MoaD subunits in yellow and orange.

Research Field 2 – Proteins in Cellular Signaling

Signaling between and within cells is key to coordinated functions in all living or-ganisms. The proteins that are involved in such signaling processes are, therefore, of fundamental importance for life, their dysregulation often causes diseases, and therapeutic drugs often target these sig-naling proteins. Receptors, which are most often localized at the cell surface, are the

most important class of signaling proteins. They receive signals from other cells (hor-mones and transmitters) and then activate signaling processes in the cell interior that ultimately cause cellular reactions.Various signaling pathways are investigat-ed at the Rudolf Virchow Center, for exam-ple those that are triggered by G-protein-coupled receptors and receptor tyrosine kinases. These systems are investigated at various levels of complexity, ranging from the molecular understanding of receptor/

ligand binding interfaces and the receptor activation process to the study of complex physiological responses. Key molecular questions that are studied in these model systems concern mechanisms of recogni-tion in signaling systems and how intra-cellular signals are patterned in space and in time. These molecular mechanisms are linked to (patho-)physiology, with a spe-cial focus on molecular mechanisms (see Figure 6) that form the basis of major car-diovascular diseases.

Groups in Research Field 2:

Stefan EngelhardtGregory HarmsHeike HermannsStephan KisslerBernhard NieswandtAlbert SickmannMartin LohseThomas HünigThomas D. MüllerManfred SchartlWalter Sebald

Fig. 6:Molecular mechanisms are linked to cardiovas-cular diseases: miR-21, a microRNA is found in failing hearts. MiR-21 RNA detected by in situ

hybridization in non-failing (wild-type) and failing (ß1-adrenergic receptor transgenic mice)

left ventricular myocardium. The miR-21-expressing cells (black arrowheads) are

localized in the interstitium between the cardiomyocytes. Scale bar: 100 µm.

Research Field 1 - Protein Structure and Function

Cellular proteins adopt defined structures, which are necessary for their specific func-tions. The fundamental mechanisms of pro-tein folding, modification and degradation are intricately linked to their molecular function and also, ultimately, to their roles in physiology and pathophysiology. In this research field, scientists utilize a variety of techniques for the study of structural as-pects of biological macromolecules, which

allow the visualization of these players at different levels of resolution and their iden-tification as well as the characterization of modifications. X-ray crystallography, high-resolution microscopy and mass spectrom-etry are the key methods in this research field. These approaches are utilized to gain insights into how newly synthesized proteins are folded in the endoplasmic reticulum, how they are targeted for deg-radation, and how large protein complexes are assembled in order to perform cellular signaling.

A common goal of this research field is the understanding of specificity and affinity in bio-molecular recognition processes. An example of how nature recycles modules with certain capabilities and alters and combines them to achieve new functions is shown in Figure 5: The ubiquitin-activating enzyme, a first element in a protein degra-dation pathway, shares an overall structure with the MoeB/D protein complex in bac-teria. This suggests that the two protein complexes of highly divergent functions share a common evolutionary ancestor.


Gregory HarmsCaroline KiskerHermann SchindelinMichael SchönAlbert SickmannManfred HeckmannStephan SigristThomas MüllerWalter Sebald

Research Program

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Rudolf Virchow CenterResearch Field 3 – Nucleic Acid Binding Proteins

Interactions between proteins and nucleic acids (DNA and RNA) are central to all as-pects of the maintenance and realization of genetic information. Again, recognition processes represent a fundamental aspect of these interactions. Several pathways that rely on protein-nucleic acid interac-tions are studied at the Rudolf Virchow Center through a combination of struc-tural, biophysical and biochemical tech-niques. They encompass the DNA damage recognition pathway (see Figure 7), which involves sophisticated mechanisms to ex-cise pieces of damaged DNA and to replace them with the original sequence; the p53 tumor suppressor family, which initiates transcriptional programs that ultimately arrest proliferation and prevent the gen-eration of genetically altered cells; several transcription factors, for example Hey, Nab and Myc, which regulate growth and differ-entiation in the cardiovascular system and in cancer; the spliceosome, which catalyzes the removal of non-coding sequences from pre-mRNAs; and finally the so-called TOP response, that regulates the level of trans-lation in response to the nutritional status

of a cell via proteins that bind to specific sequences in RNA (so-called TOP motifs).

Analysis of these pathways will provide an understanding of how the intricate interactions of individual proteins or of

Fig. 7:Model of the XPD-DNA complex, which is a key component of the DNA-damage repair machinery (Wolski et al., PLoS Biol, 2008). The different domains of the XPD protein (named after a disease that mutations in this protein may cause, Xeroderma pigmentosum) are indicated by different colors. The DNA, which is recognized and bound by the XPD protein, is shown as an orange ribbon with the bases as spikes.

Research Field 4 – Proteins in Cell-Cell Interaction and Motility

Interactions of cell surface proteins with the extracellular space and regulation of the cytoskeleton determine cellular adhe-sion and motility. Cell adhesion and mi-gration are central to diverse homeostatic processes – for example the mounting of an effective immune response or the re-pair of injured tissues. Failure of cells to migrate, or migration of cells to aberrant

locations, is intricately involved in many pathologies including vascular and inflam-matory diseases as well as tumor formation and metastasis (see Figure 8). Several tar-get proteins were identified at the Rudolf Virchow Center that regulate the interac-tions of a cell with its micro-environment. The Rudolf Virchow Center has also devel-oped methods to visualize, analyze and manipulate these dynamic processes from single molecules to protein complexes and from cultured cells to living animals. To

Fig. 8:Proteolytic digestion of collagen tissue (grey) is dependent on and promotes

collective invasion. Multicellular invasion strands form along proteolytic

tracks (blue). Abrogation of multicel-lular proteolytic invasion (left) and persistent non-proteolytic single-cell

movement in the presence of proteaseinhibitor (right). Image taken

from Wolf et al., Nature Cell Biol, 2007.


Peter FriedlAntje GohlaGregory HarmsAsparouh IlievBernhard NieswandtMichael SchönAlbert Sickmann

assess the potential importance of candi-date proteins that may provide keys to the prevention or treatment of major diseases, in vivo mouse models of cardio- and cere-brovascular diseases, (auto-)immune disorders, and malignant tumors have been established. Together, this multidisciplinary approach has not only provided fundamen-tal insights into the biology of cell-cell interactions, but has also identified target proteins that hold promise for the develop-ment of new therapeutic strategies.


Stefan EngelhardtPeter FriedlGregory HarmsCaroline KiskerAlbert SickmannThorsten StieweMartin EilersUtz FischerManfred Gessler

multi-protein complexes with nucleic acids lead to the formation of higher order com-plexes required to maintain the genomic integrity and to realize genomic programs in the cell.

Research Program

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Rudolf Virchow CenterBiomedical focus

Like any protein, target proteins are often expressed in many different cells and tis-sues and may, therefore, be involved in several, yet very different (patho-)physi-ological functions. While their basic struc-tural and functional mechanisms may be the same in all these situations, the study of a given protein can provide insight into a whole variety of physiological processes and diseases and can lead into very differ-ent biomedical fields. This is where the in-terdisciplinary nature of the Rudolf Virchow Center and the fact that it is embedded in a large network of biomedical research comes into play.The many implications that research on a given target protein may have, can best be illustrated with an example. A protein currently studied by several Rudolf Virchow Center groups is called “stromal interaction molecule 1” (STIM1). STIM1 appears to rep-resent the long postulated “missing link” that connects depletion of intracellular Ca2+ stores to the opening of a specific group of calcium channels at the cell surface, in order to “refill” the cell with calcium.

The group of Albert Sickmann observed by mass spectrometry that STIM1 is abundant-ly expressed in platelets. This prompted the group of Bernhard Nieswandt to inves-tigate its in vivo functions, and this then catalyzed studies on other functions of this protein by several groups of the Rudolf Virchow Center. Studies on a mutation in STIM1 (called Sax) indicated that STIM1 might be a critical regulator of Ca2+ entry into platelets. STIM1-deficient mice were then generated. Stim1-deficient platelets showed major functional defects in vivo, revealing that this pathway of Ca2+ entry is of paramount importance for thrombus sta-bilization. Subsequent collaborations with the Department of Neurology in Würzburg showed that the lack of STIM1-dependent Ca2+ entry protected mice from the devel-opment of experimental ischemic stroke. Further studies with a second knock-out mouse line identified the so-called Orai1 protein as the Ca2+ channel that is activat-ed by STIM1; these studies further showed that also Orai1 is essential for pathological thrombus formation.

Investigations on the role of STIM1 in immune cell function were undertaken in collaboration with the Institute of Im-munology in Würzburg and revealed that STIM1-dependent Ca2+ entry is also essen-tial for homeostatic proliferation of thymic T-cells.

The important role of STIM1 in the regu-lation of Ca2+ entry in platelets and in T-cells has prompted collaborations of sev-eral groups at the Rudolf Virchow Center to study its function in various organs and biological systems, to investigate its biochemical and cellular regulation and to search for cellular proteins that interact with STIM1.

Fig. 9:Left: Model of STIM1-dependent calcium entry in platelets. Stimulation of membrane receptors (R) triggers release of Ca2+ from the endoplasmic reticulum (ER) via the second messenger inositol trisphosphate (IP3). This causes a decline of Ca2+-concentrations in the ER, the cellular store for calcium. STIM1 detects this decline and activates a calcium channel (Orai1) in the cell membrane to allow sustained Ca2+ influx. Middle: Schematic drawing of STIM1 structure. The location of the Sax mutation is indicated. Right: Mouse embryos with the Sax-mutation in the STIM1-gene show severe hemorrhage in different regions of the body (Grosse et al., J. Clin. Invest., 2007).

Research Program

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Technologies

In order to study target proteins at various levels of complexity, a whole range of tech-nologies and methods has been established at the Rudolf Virchow Center. The special framework of the Center, with its wide spectrum of cutting edge analytical tech-nologies under one roof, strong collabora-tions between the research groups and the close connection to clinical research, offers excellent opportunities to analyze these proteins at different levels of complexity, ranging from their molecular structure and function to biochemical mechanisms, cel-lular responses, and (patho-)physiological roles. A particular focus is the visualiza-tion of biological macromolecules at dif-ferent levels of resolution – ranging from atomic structures to imaging in the whole body. The technologies that have been es-tablished and are now utilized are depicted in Figure 10.

Fig. 10:Technologies used for the visualization and imaging of target proteins at the Rudolf Virchow Center.

They range from methods for the inves-tigation of single proteins and their mo-lecular structures – such as X-ray crystal-lography and atomic force microscopy – to methods that assess complex biomedical functions such as cardiac catheterization and ultrasound, in vivo platelet function microscopy and the generation of the respective genetically modified mouse models. Optical methods have gained a particularly prominent status, with the es-tablishment of several technologies that elucidate information at the nm-scale: fluorescence resonance energy transfer (FRET) and fluorescence lifetime imaging (FLIM) measurements, stimulated emission depletion (STED) microscopy and single molecule fluorescence microscopy permit the analysis of protein-protein interactions and conformational changes as well as the direct optical resolution of larger proteins and protein complexes in living cells.

Other techniques include various mass spectrometry technologies that are essen-tial for the identification of proteins and the analysis of their modifications; trans-genic mouse technologies by classical as well as lentiviral transgenic, knock-out and RNAi-technologies; and a DNA array unit was established in collaboration with the Interdisciplinary Center for Clinical Re-search (IZKF), providing access to custom-made as well as commercial array analyses. All of these technologies are not only used intensively by research groups within the center, but also by other groups in Würz-burg and elsewhere.

Partnering with industry

Collaborations with companies of various sizes and product portfolios are an impor-tant part of research at the Rudolf Virchow Center. They are organized into multiple forms. The research group of Stefan Engelhardt is fully funded by the biotech company Procorde/Trigen, Martinsried, Sanofi-Aventis, Frankfurt, and the Bavar-ian Ministry of Economics. Other groups receive specific funding from companies, mostly in the context of projects funded by the Federal Ministry of Research and Educa-tion (BMBF) and similar sources, but also through direct collaborations.

Several new microscopic instruments were co-developed in formal collabora-tions with optical companies. For example, a multi-photon platform for optical imag-ing in vivo was constructed with LaVision BioTec (Bielefeld) by the Groups of Gregory Harms and Peter Friedl; new, rapid FLIM detectors for this microscope are currently being developed as an RVZ Network proj-ect. A new type of microscopy platform, called iMIC, is being engineered with Till Photonics (Gräfelfing), in a BMBF-funded collaboration. Formal collaborations with Leica concerned the development of a TIRF-microscope that is suitable for multi-color detection and FRET-measurements. A second collaboration with Leica, an exten-sion of a collaboration between Stephan Sigrist and Stefan Hell at the Max Planck Institute for Biophysical Chemistry in Göt-tingen, was the development of a com-mercial high-resolution STED microscope; this type of fluorescence microscopy allows

resolution below the diffraction limit and is used at the Rudolf Virchow Center most extensively in imaging of receptors and synaptic proteins.

Similarly, the Group of Albert Sickmann (Mass spectrometry) enjoys close contacts with multiple companies in order to stay at the forefront of new developments; an ex-ample is the development of new HPLC col-umn materials with Dinoex, Sunnyvale, CA.

Some of our research projects have led to patents, which are usually held by the University; in some instances, patents were applied for together with biotech, pharma-ceutical or technology companies.

A new transfer project that received ma-jor funding for technology transfer is car-ried out in collaboration with the Institute of Pharmacology and Toxicology and the Department of Medicine. This project, led by Roland Jahns, intends to develop new therapies against receptor autoantibod-ies in heart failure. It won funding as one of twelve projects in the “Exist GoBio“ competition of the BMBF and led to the foundation of a new spin-off company called CorImmun.

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Visits

Many events were organized for the gen-eral public, for students and for scientists. Politicians like representatives of the Ba-varian State Ministry of Sciences, Research and the Arts and Members of the Bavarian Parliament like Prof. Walter Eykmann and Manfred Ach visited the Center.

In February 2008, the State Minister Joachim Hermann, Bavarian State Minis- try of the Interior together with the Mayor of Würzburg, Dr. Pia Beckmann, presided over the “Richtfest” for the new building for the Rudolf Virchow Center and the Center for Infectious Disease Research. Almost 10,000 m2 of space will be generated by a total con- version of the old surgical hospital; the building approaches completion in 2009 and will provide excellent facilities.

Rudolf Virchow CenterEvents

Fig. 11:The new building for the Rudolf Virchow Center. Left: Construction work. Right: “Richtfest” in February 2008.

Workshops

A number of symposia and courses were organized at the Rudolf Virchow Center in 2008. Among recent conferences, the fifth “Proteomics Workshop“ (September 07-10),

Fig. 12:In 2008, the Rudolf Virchow Center invited scientists for several outstanding symposia and workshops: For example the “Proteomics Workshop” and the “Dynamic Microscopy” workshop.

organized by the Group of Albert Sickmann, provided an introduction into the theory and practical aspects of these techniques, with external experts and tutors from the

Rudolf Virchow Center. Renowned speakers from the field of proteomics gave lectures on the latest developments. Speakers were: Peter Roepstorff (Odense, Denmark) Kris Gevaert (Gent, Belgium), André Deelder (Leiden, the Netherlands), Friedrich Lott-speich (Martinsried), Henning Urlaub (Göt-tingen), Kai Scheffler (Thermo Scientific), Lennart Martens (Cambridge, UK), and Helmut E. Meyer (Bochum). This year, the fourth workshop “Dynamic Microscopy” was organized by the Groups of Gregory Harms and Stephan Sigrist (September 22-24). The symposium featured internationally renowned speakers in the fields of opti-cal and scanning microscopy, including: Antoine Triller (Paris, France), Stefan Hell (Göttingen), Petra Schwille (Dresden), Jan Huisken (San Francisco, USA), Andreas Zumbusch (Konstanz), and Jörg Wieden-mann (Southampton, UK).

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Science Day

In September 2008, the Rudolf Virchow Center opened its doors to children and high-school students, launching the Virchowlab and celebrating the award of its school projects as a winner in the contest “Deutschland - Land der Ideen”. Guided lab tours and several hands-on experiments showed the everyday life in a research center. Several hundred people visited the Center. As a special highlight, the “Wissenschaft Interaktiv” exhibits were on display.

Fig. 13:Science Day 2008 at the Rudolf Virchow Center: Hands on for everyone!

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Funding

Apart from support by the State of Bavaria and the University of Würzburg, our main source of support is the core funding by the DFG with €5 million per year (plus overhead costs of €1 million). The DFG funding is complemented by grants from various sources (Fig. 14) totaling more than €6.4 million in 2008 (i.e. about 130% of core funding).

Publications

Publications are the main reflection of an academic research center. Since 2002, the total number of peer reviewed publica-tions by groups and members of the Rudolf Virchow Center was 1758, with 578 com-ing directly from funding by the Center. The scientific quality of these publications is evidenced by the fact that 21% of our publications appeared in the top 1% jour-nals, and almost 85% appeared in the top 10% journals. The normalized relative field impact, a size-independent measure of sci-entific impact developed by the Center for Science and Technology Studies, Leiden, was 3.05, which is considered excellent. Members of the Rudolf Virchow Center have been cited more than 30,000 times for over 1700 papers published since 2002. Finally, benchmarking shows citation rates similar to renowned institutes of comparable ori-entation and size in Germany and in the US (Figure 15).

Although individual projects and their results are always the key element in evaluating our work, it is helpful to look at some commonly used indicators in order to gauge our overall performance. Apart from the fact that we have attracted scientists from more than 20 countries, the key figures in such analyses are grants, publications with their bibliometric analysis, awards and the careers of our scientists.

Rudolf Virchow CenterOutput and Evaluation

Fig. 14:Sources of extramural funding at the Rudolf Virchow Center (Abbreviations: DFG: German Research Foundation; BMBF: Federal Ministry of Education and Research; BayStMWIVT: Bavarian Ministry of Economic Affairs and Technology; IZKF: Interdisciplinary Center for Clinical Research; EU: European Union; NIH: US National Institutes of Health).

Fig. 15:Benchmarking of citations with institutes of comparable orientation in Germany and in the US shows similar results. (blue: Rudolf Virchow Center; yellow: Max Planck Institute for Experimen- tal Medicine, Göttingen; green: Max Planck Institute for Medical Research, Heidelberg, red: Beckman Center, Stanford University, USA).

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Collaborations

A large number of collaborations show that the Rudolf Virchow Center plays an increas-ing role in the biomedical research commu-nity, locally, nationally and in international networks. Local collaborations indicate that it fulfills its intended role as a back-bone for biomedical research in Würzburg. Most major collaborative research projects in the life sciences in Würzburg involve the active participation of the Rudolf Virchow Center (Fig. 16).

Fig. 16:Collaborations of the Rudolf Virchow Center at the University of Würzburg. Shown is the active participation (i.e. common projects with publications and funding) in collaborative projects (DFG funded projects: blue, BMBF funded projects: yellow)

To foster collaborations with other groups in Würzburg, we initiated the RVZ Network in 2006. Following review by the Scientific Advisory Board, high-risk projects are funded that are carried out by research groups in Würzburg in collaboration with

Awards

Members of the Rudolf Virchow Center re-ceived numerous prestigious national and international awards. During the past year, this included the German Cancer Award (Peter Friedl), the GlaxoSmithKline Award (Bernhard Nieswandt), and the Analytica Research Award (Albert Sickmann).

Science Careers

Many of our members, including all junior group leaders, have received offers for professorial positions in Germany and abroad, and the first generation of group leaders has by now successfully moved into senior positions. Last year, Bernhard Nieswandt, one of the first three junior group leaders, had been awarded a Re-search Professorship at the Rudolf Virchow Center; this year, responding to a compet-ing offer, he accepted the newly created Chair in Vascular Medicine.

Several group leaders of the Rudolf Virchow Center have accepted attractive offers

from leading institutions in Germany and abroad – Thorsten Stiewe became Professor for Molecular Biology and Tumor Research at the University of Marburg, Peter Friedl assumed a Professorship for Microscopical Imaging of the Cell at the Radboud Uni-versity Nijmegen, Albert Sickmann became Professor and Chair of the Leibniz Insti- tute of Analytical Sciences, Dortmund, Stefan Engelhardt is now Professor and Chair of the Institute of Pharmacology and Toxicology, TU München, Stefan Schulz is now Professor and Chair of the Institute of Pharmacology and Toxicology, University

of Jena, Stephan Sigrist accepted a Chair at the Institute of Genetics, FU Berlin, and Michael Schön became Professor and Chair of Clinical Dermatology, University of Göttingen.

It is too early for a formal evaluation of the alumni of our teaching programs, but we do stay in contact with them and follow where they go and how they fare. It is a good sign that most undergraduate as well as graduate students have stayed in sci-ence and have continued their research in institutions all over the world.

groups at the Rudolf Virchow Center. Groups of the Rudolf Virchow Center itself can also apply for these funds, thus allowing a flexi-ble allocation of resources. Seven projects have been funded in 2008, and several pub-lications from these projects are under way.

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Stefan EngelhardtHeike HermannsAsparouh IlievStephan KisslerAntje GohlaAlma Zernecke

Gregory HarmsCaroline KiskerHerrmann SchindelinAlbert Sickmann

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Peter FriedlBernhard NieswandtMichael P. Schön

Martin LohseStephan Sigrist/ Manfred Heckmann

Junior Research Groups

Core Center

Research Professors and RVZ Network

Bio-Imaging Center

Sc ience

Utz FischerManfred GesslerThomas HünigThomas D. MüllerManfred SchartlWalter SebaldMartin EilersRoland Jahns

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Core Center

Research Professors and RVZ Network

Bio-Imaging Center

17

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Stefan Engelhardt

E-mail: [email protected]: +49(0)931 201 487 10Fax: +49(0)931 201 481 23http://www.rudolf-virchow-zentrum.de/forschung/engelhardt.html

The Cardiac Target Protein group investigates cellular signaling mechanisms in the myocardium. Our current ef-forts focus on the role of microRNAs in cardiac health and disease, mechanisms of β-adrenergic receptor signal-ing and intercellular communication in the heart. Our methodological spectrum ranges from optical studies of receptor conformational changes, basic cell and molecular biology techniques to the analysis of cardiac function in living mice.

Role of microRNAs in cardiac healthand disease

MicroRNAs comprise a broad class of small non-coding RNAs that control expression of complementary target mRNAs. Dysregula-tion of microRNAs in various disease states is caused by altered transcription, muta-tion, epigenetic change, or viral infection. MicroRNAs were recently implicated in the regulation of diverse cardiac functions. Al-though these studies demonstrate roles for microRNAs in heart physiology, growth and morphogenesis, the molecular mechanisms of microRNA function in disease pathways in vivo are as yet poorly understood. Single-stranded oligonucleotide microRNA antago-nists can silence endogenous micro-RNAs in vitro and in vivo, resulting in effects on target mRNA and protein levels. These fi nd-ings have pointed to the potential utility

Fig. 1:Inhibition of miR-21 inhibits cardiac hypertrophy and fi brosis. A synthetic miR-21 antagonist (Antagomir-21, Ant-21) was applied on three consecutivedays after transverse aortic constriction (TAC) that induces pressure overload of the left ventricle. Three weeks after TAC, the myocardium was analyzedfor cardiomyocyte dimensions and interstitial fi brosis. White bars represent control treated animals, black bars represent antomir-21 treated animals.(Modifi ed from Thum, Gross et al., Nature, 2008).

of such so-called “antagomirs” as tools for validating microRNA function in vivo, and perhaps more importantly, as a novel ther-apeutic agent. We are currently focusing on the identifi cation of micro-RNAs, that are specifi cally expressed in certain car-diac cell types. Both transgenesis and tar-geted silencing approaches are employed to elucidate their function in physiologyand disease.

We recently found that microRNA-21 (miR-21) regulates the ERK-MAPkinase signaling pathway in cardiac fi broblasts, which impacts on global cardiac struc-ture and function. MiR-21 levels are in-creased selectively in fi broblasts of the failing heart, augmenting ERK-MAPkinase activity through inhibition of sprouty1.

This mechanism regulates fi broblast sur-vival and growth factor secretion appar-ently controlling the extent of interstitial fi brosis and cardiac hypertrophy. In vivo silencing of miR-21 by a specifi c antagomir in a murine pressure overload-induced dis-ease model reduces cardiac ERK-MAPkinase activity, inhibits interstitial fi brosis and attenuates cardiac dysfunction. These fi nd-ings reveal that microRNAs can contribute to myocardial disease via an effect in car-diac fi broblasts. Our results validate miR-21 as a disease target in heart failure and for the fi rst time establish the therapeutic effi cacy of microRNA therapeutic interven-tion in a cardiovascular disease setting (Fig. 1).

Since October 2008, Stefan Engelhardt is the new Director of the Institute of Pharmacology and Toxicology, Technische Universität München (TUM). The group is currently in transition to the TUM.

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Selected Publications

Thum, T., Gross, C., Fiedler, J., Fischer, T., Kissler, S., Bussen, M., Galuppo, P., Just, S., Rottbauer, W., Frantz, S., Castoldi, M., Soutschek, J., Koteliansky, V., Rosenwald, A., Basson, M.A., Licht, J.D., Pena, J.T., Rouhanifard, S.H., Mucken-thaler, M.U., Tuschl, T., Martin, G.R., Bauersachs, J., and Engelhardt, S. (2008) MicroRNA-21 contributes to myocardial disease by stimulating MAP kinase signaling in fibroblasts. Nature, 456, 980-4.

Lewin, G., Matus, M., Basu, A., Frebel., K., Rohsbach, S.P., Safronenko, A., Seidl, M.D., Stümpel, F., Buchwalow, I., König, S., Engelhardt, S., Lohse, M.J., Schmitz, W., and Müller, F.U. (2009) Critical role of transcription factor CREM in beta1-ad-renoceptor-mediated cardiac dysfunc-tion. Circulation, 119, 79-88.

Cook, A.R., Bardswell, S.C., Pretheshan, S., Dighe, K., Kanaganayagam, G.S., Jabr, R.I., Merkle, S., Marber, M.S., Engelhardt, S., and Avkiran, M. (2008) Paradoxical re-sistance to myocardial ischemia and age-related cardiomyopathy in NHE1 transgenic mice: A role for ER stress? J. Mol Cell Cardiol, in press.

Vilardaga, J.-P., Bünemann, M., Feinstein, T.M., Lambert, N., Nikolaev, V.O., Engelhardt, S, Lohse, M.J., and Hoffmann, C. (2008) GPCR and G proteins: drug efficacy and activation in live cells. Mol Endo, in press.

β-adrenergic signaling in heart failure

Heart failure is characterized by chronic ac-tivation of the sympathetic nervous system and enhanced release of catecholamines. The resulting chronic stimulation of myo-cardial β-adrenergic receptors is detrimen-tal and contributes to progression of the disease. Consequently, treatment with β-blockers has emerged as an effective thera-peutic approach to treat cardiac failure. We recently succeeded in directly assessing the conformational change of the β-adrenergic receptor protein that occurs after binding of an agonist to the receptor protein. Us-ing the FRET-technique, we succeeded in recording for the first time the activation of the human β1-adrenergic receptor in living cells in real time (Rochais et al., J Clin Invest, 2007). Currently, we are using this approach to determine the kinetics of receptor activation and deactivation, depending on receptor polymorphisms as well as repetitive receptor activation. Our data suggest that receptor polymorphisms critically determine the kinetics of receptor activation and deactivation.In addition, we aim to identify novel tar-gets downstream of the β-adrenergic recep-

Extramural Funding

Transatlantic Network of Excellence, Fondation Leducq, ParisInterdisciplinary Center for Clinical ResearchBMBF, Competence Network Cardiac Failure

Fig. 2:Generation of PI16-deficient mice. The 3rd and 4th exon were flanked with loxP-sites.

Cell-cell communication in the mammalian heart

Cardiomyocyte hypertrophy and intersti-tial fibrosis are hallmarks of congestive heart failure. Indirect evidence suggests that factors secreted from the heart are important regulators of cardiomyocte hy-pertrophy, but only few such factors have been identified. In an effort to identify larger parts of the myocardial secretome, we have recently conducted a secretion trap screen in yeast and have identified 54 proteins that are putatively secreted from the heart (Frost & Engelhardt, Circu-lation, 2007). We are currently follow-ing protease inhibitor 16 (PI16), which displays strong upregulation in cardiac failure and appears to inhibit cardiom-yocyte hypertrophy. Next to several ap-proaches in vitro and in vivo to elucidate PI16’s mechanism of action we have suc-cessfully generated mice with a floxed allele of PI16 (Fig. 2).

tor using a phosphoproteomic approach. This project is carried out in close collabo-ration with the Mass Spectrometry group.

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None of the STAT transcription factors STAT1, STAT3 or STAT5 stimulate transcription of CCL1 or CCL8. However, we identifi ed a novel negative regulatory function of activated STAT5 for the gene expression of CCL1. In the absence of STAT5, mRNA and protein levels of CCL1 were signifi cantly elevated (Fig. 2A, B). Intriguingly, not STAT5 itself, but its target protein CIS was found to be responsible for the suppression of CCL1 expression. Overex-pression of CIS in STAT5-defi cient cells re-duced the secretion of CCL1 back to the level found in wild-type cells (Fig. 2C).

Heike Hermanns

E-mail: [email protected]: +49(0)931 201 487 22Fax: +49(0)931 201 487 02http://www.rudolf-virchow-zentrum.de/forschung/hermanns.html

Dysregulated cytokine signaling is involved in the pathogenesis of a large number of diseases including chronic infl ammation, autoimmunity and cancer. Estimated costs only for the treatment of rheumatoid arthritis alone are about 10-15 billion Euro per year. Therefore, understanding of cytokine signaling specifi city is necessary to generate more specifi c therapeutic intervention methods and avoid harmful side effects. Cytokines are soluble immunomodulatory proteins secreted by tissue and immune cells. A large number of cytokines transduce sig-nals via shared cell surface receptors that form multi-molecular complexes involving ligand-specifi c and signal transducing receptors. This explains why many of the signaling cascades are common to a number of different cytokines, but leaves the question open as to how signaling specifi city is achieved. Using the family of inter-leukin-6-type cytokines, in particular IL-6 and oncostatin M, as a model system, our laboratory investigates the spatio-temporal resolution of cytokine receptor signaling and aims to identify novel target proteins involved in progression of infl ammatory diseases.

The JAK/STAT pathway as a negative reg-ulator of chemokine expression

The appropriate immune response to trau-ma and invading pathogens relies on the interaction of various cell types orches-trated by direct cell contact or solublefactors. As an initial step leukocytes are recruited to sites of tissue damage. This process is mainly controlled by members of the chemokine (= chemotactic cytokine) superfamily, in particular by inducible “in-fl ammatory” chemokines. This subfamily comprises the majority of the 50 so far known chemokines in humans and is distin-guished from the constitutively expressed “homeostatic” chemokines.

The interleukin-6-type cytokine onco-statin M (OSM) is a known pro-infl ammatory cytokine, secreted by activated monocytes, neutrophils and T lymphocytes. The human cytokine can signal through two receptor complexes: the type I receptor complex consisting of gp130, the common receptor subunit of all IL-6-type cytokines, and the leukemia inhibitory factor (LIF) receptor or the type II receptor complex composed of gp130 and the OSM receptor β subunit. OSM is one of the most potent activators of the JAK/STAT- and MAPK-pathways. Study-ing the contributions of OSM to infl amma-tory processes, we realized that OSM is a very strong inducer of chemokine expres-sion, particularly of CCL1, CCL7 and CCL8, by human primary dermal fi broblasts. The

induction of these chemokines occurs with faster kinetics than in response to the prime pro-infl ammatory cytokines interleu-kin-1β or tumor necrosis factor α (TNFα). The production of CCL1 and CCL8 is impor-tant for migration of monocytes, while blocking antibodies specifi c for CCL1 addi-tionally inhibit the migration of T lympho-cytes. We identifi ed the mitogen-activated

protein kinases ERK1/2 and p38 as crucial factors for the enhanced expression of CCL1, CCL7 and CCL8. Depletion of the ERK1/2 target genes c-Jun or c-Fos strong-ly decreases chemokine expression, while p38 MAPK prolongs the half-life of CCL1, CCL7 and CCL8 mRNA through phosphoryla-tion and degradation of tristetraproline (Fig. 1).

Fig. 1:Preincubation of dermal fi broblasts with the p38 inhibitor SB202190 abrogates OSM-induced CCL1 expression. Knock-down of the mRNA destabilizing protein TTP releases this blockade.

21

Extramural Funding

SFB 542, TP B6 (until 06/2008)SFB 542, TP C11 (in collaboration with J. Baron and H. F. Merk, until 06/2008)SFB 487, TP B9 (from 01/2009)

Fig. 3:Constitutive activation of the JAK2/STAT5 pathway in HEL leukaemia cells prevents the IL-1b-induced CCL1 secretion (A). Knock-down of CIS expression results in release of CCL1 from HEL cells due to the constitutively activated MAPK ERK1/2 (B).

Fig. 2:STAT5 deficiency leads to prolonged transcrip-tion of CCL1 (A) and elevated levels of secreted CCL1 at later time points (B). Overexpression of the STAT5 target protein CIS reduces CCL1 secretion back to wild-type levels (C).

Divergence of fibroblasts exemplified by specific expression of the chemokine CCL13/MCP4

Rheumatoid arthritis (RA) is a severe, chronic disease characterized by a pro-found inflammatory response that leads to

Fig. 4:CCL13 is secreted by synovial fibroblasts in response to OSM treatment, but not in other types of fibroblasts or chondrocytes (A). The release of CCL13 from synovial fibroblasts stimulates migration of monocytic cells. Blockade of CCL13 significantly reduces monocyte migration (B).

Chemokine expression is not only important for an appropriate immune response to in-vading pathogens, but also plays a crucial role in the immune surveillance to combat tumor progression. Interestingly, the JAK/STAT pathway is constitutively activated in a number of tumor cells where it promotes cell growth and inhibits apoptosis. We analyzed whether the constitutive activation of this pathway can also contribute to suppression of chemokine production by tumor cells. In-deed, in contrast to monocytes from healthy donors, human acute myeloid leukemia cells (HEL), which express a constitutively ac-tive version of the Janus kinase JAK2, and therefore constitutively express CIS, did not secrete CCL1 in response to IL-1β (Fig. 3A). This blockade could be released by a target-ed inhibition of CIS expression using siRNA knock-down technology (Fig. 3B).

joint destruction as well as extraarticular symptoms with a significant impact on both morbidity and mortality. The cause and pathologic processes underlying RA have not yet been fully elucidated. How-ever, it is known that both, the cellular immune system and the cytokine network are subject to profound dysregulations. The success of an anti-proinflammatory cyto-kine therapy directed against either TNFα or, to a lesser extent, IL-1β has highlighted the significance of these cytokines in the pathological progression of RA. Neverthe-less, these therapeutic strategies are not beneficial to all patients, suggesting that other players must be involved in the pathogenesis.

We identified oncostatin M as a power-ful inducer of CCL13 chemokine expression in primary synovial fibroblasts from rheu-matoid arthritis patients as well as from healthy controls, but not in other types of fibroblasts or chondrocytes (Fig. 4A). Neither IL-6 nor TNFα could stimulate the expression of CCL13 in synovial fibroblasts; IL-1β was a very weak inducer. Investigat-ing the underlying molecular mechanism we identified STAT5, ERK1/2 and p38 as critical factors involved in OSM-dependent transcription and mRNA stabilization of CCL13. Inhibition of CCL13 using block-ing antibodies strongly reduced migration of monocytes (Fig. 4B). Furthermore, we could show that synovial fibroblasts from RA patients constitutively produce low amounts of CCL13, partially dependent on autocrine stimulation by constitutively produced OSM.

In conclusion, in contrast to other promi-nent cytokines involved in the pathogen-esis of RA, OSM can strongly upregulate expression of CCL13, a chemokine recently identified in the synovial fluid of RA pa-tients. Despite potent OSM-induced sig-nal transduction in all types of fibroblasts analyzed, only synovial fibroblasts secrete CCL13, which might be indicative of tis-sue-specific imprinting of different fibro-blasts during development (Hintzen et al., in revision).


Hintzen, C., Evers, C., Lippok, B.E., Volkmer, R., Heinrich, P.C., Radtke, S.*, and Hermanns, H.M.* (2008) Box 2 re-gion of the oncostatin M receptor de-termines specificity for recruitment of Janus kinases and STAT5 activation. J Biol Chem, 283, 19465-19477.(* authors contributed equally).

Hintzen, C., Haan, C., Tuckermann, J.P., Heinrich, P.C., and Hermanns, H.M. (2008) Oncostatin M-induced and con-stitutive activation of the JAK2/STAT5/CIS pathway suppresses CCL1, but not CCL7 and CCL8, chemokine expression. J Immunol, 181, 7341-7349.

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Fig. 1:Phalloidin staining of SH-SY5Y cells at differenttime points after 0.1 µg/ml PLY treatmentrevealed fi lopodia, lamellipodia and stress fi berformation. Scale bars: 10 µm.

Now, we are trying to identify the exact mechanisms leading to these changes, using not only cell lines (SH-SY5Y human neuroblastoma cells), but also primary as-trocyte/microglia cultures and macrophage cultures, and trying to translate these changes into tissue-specifi c context. A short overview of the specifi c mechanistic questions is presented in Figure 2.

Asparouh Iliev

E-mail: [email protected]: +49(0)931 201 489 65Fax: +49(0)931 201 487 02http://www.rudolf-virchow-zentrum.de/forschung/iliev.html

Pathogenic bacteria developed adaptive mechanisms (e.g. toxins) to improve their virulence. While some toxins destroy cells, others modulate cellular functions, thus improving pathogen propagation. Small GTPase activity modulation, producing actin remodeling, is favoured in evolution. We found a link between pore formation and small GTPase activation by a member of the cholesterol-dependent cytolysins (CDC) – pneumolysin. It is a major pathogenic factor of Streptococcus pneumoniae, which causes the most frequent form of bacterial meningitis. Our work, funded by the Emmy Noether-Program of the German Research Foundation, focuses on: 1) clarifying the link between pore formation by CDCs and small GTPase activation; 2) identifying activation mechanisms; 3) examining pneumolysin effects on microtubules; 4) studying toxin effects on brain structures, mostly dependent on small GTPases, namely – the synapses; 5) clarifying the role of pneumolysin-induced small GTPase activation on phagocytosis and cell motility. Finally, we aim to defi ne possible drugable targets, which might prove useful in improving the outcome of pneumococcal infections.

Toxin-induced actin remodeling

A surprising fi nding of our work over the last few years was the discovery of small GTPase (specifi cally Rac1 and RhoA) ac-tivation by sub-lytic amounts of the cho-lesterol-dependent cytolysin pneumolysin, known as a classical pore-forming toxin. Within a very short period of time (within 16 min) after exposure, pneumolysin was capable of producing massive actin cyto-skeleton reorganization with formation of stress fi bers, lamellipodia and fi lopodia (Fig. 1).

Funded by the Emmy Noether-Program of the DFG. Since February 2008, Asporouh Ilievis associated to the Rudolf Virchow Center as a Junior Research Group leader.

Bacteria have developed specifi c mecha-nisms to facilitate their interaction with mammalian organisms during the course of their co-evolution. One of these is the pore-forming capacity of cholesterol-bind-ing cytolysins. Other toxins modulate intra-cellular signaling cascades allowing bacte-ria to overcome the pathogen defense of the host. Among these, modulation of the Rho GTPases is of particular interest. Rho GTPases belong to the large superfamily of Ras-proteins, monomeric GTP-binding pro-teins with a molecular mass between 20 and 30 kDa. Rho, Rac and Cdc42 are the most intensively studied Rho GTPase members. They mediate cell adhesion, motility, endo-/exocytosis, and phagocytosis through the regulation of actin cytoskeleton dynamics. Furthermore, the Rho GTPases affect apop-totic processes, gene expression, and can modulate the stability of the microtubule cytoskeleton.

Fig. 2:Overview of the general mechanistic questionsin the activation of RhoA and Rac1 by the pore-forming cytolysin pneumolysin.

23

Extramural Funding

Emmy Noether-Programm (DFG ENP IL 151/1-1)

Fig. 3:High-resolution confocal imaging of dendritic spines in GFP-tranfected primary cortical mouse neuron. Red arrows indicate mature (mushroom) spines, corresponding to established syn-apses. Green arrow indicated an immature (developing) dendritic spine. Scale bar: 2 µm.

Fig. 4:Microtubular bundling (α-tubulin immunostaining) in SH-SY5Y human neuroblastoma cells by sub-lytic concentration of pneumolysin (PLY; 0.1 µg/ml) at 30 and 120 min after toxin challenge. Scale bars: 10 µm.

Synaptic damage in meningitis

As a major pathogenic factor of Streptococ-cus pneumoniae, pneumolysin contributes substantially to the neurological symptoms in the course of pneumococcal meningitis. The severe patient disability contrasts with the relatively limited neuronal cell damage. We suggest that pneumolysin might dam-age synapses via its small GTPase modulat-ing effects, since synapses are structurally and functionally critically dependent on proper actin organization. For this purpose, we use high-resolution microscopy to study dendritic spine dynamics (corresponding to the post-synaptic structures) in GFP-ex-pressing dissociated primary neurons (Fig. 3) in brain slices and live animals.

Toxin-induced microtubule changes

Recently, we described strong and rapid microtubule stabilization by sub-lytic amounts of pneumolysin (Iliev et. al., Mol Microbiology, 2008), which is a unique fea-ture among bacterial toxins (Fig. 4). These effects were partially regulated by the src-family of tyrosin kinases and led to inhibition of the intracellular organelle trafficking. Such an inhibition could be relevant to the disrupted axonal trans-

In another project, we are comparing the role of pneumolysin with another member of the same toxin group – listeriolysin of Listeria monocytogenes. Although homolo-gous to pneumolysin, this toxin is released by bacteria within host cells. It demon-strates pore-forming abilities only at lower pH (in the phagosomes). Thus, comparison between pneumolysin and listeriolysin might allow us to examine the importance of the outer cell membrane pore formation, as a factor leading to small GTPase activa-tion.

Outlook

The expected scientific milestones of our work are: 1) Defining the mechanisms, by which cholesterol-dependent cytolysins might lead to small GTPase activation; 2) Determining the role of pore formation in the small GTPase activation and mapping active toxin domains; 3) Defining aspects of dendritic spine/synapse collapse in the course of meningitis; 4) Studying the in-hibitory role of pneumolysin in phagocy-tosis and immune cell motility; 5) Deter-mining the role of membrane cholesterol clustering by pneumolysin in Toll-like re-ceptor 4 (and TLRs generally) activation; and 6) Establishing a pharmacological ap-proach to ameliorate the damage by cho-lesterol-dependent cytolysins by applying cholesterol-lowering drugs and ion channel blockers. The long-term aim of the project is to integrate the observed membrane-associated effects (pore formation) and following downstream molecular cascades leading to RhoA and Rac1 activation in order to translate these findings into higher organisms (mouse models) in an ultimate effort to define specific therapeutic options, relevant to brain and im-mune function.


Iliev, A.I., Djannatian, J.R., Opazo, F., Gerber, J., Nau, R., Mitchell, T.J., and Wouters, F.S. (2008) Rapid microtubule bundling and stabilization by the Strep-tococcus pneumoniae neurotoxin pneu-molysin in a cholesterol-dependent, non-lytic and Src-kinase dependent manner inhibits intracellular traffick-ing. Mol Microbiol, in press.

port, observed in human brain tissue in the course of pneumococcal meningitis. It is logical to suggest that pneumolysin modulates cellular cytoskeletal structures in an effort to improve pneumococcal inva-sion through the blood brain barrier, and in the brain, thus impairing the adaptive abilities of the organism as a whole and allowing transmission of the pathogen to new hosts.

24

In previous work, we have successfully si-lenced the expression of the candidate gene Nramp1 in NOD mice and determined that this gene is associated with the disease. The positive outcome of this fi rst study led us to initiate several new projects investi-gating further candidate genes associated with type 1 diabetes in this mouse model. These genetic studies are being carriedout in collaboration with John Todd and Linda Wicker (Cambridge University, UK). Their extensive studies of type 1 diabe-tes genetics in both humans and the NOD model have resulted in identifying several more promising candidate genes awaiting evaluation.

Importantly, many disease-associated gene polymorphisms in humans do not lead to the complete absence of a gene product, but instead result in altered splic-ing and/or reduced expression. RNAi is exquisitely suited to model more subtle variations in gene expression compared to conventional KO technology. For example, the mutation of the gene CTLA-4 that as-sociates with type 1 diabetes in humans is known to alter splicing and reduce expres-sion of a shorter form of the gene called sCTLA-4. We have now generated NOD ani-mals where this shorter splice variant, but not the full-length isoform, is reduced byRNAi (Fig. 1).

Stephan Kissler

E-mail: [email protected]: +49(0)931 201 440 65Fax: +49(0)931 201 440 68http://www.rudolf-virchow-zentrum.de/forschung/kissler.html

The primary function of the immune system is to recognize and eliminate pathogens. This task requires immune cells to be reactive to a wide range of antigens. While the immune system is tightly regulated to prevent activa-tion by innocuous antigens, including self-antigens, a signifi cant number of people develop autoimmune diseas-es. Our laboratory seeks to understand the genetic polymorphisms that predispose individuals to autoimmunity and the regulatory pathways that fail during onset of disease. Our main approach is the genetic manipulation of model organisms by RNA interference (RNAi). We use lentiviral transgenesis to generate animals in which target genes are constitutively silenced by RNAi. After pioneering this strategy in the most widely-used model for type 1 diabetes, we are now refi ning lentiviral technology to make it more versatile and specifi c for studying immune tolerance. Understanding genetic factors and functional pathways involved in autoimmunity should ultimately help develop new therapeutic approaches.

Immune tolerance and autoimmunity

The immune system evolved to protect us against pathogenic viruses, bacteria, parasites and fungi. Due to the diversity of existing pathogens, the immune system developed complex mechanisms to ensure it would be capable of recognizing virtually any invading pathogen. This high degree of reactivity, however, required the simulta-neous evolution of regulatory pathways to ensure immune tolerance of self-antigens, or antigens whose presence is innocuous, particularly on mucosal surfaces (airways and digestive system).

Despite these regulatory mechanisms, a signifi cant percentage of the population develops autoimmunity, in most cases char-acterized by immune responses against a specifi c tissue or organ (e.g. type 1 diabe-tes, multiple sclerosis). Many of these auto-immune diseases can be studied in animal models. This is particularly true for type 1 diabetes, for which the non-obese diabetic (NOD) mouse strain constitutes the most widely-studied and relevant experimental model. Numerous genetic loci have been associated with type 1 diabetes in the NOD mouse, yet the exact gene polymorphisms that affect disease susceptibility are mostly unknown. Of those susceptibility genes that have been identifi ed in the mouseto date, most correspond to human candi-date genes, reinforcing the validity of this animal model.

Identifi cation of causal gene variants is particularly challenging in the NOD model, since this strain has long been refractory to the generation of embryonic stem cells that could be used for targeted gene muta-tion. To test candidate genes, mutant al-leles have to be generated in a different genetic background (e.g. the widely used 129Sv mouse strain) and crossed into the NOD background by selective breeding. This approach is not only extremely time con-suming, but it also carries the risk of intro-ducing polymorphic regions that fl ank the mutant allele, thereby obscuring or falsify-ing conclusive phenotypes. Together, these problems have long hindered the validation of candidate genes in the NOD model.

Studying disease genetics by RNA inter-ference

To circumvent the diffi culties of gene-dele-tion in the NOD mouse, we have pioneered lentiviral transgenesis in conjunction with RNAi in this disease model. Generating transgenic mice by single-cell embryo in-fection with lentivirus has proven highly effi cient and technically simpler than conventional transgenesis methods. In ad-dition, this method allows the rapid gen-eration of transgenic mice directly in theNOD background.

25

Fig. 1:Expression of sCTLA-4 mRNA, but not flCTLA-4, liCTLA, or the unrelated flIcos mRNA, is reduced in sCTLA-4 knockdown animals. RNA was extracted from splenocytes, reverse-transcribed and quantified using Taqman qPCR (difference in cycles between target mRNA and beta-actin mRNA is shown).

Fig. 2:Flow cytometry analysis of lymph node cells from wild-type (wt) or sCTLA-4 knock- down (sCTLA-4) animals. Top row: CD3 expression in total lymph node population. Middle row: CD4 vs. CD8 expression within the CD3-positive population. Botom row: FoxP3 and CD25 expression within CD3-positive/CD4-positive cells.

Following disease pathology in vivo by bioluminescence

In our first study involving lentiviral RNAi, we employed a construct where shRNA ex-pression is driven by the ubiquitous U6 promoter. While this led to consistent and effective gene silencing, we have now ad-opted a new strategy that allows shRNA expression to be restricted to particular cell types. Using tissue-specific promoters and a new shRNA design, our laboratory has engineered lentiviral vectors to silence genes exclusively in beta islet cells of the pancreas (Fig. 3).

We have started characterizing this new transgenic line: the reduction of sCTLA-4 in the NOD mouse, which mimics the hu-man CTLA-4 polymorphism associated with autoimmunity, does not have any gross ef-fects on the development of the immune system. All populations of T lymphocytes analyzed are largely unaffected by sCTLA-4 knockdown (Fig. 2).

We have however started to characterize subtle differences in the regulatory T-cell compartment of these mice. These differ-ences, if they affect disease on a systemic level (which we are yet to determine), could help explain how the human genetic polymorphism of CTLA-4 contributes to dis-ease susceptibility.

Extramural Funding

Juvenile Diabetes Research Foundation International:Innovative Grant 5-2008-331Innovative Grant 5-2008-369Pilot Award 26-2008-881

Fig. 3:The rat insulinoma cell line Ins-1E was infected with lentivirus encoding the firefly luciferase under the control of the rat insulin promoter (RIP). Luminescence was meas-ured in cell lysates, and is shown as relative luminescence units (RLU). No expression was detected in infected HEK293 cells that do not express insulin (data not shown).

Fig. 4:Single cell embryos were injected with lentivirus encoding the luciferase gene under the control of the CMV promoter. Five of the embryos reimplant-ed into pseudopregnant females survived until birth. At 4 weeks of age, these mice were anaes-thetised, injected with D-luciferin substrate and imaged using a NightOwl bioimaging system. One mouse shows strong expression of the luciferase gene, particularly apparent in regions where the dark hair does not obscure bioluminescence.

In addition, we have incorporated the fire-fly luciferase gene into the lentiviral vector as a cell-specific marker (Fig. 4) to allow us to monitor cells targeted by RNAi in vivo by non-invasive bioimaging. This will enable us to evaluate the effect of a particular gene knock-down on beta cell survival over time. The beta cell mass can be measured at bi-weekly intervals, for example, for the entire duration of a transgenic animal‘s life. This approach will facilitate assessing treatments that promote beta cell survival or regeneration.

Overall, our novel approach to studying disease genetics and pathways by RNAi in the NOD model of type 1 diabetes should help elucidate the parameters that influ-ence the onset and development of auto-immunity. At the same time, we will be ex-ploring therapeutic strategies to promote beta cell survival and regeneration in af-fected animals.


Thum, T., Gross, C., Fiedler, J., Fischer, T., Kissler, S., Bussen, M., Galuppo, P., Just, S., Rottbauer, W., Frantz, S., Castoldi, M., Soutschek, J., Koteliansky, V., Rosenwald, A., Basson, M.A., Licht, J.D., Pena, J.T., Rouhanifard, S.H., Mucken-thaler, M.U., Tuschl, T., Martin, G.R., Bauersachs, J., and Engelhardt, S. (2008) MicroRNA-21 contributes to myocardial disease by stimulating MAP kinase signaling in fibroblasts. Nature, 456, 980-4.

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Fig. 1:Chronophin partiallycolocalizes with F-actinin areas of dynamicactin cytoskeletalremodeling. Shown isa GC-1 cell undergoing cytokinesis. Chronophin(green) and fi lamen-tous actin (red) signalsoverlap (yellow) in the ingressing cleavage fur-rowand in membraneruffl es. Nuclei arestained in blue.

Regulated cell adhesion and directional cell migration are essential for human biology from conception to death. In the adult organism, cell adhesion and migration are central to homeostatic processes such as mounting an effective immune response or repairing injured tissues. Failure of cells to migrate, or migration of cells toaberrant locations, is intricately involved in pathologies including vascular and infl ammatory diseases as wellas in tumor formation and metastasis. Effective cell adhesion and migration are based on the preciseintegration of localized, transient signaling events with changes in the cytoskeleton and appropriate cell-cell and cell-matrix interactions. Our goal is to understand the physiological and pathological functions of thenewly identifi ed, Rho-GTPase-dependent phosphatases Chronophin and AUM, that have emerged asmajor regulators of cytoskeletal dynamics. We will focus our studies on the roles of CIN and AUM for cell-cell interactions in the cardiovascular system.

Antje Gohla

E-mail: [email protected]: +49(0)211 811 570 7Fax: +49(0)211 811 272 6http://www.uniklinik-duesseldorf.de/biochemieundmolekularbiologiezwei

The actin depolymerizing factor (ADF) and cofi lin are closely related proteins that control stimulus-induced actin cytoskel-etal remodeling in virtually all cells. Their actin-reorganizing activities are tightly controlled by reversible phosphorylation of a single serine residue. In recent years, the signaling pathways leading to ADF/co-fi lin inactivation by phosphorylation have been studied in great detail. However, de-spite substantial efforts directed toward identifying the molecular mechanisms of ADF/cofi lin activation, ADF/cofi lin-spe-cifi c phosphatases have long remained elusive. Using an activity-based biochemi-cal screening approach, we have identifi ed Chronophin (CIN), a novel ADF/cofi lin-spe-cifi c phosphatase with unique structural and biochemical properties (Gohla et al., Nat Cell Biol, 2005). CIN is a member of the largely unexplored family of haloacid dehalogenase (HAD)-type phosphatases. Interestingly, depletion of CIN by RNA in-terference technology in various cell cul-ture models results in aberrant cytoskeletal dynamics during mitotic cell division and cell adhesion, as well as in defective cell motility. Thus, CIN is a promising target protein that regulates ADF/cofi lin-depen-dent actin remodeling relevant to cell-cell interactions and motility (Fig. 1).

Antje Gohla will start as a Junior Research Group leader in March 2009.

As a fi rst step towards deciphering the regulation of CIN activity in response to extracellular cues, we used yeast two-hy-brid and biochemical approaches to dem-onstrate that CIN functions as a major Ca2+/Calmodulin-dependent regulator of

actin dynamics when bound to the Ca2+-and integrin-binding protein 1 (CIB1). The composition of the CIB1/CIN com-plex bears a striking resemblance withthe serine/threonine-phosphatase calci-neurin, which has so far been believed to be unique among phosphatases in its abil-ity to sense Ca2+ through its activation by calmodulin. Calcineurin is a master regula-tor of signaling cascades that govern the development and function of e.g. the im-mune, nervous and cardiovascular systems, and was catapulted to center stage with the discovery that it is the target of the immunosuppressants cyclosporine A and FK506. In addition to characterizing the biochemical details of the protein/pro-tein-interactions in the CIN phosphatase complex, we are systematically testing the hypothesis that CIN meets the criteria of the long-postulated, non-calcineurin, Ca2+-

dependent phosphatase involved in actin cytoskeletal remodeling.

Employing a bioinformatics approach, we have recently discovered a novel CIN-re-lated phosphatase that we named AUM (for actin-remodeling, ubiquitously expressed, Mg2+-dependent HAD phosphatase). Unex-pectedly, AUM does not activate ADF/co-fi lin-dependent actin dynamics, but rather functions as a specifi c tyrosine phospha-tase that is crucial for the regulation of cell morphology, adhesion and differentiation. Studies on the regulation of AUM activity by extracellular signals and on physiologi-cal AUM substrates are ongoing.

In addition to investigating the role and regulation of CIN and AUM at the structural, biochemical and cell biological level, we will also analyze their in vivo functions for cell-cell interactions in the cardiovascular system using CIN- and AUM-defi cient mouse models.

Outlook

27

Alma Zernecke

E-mail: [email protected]: +49(0)241 803 598 5Fax: +49(0)241 808 271 6http://www.ukaachen.de

Alma Zernecke will start as a Junior Research Group leader in March 2009.

Atherosclerosis, with its clinical manifestations of myocardial infarction, stroke and peripheral artery disease, is imminently becoming the leading cause of death worldwide. Infl ammation has emerged as a crucial force driving the initiation and progression of atherosclerotic lesion formation. Initiated by the activation and dysfunction of endothelial cells, leukocyte subsets are recruited and accumulate in atherosclerotic lesions. Details regarding the involvement of different leukocyte subpopulations in the pathology of this disease are emerging. While mononu-clear cells found in the lesions are predominantly comprised of monocyte-derived macrophages, which transform into foam cells characteristic for fatty-streak lesions, T lymphocytes and dendritic cells have also been revealed in close proximity. Moreover, immune responses are described to participate in all phases of atherosclerosis, and pro-atherogenic and atheroprotective cytokines and T-cell subpopulations have been defi ned. The delicately adjusted two-edged immune balance and the exact function of these cell types remain elusive to date.

The non-random attraction of mononuclear cells to specifi c tissue targets is governed by sequential, and also mutually overlap-ping steps in the interaction with the vessel wall: namely rolling interactions, followed by integrin-dependent arrest and chemokine-triggered transendothelial dia-pedesis. In the past, we identifi ed several adhesion molecules and chemokines/recep-tors that are important in the accumula-tion of monocytes/macrophages at sites of infl ammation. In addition, it recently be-came clear that chemokines are important in controlling survival and cell homeostasis in different cell subpopulations. For exam-ple, disruption of the interaction between CXC-chemokine receptor 4 (CXCR4) and its ligand CXCL12 aggravated diet-induced atherosclerosis in mice. This was linked to deranged neutrophil homeostasis with an expansion of this cell population in the cir-culation and an increased neutrophil con-tent in plaques. In contrast, the absence of the receptor CX3CR1 resulted in a sig-nifi cant reduction in monocyte survival and blood monocyte levels, and protected from atherosclerosis development. In this study, knock-in mice were employed that carry a targeted replacement of the cx3cr1 gene by a gene encoding green fl uorescent protein (gfp) to visualize monocytes/macrophages by their GFP-expression (Fig. 1).

Less is known about the functions of T-cell and dendritic cell subsets in ath-erosclerosis. By targeting specifi c che-mokines/cytokines and their receptors in atherosclerosis-prone apolipoprotein E-de-

fi cient mice, we will address the functions of different immune cell subpopulations in atherosclerosis. A particular focus will be on their interactions at sites of infl amma-tion, but also within lymphatic tissue, and their role in shaping specialized immune responses that control the development of atherosclerosis. Furthermore, we will inves-tigate the localization of these cells in the vessel wall and their routes of entry during lesion formation.

Given the remarkable role of adaptive and innate immunity in atherosclerosis, tar-geting of its cellular constituents and un-derstanding the complex equilibrium and interplay between immune-cell subpopu-lations that contribute to the process of atherosclerosis will be important to iden-tify new therapeutic approaches to treating this disease.

Fig. 1:Lipid deposits stained by Oil-red-O in an athero-sclerotic lesion in the aortic root of an apoli-poprotein E-defi cient mouse transplanted with cx3cr1+/gfp bone marrow and fed a high fat diet (upper panel). Immunfl uorescence stain-ing was performed for monocytes/macrophages (using the marker MOMA-2, red) in co-localiza-tion with gfp+ cells (green), cell nuclei were counter-stained by DAPI (blue, lower panel).

Outlook

28

Gregory Harms

E-mail: [email protected]: +49(0)931 201 487 17Fax: +49(0)931 201 487 02http://www.rudolf-virchow-zentrum.de/forschung/harms.html

The research group Biomedical Molecular Microscopy studies molecular interactions in cell signaling, membrane proteins and cytosolic messengers. We apply a wide range of different techniques, such as fl uorescence resonance energy transfer (FRET) microscopy, single-molecule microscopy and dynamic confocal microscopy. Such methods require by using custom-built wide-fi eld and confocal microscopes capable of ratiometric FRET, fl uorescence recovery after photo-bleaching (FRAP), fl uorescence correlation spectroscopy (FCS), and single-molecule track-ing (SMT). These microscopes allow the detection of low, endogenous levels of proteins in and on living cells. Key objectives are the development of biosenors and imaging techniques to study different biological problems. We study the biological aspects initially using optical microscopy and further apply the dynamic techniques to determine the temporal distribution of cellular events.

Fig. 1:Microscopy Systems in the Rudolf Virchow Center.

Our research focuses on cell adhesion, migration and cancer progression. Cell ad-hesion and migration are investigated via growth and development as well as mo-lecular signaling pathways, e.g. Src kinase family. Cancer progression is studied with emphasis on the role of the growth factors, such as the Bone Morphogenetic Protein (BMP)/Smad pathway.

FRET microscopy allows us to observe both the dynamics and cellular localization of protein conformational changes and protein-protein interactions with improved interpretation based on both anisotropy and fl uorescence lifetime. We observe the diffusion dynamics of lipids and proteins by long-range techniques such as FRAP, to complement short-range methods such as FCS. We also measure single-molecules by wide-fi eld imaging and TIRF, since we now have the latest technologies to monitor long and short diffusion ranges with track-ing, FRET, and co-localization events.

Sheet Illumination Microscopy for improved, sensitive Imaging in Tissues and Organisms

We developed our versions of selective plane illumination microscopy (SPIM) and ultramicroscopy. SPIM and ultramicroscopy use light sheet illumination parallel tothe focal image plane of a microscopyobjective, a concept introduced bySiedentopf in 1903. Our setup enables penetration depths beyond 1 mm in liv-ing embryos with resolution comparable to

confocal microscopy. The growing line of evidence points to the importance of the tracking low numbers of proteins in tissues and living organisms. We successfully ap-plied our SPIM setup to detect single pro-teins with single nano-crystals.

Ultramicroscopy applies sheet illumina-tion to image large tissue sections cleared to achieve optical transparency by methods developed by Spateholz in 1914. To over-come the limitations of only 0.4 mm pen-

etration in mice of up to 5 weeks old, we improved the clearing procedures, instru-mentation, and analysis to perform imaging in adult mouse tissue (> 2 years) yielding cellular and anatomical details of disease models. As a fi rst model, we used an AAV-DsRed expression system and showed that our improved setup can achieve over more than 4 millimeter penetration with subcel-lular resolution in various tissues of the central nervous system (CNS).

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Fig. 2:SPIM and Ultramicroscopy. Diagram, AAV-DsRed Image of a mouse spinal cord section, and transmission/QuantumDotFluorescence Image of drosophila larvae.

Fig. 3:FRET and Anisotropy platform diagram and imaging calculation diagram.

Fig. 4:Fluorescence image of actin in an XIAP knock-down cell.

Anisotropy and FRET Microscopy: PH do-main and PTH Receptor

We developed a method and instrument for polarized fluorescence resonance energy transfer (FRET) and anisotropy imaging mi-croscopy done in parallel for improved in-terpretation of the photophysical interac-tions. This apparatus was shown to better determine the protein-protein interactions of the pleckstrin homology domain and the conformational changes in the Parathyroid Hormone Receptor, a G-protein coupled receptor, both fused to the cyan and yel-low fluorescent proteins for either inter- or intra-molecular FRET. From the anisotropy measurements of donor and acceptor of these FRET interactions, we find that our instrumentation and method also charac-terizes crucial effects from homo-transfer, polarization specific photobleaching and background molecules.

X-linked and cellular IAPs modulate the stability of C-RAF kinase and cell motility

As a part of a collaboration with Krishna Rajalingam and Ulf Rapp, we were able to demonstrate that Inhibitor of Apoptosis Proteins (IAP) modulate mitogen activated protein kinases, by directly binding to C-RAF kinase. Moreover, short interfering RNA (siRNA)-mediated silencing of IAPs leads to stabilization of C-RAF in human cells. XIAP binds strongly to C-RAF and promotes the ubiquitination of C-RAF. Cells with reduced XIAP levels addtionally exhibited enhanced spreading, migration and wound healing. The data show an unexpected role of XIAP and c-IAPs in the turnover of C-RAF, where it modulates the MAP kinase signaling pathway and cell migration, rather than its traditional anti-apoptotic role, casting the targeting of XIAP during cancer treatment in a different light.


Dogan,T., Harms, G.S., Hekman, M., Karremann, C., Alnemri, E.S., Rapp, U.R., and Rajalingam, K. (2008) X-linked and cellular IAPs modulate the stabil-ity of C-RAF kinase and cell motility, Nat Cell Biol, 10, 1447-1455.

Sauer, M., Bretz, A., Beinoraviciute-Kellner, R., Beitzinger, M., Burek, C., Rosenwald, A., Harms, G., and Stiewe, T., (2008) C-terminal diver-sity within the p53 family accounts for differences in DNA binding and transcriptional activity. Nuc Acid Res, 36, 1900-1912.

Schlickum, S., Sennefelder, H., Friedrich, M., Harms, G., Lohse, M.J., Kilshaw, P., Schön, M.P. (2008) Integrin αE(CD103)β7 influences cellular shape and motil-ity in a ligand-dependent fashion. Blood, 112, 619-625.

Steinmeyer, R., and Harms G.S. (2008) Fluorescence Resonance Energy Trans-fer and Anisotropy Reveals Both Het-ero- and Homo-Energy Transfer in the Pleckstrin Homology-Domain and the Parathyroid Hormone-Receptor. Micros Res Tech, 10.1002/jemt.20632.

Extramural Funding

DFG GK 1048DFG GK 1342

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DNA is a dynamic molecule that is constantly compromised in its structural integrity. It has been estimated that approximately 200,000 damage events occur daily in each human cell. Thus, organisms require effi cient DNA damage response pathways to maintain their genomes in a functional state. Among the various DNA re-pair mechanisms available to the cell, nucleotide excision repair (NER) stands out due to its broad substrate specifi city. This system recognizes damages such as the carcinogenic pyrimidine dimers induced by UV radiation, benzo[a]pyrene-guanine adducts caused by smoking, as well as guanine-cisplatinum adducts formed during chemotherapy. Through a combination of structural, microscopic and biochemical studies involving the discrete steps of the NER cascade, a general understanding of the sequential process of damage recognition, followed by excision can be obtained. Our second focus is on structure-based drug design to identify new therapeutics against infectious diseases. Our main targets are essential bacterial enzymes involved in fatty acid biosynthesis.

Fig. 1:(Left) Two views of the model of the XPD-DNA complex.(Right) The XPD-DNA complex visualized by atomic force microscopy.

Caroline Kisker

E-mail: [email protected]: +49(0)931 201 483 00Fax: +49(0)931 201 483 09http://www.rudolf-virchow-zentrum.de/forschung/kisker.html

Nucleotide Excision Repair

The importance of this repair mechanism is refl ected by three severe inherited diseases that are due to defects in NER:xeroderma pigmentosum, Cockayne syn-drome and trichothiodystrophy. Three major aspects in the “recognition” and “repair” events of NER are still not understood: (1) What are the structural determinants of theDNA required for damage recognition? (2) How are damage-induced conformational changes in the DNA perceived by a DNA repair protein complex, and how does the recognition of protein-DNA contacts translate into high binding affi nities? (3) How does the initial binding event affect subsequent binding of additional proteins, and thereby, allow the cascade of events to proceed?

The XPD Protein

Over 30 proteins have been identifi ed in humans that are critical for mediating the individual steps leading from damage rec-ognition to incision and repair. However, due to the paucity of specifi c structural intermediates, the precise role of each protein has not been fully delineated. NER has been proposed to proceed through “bi-partite substrate discrimination” or “multi-partite damage recognition” models. The repair process is initiated by the combined action of XPC and HR23B, which recognize a general disruption of Watson-Crick base-pairing created in the vicinity of the dam-aged nucleotide. Both proteins are required to recruit the 10-subunit transcription fac-tor TFIIH to this site. XPD and XPB, two helicase subunits of TFIIH, open the DNA

around the lesion in an ATP-dependent fashion. This is the fi rst catalytic step in this reaction pathway, leading to a con-formational change that allows recruitment of additional NER factors. A second more important function of the two helicases is damage verifi cation. XPD uses its helicase activity to verify the damage and ensures that the backbone distortion is not the result of an unusual DNA sequence. This process was termed “enzymatic proofread-ing”. We solved the structure of XPD from Thermoplasma acidophilum, which shares high sequence identity to its eukaryotic homologues. The structure of XPD revealed that the fi rst three domains of the protein form a donut-shaped structure with a cen-tral opening, while the C-terminal domain is positioned adjacent to the N-terminal domain. A model of the protein-DNA com-plex has been proposed where one of the ssDNA strands passes through the central hole of the donut. Unlike any DNA or RNA helicase analyzed so far, XPD also contains a domain harboring a 4Fe4S cluster, which has been proposed to play a role in the damage recognition process.

The analysis of mutations leading to one of the three severe human diseases, xero-derma pigmentosum, Cockayne syndrome or trichothiodystrophy, provided the basis for a more detailed understanding of how the combined action of the helicase and 4Fe4S cluster domains manage to verify damage

31

Fig. 2: Mutations in of the XPD protein leading to xero-derma pigmentosum (XP), Cockayne syndrome (CS) or trichothiodystrophy (TTD). Point muta-tions leading to either XP, CS or TTD are shown in a space filling representation and colored according to the disease they cause in human XPD, gray (XP), blue (TTD)and green (XP/CS).

Fig. 3: Structure of the KasA dimer (middle). Orange spheres depict the substrate mimic in the acyl channel (left). TLM in the binding pocket is shown in black (right).


Dietzel, U., Kuper, J., Doebbler, J.A., Schulte, A., Truglio, J.J., Leimkühler, S., and Kisker, C. (2008) Mechanism of Substrate and Inhibitor Binding of Rhodobacter capsulatus Xanthine De-hydrogenase. J Biol Chem, in press.

Lu, H., England, K., amEnde, C., Truglio, J.J., Luckner, S., Marlenee, N., Knudson, S.E., Knudson, D.L., Bowen, R.A., Kisker, C., Slayden, R.A., and Tonge, P.J (2008) Slow-Onset Inhibition of the FabI Enoyl Reductase from Francisella Tularensis: Residence Time and In Vivo Activity. ACS Chem Biol, in press.

Respicio, L., Nair, P.A., Huang, Q., Anil, B., Tracz, S., Truglio, J.J., Kisker, C., Raleigh, D.P., Ojima, I., Knudson, D.L., Tonge, P.J., and Slayden, R.A. (2008) Characterizing septum inhibition in Mycobacterium tuberculosis for novel drug discovery. Tuberculosis, 88, 420-429.

Wolski, S.C., Kuper, J., Hänzelmann, P., Truglio, J.J., Croteau, D.L., Van Houten, B., and Kisker, C. (2008) Crystal structure of the FeS cluster-containing nucleo-tide excision repair helicase XPD. PloS Biol, 6, 1332-1342.

Extramural Funding

DFG (SFB 630, TP B7), (KI-562/2-1)NIH R01 GM070873NCI 5PO1 1CAO4799514

within the NER repair cascade. Xeroderma pigmentosum mutations are mostly located in both helicase domains and therefore drastically reduce helicase activity and DNA binding. Cockayne syndrome-causing mutations cluster around the interface re-sponsible for ATP binding, resulting in two different effects. First they prevent ATP binding but secondly they also inhibit the associated conformational changes that occur upon ATP binding and hydrolysis. The trichothiodystrophy variants are more like-ly to destabilize the entire protein since most of them interfere with the fold of a domain. Single molecule AFM experiments combined with structural biology on com-plexes of wild-type as well as mutant XPD with damaged DNA substrates are currently underway to determine the contributions of the individual conserved amino acids impli-cated in the different disease phenotypes.

Structure-Based Drug Design

Tuberculosis (TB), the infectious disease caused by Mycobacterium tuberculosis, re-mains one of the leading causes of death in the world. The rapid emergences of danger-ous new strains, which are resistant against most known antibiotics, make it a global health threat. Consequently, the identifica-tion of new drug targets and the develop-ment of novel TB chemotherapeutics that circumvent existing drug resistance mecha-nisms are urgently needed.

Mycobacteria have a unique cell wall containing mycolic acids, very long-chain lipids that provide protection and allow

the bacteria to persist in the human macro-phage. Inhibition of mycolic acid biosyn-thesis impairs the integrity of the cell wall, which is essential for the viability of the bacteria. KasA and InhA are key enzymes involved in the biosynthesis of longchain fatty acids and mycolic acids.Recently, we determined the first structure of KasA in the apo form and bound to Thio-lactomycin (TLM), a natural product inhibi-tor. Our structural studies provide detailed insight into the interaction of the inhibitor with KasA and explain the importance of the TLM isoprene group for KasA inhibition. TLM binds to the malonyl binding pocket and forms two strong hydrogen bonds to the catalytic site histidines. The intercala-tion of the isoprenoid tail into the space between two peptide bonds further stabi-lizes TLM binding.

Fatty acid biosynthesis in M. tuberculo-sis generates fatty acids up to C54-C56 in length, in contrast to the much shorter lipids produced by organisms such as E. coli. Significantly, binding of a long PEG chain in the acyl channel of the KasA structures allowed us to unambiguously as-sign the mode of substrate binding and the residues important for fatty acid binding. This channel, which is truncated in the homologous protein from E. coli, accommo-dates an acyl chain, which can contain up to 56 carbon atoms and thereby efficiently build the mycolic acids required for its cell wall.

Our structures not only provide the first view of a KasA protein involved in the synthesis of long chain fatty acids, but also a robust platform for the development of novel TLM analogs with high affinity for KasA.

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The general aim of our research is to understand the functions of biologically important proteins. To this end we focus on two general topics: First, protein folding in the endoplasmic reticulum (ER) and degradation of misfolded proteins via the ubiquitin-dependent protein degradation pathway. Second, we are interested in the structure and function of inhibitory neuronal receptors and the mechanism of their anchoring at the postsynaptic membrane. Our intention is to understand these proteins and the processes they participate in at the molecular level. Besides X-ray crystallography, we use a combination of complementary techniques for the biochemical and biophysical characterization of these target proteins. These studies have direct medical relevance, for example, misfolding and aggregation due to defi ciencies in the endoplasmic reticulum associated degradation (ERAD) pathway lead to a variety of pathophysiological states such as the neurodegenerative disorders Alzheimer’s and Parkinson’s disease.

Fig. 2:Split GFP assay. (Top) Fluorescence microscopy for two selected yeast strains. (B) GFP fl uorescence intensity distribution curves (MFI, mean fl uorescence intensity) for the split GFP combinations and controls (GFP and non-GFP).

Protein folding and maturation in the endoplasmic reticulum

Secretory proteins are translocated into the endoplasmic reticulum where a sophisti-cated machinery assists them in achieving their native conformations. Many of these newly synthesized proteins are N-glycosyl-ated and/or contain disulfi de bonds, which stabilize their structures. We are studying the enzymes oligosaccharyl transferase and protein disulfi de isomerase (PDI), which are essential for the folding and maturation of proteins passing through the ER.

Protein disulfi de isomerase catalyzes both the formation of disulfi de bonds in newly synthesized proteins and the isomerization of incorrectly formed disulfi de bonds. The enzyme features a modular architecture with four thioredoxin domains, a, b, b’ and a’, and a C-terminal tail (referred to as c). We have now determined two crystal structures of protein disulfi de isomerase from baker’s yeast, which we refer to as the twisted “U” and “boat” conformations. The rigid b and b’ domains form a common platform in both structures, to which the a and a’ domains are attached via fl exible linkers. Signifi cant conformational changes exist between the two structures, in par-ticular, the a domain undergoes a drastic rotational motion. In the second crystal form, we also observed a PDI dimer (Fig. 1), in which a signifi cant amount of sur-face area is buried.

Fig. 1:Structure of the PDI dimer. One subunit is in ribbonand the other in surface representation with the a, b, b’ and a’ domains in magenta, cyan, orange/yellow and red, respectively, and the C-terminal domain in green.

Hermann Schindelin

E-mail: [email protected]: +49(0)931 201 483 20Fax: +49(0)931 201 483 09http://www.rudolf-virchow-zentrum.de/forschung/schindelin.html

We addressed the signifi cance of the ob-served dimer by in vitro and in vivo studies. Analytical ultracentfi guation indicated a dimer population at high protein concen-

trations with an estimated dimer dissocia-tion constant of around 0.1 mM. Given the high abundance of the enzyme in the ER, with a reported concentration of ~0.2-0.5 mM, the PDI dimer would account for more than 50% of the total protein based on the estimated KD. To address the relevance of the dimer in vivo, a haploid PDI strain was generated containing two plasmid-borne copies of PDI one with a HA3-tag and one with a Myc3-tag. Microsomes from this strain were exposed to a crosslinker, followed by immunoprecipiation with anti-HA antibody and Western blot analyses. Myc3-tagged PDI was found to crosslink with HA3-tagged PDI, indicating that PDI in the ER can form either a dimer or higher oligomers. Finally, the so-called “split-GFP” approach was used to confi rm PDI dimer-ization in the ER. GFP was divided into an N-terminal (NGFP) and C-terminal half (CGFP), which were individually attached to PDI. Assuming that PDI dimerizes, the two halves of GFP will be brought into close spatial proximity to reconstitute in-tact GFP, as characterized by its ability to

emit fl uorescence. To minimize constraints during the reassembly of split-GFP, the two halves of GFP were attached to either ter-minus of PDI and the following combina-tions were explored: NGFP-PDI+CGFP-PDI, PDI-NGFP+CGFP-PDI, NGFP-PDI+PDI-CGFP and PDI-NGFP+PDI-CGFP. For all combina-tions, a subset of cells was observed to emit fl uorescence (Fig. 2), which indicated the successful reassembly of the GFP halves into intact GFP. In the dimer observed in the 22° C structure, both the active site in the a’ domain and the potential substrate-binding site in the b’ domain are buried. This conformation of the dimer therefore presumably represents an “inactive” state of PDI since most of its catalytic elements are inaccessible.

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Lee, I., and Schindelin, H. (2008) Structural insights into E1-catalyzed ubiquitin activation and transfer to conjugating enzymes. Cell, 134, 268-78.

Li, H., Chavan, M., Schindelin, H., Lennarz, W.J., and Li, H. (2008) Structure of the oligosaccharyl trans-ferase complex at 12 A resolution. Structure, 16, 432-40.

Li, G., Zhao, G., Schindelin, H., and Lennarz, W.J. (2008) Tyrosine phos-phorylation of ATPase p97 regulates its activity during ERAD. Biochem Biophys Res Commun., 375, 247-51.

Tian, G., Kober, F.X., Lewandrowski, U., Sickmann A., Lennarz, W.J., and Schindelin, H. (2008) The catalytic activity of protein-disulfide isomer-ase requires a conformationally flex-ible molecule. J Biol Chem, 283, 33630-40.

Zhao, G., Li, G., Zhou, X., Matsuo, I., Ito, Y., Suzuki, T., Lennarz, W.J., and Schindelin, H. (2008) Structural and mutational studies on the importance of oligosaccharide binding for the ac-tivity of yeast PNGase. Glycobiology, in press.

Extramural Funding

DFG Schi 425/2-1, PI Hermann SchindelinDFG Schi 425/3-1, PI Hermann SchindelinDFG SFB487, C7, PI Hermann Schindelin

Fig. 4: Dlc-gephyrin interaction. (Top) Surface representa-tion of the Dlc dimer (hydrophobic residues in green) with the bound gephyrin peptide (yellow C-atoms) and the corresponding electron density. (bottom) Detailed representation of interacting residues with the Dlc monomers in green or blue.

Fig. 3: Substrate-binding by PNGase. (Top) Ribbon dia-gram with chitobiose (yellow) covalently linkedto the active site cysteine (magenta). (Bottom) Sur-face representation of PNGase modeled in complex with a glycopeptide (with peptide and carbohy-drate C-atoms in green and yellow, respectively).

Endoplasmic reticulum associated pro-tein degradation

Endoplasmic reticulum associated protein degradation (ERAD) is an essential cellular pathway which prevents the accumulation of misfolded (glyco)proteins in the ER and entails their recognition, retrotransloca-tion to the cytosol, ubiquitination and degradation by the proteasome. Peptide:N-glycanase (PNGase) is an important ERAD component since it deglycosylates mis-folded glycoproteins, thus facilitating their proteasomal degradation. PNGase belongs to the transglutaminase superfamily and features a Cys, His, and Asp catalytic triad that is essential for its enzymatic activity. An elongated substrate-binding groove centered on the active site Cys191 was vi-sualized in earlier crystal structures, which on one end binds the peptide moiety of the substrate as demonstrated by a complex with Z-VAD-fmk, a peptide-based inhibitor targeting the active site cysteine. Cys191 can also be labeled with haloacetamidyl-containing carbohydrate-based inhibi-tors, and we have determined the crystal structure of yeast PNGase in complex with N,N‘-diacetylchitobiose. We found that the disaccharide binds on the side opposite to the peptide-binding region with the active site Cys191 being located approximately midway between the carbohydrate and peptide binding sites (Fig. 3). Mutagenesis studies confirmed the critical role of the chitobiose-interacting residues in substrate binding and suggested that efficient oligo-saccharide binding is required for PNGase activity. In addition, the N-terminus of a symmetry-related PNGase was found to bind to the proposed peptide-binding site of PNGase. Together with the bound chito-biose, this enabled us to propose a model for glycoprotein binding to yeast PNGase.

Following up on earlier observations that the N-terminal domain of mammalian PNGases interacts with the C-terminus of the AAA-ATPase p97, in particular its last 10 residues, and that phosphorylation of the penultimate tyrosine residue of p97 abrogates this interaction. To follow this up we investigated the physiological con-sequences of this posttranslational modi-fication. We found that the c-Src kinase directly and selectively phosphorylated the penultimate tyrosine of p97 in vitro. Fur-ther experiments showed that overexpres-sion of c-Src significantly increased the phosphorylation level of p97 in cells and caused accumulation of the GFP-labeled ERAD substrate TCRα, as well as ubiquitin-conjugated substrates. All these results point toward a possible role of p97 phos-phorylation in misfolded glycoprotein deg-radation. Since phosphorylation of p97’s highly conserved penultimate tyrosine residue not only blocks binding of PNGase to p97, but also the substrate-processing p97 cofactor Ufd3, and possibly other co-factors, it is currently not clear which of these perturbed interactions leads to the observed phenotype.

Neuroreceptor anchoring

The neuroreceptor anchoring protein ge-phyrin is essential for the proper location of inhibitory neuronal glycine and GABAA receptors. Gephyrin presumably forms a hexagonal scaffold below the postsynaptic membrane, thus allowing it to cluster gly-cine and GABAA receptors and also bridges them to cytoskeletal elements. Gephyrin is also involved in the transport of glycine receptors. In this context, the gephy-rin-GlyR complex interacts with the light chains of the dynein motor complex, an in-teraction mediated by a binding motif lo-cated in the central linker of gephyrin. We have determined the crystal structures of the light chains I and II in their apo-state, and in complex with a peptide derived from the gephyrin linker. Two of these peptides bind in a symmetric fashion to the light chain dimer, each extending two existing β-sheets by an additional strand (Fig. 4). We have analyzed the contribution of resi-dues in the interface by site-directed muta-genesis and isothermal titration calorimetry. These studies revealed that the C-terminal half of the gephyrin peptide, including the Gly-Val-Gln tripeptide, is primarily recognized by the dynein light chains.

34

Albert Sickmann

E-mail: [email protected]: +49(0)931 201 487 30Fax: +49(0)931 201 481 23http://www.rudolf-virchow-zentrum.de/forschung/sickmann.htmlhttp://www.protein-ms.de

Fig. 1:Comparison of a current membrane survey and pub-lished platelet proteome data.

Since September 2008, Albert Sickmann is the new Director of the Department of Proteomics at the Institute for Analytical Sciences (ISAS), Dortmund. The group is currently in transition to Dortmund.

Mass spectrometry based methods increasingly replace and bolster traditional techniques for protein characteriza-tion. Therefore, proteomics has gained a premiere position within numerous ongoing research projects. Although initial attempts with whole cell proteomic approaches have shown the dire need for further development of ana-lytical approaches, the fi rst datasets also delivered scores of novel candidates, e.g. for cardiovascular research. However, a distinct shift away from large-scale proteomics towards subject-driven, more focused approaches is taking place. Moreover, analyses of post-translational modifi cations like phosphorylation and glycosylation as well as methods for quantifi cation are hot topics of current research. Applied to human platelets, our studies function as mandatory steps for the analysis of major signaling networks and are thus of benefi t for the fi eld of platelet-related cardiovascular research.

The Platelet Proteome

Platelets represent a unique cell type re-sponsible for numerous functions in throm-botic, hemostatic and lately infl ammatory events. However, the whole complexity of platelet involvement remains elusive in many cases. This is partially due to the un-known protein content of human platelets, rendering functional analysis a challenging task. For detailed analysis of the platelet proteome, we employ several strategies to elucidate whole-cell and subcellular pro-teomes, as well as their post-translational modifi cations such as phosphorylation and glycosylation.

Platelet Plasma Membrane Proteomics

Platelet plasma membrane proteins are of immediate importance for outside-in and inside-out signaling cascades, e.g. during activation and aggregation of platelets during hemostatic and thrombotic events. Already, a fi rst selection of membrane span-ning proteins has been established, includ-ing proteomic approaches as shown by Moebius et al in 2005. Nevertheless, many of these approaches were unable to iden-tify low abundant or hardly accessible pro-teins such as seven-transmembrane span-ning receptors. Although antibody-based methods succeed in detecting them, they are of limited use regarding unknown spe-cies. In sharp contrast, proteomic methods

with suitable sample preparation can over-come this pitfall due to their unique and effi cient mode of peptide sequencing.

We therefore adopted an alternative preparation method for plasma membrane enrichment by aqueous two-phase parti-tioning (Schindler et al., J Proteome Res, 2008) and applied this in a subsequent large-scale analysis of platelet plasma membrane proteins. Roughly 1300 proteins were identifi ed in platelet preparations with more than half being intrinsic mem-brane proteins. Thereof, 362 proteins could be directly linked to the plasma membrane by gene ontology (GO) annotations, leaving a vast number of membrane proteins with unknown subcellular distribution and func-tion. Moreover, using GO terms, approxi-mately 300 proteins could be identifi ed as being involved in signal transduction and cell communication. This gives but a mere hint about the opportunities this vast and valuable collection of platelet proteins could offer to the functional research com-munity. In comparison to other approach-es, we identifi ed more proteins than any other singular platelet proteome study to date (compare Fig. 1), despite our focus on membrane samples. The accumulated data is estimated to trigger research efforts of other research groups and might lead to a deeper understanding of the complex events accompanying platelet function.

Glycoslations

Glycosylation of membrane proteins as well as secreted components is known to have a major impact on the synthesis, structure and thereby function as well as activity of protein isoforms. Although initial glyco-sylation site studies have been conducted on platelets, still a large number of modi-fi cation sites remain yet unknown, since 50% of the proteins are estimated to be glycosylated. In this context, most strate-gies for glycosylation site analysis rely on enrichment of glycopeptides by various means. While titaniumdioxide has already been demonstrated to enrich for sialylated glycopeptides, we explored the enrichment effect of electrostatic repulsion hydro-

35

Fig. 2:Differential quantification of phosphopeptides by multiple reaction monitoring.

Fig. 3:N-terminal combined fractional diagonalchromatography (COFRADIC).

Phosphorylations

In contrast to the more static glycopro-teome of platelets, phosphorylation-de-pendent protein modulation is a highly dynamic, but concerted process during platelet activation, which possibly involves hundreds of regulations. Based on an exten-

Outlook

While the platelet proteome project represents one of the major research top-ics of the group, other projects in the cardiovascular field include the phosphor-ylation-dependent analysis of the VASP-interactome in platelets and endothelial cells using affinity purification and mass spectrometric identification. Further ma-jor research topics include e.g. stoichi-ometry analysis of the SMN-complex via mass spectrometric label-free strategies. Upon its incorrect assembly, this complex is involved in the development of spinal muscular atrophy. Its in-depth analysis


Kroiss, M., Schultz, J., Wiesner, J., Chari, A., Sickmann, A., and Fischer, U. (2008) Evolution of an RNP assem-bly system: a minimal SMN complex facilitates formation of UsnRNPs in Drosophila melanogaster. Proc Natl Acad Sci U S A, 105, 10045-10050.

Lewandrowski, U., Sickmann, A., Ce-saro, L., Brunati, A.M., Toninello, A., and Salvi, M. (2008) Identification of new tyrosine phosphorylated proteins in rat brain mitochondria. FEBS Lett, 582, 1104-1110.

Meisinger, C., Sickmann, A., and Pfanner, N. (2008) The mitochondrial proteome: from inventory to func-tion. Cell, 134, 22-24.

Schindler, J., Lewandrowski, U., Sickmann, A., and Friauf, E. (2008) Aqueous polymer two-phase systems for the proteomic analysis of plasma membranes from minute brain sam-ples. J Proteome Res, 7, 432-442.

Zahedi, R.P., Lewandrowski, U., Wi-esner, J., Wortelkamp, S., Moebius, J., Schutz, C., Walter, U., Gambaryan, S., and Sickmann, A. (2008) Phospho-proteome of resting human platelets. J Proteome Res, 7, 526-534.

philic interaction chromatography (ERLIC). We were able to efficiently enrich glycan bearing peptides, thus enabling the sepa-ration and identification of 125 glycosyl-ation sites on 66 platelet proteins with a potential for future isoform resolution of glycopeptides.

sive survey of phosphorylation sites from resting platelets with over 1000 identified phosphorylation sites to date (Zahedi et al., J Proteome Res, 2008), a comparative approach was started to detect relative dif-ferences, between individual phosphoryla-tion sites. Therefore, resting and Iloprost stimulated platelet samples were subjected to phosphopeptide enrichment and label-free relative quantification via multiple reaction monitoring as shown in Figure 2. So far, the ratio of two-fold regulated to unchanged sites is roughly 1:10 within this ongoing work. The derived information will be introduced into future bioinformatic models of platelet regulatory networks.

including subunit composition, will help to further clarify its detailed function-al mechanisms (Kroiss et al., Proc Natl Acad Sci U S A, 2008). In addition, a premier focus is set on mitochondrial proteomics. This includes the search for mitochondrial phosphopeptides as dem-onstrated for platelets or yeast (Lewand- rowski et al., FEBS Lett, 2008) as well as the functional analysis of the mitochondrial protein import machinery (Meisinger et al., Cell, 2008), e.g. by de-dicated N-terminal peptide sorting using COFRADIC approaches.

Extramural Funding

DFG (Si 835/2-1), (Si 835/5-1), (Trans-regio 17), (SFB 688), (GK 1342), BMBF (QuantPro Project C)

36

Role of membrane-type 1 matrix metal-loproteinase (MMP-14) in invasion

The invasion and metastasis of cancer cells from the primary tumor to other organs in the body are the main cause for compro-mised life expectancy and quality in cancer patients. As a prerequisite to metastasis, cancer cells disseminate into the adjacent tumor stroma and degrade extracellular matrix (ECM), thereby causing destructive tissue remodeling. Recent progress in high-resolution multimodal microscopy recently provided a detailed four-dimensional map on where and how cell surface-localized proteases, particularly membrane-type 1-matrix metalloproteinase (MT1-MMP, MMP-14) control two distinct types of 3D tissue invasion by ECM cleavage. Thereby, indi-vidually moving cells (“pathfi nder cells”) cleave collagen fi bers several micrometers rearward from the leading edge and re-align these fi bers into parallel bundles that bor-der tube-like ECM microtracks. These micro-tracks are wide enough to encompass an individual cell body (10 µm) (1). Next, the uniform large-scale clearance of a directly bound ECM interface layer is achieved by multiple cells that that follow along this track and remain connected which leads to multicellular invasion along wider macro-

The Cell Dynamics group focuses on the visualization of cell-matrix interactions and dynamic cell patterning during immune cell interactions and tumor invasion. We use 3D extracellular matrix (ECM) based cell culture models and advanced imaging procedures to monitor cellular and molecular events during tissue invasion, im-mune cell interactions and tissue remodeling. To validate in vitro fi ndings, in vivo-imaging of tumor and immune cell migration is performed by multiphoton microscopy. Key topics are the diversity of tumor invasion and inter-action with the tumor stroma; novel escape responses of tumor progression during targeted experimental thera-py; the serial dynamics of T-cell scanning across antigen-presenting cells during immune cell activation and effector function; and the regeneration of epithelial and interstitial tissue using tissue engineering.

Fig. 1:Set-up (A) and resolution (B) of near-infrared and infrared multiphoton microscope.

Peter Friedl

E-mail: [email protected]: +49(0)931 201 267 37Fax: +49(0)931 201 267 00http://www.rudolf-virchow-zentrum.de/forschung/friedl.html

Since October 2007, Peter Friedl holds the Chair for Microscopical Imaging of the Cell at the Nijmegen Centre for Molecular Life Sciences, Radboud University, Nijmegen, Netherlands, but remains an active member of the Rudolf Virchow Center and the Department of Dermatology in Würzburg.

tracks. By yet unclear mechanisms, cells retain cell-cell junctions and invade collec-tively (2). The transition from proteolytic to nonproteolytic dissemination represents a molecular switch of cancer cell migra-tion mode, and thus, highlights an escape strategy of cancer dissemination through tissue after molecular interference (1). The understanding of such plasticity and the mechanisms of cell-cell communication during collective invasion will be impor-tant in elucidating combination interfer-ence strategies against the metastaticcascade of cancer.

Using a tunable optical parametric oscil-lator as excitation light source we show that infrared-excited (IR-)MPM at wave-lengths above 1080 nm doubles the imag-ing depth, enables effi cient excitation of red fl uorophores and fl uorescent proteins

Intravital multiphoton microscopy of cancer invasion in vivo

Multiphoton microscopy (MPM) has become the method of choice for investigating cell structure and function in tissues and or-gans, including the invasion and progres-sion of cancer lesions. We have set up a prototype of an infrared-excited MPM plat-form that provides high-resolution micro-scopy (Fig. 1).

37


Alexander, S., Koehl, G.E., Hirschberg, M., Geissler, E.K., and Friedl, P. (2008) Dynamic imaging of cancer growth and invasion: a modified skin-fold cham-ber model. Histochem. Cell Biol, 130, 1147-1155.

Andresen, V., Alexander S., Heupel W.-M., Hirschberg M., and Friedl, P. (2008) Infrared multiphoton microscopy: sub-cellular-resolved deep tissue imaging. Curr Opin Biotechnol, in press.

Friedl, P., and Wolf, K. (2008) Tube travel: protease functions in individual and collective cancer invasion. Cancer Res, 68:7247-7249.

Friedl, P., and Weigelin, B. (2008) Inter-stitial leukocyte trafficking and immune function. Nat Immunol, 9, 839-848.

Extramural Funding

DFG FR1155 (6-3, 7-1, 8-1, 8-2)German Cancer Foundation 106950Dutch Cancer Foundation (KWF 2008-4031)FP6 NoE EMIL – LSHC-CT – 2004-503569FP7 Project ENCITE HEALTH TH-15-2008-208142

Fig. 2:Improved excitation (A) and detection (B) of red fluorophoes by IR MPM.

Fig. 3:Intravital multiphoton microscopy of live tumor xenograft in nude mouse.

Awards

German Cancer Award 2008

The dynamic immunological synapse

The migration of leukocytes is integral to their function, including activation and ef-fector function (4). For their homeostasis, T-cells have to integrate non-cognate TCR-dependent and –independent signals to survive and weakly proliferate. In contrast to antigen-specific engagement, homeo-static interactions are migratory and lack stable T-cell-binding to the antigen-pre-senting cell (APC). We show that T-cells contacting dendritic cells (DC) in the ab-sence of cognate antigen maintain amoe-boid crawling, polarize toward the DC and form a receptor- and signal-rich interaction plane composed of three zones: the actin-rich leading edge poor in signal but driving migration; a mid-zone mediating TCR/MHC-

class II signaling and proliferation; and the rear uropod generating MHC-independent signals implicated in survival (J. Storim and P. Friedl, submitted). Although short-lived, this dynamic immunological synapse mediates TCR-dependent and -independent signals via distinct domains, whereby amoeboid-moving T-cells serially sample APC without a stop signal. Similar contact dynamics are maintained by cytolytic ef-fector cells engaged with antigen-present-ing target cells. An understanding of how migration and stable cell-cell interactions contibute to the tuning of immune re-sponse will be important to balance migra-tion-targeting therapy in immune interven-tion and vaccination.

(Fig. 2), and reduces phototoxicity and photobleaching, compared to conventional MPM (3).

Cancer invasion and metastasis are the result of pro-migratory activity in tumor cells which is in part induced and directed by structural and molecular signals pro-vided by the tumor microenvironment (Fig. 3). IR-MPM of orthotopic HT-1080 fibrosar-coma xenografts revealed deep collective invasion strands of several hundred con-nected cells. Collective invasion is a spe-cial invasion type of cell masses that are connected by cell-cell junctions, thereby forming an ill-fated, abortive variant of migration seen during embryological devel-opment. In future studies, intravital deep tumor imaging by IR-MPM will be used to address molecular therapy in order to over-come radioresistance.

38

Platelets are small anuclear cells that are produced by bone marrow megakaryocytes and circulate for approxi-mately 10 days in the blood stream before they are cleared by cells of the reticuloendothelial system. During their lifetime, most platelets never undergo fi rm adhesion. As a response to vascular injury, however, platelets rapidly adhere to tissue and to one another to form a platelet plug, which, in combination with the coagulation system, allows re-establishment of normal blood fl ow in the disrupted vasculature. If this process occurs in an uncontrolled manner in diseased vessels, e.g. upon rupture of an atherosclerotic plaque it may lead to arterial occlusion and infarction of vital organs. Such acute ischemic cardiovascular and cerebrovascular syndromes are still a major cause of death or serious pathological complications in Western societies. Our group uses genetically modifi ed mouse lines in combination with disease models to identify new strategies to inhibit the thrombotic and/or pro-infl ammatory activity of the cells while preserving their hemostatic function. Since platelets and other cells of hematopoietic origin share many signaling pathways, we also study mechanisms of immune cell activation in order to defi ne the function of proteins of interest. During the last year, the mechanisms of Ca2+ entry in platelets and immune cells became a focus of our research.

Fig. 1:STIM1 and Orai1 are essential for SOCE in platelets. (A) Structure of STIM1. SAM indicates sterileα-motif; TM, transmembrane domain; CC, coiled-coil domain. (B) Store-operated calcium entry (SOCE)in platelets. G-protein coupled receptors (GPCRs) activate PLCβ through Gq, whereas some adhesion receptors activate PLCγ2 leading to calcium store release through inositol-1,4,5-trisphosphate receptors (IP3-R) in the endoplasmic reticulum (ER) membrane. This disrupts the calcium binding of the EF hand domain of STIM1 in the ER lumen and leads to the activation and redistribution of STIM1 to plasma membrane (PM) near puncta where it opens Orai1, the major store-operated calcium (SOC) channel in the PM, to allow calcium entry. (C) Store release and SOCE was studied in platelets of the indicated mouse lines using Fura-2 loaded platelets and fl uorimetric intracellular calcium measure-ments. The store content was estimated by the use of the SERCA inhibitor thapsigargin, whereas SOCE was measured by the subsequent addition of extracellular calcium. Representative curves and mean changes in the intracellular calcium concentration (Δ[Ca2+]i) plus or minus SD.

Bernhard Nieswandt

E-mail: [email protected]: +49(0)931 201 440 60Fax: +49(0)931 201 440 68http://www.rudolf-virchow-zentrum.de/forschung/nieswandt.html

Orai1, but not transient receptor potential channel 1 is the platelet SOC channel

Although SOCE has long been thought to be a major pathway for Ca2+ entry in platelets, the identity of the SOC channel in platelets has been controversially debated. Some investigators proposed transient receptor potential (TRP) C1 to fulfi l this function based on the observation that antibod-ies against the channel impaired SOCE in platelets. However, others could not detect TRPC1 in the plasma membrane of plate-lets and raised doubts about the specifi c-ity of the inhibiting anti-TRPC1 antibod-ies. To address the role of TRPC1 in SOCE in platelets, we analyzed mice lacking TRPC1 in cooperation with Alexander Dietrich and Thomas Gudermann (Marburg). Platelets from these mice display fully intact SOCE and also otherwise unaltered calcium ho-

Stromal interaction molecule 1 is essen-tial for store-operated calcium entry in platelets.

Agonist-induced elevation of [Ca2+]i is a central step in platelet activation, but the underlying mechanisms are not fully under-stood. A major pathway for Ca2+ entry in non-excitable cells involves receptor-medi-ated release of intracellular Ca2+ stores fol-lowed by activation of store-operated cal-cium (SOC) channels in the plasma membrane. Stromal interaction molecule 1

(STIM1) is a Ca2+ sensor in the sarcoplas-mic/endoplasmic reticulum (SR/ER) that activates Ca2+ release activated channels (CRAC) in T-cells. However, its function in mammalian physiology in general, and platelets specifi cally, is unknown. We have generated mice constitutively lacking STIM1. These mice display early postnatal lethality and marked growth retardation. For studies on platelet function, bone mar-row from these mice was transplanted into lethally irradiated wild-type recipients and these animals were analyzed after 4-6

weeks. Chimeras with STIM1-defi cient platelets displayed a marked defect in ago-nist-induced Ca2+ responses, as well as im-paired activation and thrombus formation under fl ow in vitro. Importantly, this defect translated into effective protection from arterial thrombosis and ischemic brain in-farction, but only led to mild bleedingtime prolongation. These results establish STIM1 as an important mediator in the pathogenesis of ischemic cardio- and cere-brovascular events (Varga-Szabo et al., J Exp Med, 2008).

39


Braun, A., Gessner, J.E., Varga-Szabo, D., Syed, S.N., Konrad, S., Stegner, D., Vögtle, T., Schmidt, R.E., and Nieswandt, B. (2008) STIM1 is essential for Fcγ re-ceptor activation and autoimmune in-flammation. Blood, in press.

Braun, A., Varga-Szabo, D., Kleinschnitz, C., Pleines, I., Bender, M., Austinat, M., Bösl, M., Stoll, G., and Nieswandt, B. (2008) Orai1 (CRACM1) is the platelet SOC channel and essential for patho-logical thrombus formation. Blood, in press.

Stoll, G., Kleinschnitz, C., and Nieswandt, B. (2008) Molecular mechanisms of thrombus formation in ischemic stroke: Novel insights and targets for treatment. Blood, 112, 3555-3562.

Varga-Szabo, D., Authi, K., Braun, A., Bender, M., Ambily, A., Hassock, S.R., Gudermann, T., Dietrich, A., and Nieswandt, B. (2008) Store-operated Ca2+ entry in platelets occurs indepen-dently of transient receptor potential (TRP) C1. Pflug Arch, 457, 377-387.

Varga-Szabo, D., Braun, A., Kleinschnitz, C., Bender, M., Pleines, I., Pham, M., Renne, T., Stoll, G., and Nieswandt, B. (2008) The calcium sensor STIM1 is an essential mediator of arterial thrombo-sis and ischemic brain infarction. J Exp Med, 205, 1583-1591.

Extramural Funding

IZKF Würzburg (E 30) DFG (SFB 688 TP A1; TP A3; TP B1), (Ni 556/7-1)

Fig. 2:STIM1 is required for the FcγR-induced generation of C5a, which itself is essential to induce neutrophil accumulation at extravascular sites in pneumonitis. Left panel: the signaling cascades leading to immune complex-induced pulmonary inflammation in the wild-type. Right panel: Generation of C5a and in consequence neutrophil influx and hemorrhage are blocked in Stim1-/- bone marrow-chimeric mice.

meostasis as compared to wild-type. Fur-thermore, platelet function in vitro and in vivo was not altered in the absence of TRPC1. Finally, studies on human platelets revealed that the presumed inhibitory anti-TRPC1 antibodies have no specific effect on SOCE and fail to bind to the protein. Together, these results provided evidence that SOCE in platelets is mediated by chan-nels other than TRPC1 (Varga-Szabo et al., Pflug Arch, 2008).

Another platelet SOC channel candidate was Orai1 (CRACM1), the recently discov-ered SOC (CRAC) channel in T-cells and mast cells, but its role in mammalian physiology was unknown. Indeed, we found Orai1 to be strongly expressed in human and mouse platelets. To test its role in blood clotting, we generated Orai1-deficient mice. Similar to Stim1-/- mice, these animals showed a high incidence of perinatal lethality and marked growth retardation. Orai1-/- plate-lets displayed severely defective SOCE (Fig. 1), agonist-induced Ca2+ responses, and impaired activation and thrombus forma-tion under flow in vitro. As a direct conse-quence, Orai1-deficiency in mice resulted in resistance to pulmonary thromboem-bolism, arterial thrombosis and ischemic brain infarction, but only mild bleeding time prolongation. These results estab-lish Orai1 as the long-sought platelet SOC channel and a crucial mediator of ischemic cardio- and cerebrovascular events (Braun et al., Blood, 2008a).

STIM1 is essential for FcγR activation and autoimmune inflammation

Fcγ receptors (FcγRs) on mononuclear phagocytes trigger autoantibody and im-mune complex-induced diseases through coupling the self-reactive IgG response to innate effector pathways, such as phago-cytosis, and the recruitment of inflamma-tory cells. FcγR-based activation is critical in the pathogenesis of these diseases, al-though the contribution of FcγR-mediated calcium signaling in autoimmune injury is unclear. We analyzed the role of store-oper-ated calcium entry for FcγR-mediated mac-rophage activation using STIM1-deficient bone marrow chimeric mice. Stim1-/- mar-crophages were unable to activate FcγR-in-duced Ca2+ entry and phagocytosis whereas the associated induction of inflammatory cytokines was only mildly affected. As a direct consequence of this defined defect, STIM1 deficiency resulted in resistance to experimental immune thrombocytopenia

STIM1-independent T-cell development and effector function in vivo

SOCE is believed to be of pivotal impor-tance in T-cell physiology. To test this hypothesis, we analyzed T-cell function in STIM1-deficient mice in collaboration with Thomas Kerkau (Immunology Department, Würzburg). In vitro analyses showed that SOCE and antigen receptor complex-trig-gered Ca2+ flux into STIM1-deficient T-cells was virtually abolished. In vivo, STIM1-de-ficient mice developed a lymphoprolifera-tive disease despite normal thymic T-cell maturation and normal frequencies of CD4+ Foxp3+ regulatory T-cells. Unexpectedly, STIM1-deficient bone marrow chimeric mice mounted humoral immune responses after vaccination and STIM1-deficient T- cells were capable of inducing acute graft-versus-host disease following adoptive transfer into allogeneic hosts. These results demonstrate that STIM1-dependent SOCE is crucial for homeostatic T-cell proliferation, but of much lesser importance for thymic T-cell differentiation or T-cell effector functions (Beyersdorf et al., J Immunol, 2009).

and anaphylaxis as well as autoimmune hemolytic anemia. In a model of immune complex-triggered acute pneumonitis the mice were completely protected, most like-ly due to a highly selective defect in the generation of biocactive anaphylatoxin C5a (Fig. 2; Braun et al., Blood, 2008b). These results establish STIM1 as a novel and es-sential component of FcγR activation and also indicate that inhibition of STIM1-dependent signaling might become a new strategy to prevent or treat IgG-dependent immunological diseases.

40

Adhesion molecules mediate tissue-specifi c recruitment of leukocytes, a key step in the pathogenesis of infl am-matory disorders. We investigate the role of adhesion molecules and whether they can be exploited as therapeu-tic target structures in infl ammatory disorders. In addition, we investigate how tumors progress, why they are resistant to chemotherapy and how novel therapeutic compounds can overcome mechanisms of resistance.

Adhesion molecules: mediators of recruit-ment and morphogenesis of immune cells

Proper immune surveillance as well as the pathogenesis of infl ammatory disordersrequire the specifi c recruitment of lympho-cytes and other cells of the immune system to their target tissues. In all tissues, the multistep cascade of lymphocyte recruit-ment starts with rolling along the vessel wall, a process that is primarily mediated by adhesion molecules of the selectin fam-ily. The subsequent steps are fi rm attach-ment and extravasation.

Three members of the selectin family are known: L-selectin is expressed on leuko-cytes, and P- and E-selectin are expressed by endothelial cells. Selectins have been implicated in a variety of disorders whose initial steps depend on interactions be-tween circulating blood cells and endo-thelial cells, thus rendering selectins attrac-tive targets for specifi c therapies against infl ammatory disorders. Several other ad-hesion molecules, such as some integrins or members of the Immunoglobulin-like su-perfamily including, expressed by circulat-ing immune cells or endothelial cells also contribute to leukocyte extravasation.

In close collaboration with groups from the Universities of Frankfurt, Lübeck, Kiel, Erlangen and Cambridge (UK), we have contributed to demonstrating that specifi -cally blocking functions of P- and L-selec-tin reduces allergic responses in preclinical models. In addition, we have demonstrated that selectins are pivotally involved in the recruitment of dendritic cells to lymph

Fig. 1:Integrin αE(CD103)ß7 affects morphogenesis of leukocytes. Epidermal sheets were separated from the dermis and plated upside-down. K562 cells transfected with YFP-fused integrin αE–constructs and wildtype ß7 (αE–open/YFP and ß7) were seeded on the exposed undersurface of the epidermis, and the formation of dendrites was visualized by confocal microscopy (left panel). Examples of fi lopodia extending across or between epidermal keratinocytes are indicated by arrows. The right panel depicts a control cell transfected with GFP (Schlickum et al., Blood, 2008).

Michael P. Schön

E-mail: [email protected]: +49(0)551 396 401Fax: +49(0)551 396 841http://www.rudolf-virchow-zentrum.de/forschung/schoen.html

Since April 2008, Michael Schön is the new Director of the Department of Dermatology and Venerology, Universitätsmedizin Göttingen.

nodes, thus presumably increasing the mounting of cellular immune responses. Fi-nally, we have contributed to the showing that polymorphisms of PECAM-1 (platelet endothelial cell adhesion molecule-1, a member of the Immunoglobulin-like super-family of adhesion molecules) affect mono-cyte adhesion to endothelial cells.

Once lymphocytes are extravasated (i.e. have left the blood vessels), they localize into the surrounding connective tissue, and eventually, into epithelia of the cor-responding organs. Within these compart-ments, lymphocytes exert their pathologic functions, such as secretion of cytokines, cytotoxicity and stimulation of other im-mune cells and resident tissue cells. It is

therefore, conceivable that the proper positioning and shaping of lymphocytes (morphogenesis) within epithelial tissues plays a crucial role for these functions, and receptors that mediate the intraepithelial localization of lymphocytes may be useful target structures in the therapy of infl am-matory disorders.

The integrin αE(CD103)β7, a heterodimeric adhesion molecule that is preferentially ex-pressed on intraepithelial lymphocytes, has been implicated in epithelial localization of T-cells through binding to E-cadherin. Thus, this receptor is a promising candi-date for the exertion of such functions. In close collaboration with a group from Cambridge, we could show by time-lapse

41


Alban, S., Ludwig, R.J., Bendas, G., Schön, M.P., Oostingh, G.J., Radeke, H.H., Fritzsche, J., Pfeilschifter, J., Kaufmann, R., and Boehncke, W.H. (2008) PS3, a semi-synthetic b-1,3-glucan sulfate, dimin-ishes contact hypersensitivity responses through inhibition of L- and P-selectin functions. J Invest Dermatol, in press.

Geserick, P., Drewniok, C., Hupe, M., Haas, T.L., Diessenbacher, P., Sprick, M.R., Schön, M.P., Henkler, F., Wajant, H., Gollnick, H., Walczak, H., and Leverkus, M. (2008) cFLIP is a critical regulator of TRAIL- or CD95-mediated apoptosis in human melanoma. Oncogene, 27, 3211-3220.

Schlickum, S., Sennefelder, H., Friedrich, M., Harms, G., Lohse, M.J., Kilshaw, P., and Schön, M.P. (2008) Integrin αE(CD103)β7 influences cellular shape and motility in a ligand-dependent fashion. Blood, 112, 619-625.

Schön, M.P., and Schön, M. (2008) TLR7 and TLR8 as targets in cancer therapy. Oncogene, 27, 190-199.

Schön, M., Wienrich, B.G., Kneitz, S., Schlickum, S., Sennefelder, H., Vöhringer, V., Hüttinger-Kirchhof, N., Stiewe, T., Ziegel- bauer, K., and Schön M.P. (2008) KINK-1, a novel small-molecule inhibitor of IKKβ, and the susceptibility of mela-noma cells to antitumoral treatment. J Natl Cancer Inst, 100, 862-875.

Extramural Funding

Deutsche Krebshilfe/Dr. Mildred Scheel-StiftungDFG EU (Angioskin)IZKFIndustry

Tumor progression and antitumoral-ther-apies

Resistance of malignant tumors to anti-tu-mor therapies is one of the central chal-lenges in the treatment of cancer patients. This problem is vividly illustrated by mela-noma, a tumor that is almost universally resistant to chemotherapy, resulting in a very poor prognosis once the tumors are metastasized. Therefore, understanding and targeting mechanisms that underlie the chemoresistance of malignant tumors, and/or the development of strategies by-passing mechanisms of resistance will be of great use for cancer therapy.

We have studied the molecular mode of action of small-molecular anti-tumor im-mune response modifiers. Imiquimod, a prominent example of such small mole-cules, which has already been approved for topical treatment of some forms of epithe-lial skin cancer, is known as an agonist at toll-like receptors (TLR) 7 and 8, and – as demonstrated previously by us – has direct pro-apoptotic effects at higher concentra-tions. We have then detected that imiqui-mod exerts novel off-target effects, which may enhance the overall clinical efficacy of the compound: The compound interferes with adenosine receptor signaling. This novel activity presumably results in inhi-bition of a negative feedback mechanism of inflammation at the interface of innate and adaptive immunity, and consequently increases inflammatory responses, which are the basis of the anti-tumor effects.

In another project, we have demonstrat-ed that specific inhibition of the IκB-ki-nase-β (IKKβ) by a novel small-molecular

compound may enhance the anti-tumor activity of cytostatics. Based on the ob-servation that the highly selective activity of the novel compound results in profound inhibition of the activation of nuclear factor-κB (NF-κB), a central transcription factor implicated in tumor progression and inducible chemoresistance, we have termed the compound KINK-1 (kinase inhibitor of NF-κB-1). The activity of KINK-1 leads to diminished activation and nuclear trans-location of NF-κB. Blocking IKKβ resulted in down-regulation of many apoptosis- and proliferation-related gene products, but did not by itself affect proliferation or apopto-sis of melanoma cells. However, significant inhibition of proliferation and increase of tumor cell apoptosis were observed when the IKKβ inhibitor was combined with some cytostatics. In vivo, IKKβ inhibition or certain cytostatics at low doses had no therapeutic effect, but a combination of the two significantly diminished pulmonary metastases in a preclinical model. Thus, the principle of IKKβ inhibition may enhance the efficacy of anti-tumor therapies and overcome mechanisms of chemoresistance.

Awards

Serono Award for Inflammation Research 2008Molecular Targeted Therapy of Cancer Award 2008

microscopy that expression of αE(CD103)β7

significantly increased the active move-ment of transfected cells. In addition, ex-pression of αE(CD103)β7 facilitated the for-mation of cellular protrusions and filopodia in a ligand-dependent fashion, both in transfection-based systems in vitro and in a naturally-occurring lymphocyte popula-tion, dendritic epidermal T-cells within mu-rine epidermis (the epithelium of the skin), in vivo. Based on these results, we propose that αE(CD103)β7 is involved in locomotion and shaping of intraepithelial lymphocytes and that it may be a promising therapeutic target in epithelial inflammatory disorders (Figure 1).

42

Our studies focus on the identifi cation and characterization of factors affecting eukaryotic gene expression at various stages. Studies on the early phase of mRNA transcription lead to the identifi cation of the Larp7 protein. This factor is part of the 7SK RNA-protein complex and regulates the production of mRNA by RNA polymerase II. The TOP-mRNAs, which encode proteins of the translation apparatus, are regulated at the translational level and hence at a late stage of gene expression. These mRNAs contain a 5’ Terminal OligoPyrimidine tract (TOP motif), which allows for the regulation in response to altered growth conditions. Factors interacting with the TOP-motif have been identifi ed and their function in TOP-mRNA regulation is currently being investigated. Lastly, we have identifi ed a fascinating chaperone system that generates U snRNPs of the spliceosome. This machinery affects gene expression by ensuring the accurate processing of mRNAs.

A role for Larp7 in 7SK RNP-mediated regulation of polymerase II

Based on its affi nity to oligoU-stretches, we identifi ed a thus far uncharacterized factor termed La-related protein (Larp7). This ubiquitous protein forms a distinct RNP complex of approx. 20S within the nucleus. Affi nity purifi cations revealed that Larp7 is part of the 7SK RNP, consisting of the non-coding 7SK RNA and the pro-tein factors Cdk9, CyclinT1 (together also termed pTEFb) and HEXIM. pTEFb stimu-lates transcription elongation by phos-phorylation of polymerase II, a process that is tightly controlled by the 7SK RNP. The sequestration of pTEFb by 7SK snRNA and HEXIM inactivates the Cdk9 kinase and hence prevents stimulation of pol II tran-scription. We have now shown that Larp7 binds to a U-stretch in the 3’terminus of 7SK snRNA and provides a bridge to Cdk9 and HEXIM. Importantly, reduced expres-sion of Larp7 strongly affects transcription from basal and HIV1-Tat-dependent pol II templates. Hence, Larp7 acts as a crucial factor in the regulation of P-TEFb via the 7SK RNP system (Markert et al., EMBO Rep, 2008, see also Fig. 1).

Fig. 1:7SK RNA controls the activity of pTEFb (composed of Cdk7 and Cyclin T1). Upon binding to pTEFb is sequestered in the RNP in an inactive form, which cannot stimulate polII transcription. Certain stimuli can release pTEFb from 7SK RNP and allow stimulation of transcription. A model illustrating the interactions within the 7SK RNP in its inhibitory state (left) and after stimulation (right) is shown.

Utz Fischer

E-mail: utz.fi [email protected]: +49(0)931 888 402 9Fax: +49(0)931 888 402 8http://www.biochem.biozentrum.uni-wuerzburg.de

RVZ Network Project

43


Chari, A., Golas, M.M., Klingenhäger, M., Neuenkirchen, N., Sander, B., Engl-brecht, C., Sickmann, A., Stark, H. and Fischer, U. (2008) An assembly chaper-one collaborates with the SMN-complex to generate spliceosomal SnRNPs. Cell, 135, 497-509.

Kroiss, M., Schultz, J., Wiesner, J., Chari, A., Sickmann, A., and Fischer, U. (2008) Evolution of an RNP assembly system: a minimal SMN complex facilitates forma-tion of UsnRNPs in Drosophila melano-gaster. Proc Natl Acad Sci U S A, 105, 10045-10050.

Markert, A., Grimm, M., Martinez, J., Wiesner, J., Meyerhans, A., Meyuhas, O., Sickmann, A., and Fischer, U. (2008) The La-related protein LARP7 is a compo-nent of the 7SK ribonucleoprotein and affects transcription of cellular and vi-ral polymerase II genes. EMBO Rep, 9, 569-575.

Extramural Funding related to project

FOR 855

Fig. 2:Model of the assisted assembly of spliceosomal U snRNPs and structures of key intermediates as determined by electron microscopy (lower part). The chaperone pICln organizes the Sm proteins in specific heterooligomeric complexes (6S and pICln-B/D3). The SMN-complex contacts these units (8S) and expels the chaperone. This generates the open ring conformation(7S), which allows U snRNA binding and assembly of the Sm core (Sm).

Identification of TOP-mRNA interacting proteins

TOP-mRNAs are an abundant class of mRNAs that encode ribosomal proteins and trans-lation factors and hence ensure the produc-tion of the translation machinery. Although it has been known for several decades that this class of mRNAs is under tight transla-tional control, the underlying mechanism is still completely unknown. One feature that distinguishes TOP-mRNAs from “normal” mRNAs is their unusual cap structure, m7GpppC. Assuming that this structure is recognized by a factor involved in TOP-mRNA regulation, we analyzed whether translation of a TOP-mRNA-report-er could be specifically inhibited with the synthetic cap analogue m7GpppC. We ob-served that this cap inhibited translation of the TOP-mRNA reporter very efficiently, whereas translation from a control mRNA lacking the TOP-motif and containing the normal m7GpppG-cap was unaffected. Con-versely, the dinucleotide m7GpppG reduced translation of non-TOP-mRNAs but failed to inhibit translation of the TOP-mRNA. These data postulate the existence of a cellular paralogue of eIF4E (i.e. the m7GpppG/A cap recognizing factor of “normal” mRNAs) that specifically recognizes the m7GpppC cap, and is required exclusively for the translation of TOP-mRNAs. Using affinity purification strategies we have purified candidate proteins that may interact with the cap of TOP-mRNAs. Their functional analysis is currently underway.

U snRNP assembly and pre-mRNA pro-cessing

Spliceosomal U snRNPs are major compo-nents of the so-called spliceosome, which catalyzes the removal of non-coding se-quences from pre-mRNAs, and hence an essential step in the realization of the ge-netic code. Spliceosomal U snRNPs contain a ring shaped core structure consisting of seven Sm proteins that assemble on a single stranded RNA-sequence found in U snRNAs. Although this “Sm core domain” assembles spontaneously in vitro, it has become clear in the last few years that this process requires a large number of trans-acting assembly factors. These factors are organized in two units, termed the SMN (survival motor neuron) complex and the PRMT5 (protein arginine methyltransferase 5) complex. Reduced expression of the SMN protein, a key protein in the SMN complex

is the underlying cause for Spinal Muscu-lar Atrophy, thus further demonstrating the importance of this pathway. Through a combination of structural and biochemi-cal studies the pathway leading to the as-sembly of the Sm core has been deciphered (Chari et al., Cell, 2008). pICln, a compo-nent of the PRMT5 complex, functions in the early phase of the pathway as an assembly chaperone that induces the formation of a higher order Sm protein unit. Under these conditions, Sm protein binding to snRNA is prevented, due to the induction of a kinetic trap. Subsequently, the SMN complex binds to this Sm unit and removes pICln, thereby allowing ring closure on snRNA. This sug-gests a mechanism similar to the one ob-served for the clamp loading reaction in DNA replication (Fig. 2).

44

Fig. 2:Ventricular septum defect in Tie2-cre Hey1fl ox/fl ox HeyL-/- embryos at E15.5, but not in Tie2-cre Hey1fl ox/wt HeyL-/- controls carrying a single functional Hey1 allele.

Development of complex biological structures such as the human heart or the vascular system depends on the coordinated interplay of multiple signaling pathways that control cellular differentiation. The Notch signaling pathway represents a central molecular switch for cell fate decisions in a large variety of tissues during develop-ment as well as in later organ homeostasis. This pathway conveys many of its effects through the Hey and Hes bHLH transcription factors, which represent the key transcriptional targets. Hey genes are critical for correct development of the heart, as refl ected by the fact that Hey2-/- as well as Hey1-/- / HeyL-/- mice suffer from severe congenital heart defects. Hey1 and Hey2 are also essential for angiogenesis and arterial fate decision, since a combined knock-out in the mouse leads to early embryonic lethality. We are interested in understanding the role of Notch signals and more specifi cally its downstream transcriptional effectors in these processes. For this we need to identify cell type specifi c targets of Hey proteins that execute the transcriptional program to modulate cell fate and behavior.

Hey genes control cardiovascular devel-opment

Notch signals control many binary and in-ductive signaling steps in development and differentiation (Fig. 1). During heart de-velopment Notch1 signaling is critical for the formation of the septum and valves. We could show that loss of the Notch target gene Hey2 in mice likewise leads to fatal cardiac septum and valve defects, indi-cating that Hey2 transmits Notch signals in cardiac development. Surprisingly, we observed very similar cardiac defects in Hey1/HeyL double knock-out mice, but not when only one of the genes was affected, suggesting that the three genes perform similar and at least partly overlapping functions in cardiac development. Expres-sion analysis identifi ed overlapping expres-sion only in the endocardium, which likely represents the critical site of action.

Fig. 1:Hey bHLH factors transduce signals of the Delta-Notch signaling pathway.

Manfred Gessler

E-mail: [email protected]: +49(0)931 888 415 9Fax: +49(0)931 888 703 8http://www.biozentrum.uni-wuerzburg.de/pc1/gessler

RVZ Network Project

Endocardial Hey expression is essential for cardiac development

We have now generated HeyL knock-out mice that carry in addition fl oxed Hey1alleles. While these mice do not showany overt heart phenotype, we could rep-licate the cardiac developmental defects as described above when a Tie2-drivencre transgene was introduced. Since Tie2is only expressed in the endocardial cells this clearly identifi es this cell popula-

tion as a key effector of Notch signaling for septum and valve development. Themorphogenetic problem lies in a reduced epithelial to mesenchymal transition (EMT) of endocardial cells in the atrio-ventricular canal, since we had shownearlier that these endocardium-derived cells are reduced in number and lackmesenchymal characteristics.

45


Doetzlhofer, A., Basch, M.L., Ohyama, T., Gessler, M., Groves, A.K., and Segil, N. (2008) Hey2 regulation by FGF provides a Notch-independent mechanism for maintaining pillar cell fate in the organ of Corti. Developmental Cell, in press.

Hu, X., Chung, A.Y., Wu, I., Foldi, J., Chen, J., Ji, J.D., Tateya, T., Kang, Y.J., Han, J., Gessler, M., Kageyama, R., and Ivashkiv, L.B. (2008) Integrated Regula-tion of Toll-like Receptor Responses by Notch and Interferon-gamma Pathways. Immunity, 29, 691-703.

Fig. 3:Hey1 represses its target genes (qRT-PCR). Examples are Hey1 itself and Bmp2.


DFG GK 1048, DFG Ge539/9-3Titratable Hey1 and Hey2 overexpression

Hey proteins have been shown to repress transcription of a small number of puta-tive target genes. Nevertheless, it is still unclear how many genes are responsive to Hey proteins and how these bHLH factors can influence transcription of their targets. To start with we generated stable HEK293 cells that express Flag-tagged Hey1 and Hey2 proteins in a doxicycline dependent manner. Comparison of uninduced and in-

Chromatin IP verifies target genes

There is evidence for both direct and indirect DNA binding of Hey1 from pri-or experiments. Using chromatin IP we could identify regions close to the tran-scriptional start site that are selectively bound by Hey1 protein for each of the Hey1 regulated target genes tested so far. This is exemplified by the strong bind-ing of Hey1 to its own promoter, shown

Large scale Hey target search

Whole genome chromatin IP sequence analysis (ChIPseq) has been employed in collaboration with C.L.Wei (Genome Insti-tute of Singapore) to identify the complete repertoire of Hey binding sites in the ge-nome. Clear and strong enrichment could be demonstrated in many putative target genes implicated in vascular development. Novel genomic binding motifs could be ex-tracted from these data by bioinformatic analysis and these have been verified by electrophoretic mobility shift assays (EMSA) in vitro using purified Hey proteins. These experiments will define the complete repertoire of Hey actions on a transcrip-tional level, which will be validated in our knock-out animals. Elucidating the func-tion of Hey proteins in cells and animal models will allow us to better understand embryonic developmental processes and to gain novel insight into the pathogenesis of cardiovascular diseases.

Fig. 4:Direct binding of Hey1 on its own promoter as tested by ChIP analysis (alphaFlag-Hey1, +Dox). A conserved intronic region is not bound (negative control, left).

duced states identified 30-100 strong can-didate genes by microarray analysis. There was a striking overlap of targets for both genes, which may reflect the strong homol-ogy especially in the DNA-binding domain of both proteins. In each case Hey proteins act as transcriptional repressors. They re-press their own promoter as well as differ-ent upstream genes, pointing to important negative feedback control.

in Figure 4. Very similar results were again obtained for Hey2. This suggests that Hey proteins function through direct repression of target promoters. A Hey1 mutant lacking the basic domain does not show evidence of DNA binding. Therefore, direct and sequence-specific DNA binding of Hey proteins seems to be the prevalent mode of action.

46

The identifi cation of functionally distinct leukocyte subsets was revolutionized about 30 years ago with the advent of monoclonal antibodies. Beyond defi ning “markers” which are selectively expressed on subpopulations of leukocytes, such mAbs are powerful tools for the isolation and structural elucidation of these cell surface receptors, as well as for studying their physiological function.

Thomas Hünig

E-mail: [email protected]: +49(0)931 201 499 51Fax: +49(0)931 201 492 43http://viminfo.virologie.uni-wuerzburg.de

RVZ Network Project

Table 1:mAbs with differential binding to conventional versus regulatory CD4 T-cells.

Our group has a long track record in iden-tifying functionally important cell surface receptors using mAb technology. In the current project, we hope to extend this work to the recently identifi ed subset of suppressor or regulatory T-cells (Treg cells). This subset of CD4 T-cells is essen-tial for the maintenance of self-tolerance and for containment of overshooting im-mune responses to infectious agents and environmental antigens. While at least in rodents, expression of the transcription factor Foxp3 can be used to distinguish Treg cells from “conventional” CD4 T-cells, no cell surface receptor has been described to date which is truly Treg specifi c. Thus, the cell surface receptors commonly used to identify Treg cells, such as CD25, CTLA-4, GITR, LAG-3, neuropilin-1 and folate re-

ceptor-4, are expressed on this cell typein an enhanced or more stable fashion compared to conventional CD4 T-cells, but are not unique. Conversely, the alpha chain of the IL-7 receptor, CD127, is downregu-lated on Treg compared to conventional CD4 T-cells, but again is not fully absent on the former.

We have developed two strategies toidentify cell surface receptors differentially expressed by conventional and Treg cells. In the fi rst approach, we bred mice defi cient for the Treg master regulator Foxp3 to the Ovalbumin/MHC II-specifi c TCR transgenic mouse line OT-II, and immunized these mice with membrane preparations from activated Treg cells spiked with ovalbumin (to provide T-cell help). Unfortunately, al-though such mice should theoretically lack

Clone Isotype Specifi city Reactivity Remarks

134 IgM ? All cells Tregs

96 IgG1,κ ? Tregs IP/MS currentlyrepeated

16B IgG1,κ phospholipids Activated Tregs

25D IgG1,κ ? Tregs IP/MS inpreparation

210 IgG1,κ CD62L Tregs

7A IgM phospholipids Activated Tregs

6G10 IgM phospholipids Activated Tregs

47

Fig. 1:Two-dimensional FACS analysis of mAbs 96 (left) and 25D (right) on CD28SA-activated lymph- node T-cells. Both show reduced expression on Treg cells as defined by Foxp3-expression (lower right quadrant) as compared to non-Treg cells (left quadrants).


Beyersdorf, N., Ding, X., Blank, G., Dennehy, K.M., Kerkau, T., and Hunig, T. (2008) Protection from graft-versus-host disease with a novel B7 binding site-specific mouse anti-mouse CD28 monoclonal antibody. Blood, 112, 4328-4336.

Gogishvili, T., Elias, F., Emery, J.L., McPherson, K., Okkenhaug, K., Hunig, T., and Dennehy, K.M. (2008) Proliferative signals mediated by CD28 superagonists require the exchange factor Vav1 but not phosphoinositide 3-kinase in primary peripheral T-cells. Eur J Immunol, 38, 2528-2533.

Guilliams, M., Bosschaerts, T., Herin, M., Hunig, T., Loi, P., Flamand, V., De Baetselier, P., and Beschin, A. (2008) Experimental expansion of the regulatory T-cell population increases resistance to African trypanosomiasis. J Infect Dis, 198, 781-791.

Kitazawa, Y., Fujino, M., Sakai, T., Azuma, H., Kimura, H., Isaka, Y., Takahara, S., Hunig, T., Abe, R., and Li, X.K. (2008) Foxp3-expressing regulatory T-cells expanded with CD28 superagonist antibody can prevent rat cardiac allograft rejection. J Heart Lung Transplant, 27, 362-371.

Na, S.Y., Cao, Y., Toben, C., Nitschke, L., Stadelmann, C., Gold, R., Schimpl, A., and Hunig, T. (2008b) Naive CD8 T-cells initiate spontaneous autoim-munity to a sequestered model antigen of the central nervous system. Brain, 131, 2353-2365.


DFG (SFB 479-TPC6, SFB 581-TPA5, HU295/8-1), Hertie1.01.1/06/00, Sander-Stiftung 2005.133.1

Of these, three produced mAbs that selectively stained activated Treg cells. However, all lymphocytes strongly reac-ted when staining was performed on per-meabilized cells, suggesting that the mAb recognize phospholipids that are exposed on pre-apoptotic cells. This hypothesis was confirmed by an ELISA performed at Prof. Martin Herrmann’s laboratory in Erlangen, which identified phosphatidyl-cholin, phosphatidylethanolamin, phos-phatidylserin, and cardiolipin as target antigens recognized by all of these mAb. Indeed, activated Treg cells rapidly un-dergo apoptosis, providing an explanation for this selective reactivity. It is currently being investigated in Prof. Herrmann’s group whether these mAb are of any use in studying cell death.

Based on its staining profile on vari-ous lymphocyte subpopulations, another clone, 210, was hypothesized to bind a cell surface marker called CD62L (L-selectin), as subsequently confirmed by competitive staining.

tolerance for Treg specific proteins, they never developed Treg specific serum titers, and did not yield any Treg- (or even Oval-bumin-) specific monoclonal antibodies.

In a more classical approach, we im-munized BALB/c mice with membrane preparations from CD28 superagonist ac-tivated rat Treg cells (see Annual Reports 2006/2007), followed by boosts with such activated, heat-treated (to destroy sup-pressive function) Treg cells. In a total of 9 fusions carried out with such hyperim-munized mice, more than 5,000 hybridoma lines have been tested for differential mAb binding to conventional and regulatory rat T-cells. Differential reactivity was observed with the 8 clones listed in Table 1.

Two interesting mAbs are currently being pursued further: clone 96 identifies a cell surface molecule that is highly expressed on conventional, but strongly downregu-lated on regulatory T-cells (Fig. 1 left). In a preliminary IP/MS experiment carried out together with Albert Sickmann’s group at the Rudolf Virchow Center, some interesting candidate sequences were identified. We are currently repeating this experiment in the hope to come up with a novel selectively downregulated molecule. Finally, mAb 25D binds to a receptor that is even more se-lectively downregulated on Treg cells (Fig. 1, right). We are currently preparing sufficient mAb for immunoprecipitation. Be-yond these potentially interesting cell surface receptors, we are extending our search in ongoing fusions.

48

In contrast to previous assumptions, structural and functional analyses of protein-protein interactions have shown that these interactions are promiscuous. Thus a particular protein can usually interact with more than one binding partner, often addressing the very same binding site. More and more proteins, in particular protein hormones/ligands exhibit this property, contradicting the old view that most biomolecule interactions would be only specifi c if they address a single binding partner. This feature possibly explains the frequently observed manifold functions of a single protein as various signaling cascades can be activated. Also the high redundancy of many proteins observed in vivo might be due to the overlapping use of signaling components. From a more mechanistic point of view the promiscuity of protein-protein interactions presents a dilemma as to how bind-ing specifi city as well as binding affi nity/strength is generated and modulated for a protein exhibiting limited specifi city. From studying diseases we know that proteins must recognize and bind their partners specifi cally. In an environment where thousands of biomolecules are present, generating binding specifi city for a group of binding partners with limited similarity rather than a single partner seems a delicate task.

IL-4, IL-5 and IL-13 as key regulators of allergies

Structural and functional investigations of protein-protein interactions in the past ten years have drawn a new picture of how biomolecules recognize and bind each other. Inherent fl exibility of the participating molecules at the side- and also main-chain level, the integration of solvent molecules as part of the binding interface create a high degree of adaptability, such that protein interfaces are able to accommodate different partner surfaces, chemically and geometrically. We are studying two protein families of secreted growth factors that represent prime examples of such adaptability: fi rst the cytokines IL-4, -5 and -13, which are involved in the development and progression of allergic diseases and asthma; and second the bone morphogenetic proteins (in collaboration with W. Sebald), which play an important role in differentiation processes during embryonic development and control homeostasis of many organs and tissues in the adult organism.

Thomas D. Müller

E-mail: [email protected]: +49(0)931 888 614 6Fax: +49(0)931 888 615 8http://www.bot1.biozentrum.uni-wuerzburg.de

IL-5 binding to IL-5Rα presents a new cytokine receptor complex architecture

Ten years of research have established interleukin-5 (IL-5) as a key cytokine involved in the onset and progression of allergic asthma. IL-5 represents the major regulator of eosinophils being involved in all steps of their differentiation,

maturation, migration and activation. Infi ltration of these eosinophils into lung tissue and their local activation leads to the classical asthma symptoms such as airway remodeling and airway hyper-responsiveness. Hence, IL-5 represents a highly interesting drug target for the treatment of asthma diseases. In terms of protein recognition, IL-5 - similar to other cytokines - shares receptor subunits with other cytokines. Following the classical path of single transmembrane receptor activation IL-5 dimerizes two receptor subunits - the IL-5 receptor IL-5Rα and the receptor βc (CD131) - to initiate downstream signaling. The latter subunit βc (CD131), also called common beta, is shared between the cytokines GM-CSF, IL-3 and IL-5, although similarities with respect to structure and amino acid sequence are limited. In contrast, within this cytokine family IL-5 exhibits several unique features. First, IL-5 forms a disulfi de-linked homodimer, whereas GM-CSF and IL-3 present monomers. The forth helix of the IL-5 monomeric subunit forms a four-helical bundle together with the three helices of the second subunit. Secondly, the crystal structure of the ternary complex of GM-CSF bound to its receptors GM-CSFRα and βc indicates that only the two membrane-proximal Fibronectin type III (Fn-III) domains forming the prototypical cytokine receptor architecture are necessary for ligand binding. Mutagenesis data on IL-5Rα however clearly show that for IL-5 ligand-receptor interaction, the fi rst of the three

The new architecture confi rms the muta-genesis data, which implied the involve-ment of the Fn-III domain 1 in ligand bind-ing. However, the structure clearly deviates from that of gp130 in the IL-6:IL-6Rα:gp130 complex, where the Fn-III domain 1 “swaps” out and forms IL6-gp130 contacts in a “dimeric” assembly.

The ligand-receptor arrangement also ex-plains the 1:1 ligand-receptor stoichiom-etry, which was considered unusual since the ligand IL-5 is a homodimer. However, the putative second binding site in the IL-5 dimer is partially blocked by the fi rst receptor molecule bound, thus not leaving enough space for two receptor molecules to be bound. Currently, we are obtaining fi nal functional data for the IL-5:IL-5Rα interaction to support the structural analy-sis. The fi nal high-resolution structure will clearly facilitate rational design of small

Fn-III domains of IL-5Rα is absolutely required for IL-5 binding. However, details about the molecular mechanism of theIL-5 ligand-receptor interaction have remained obscured due to the lack of structural data.

We have now crystallized the binary ligand-receptor complex of IL-5 bound to the extracellular part of its high-affi nity receptor IL-5Rα and determined its structure to high-resolution. The structure of the IL-5 receptor IL-5Rα reveals a new cytokine receptor architecture similar to the interleukin-13 receptor IL-13Rα1; the three Fn-III domains of IL-5Rα wrap around the ligand like a wrench.

RVZ Network Project

49

Fig. 1:Structure of the IL-5:IL-5Rα complex. (a) Ribbon representation of the complex, side view (b) front view; the N-terminal Fibronectin type III (Fn-III) domain is shown in green, the classical cytokine-recognition motif composed of the two membrane-proximal Fn-III domains is shown in cyan. (c) The 1:1 ligand receptor stoichiometry is due to overlap of the receptor binding site with the two possible binding sites in the IL-5 homodimer (marked in blue and magenta).

Fig. 2:NMR solution structure of the BMP receptor BMPR-IA. (a) Ribbon representation of BMPR-IA. (b) Overlay of a structure ensemble of BMPR-IA showing disordered region for the termini and the β4β5-loop. (c) Sausage plot of BMPR-IA.

Fig. 3:Superposition of BMPR-IA in its bound (blue) and unbound (brown) conformation. The „unbound“ conformer of BMPR-IA lacks the helix α1 in the β4β5-loop. (b) Docking of the unbound conforma-tion of BMPR-IA onto BMP-2. Upon complex formation the β4β5-loop of BMPR-IA passes through a conformation rearrangement to form helix α1.


Klages, J., Kotzsch, A., Coles, M., Sebald, W., Nickel, J., Mueller, T., and Kessler, H. (2008) The solution structure of BMPR-IA reveals a local disorder-to-order transition upon BMP-2 binding. Biochemistry, 47, 11930-11939.

Kotzsch, A., Nickel, J., Seher, A., Heinecke, K., van Geersdaele, L., Herrmann, T., Sebald, W., and Mueller, T.D. (2008) Structure Analysis of Bone Mor-phogenetic Protein-2 Type I Receptor Complexes Reveals a Mechanism of Re-ceptor Inactivation in Juvenile Polypo-sis Syndrome. J Biol Chem, 283, 5876-5887.

Saremba, S., Nickel, J., Seher, A., Kotzsch, A., Sebald, W., and Mueller, T.D. (2008) Type I receptor binding of bone morphogenetic protein 6 is dependent on N-glycosylation of the ligand. Febs J, 275, 172-183.

Zhang, J.L., Qiu, L.Y., Kotzsch, A., Weidauer, S., Patterson, L., Hammerschmidt, M., Sebald, W., and Mueller, T.D. (2008) Crystal structure analysis reveals how the Chordin family member Crossvein-less 2 blocks BMP-2 receptor binding. Dev Cell, 14, 739-750.


SFB 487 Teilprojekt B2DFG (MU1095/1-1, MU1095/3-1und MU1095/3-2)GK520 Teilprojekt C1

A local disorder-to-order transition in the BMP receptor IA fold upon ligand binding

The bone morphogentic proteins (BMPs) together with growth and differentiation factors (GDFs) are part of the large family of TGF-β superfamily. They control many aspects of proliferation and differentiation during embryonal development and tissue homeostasis besides their well-known name-endowing function to induce ectopic bone growth. Receptor activation of these factors is induced by ligand-dependent receptor oligomerisation similar to the above-mentioned receptors of the cytokine family. Two different subsets of receptors termed type I and type II are required for signaling. One hallmark is the very limited number of receptors available for a comparatively large number of ligands. About 18 BMP/GDF members signal through using only three different type I and three different type II receptors. In addition, various ligands can bind and signal through all of these six receptors. This raises the question of how many diverse functions can be generated by so few receptor combinations. On a molecular level, this protein family represents a prime example of promiscuous protein-protein interactions.

Earlier structure studies in our lab pointed towards inherent molecule flexibility in the ligand members as a possible factor allowing protein surfaces of different interaction partners to adapt and thus compensate for differences in interface geometries and chemistries. It was however unclear whether one binding partner adopts a rigid scaffold on its interface surface on which the other partner can adapt and fold or whether both binding partners are flexible. In collaboration with the group of Horst Kessler from the TU München we determined therefore the structure of the BMP receptor BMPR-IA in solution by NMR spectroscopy.

Interestingly, the structure of unbound BMPR-IA lacks a short helical element called helix α1. This is located in the cen-ter of the ligand-receptor interface upon complex formation and carries the hot spot of binding for the BMP-2:BMPR-IA interac-tion. Although the generation of helix α1 in BMPR-IA just upon complex formation seems incompatible with the fact that resi-dues of this element contribute the major-ity of binding free energy of the BMP-2:BMPR-IA interaction, a network of intra- and intermolecular interactions emanating from residues of helix α1 likely compensate for the relatively large entropy loss. Addi-tional experiments suggest that this helix element in BMPR-IA is preformed and can form spontaneously upon changes in the environment, e.g. ligand binding. On the other hand the built-in flexibility in ligand as well as receptor interfaces allows for a maximum of adaptability between differ-ent binding partners and thus represents a likely source for the ligand-receptor pro-miscuity observed in the BMP-family.

molecule IL-5 inhibitors. Such inhibitors could be better used to treat asthma and hypereosinophilia than current antibody approaches.

50

Tumor modifi er genes are genetic factors, which are not involved in the primary steps of neoplastic transforma-tion and initiation of the process of tumorigenesis process but not critical in determining the malignant pheno-type of the tumor and the course of the cancerous disease. Such genes have been identifi ed in various organisms, but knowledge about their molecular identity and their biochemical functions is almost completely lacking. We use melanoma-developing transgenic fi sh as model systems for identifying and isolating such tumor modifi er genes through an ENU mutagenesis screen and by a candidate gene approach. By utilizing forward genetics and advanced transgenic methods we search for genes that lead to either a more benign or a more malignant phe-notype of the melanoma.

Manfred Schartl

E-mail: [email protected]: +49(0)931 888 414 8Fax: +49(0)931 888 415 0http://pch1.biozentrum.uni-wuerzburg.de/

Fig. 1:Different genetic backgrounds give different tumor phenotypes. On the left side: mitf:xmrk expression in the Carbio strain developing cutaneous exophytic xanthoerythrophoroma. On the right side: mitf Xmrk expression in the HB32C background leading to invasive melanoma.

As primary oncogene the xmrk gene is used, a mutated version of the epidermal growth factor receptor egfrb, which is responsible for tumor formation in the Xiphophorus melanoma model. Two mutations in the extracellular domain of this receptor ty-rosine kinase result in the formation of an intermolecular disulfi de bond between two monomers. This ligand independent dimerization of Xmrk molecules results in a constitutive activation of the receptor and in permanent signaling. The signal transduction events downstream of Xmrk are reasonably well understood in vitro. To get a broader and more complete insight into oncogenic receptor function in vivo and to identify further proteins interfering with the activated pathways downstream of Xmrk, we utilize the medakafi sh. Me-daka has in contrast to Xiphophorus many

advantages, like a completely sequenced genome, daily egg spawning, extracorpo-rally developing transparent embryos and the availability of a large collection of mu-tants, which make medaka ideal for devel-opmental and genetic studies, as well as high throughput approaches.

To transfer the melanoma system from Xiphophorus to medaka, the oncogenic re-ceptor Xmrk was stably expressed under the medaka mitf promoter, which resulted in pigment cell tumors of various types. Be-sides black, melanin-containing melanoma, red to yellow XE-tumors (xanthoerythroph-oroma) originating from other fi sh specifi c pigment cell types (xanthophores or eryth-rophores) were detected. The melanotic tumors displayed highly invasive growth, penetration mainly into the musculature and internal organs. Appearance of lesions

RVZ Network Project

in inner organs without connection to the main tumor shows the potential of the melanoma for metastasis formation. The XE-tumors reside on the skin and are able to populate huge areas of the fi sh´s sur-face. Additional expression of xmrk in p53-negative medaka led to the appearance of giant focal pigment cell tumors, while tu-mor onset was unchanged compared to p53 wildtype medaka.

Importantly, all homozygous mitf::xmrk transgenic fi sh displayed a tumor pen-etrance of 100%. Most interestingly, de-pending on the genetic background of the medaka strain used, exophytic XE-tumors or invasive melanoma originating from cu-taneous or extracutaneous sites occurred (Fig 1). Thus, tumor modifi er genes become apparent, as factors regulating tumor char-acteristics but not tumor incidence. The

51


DFG SFB-TR 17


Herpin, A., Fischer, P., Liedtke, D., Klüver, N., Neuner, C., Raz, E., and Schartl, M. (2008) Active Sdf1a and b-induced mobility guides Medaka PGC migration. Dev Biol, 320, 319-27.

Klüver, N., Herpin, A., Braasch, I., Drießle, J., and Schartl, M. (2009) Regulatory back-up circuit of medaka wt1 co-orthologs ensures PGC mainte-nance. Dev Biol, 325, 179-88.

Leikam, C., Hufnagel, A., Schartl, M., and Meierjohann, S. (2008) Oncogene activation in melanocytes links reactive oxygen to multinucleated phenotype and senescence. Oncogene, in press.

Schartl, M. (2008) Evolution of Xmrk: an oncogene, but also a speciation gene? Bioessays, 30, 822-32.

Schartl, M., and Herpin, A. (2008) Regulatory putsches create new ways of determining sexual development. EMBO Rep, 9, 966-8.

Fig. 2:Proliferating and highly invasive melanoma colonies (T = tumor) located in the body cavity (I = intestine, M = muscle) in a mitf::Xmrk transgenic fish are characterized by enhanced numbers of phospho-histone (mitosis-specific) H3 positive cells.

It is expected that genes modifying the malignant phenotype are involved in pro-cesses such as transition from the benign to the malignant state, tumor cell migra-tion, invasion, proliferation and metasta-sis (Fig. 2). In molecular terms, it is an-ticipated that these genes are involved in modulating the intracellular signals elicit-ed by the activity of the melanoma-induc-ing transmembrane receptor. To test this hypothesis, we investigated the intracel-lular signal transduction in different xmrk induced tumors of medaka. All sampled tumors showed a high expression of xmrk. Strong activation of phosphatidylinositol 3-kinase (PI3 kinase) and the downstream factor Akt was detected in those tumors. The PI3 kinase signaling pathway has been well established as a mediator of Xmrk sig-nals contributing to mitogenic signals and apoptosis of melanoma in vitro. Activation of the MEK and MAPK pathways were not evident in medaka tumors, due to a consid-erable basal activity of both pathways in the normal medaka skin. Most remarkable, besides PI3 kinase, was the strong activa-tion of the signal transducer and activa-tor of transcription Stat5, a known tran-scription factor in tumors of Xiphophorus and human melanoma. Activation of Stat5 is directly correlated with the expression level of Xmrk in the medaka melanoma. This observation in the medaka tumors is supported by the anti-apoptotic and pro-liferation activity of Stat5 previously de-scribed for Xmrk in Xiphophorus tumors. More importantly, activation of Stat5 in human melanoma not only enhances tumor survival, but also mediates interferon re-sistance. Further studies will show if Stat5 signals are mainly involved in maintenance of the malignant state or if they are also necessary for early stages of tumor devel-opment in the medaka tumor model.

stable xmrk transgenic lines give a very stereotyped tumor development with an early onset during larval stages and com-parable tumor growth characteristics. They are therefore well suited for large scale testing of chemical compound libraries and for mutagenesis screens to identify tumor modifier genes.

52

Walter Sebald

E-mail: [email protected]: +49(0)931 888 411 1Fax: +49(0)931 888 411 3http://pch2.biozentrum.uni-wuerzburg.de/

BMP signaling

BMPs are a family of dimeric extracellular proteins that signal into cells by as-sembling two types of single-span serine/threonine kinase receptors in the plasma membrane. BMP-2 is a prototypical BMP which determines multiple steps during embryonal development and regulates bone regeneration in the adult organism. The type I chains (BMPR-IA, BMPR-IB, ActR-IA) bind BMP-2 with high affi nity and initiate the intracellular Smad-dependent pathways. The type II chains (BMPR-II, ActR-II, ActR-IIB), which bind BMP-2 with low affi nity, transactivate the type I chain by phorphorylation at their gylcine-/serine-rich GS box. Dimeric BMP-2 can bind two type I and two type II receptors. The receptors exist in the membrane as monomeric as well as preassembled homo- and heterodimeric forms.

Bone morphogenetic proteins (BMPs) and BMP-like proteins are key regulators of organ development and tissue regeneration. Dysregulation of BMP signaling results in tumor formation as well as other disorders including cardiovascular, musculoskeletal and urogenital diseases. To understand how BMPs bind and activate their recep-tors and how they are regulated by extracellular modulator proteins, we study the structure of ligand receptor complexes and the energetics and kinetics of BMP interactions with receptors and modulator proteins. We are generating BMP mutants that mimic mutations in familial disorders and which have useful properties for applica-tions in regenerative medicine and musculoskeletal diseases. In 2008, we focused on the interaction of BMP-2 with its high-affi nity receptor BMPR-IA and with VWC (Von Willebrand factor type C) domains of Chordin-like proteins.

A local disorder-to-order transition is a salient feature of the interaction be-tween BMP-2 and its high-affi nity recep-tor BMPR-IA

Contact between BMP-2 and BMPR-IA is mostly hydrophobic with a “knob-into-hole” motif, i.e. a protruding receptor phenylalanine that fi ts into a deep hole in the BMP-2 dimer. Only two hydrogen bonds are critical for binding affi nity. The main binding determinants of the receptor are two juxtaposed side-chains – the “knob” phe85 and a gln86 which establishes one of the important hydrogen bonds. In

the crystal structure of the complex, these binding determinants are located in a short α-helix.

Surprisingly, this α-helix is not seen in the free receptor in solution analyzed by NMR spectroscopy (Fig. 1). It is also not present in a crystal structure of the receptor in a complex with a neutralizing recombinant antibody. This strongly suggests that the helix is formed in the contact process with BMP-2. However, it is not known at this time if the helical form of the receptor already occurs as a minor conformation of

the free receptor in equilibrium with the non-helical form. BMP-2 binding would then draw the population into the helical form. Alternatively, BMP-2 may simply induce the helical form by a classical “induced fi t” mechanism.

Nevertheless, it is clear that a pronounced local disorder-to-order transition occurs in the receptor binding epitope during complex formation with BMP-2.

Remarkably, the BMP-2 epitope carries the two major polar binding determinants in a so-called “pre-helix loop”. Several lines of

Fig. 1:Disorder-to-order transition in the β4-5 loop of free and bound BMP receptor BMPR-IA might be important for promiscuous interaction between BMPs and the receptor.

RVZ Network Project

53


SFB487, TP 1KFO103, TP CDFGRVZIndustry


Klages, J., Kotzsch, A., Coles, M., Se-bald, W., Nickel, J., Muller, T., and Kes-sler, H. (2008) The solution structure of BMPR-IA reveals a local disorder-to-order transition upon BMP-2 binding. Biochemistry, 47, 11930-11939.

Kotzsch, A., Nickel, J., Seher, A., Hei-necke, K., van Geersdaele, L., Herrmann, T., Sebald, W., and Mueller, T.D. (2008) Structure analysis of bone morphogenet-ic protein-2 type I receptor complexes reveals a mechanism of receptor inacti-vation in juvenile polyposis syndrome. J Biol Chem, 283, 5876-5887.


Zhang, J.L., Qiu, L.Y., Kotzsch, A., Wei-dauer, S., Patterson, L., Hammerschmidt, M., Sebald, W., and Mueller, T. D. (2008) Crystal structure analysis reveals how the Chordin family member crossvein-less 2 blocks BMP-2 receptor binding. Dev Cell, 14, 739-750.

evidence indicate that this loop is mobile and can exist in different orientations, only one of which is found in the complex with the BMPR-IA receptor. The structural flexibility in the binding epitopes of both BMP-2 and the BMPR-IA receptor seems to be responsible for the slow on-rate constants of the association between the two proteins (kon = 5 x 105). Otherwise, this flexibility seems to allow the promiscuity of BMP/type I receptor interactions. BMP-2 can bind three different type I receptors and BMPR-IA can bind to a variety of BMPs and BMP-like proteins.

Crystal structure analysis reveals a mech-anism of BMPR-IA receptor inactivation in juvenile polyposis syndrome

BMP-2, via signaling through BMPR-IA, exhibits a tumor suppressor function in epithelial cells. Mutations leading to JPS (juvenile polyposis syndrome) are linked to inactivation of the BMPR-IA receptor. This autosomal dominant disease is characterized by excessive colorectal polyp growth, creating a predisposition for colorectal cancer. The mechanism of how the known missense mutations in the extracellular BMP-2 binding domain of BMPR-IA inactivate the receptor was not understood due to their location outside the ligand binding epitope. On the basis of our biochemical and biophysical analyses, we can now show that these mutations alter the local or global folding of the receptor, thereby inactivating BMPR-IA and causing loss of the BMP-2 tumor suppressor function in colon epithelial cells (Fig. 2).

The first structure of a Von-Willebrand factor type C (VWC) domain in complex with BMP-2

The activity of BMPs is regulated by numerous modulator proteins in the extracellular space. Many of these proteins, such as Chordin or CV2 (crossveinless-2), contain one or multiple VWCs (Von Willebrand factor type C) domains that at least in some instances can bind BMPs. The affinity of CV2 for BMP-2 is comparable to that of the high affinity BMPR-IA receptor. It therefore seems possible that CV2 functions as a competitive inhibitor as well as a storage and/or delivery system for BMPs.

VWC1, the first of the five VWC domains in CV2, is the only one that binds BMP-2. The high affinity of this interaction allowed the isolation and crystallization of the complex. It consists of two VWC1 domains and a dimeric BMP-2. The estab-lished structure revealed for the first time the unusual and new architecture of the VWC domain and the mechanism for high affinity binding of BMP-2 (Fig. 3). The N-terminal clip binds within the wrist epitope for type I receptor interaction. Its deletion lowered affinity 2000–fold, indicating its role in high-affinity binding to BMP-2. Subdomain 1 binds within the knuckle epitope of BMP-2 involved in type II receptor binding. Subdomain 2, surprisingly, has no contacts with BMP-2 and does not contribute to binding affinity. In conclusion, by sitting like a paper clip on BMP-2, VWC1 interferes with both type I and type II receptor binding using only the 42 residues provided by the N-terminal clip and SD1.

Fig. 2:Some mutations in the extracellular domain of BMPR-IA leading to juvenile polyposis syndrome (JPS) are located outside of the binding epitope for BMP (upper part a, b). The mutant receptors are efficiently transported and integrated in the cell membrane (lower part, a-d). This suggests, that disruption of BMPR-IA function results from local or global conformational changes.

Fig. 2:The Van-Willebrand factor type C (VWC) domain of crossveinless 2 (CV2) binds with high affinity to BMP-2 employing a paperclip-like mechanism.

54

Martin Eilers

E-mail: [email protected] Phone: +49(0)931 888 416 0Fax: +49(0)931 888 424 2http://pch2.biozentrum.uni-wuerzburg.de/

RVZ Network Project

Over the past 25 years, we have come to understand that cancer is a genetic disease where healthy cells turn into tumor cells as a result of four to six mutations in a subset of cellular genes. These mutations occur in two groups of genes: one, called proto-oncogenes, are positive regulators of cell proliferation and cell survival, and their function is tightly controlled during normal development. The mutations that occur in tumor cells unleash the function or expression of these genes from their normal control, turning them into onco-genes. The other group, called tumor sup-pressor genes, normally restrict cell prolif-eration and survival, and it is the loss of these genes that promotes tumor forma-tion. Many examples from both classes of genes are now known: from these studies, a relatively small central group of genes emerges that are deregulated in the ma-jority of all human cancers. One example of such a “central player” in oncogenesis is the MYC family of genes. The family has fi ve members, and three of them, c-, L- and N-Myc, are causally involved in the genesis of a multitude of human tumors.

Our group studies the function and regu-lation of the proteins encoded by these genes. Myc proteins are nuclear proteins and they act at least in part by regulating the expression of target genes. Interest-ingly, they can both activate and repress target genes after binding to specifi c DNA sequences located in the promoter of their targets. One issue we try to understand is why binding of Myc to DNA sometimes leads to activation and in other cases re-pression of target genes. We have found that Myc represses transcription when binding to a partner protein that we have called Miz1 and we are using both tissue culture models to understand mechanistic aspects of Myc/Miz1-mediated gene repres-sion and mouse models to understand the function of this complex in the biology of Myc proteins.

Recent work from a number of laborato-ries has implicated Myc in the regulation of self-renewal of stem cells, both during normal development and during tumorigen-esis. Specifi cally, several observations sug-gest that Myc can repress the expression of genes that control the interaction of stem

Fig. 1:The picture shows a graphic illustration of complex formation between Miz1 and its co-activator, nucleophosmin (NPM); NPM is normally localized in the nucleolus, but expres-sion of Miz1 recruits it to the nucleus, where it simulates expression of Miz1-dependent genes (Wanzel et al., Nature Cell Biology, 2008).

Cancer affects 1,600,000 people every year in the EU, resulting in approximately 900,000 deaths. Trends indi-cate that the incidence will continue to rise in the years to come, primarily as a result of the aging population. Although most cancers occur in the elderly population, cancer has become the prevalent cause of death in the age group under 75, with devastating effects on many young and middle age individuals, and their families.

Martin Eilers is associated as RVZ Network Member from 2009 on.

cells with their environment, the stem cell “niche”. This may be an important mecha-nism that restricts the emergence of stem cell tumors, since most oncogenic muta-tions lead to enhanced levels of Myc and would thereby lead to the loss of stem cells from their niche. As part of our work within the Rudolf Virchow Center, we are using multiple approaches to test this model and test its implications for Myc biology.

Outlook

55

Roland Jahns

E-mail: [email protected] Phone: +49(0)931 201 702 30Fax: +49(0)931 201 480 2http://www.rudolf-virchow-zentrum.de/forschung/jahns.html

Fig. 1:Schema depicting antibodies (Ab) targeting the second extracellular loop of the β1-AR. Binding of the antibody induces or stabilizes an active receptor conformation and thereby engenders an intracellular signaling cascade leading to activation and subsequent dissociation of the heterotrimeric Gs protein in Gαs and Gβγ subunits. Gαs activates both adenylyl cyclase (AC), which catalyzes cAMP formation (ATP to cAMP + Pi) and the L-type calcium channel (not depicted); cAMP in turn activates protein kinase A, which then phosphorylates various substrates critically involved in the regulation of intracellular/sarcoplas-mic calcium levels (not shown).

Previously, in a human-homologous rat model we demonstrated that stimulatory antibodies directed against the second extracellular receptor-loop of the beta1-re-ceptor (anti-beta1-ECII) may in fact cause cardiac dilatation and progressive pump failure. In this model, our novel beta1-ECII-homologous cyclopeptides (beta1-ECII-CP), originally designed to scavenge circulating receptor-antibodies, not only prevented antibody-induced development of cardiac dilatation and failure, but also reversed overt heart failure. After 4-5 i.v. injections, the cyclopeptides evoked asignifi cant decrease in, and fi nally ces-sation of anti-beta1-ECII antibody-pro-duction, in spite of continued boosts withthe immunogen.

So far, the accorded GoBio grant in the fi rst funding period has served to optimize biochemical structure and biological effi -cacy of our novel cyclopeptides, including the execution of numerous immunological and preclinical studies. The second fund-ing period will mainly serve to advance and translate our promising data obtained in animals towards a fi rst application in humans (phase I study with the mosteffi cient cyclopeptides). In the end, we hope to gain a novel powerful therapeu-tic tool with no apparent side-effects to combat heart failure in anti-beta1-ECII-positive patients.

In parallel, we arre currently developing a novel fl uorescence-based screening strat-egy for the large scale diagnostic detection of such stimulatory antibodies. For this purpose, we are using a new highly sensi-tive and specifi c fl uorescent cAMP-sensor, which allows us to visualize and quantify receptor-mediated increases in cellular cAMP by fl uorescence resonance energy

Heart failure is one of the most frequent diseases and causes of death in Germany and other industrializedcountries. Cardiac pump failure results in insuffi cient blood supply to the body resulting in shortness of breath and fl uid retention. In addition to complex hormonal compensatory mechanisms, recent clinical and experimen-tal data point towards an important role of autoimmunity in the pathogenesis of heart failure: In up to 30% of patients with heart failure, functionally active autoantibodies stimulating the beta1-adrenergic membrane receptor – which represents the key receptor for cardiac excitation/contraction coupling – appear to be causally involved in the induction and course of the disease.

Research Program GoBio, Federal Ministry of Education and Research (BMBF)Associated at the Rudolf Virchow Center.

transfer (FRET). In this diagnostic assay, the receptor-cyclopeptides will be used to ascertain the specifi city of anti-beta1-AR-mediated receptor activation.

Both, our novel diagnostic and therapeu-tic strategies and the tools we have devel-oped are protected by patents, and served as a basis for founding a spin-off biotech company of the University.

RVZ associated Project

56

Design of sensors for receptor activation and cyclic nucleotides

The strategy to create sensors for receptor-cyclic nucleotide signaling is based on a technique called fl uorescence resonance energy transfer (FRET). FRET is the trans-fer of energy from one fl uorescent moietyto another one in close vicinity. In our sensors, these moieties are generally cyan (CFP) and yellow (YFP) fl uorescent proteins.

E-mail: [email protected]: +49(0)931 201 484 00Fax: +49(0)931 201 484 11http://www.rudolf-virchow-zentrum.de/forschung/bioimagingcenter/lohse.html

Martin Lohse

Cyclic nucleotides – cyclic AMP (cAMP) and cyclic GMP (cGMP) – are ubiquitous intracellular messengers that are produced in response to multiple stimuli, act on several intracellular targets and thereby regulate many biological functions.We aim to identify the processes of how receptors trigger the activation of cyclic nucleotide production, to de-

scribe the kinetics of on- and off-rates of signaling and to elucidate how specifi city in such systems is generated. To do so, we have generated a number of fl uorescent sensors for receptors and G-protein-mediated signaling. In addition, we develop fl uorescent sensors for cAMP and cGMP. These sensors can now be used to generate images of cAMP and cGMP in intact cells, resolved in space and in time.

Fig. 1:(A) Principle of a FRET-sensor for camp: The sensor comprises a cAMP-binding domain (grey) fused to cyan (CFP) and yellow fl uorescent proteins (YFP). Binding of cAMP changes the FRET between CFP and YFP; therefore, cyan emission increases and yellow emission decreases. (B) Fusion of a cAMP-sensor to specifi c phosphodiesterases (PDEs) permits selective testing of inhibitors. In this example, the PDE4-inhibitor rolipram enhances a cAMP-signal.

Funds from this project were initially used mostly to establish the research group led by Stefan Schulz, who held a DFG Heisenberg Profes-sorship and a joint appointment in Pharmacology. His group studies receptors for opiates and somatostatin, in particular their regulation and their intracellular traffi cking. Stefan Schulz has now accepted an offer for the Chair in Pharmacology and Toxicology at the University of Jena.

Spatial patterning of cAMP signals

Increasing evidence suggests that cAMP signals may show complex patterns in space and time. Our FRET sensors permit one to image changes in intracellular cAMP with a CCD-camera and monitor spatial and tem-poral patterns of cAMP-signals. Where and when a cell the concentrations of cAMP (or cGMP) change in response to external stimuli can be captured like a fi lm. Figure 2

FRET can cause a YFP to emit yellow light when a nearby CFP is excited. FRET is very sensitive to changes in the distance be-tween the two fl uorescent moieties: even small increases in the distance lead to a large loss in FRET. For example, sensorsfor cAMP are built with three elements (Fig. 1A): CFP, a binding domain for cAMP, and YFP. When cAMP binds to the bind-ing domain, this results in movement of the two helices fl anking the binding site.

This movement changes the distancebetween CFP and YFP and causes achange in FRET. Such changes in FRETcan be monitored by measuring the signal intensities of the CFP- and YFP-emissions (and their ratio).

We have directly fused such cAMP-sensors to specifi c phosphodiesterases (PDEs; Fig. 1B). This allows very specifi c analysis of inhibitors for defi ned PDEs, a major class of target proteins for drugs.

shows how a cAMP-signal propagates from the site of receptor stimulation and quickly reaches the entire cell.

Fig. 2:Imaging of a cAMP-signal to local recep-

tor stimulation. The images are taken sequentially before (left) and after local

stimulation of the cell (arrow).

A B

57


Vilardaga, J.P., Nikolaev, V.O., Lorenz, K., Zhuang, Z., and Lohse, M.J. (2008) Direct inhibition of G protein signal-ing by cross-conformational switches between α2A-adrenergic and µ-opioid receptors. Nature Chemical Biology, 4, 126-131.

Shafer, O.T., Kim, D.J., Dunbar-Yaffe, R., Nikolaev, V.O., Lohse, M.J., and Taghert, P.H. (2008) Widespread receptivity to neuropeptide PDF throughout the neuronal circadian clock network of Drosophila revealed by real-time cyclic AMP imaging. Neuron, 58, 223-237.

Herget, S., Lohse, M.J., and Nikolaev, V.O. (2008) Real-time monitoring of phosphodiesterase inhibition in in-tact cells. Cell Signal, 20, 1423-31.

Lesche, S., Lehmann, D., Nagel, F., Schmid, H.A., and Schulz, S. (2008) Differential effects of octreotide and pasireotide on somatostatin receptor internalization and trafficking in vitro. J Clin Endocrinol Metab, in press.

Lorenz, K., Schmitt, J.P., Schmitt-eckert, E.M., and Lohse, M.J. (2008) A new type of ERK1/2-autophosphor-ylation causes cardiac hypertrophy. Nature Medicine, in press.

Extramural Funding

BMBF-Biophotonics: LiveCell ScreeningDFG-SFB487, TPA1DFG-SFB688, TPB6Leducq-Foundation, Transatlantic Network of Excellence CAERUSHumboldt Fellowship to Davide CalebiroEMBO Fellowship to Veronika Hlavackova From 2009: European Research Council Advanced Investigator Grant TOPAS

Stefan Schulz:DFG-Heisenberg ProfessorshipDFG Schu 924/10-1, 11-1, 12-2

Mechanisms of receptor activation

Activation of receptors is triggered by binding of agonists and a subsequent conformational change. Last year saw the elucidation of several G-protein-coupled receptor structures and – together with older biochemical and biophysical data – one can develop concepts about the “mechanics” of receptor activation. We have developed FRET-based sensors con-sisting of receptors carrying two fluores-cent labels. Such modified receptors have permitted detailed analysis of the kinetics of receptor activation.

In recent years, evidence has accumulat-ed that G-protein-coupled receptors may form dimers. We have found a functional dimer between the μ-opiate and the α2A-adrenergic receptors. With an α2A-adrener-gic receptor suitable for FRET-measurement we found that its conformation is rapidly (~500 ms) altered, when a co-expressed μ-opiate receptor is activated. Studies inves-tigating this “cross-talk” between the two

De-activation and internalization of receptors

Two major mechanisms contribute to the de-activation of receptors: recruitment of β-arrestins and receptor internalization. We investigated these mechanisms for sev-eral G-protein-coupled receptors: adren-ergic, purinergic and somatostatin. These experiments revealed important differences in the ability of compounds to induce ei-ther receptor signaling or receptor inter-nalization, and thus provide evidence for multiple active conformations.

For example, the clinically used soma-tostatin analogs, octreotide and lanreotide, act primarily by binding to somatostatin receptor 2 (sst2). A new ligand, pasire-otide, which is under clinical development for multiple endocrine diseases (acromega-ly, Cushing‘s disease and carcinoid tumors) binds with high affinity to sst2 but also to other somatostatin receptors. Octreotide-mediated receptor activation leads to the formation of stable complexes facilitating the internalization of sst2 and β-arrestin-2 into the same endocytic vesicles. In con-trast, pasireotide led to the formation of unstable complexes that dissociated at or near the plasma membrane. Thus, sst2

Fig. 3.Imaging cAMP-signals in individual neurons of the Drosophila brain. Individual neurons express the cAMP-sensor (left) and show very variable responses to stimulation by the neuro-mediator octopamine (right).

Fig. 4.Model of the dimer between an α2A-adrenergic and a µ-opiate receptor. The two receptors show direct interactions so that only one of the two can be active.

receptors recycled rapidly to the plasma membrane after endocytosis in pasireotide – but not in octreotide-treated cells. Thus, different agonists modulate soma-tostatin receptors in a clearly distinct manner. These findings may provide an explanation for the differential regulation of somatostatin receptor responsiveness during long-term administration of stable somatostatin analogs.

Another application of these sensors is their use in vivo. Collaboration with the laboratory of Paul Taghert (Washington University, St. Louis) led to the genera-tion of transgenic Drosophila that express a cAMP-sensor in defined neurons of the

brain. This permits the simultaneous opti-cal recording of cAMP signals in response to specific stimuli in multiple neurons and reveals a marked heterogeneity of such re-sponses (Fig. 3).

different receptors suggest that in such a dimer only one of the two receptors is ac-tive (Fig. 4). This mechanism may explain complex pharmacological interactions be-tween drugs acting at different receptors. It may also provide a means of indirectly activating receptors. These effects may have immense practical consequences for drug therapy.

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E-mail: [email protected]: +49(0)931 201 489 89Fax: +49(0)931 201 489 78http://www.rudolf-virchow-zentrum.de/forschung/bioimagingcenter/sigrist.html

Stephan Sigrist

During brain development, the formation of functional neuronal circuits requires tightly coordinated synapse for-mation of synaptic connections (“synaptogenesis”) – defi cits during this stage can lead to autism. Synapses form by an asymmetric association of highly specialized membrane domains (presynaptic active zone, postsynaptic receptor fi eld). Active zones (AZs), which often are associated with macromolecular electron dense assemblies control synaptic release function e.g. during learning and memory processes. However, their composition and exact functional role remain to be explored. Methodologically, our group capitalizes on recent developments in light microscopy to investigate mechanisms organizing assembly and plasticity of synapses under in vivo set-tings. Imaging is combined with genetics and ultrastructural methods to analyze both Drosophila neuromuscular and murine hippocampal synapses. In this way, we also seek to contribute to a functional characterization of mutations in active zone proteins from autistic patients.

Fig. 1:In vivo analysis of protein accumulation during developmental addition of synaptic sites. Shown are developing NMJs of intact living third instar Drosophila larvae. Confocal stacks of sequentially in vivo-imaged NMJs (muscle 26), ∆t = 12 h. NMJs co-expressing the indicated labels, GFP con-structs green, mRFP constructs magenta. Upper panels show individual in vivo-imaged synapses (arrow head) positive for only one label at t = 0 h, but positive for both labels at t = 12 h. Lower panels show a prospective synapse (arrow) positive for only one label at t = 12 h. Scale bars: 1 µm. A) BRP-shortGFP/DGluRIIAmRFP; B) DLiprin-αGFP/DGluRIIAmStraw; C) DLiprin-αGFP/BRP- Scale bars: 1 µm.

In vivo imaging of AZ assembly

Our group established protocols to study molecular dynamics during synapse assem-bly and plasticity in living animals, combin-

Since August 2008, Stephan Sigrist is Professor for Molecular Developmental Genetics of Animals, Freie Universität Berlin. Manfred Heckmann is now leading the group since November 2008.

Chemical synapses are specialized for rap-id directional signaling and are arguably the most elaborate signaling machines of cells. Within the development of neuronal circuits, the formation of synapses (“syn-aptogenesis”) is obviously an essential step. Specialized presynaptic sites called active zones (AZs) feature synaptic vesicle release that is unmatched by any other cellular locations in terms of speed and accuracy. AZs are characterized by mac-romolecular architectures often observable by electron microscopy (“dense bodies”). Overall, recent work suggests that in vivo synaptogenesis is a long and intricate pro-cess involving multiple interrelated steps with reciprocal induction, as well as inde-pendent assembly of pre- and postsynaptic structures. Despite the increasing amounts of genetic and molecular data, the exact spatio-temporal sequence of in vivo AZ assembly during development of synaptic circuits remains a challenging problem. This is important, since comprehensive understanding of AZ assembly, which is complicated by the partly redundant and cooperative interactions involved, can only be understood in the context of an in vivo sequence.

ing confocal and 2-photon micro-scopy in vivo (Ataman et al., Neuron, 2008; Fuger et al., Nature Protoc, 2007; Rasse et al., Nat Neurosc, 2005; Schmid et al., Nat Neurosc, 2008). Here, “fl uorescence recovery after photobleaching” (FRAP) und photo-activa-tion experiments allowed us to quantify the in vivo mobility of relevant synaptic pro-teins at identifi ed populations of synapses. We recently used these protocols to study the developmental formation of AZs in vivo (Fouquet, Owald et al, submitted; see Fig. 1). We recently identifi ed the large coiled-

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Ataman, B., Ashley, J., Gorczyca, M., Ramachandran, P., Fouquet, W., Sig-rist, S.J., and Budnik, V. (2008) Rapid activity-dependent modifications in synaptic structure and function re-quire bidirectional Wnt signaling. Neuron, 57, 705-718.

Bogdanik, L., Framery, B., Frolich, A., Franco, B., Mornet, D., Bockaert, J., Sigrist, S.J., Grau, Y., and Parmen-tier, M.L. (2008) Muscle dystroglycan organizes the postsynapse and regu-lates presynaptic neurotransmitter re-lease at the Drosophila neuromuscular junction. PLoS ONE, 3, e2084.

Schmid, A., Hallermann, S., Kittel, R. J., Khorramshahi, O., Frolich, A. M., Quentin, C., Rasse, T.M., Mertel, S., Heckmann, M., and Sigrist, S.J. (2008) Activity-dependent site-spe-cific changes of glutamate receptor composition in vivo. Nat Neurosci, 11, 659-666.

Schmid, A., and Sigrist, S.J. (2008) Analysis of neuromuscular junc-tions: histology and in vivo imaging. Methods Mol Biol, 420, 239-251.

Extramural Funding

SFB 554, 581, 487DFG Project SI849/2-1 and SI849/2-2

Fig. 2:Discrete DLiprin-αGF clusters sur-rounding the AZ core. A) STED image of DLiprin-αGFP displays struc-tures beyond diffraction-limited res-olution obtained with confocal mi-croscope (arrow heads). Scale bar: 1 µm. B) Predicted sequence of AZ assembly based on STED images. C) A model of AZ molecular architec-ture at Drosophila NMJ synapses.

STED analysis of AZ architecture

The size of individual AZs is in the range of a few hundred nanometers, making light microscopic analysis of synapse substruc-ture difficult. Recently, stimulated emis-sion depletion microscopy (STED) (Hell, 2007) has proven valuable for dissecting AZ architecture and synaptic vesicle move-ment (Jin and Garner, 2008; Kittel et al., Science, 2006; Westphal et al., 2008). Us-ing STED, we previously uncovered a do-nut-shaped distribution of BRP when using the monoclonal antibody Nc82 (MAB Nc82, (Kittel et al., 2006)). We have now further analyzed AZ assembly and maturation. At mature AZs, a defined number of “quantal” DLiprin-α clusters at the edge of the AZ co-ordinated this BRP core in the AZ center. Our analysis demonstrates the increased resolution possible with STED imaging, un-covering discrete dots of DLiprin-α located here (Fig.2A). These dots (green) coordi-nate the inner AZ core (Fig. 2B), which is organized by BRP (magenta). On the basis of these data and our previous studies, we can propose a molecular assembly sequence (Fig. 2B) as well as molecular architecture models for AZs (Fig. 2C), taking into ac-count our studies on the accumulation of postsynaptic glutamate receptors (Schmid et al., Nat Neurosc, 2008).

Active zone assembly and autism

In recent years, autism-spectrum disease (ASD) has been linked to mutations of the synaptic proteins (“synaptopathies”). Prominent examples here are Neurexin and Neuroligin. Neurexin is an AZ-localized cell adhesion molecule, transsynaptically inter-acting with its postsynaptic partner Neu-roligin. Generally, it appears highly likely that other AZ proteins might contribute in a similar way (for review see: Garber, K., Neuroscience. Autism‘s cause may reside in abnormalities at the synapse. Science 307, 190ff, 2007). Our hope is to contribute to the understanding of pathogenetic mecha-nisms at AZs in the course of ASD, capitaliz-ing on the possibility to quantify numbers as well as the ultrastructural, molecular and functional status of AZs in an efficient genetic system.

coil domain protein Bruchpilot (BRP) as a master organizer of dense body formation in Drosophila. This protein also is needed for proper clustering of Ca2+-channels (Kit-tel et al., Science, 2006; Schoch and Gun-delfinger, 2006). Moreover, we included Lip-rin-α, another major conserved organizer of AZs. Notably, we show that newly forming AZs, similar to PSDs (Rasse et al., Nat Neu-rosc, 2005), started small and increased in size over many hours in vivo, before they accumulated detectable levels of BRP and reached a final “mature” size at developing NMJs. This assembly process of individual new synaptic sites is protracted over hours, with an overlapping, defined sequence of pre- and postsynaptic proteins joining in. Here, DLiprin-α appears to be a very early player involved in initializing AZ assembly, while BRP together with Ca2+-channel in-corporation clearly followed postsynaptic postsynaptic glutamate receptors (Schmid et al., Nat Neurosc, 2008).

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Teaching &TrainingUndergraduate and Graduate Programs

Coordinator: Bw. (VWA) Carmen DengelE-mail: [email protected]: +49(0)931 201 487 13Fax: +49(0)931 201 489 78http://www.rudolf-virchow-zentrum.de/ausbildung/ausbildung.html

The Rudolf Virchow Center is not only dedicated to excellent research but also actively involved in numerous educational programs for both undergraduate and graduate students. The Center has developed, and is hosting, the undergraduate BSc/MSc-program Biomedicine and the Virchow Graduate Program, with the latter being part of the Graduate School of Life Sciences (GSLS) of the University.By combining both research and training, the Rudolf Virchow Center has adopted the Humboldt tradition, in

which both are inseparable. The Center‘s training programs are fully integrated into University-wide programs and have notably been the seed for restructuring other undergraduate and graduate programs at the University of Würzburg. The Center thus provides a stimulating and nurturing environment for researchers and students alike.The Rudolf Virchow Center strives to achieve excellence as much in its undergraduate and graduate training

programs as in its research program. Both training programs specifically target future researchers who will work at the interface between the life sciences and medicine. The undergraduate program in Biomedicine was initiated in 2001, and five classes have already graduated with the Bachelor of Science. Three classes have also success-fully completed the Master‘s course. The positions held by alumni of this program are testament to the excellent training they received and prepared them for successful careers.The development of the Virchow Graduate Program reflects the Center‘s dedication towards graduate

training. Together with several other DFG-funded graduate programs, the Virchow Graduate Program has become the nucleus for large-scale reform in graduate training at the University of Würzburg. This reform culminated in the founding of the Graduate School of Life Sciences that was awarded funding by the national “Exzellenz Initiative” in 2006.

Fig. 1:Students who finished their Bachelor´s degree in Biomedince in 2008:(From left) Yasmin Habbaba, Anja Thiessen, Friederike Mühlpfordt, Katrin Strecker, Sabine Fraschka, Mirjam Otto, Andrea Imle, Karla Schraut, Wiebke Nahrendorf, Jakob Burkhard, Anna Damm, Jorg Körner, Claudia Baumann, Julia Auinger, Katharina Godzik, and Marianne Frings with the Dean of the faculty of Biology, Martin Müller (left), and the Deputy Director of the Examination Committee, Thomas Brand (right) (not on picture: Julia Erb, Lars-Uwe Klöckl, Valentin Bruttel, Stefanie Kraft, Anke Hellrung, Fabian Kaiser).

Fig. 2:Students who finished their Master´s degree in Biomedicine in 2008: (From top and left) Miriam Alb, Janine Regneri, Daniela Endt, Christin Luft, Peggy Synwoldt, Elisa Opitz, Simon Leierseder, Silke Mühlstedt, Stefanie Lidl, Harald Depner with the Deputy Director of the Examination Committee, Thomas Brand (left), and the Dean of the faculty of Biology, Thomas Müller (right).(not on picture: Veit Althoff, Natalie Burkard, Cornelia Burkert, Julia Drießle, Kerstin Engelmann-Pilger, Claudia Gebhardt, Martin Graf, Eva Thoma, Stefan Troppens, Christopher Vollmers, Stefanie Wegener, Timo Vögtle).

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Teaching ActivitiesThe undergraduate program in biomedicine is a small, research-oriented program that enrolls 25-30 students each year. Its main focus is research-based training at the interface between the life sciences and medicine. Members of the Rudolf Virchow Center carry more than half of the teaching load and also provide opportunities for, and supervision of the majority of theses.

Undergraduate Program in Biomedicine

Manfred Schartl(Chairman of the Examination Committee)

Theodor-Boveri-Institute, Physiological Chemistry IBachelor Program (BSc – 6 semesters)

AdmissionAdmissions are based on grades in the final high school ex-amination.Structure and contentThe three-year BSc curriculum combines elements of under-graduate programs in natural sciences (mathematics, physics, chemistry, biochemistry, molecular biology, cell biology) with key modules in preclinical medicine (anatomy, physiology, microbiology, immunology, virology, pharmacology, toxicology, and pathology). Many of the modules have been specifically developed for this course, while others were adapted from the curricula of the faculties of Biology and Medicine.

The curriculum has a strong focus on practical laboratory work in order to prepare the students for research. The topics are weighted to reflect direct relevance to state-of-the-art biomedi-cal research. The curriculum also includes modules on scientific regulatory matters to legally qualify students for chemical, ra-dioactive and genetic engineering work, as well as for animal experimentation.

To facilitate international exchange, the curriculum complies with the European Credit Transfer System (ECTS). Credit points (EP) and corresponding grades are collected during the course and included in the final grade. Each module includes an ex-amination which can be a written or a practical tests, a presen-tation of research results, or a piece of scientific writing. Most students do particularly well in research-based examinations. The structured and modular nature of the curriculum allows rapid progression. A thesis, written in English and based on the student‘s own laboratory work, is publicly defended in a final examination that concludes the course.

Master Program (MSc – 1.5 years)

AdmissionAdmission to the MSc-program is based either on a Bachelor degree in Biomedicine from the University of Würzburg or an equivalent degree from another university.Structure and contentThe three-semester MSc curriculum allows for much more free-dom than the structured BSc-program. All students start with a six-week laboratory course on model organisms used in biologi-cal and medical research (Viruses, E. coli, Candida, S. cerevisiae, Drosophila, zebrafish, mouse/rat).

Management of the programs

Two committees that include members of the faculties of Biolo-gy and of Medicine as well as a coordinator, Carmen Dengel from the Rudolf Virchow Center, share the responsibility for content and organization of the BSc/MSc-program.

The examination committee, chaired by Manfred Schartl, su-pervises the organization of examinations and decides on ad-missions, transfers and accreditation of courses taken at other universities or research institutions. The study committee, chaired by Werner Lutz, is responsible for the study program and supervises quality and content of teaching.ResultsThe BSc curriculum started in 2001, with a new class starting every winter semester. The number of applications has remained high with over 600 applications each year. To date, we have had (including current students) 258 BSc students and 79 MSc stu-dents, of which 204 and 59 were female, respectively. Students have come from all over Germany, and even abroad.

Overall, the performance of BSc and MSc students has been excellent so far. Two key features of these structured training programs may be responsible for their popularity and success: first, our students acquire a particular ability to address a re-search problem, then design and present a relevant research project. Second, more than half of the students take the oppor-tunity to spend study-time abroad. Most BSc graduates decided to continue their studies with the MSc-program.

The students then carry out two six-week rotations in labo-ratories of their choice, with the possibility to spend time in external institutions and even abroad. Accompanying lectures cover molecular pathology, biomaterials, neurobiology and car-diovascular biology.

The final part of the course is dedicated to a 9-month research project. The MSc-program is concluded with a public defense of the student‘s MSc thesis, again written in English and based on the student‘s research. The MSc qualification can lead directly into doctoral training and the thesis can be credited towards the PhD.

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Graduate Program

Training Activities

Key Elements of Training in the Graduate Schools The traditional single advisor (“Doktorvater“) is replaced by a three-person committee. A panel of training activities is offered, from which an individual program is tailored to each graduate student. Graduate students actively participate in the program by offering and organizing courses and symposia. A set of requirements has to be met to guarantee a common quality standard.

Mentoring SystemEach student has an individual supervisory committee, which meets with the doctoral student at regular intervals to monitor progress and adjust the research and training activities. Addi-tionally, the graduate students report the status of their project within the research groups and programs, exchanging ideas and obtaining feedback within their peer-group.

Training ActivitiesThe training activities total a minimum of 150 hours per year and consist of laboratory seminars, journal clubs, program-sem-inars, methods courses and transferable skills workshops as well as retreats and international conferences.

Common Graduation Commission The participating faculties form a new common Graduation Com-mission within the Graduate School. The Commission is respon-sible for the conferral of all doctoral degrees within the Gradu-ate School. This enforces common standards across disciplines and fosters interdisciplinary cooperation in graduate training.

Since its inception, the Rudolf Virchow Center has aimed to offer a structured doctoral training program of the highest quality. The program is largely based on earlier experiences with doctoral training at the University of Würzburg, notably in the context of several DFG-funded Research Training Groups (Graduiertenkollegs). Another model for the Center‘s own program was the MD/PhD-program that was initiated by the faculties of Biology and Medicine in 1996/97 as the first such program in Germany. These programs, after training several generations of basic and clinical scientists, have shown the effectiveness and success of a more structured training concept. Accordingly, the Rudolf Virchow Center is running its own Graduate Program (see Page 65).Most notably, the Rudolf Virchow Center successfully catalyzed the introduction of a structured graduate train-

ing in the context of a Graduate School as the standard model throughout the whole university by developing key elements and by helping to build the necessary structures.

The Graduate School of Life Sciences (GSLS) is the culmination of an Rudolf Virchow Center initiative dating back to 2001/2 to implement structured doctoral training on a larger scale. The GSLS was founded in 2006 and was awarded funding by the “Exzellenz Initiative” that same year. The concept of Graduate Schools bringing together broad fields of research has since been extended to the entire University. Three more Graduate Schools have started or are under way in the Humanities, Science and Technology and Law, Economics and Society, respectively. All of them operate independently with respect to their research and training activities. Still, they are part of a single central institu-tion, the “University of Würzburg Graduate Schools” (UWGS). The UWGS serves as a holding and monitors basic rules and stan-dards, in addition to delivering general services. Martin Lohse, speaker of the Rudolf Virchow Center, was elected director of the UWGS in 2008.

The Graduate School of Life Sciences is jointly supported by the faculties of Medicine, Biology, Chemistry & Pharmacy, Physics & Astronomy as well as the Philosophical Faculty II. All doctoral students enroll in the study program “Life Sciences”. A Common Graduation Commission of all participating faculties graduates doctoral students with the degree Dr. rer. nat. or Ph.D.

The School has been growing rapidly since 2006. It started with about 60 doctoral students and 28 founding members. Now the numbers are close to 250 and 150, respectively, and still rising.

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Virchow Graduate Program

SFB 487 Regulatory Membrane Proteins

SFB 688Cellular Inter-actions in the Cardiovascular

System

TR 17Ras-dependent

Cancer

GK 1048 Organ Develop.

Graduate School ofLife Sciences

Graduate School ofScience & Technology

Graduate School ofHumanities

Graduate School of Law, Economics &

Society

University of Würzburg Graduate Schools

SectionInfection &Immunity

SectionBiomedicine

SectionIntegrative

Biology

SectionNeuroscience

SectionMD/PhD

The Graduate School of Life Sciences comprises four scientific sections and a MD/PhD program, reflecting the research foci in the Life Sciences at our University. The section “Biomedicine” was initiated by the Rudolf Virchow Center and embodies the nucleus of the GSLS. This section is still the largest with a to-tal of 86 doctoral students. 32 of the doctoral students of this section belong to the Virchow Graduate Program, documenting the pivotal role of this institution in its section. Other doc-toral students working at the Rudolf Virchow Center have joined other sections such as Infection and Immunity or Neuroscience according to the main focus of their research work.

A special fellowship program of the GSLS is the core element of funding by the “Exzellenz Initiative”. Almost 1000

Fig. 3:Structure of the University of Würzburg Graduate Schools.

Training ActivitiesGraduate Program

standardized written applications have been evaluated so far in a staged process involving interviews, of which more than 150 were conducted by the admission board – either in Würzburg, by means of video conferencing or abroad. 37 fellows were recruited in total. They originate from 11 different countries, with 60% of the fellows being from abroad. The fellowships are portable and the fellows can freely choose a laboratory and project among the member-laboratories of the GSLS. The Rudolf Virchow Center proved to be exceptionally compe-titive in attracting fellowship recipients: 7 fellows are working at the Center.

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Caroline Kisker(Chairperson Section Biomedicine)Rudolf Virchow Center

Training activities

In addition to the training activities offered by the individual programs and their research groups, a number of activities were organized for all graduate students in Biomedicine and the Life Sciences. Training activities and events in 2008, organized by Carmen Dengel, coordinator at the Rudolf Virchow Center, in-cluded:

Workshops “Effective scientific writing” Three-day workshops were held by a professional science writer to provide tools to organize, structure and write research papers.

Workshops “Oral presentation” Two-day workshops provided opportunities to learn and to test strategies for effective and concise oral presentations.

Workshops “Poster presentations” Two-day workshops focused on the key elements for effective poster design.

Meetings and events

In addition to many international scientific conferences and meetings that the graduate students attended, a number of events were organized specifically for the graduate students. Highlights were:

Junior Faculty Lectures “Hottest Life Science” (July 5, 2008)This annual event is a symposium at which selected junior faculty members from the different sections – including bio-medicine – presented their work to a broad audience of stu-dents and researchers from different disciplines. The event has been followed by a barbecue to provide an informal setting for presenters to discuss their work with students.

The Section Biomedicine provides a training program that is tailored to graduate students‘ individual needs within a defined framework. The Section not only serves as an interdisciplinary link between the different graduate programs but also between scientific research and practical experience, such that graduate students have the possibility to work in international teams with scientists from various research areas.

Program Section Biomedicine

Training Activities

Graduate student-organized activities

Graduate students organized a number of informal laboratory courses as well as seminars. Since 2005, the graduate students from the Section Biomedicine, together with students from the MD/PhD program, have organized a yearly international symposium with high-profile speakers from around the world. Remarkably, the students are responsible for all scientific and administrative aspects, such as the selection and invitation of international speakers, raising sponsorship funds from compa-nies, and organizing the day‘s event. Since the schedule of this event has been switched from a fall-event to a spring-sympo-sium, no symposium was organized in 2008. Instead, the stu-dents are currently preparing for the spring 2009 event.

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Events

Annual RetreatThis year’s annual Graduate retreat was again held in conjunc-tion with the annual Rudolf Virchow Center retreat, which took place in the Wildbad Conference Center, near Rothenburg ob der Tauber from September 18th to 20th. Each student presented his work either orally or as a poster, and for the second year running, the hotly contested award competition constituted a highlight of the meeting. This year‘s winners were Claudia Jentzsch from the Engelhardt laboratory for the Best Talk and Christoph Hintzen of the Hermanns group for the Best Poster.

Virchow Graduate Program

Training Activities

Stephan Kissler(Coordinator of the Virchow Graduate Program)


The Virchow Graduate Program is the Rudolf Virchow Center‘s own graduate program and is part of the Biomedicine Section of the Graduate School of Life Sciences. The concept of this program is to bring together all the graduate students working in the Center, regardless of their source of funding and external affiliations, and to generate a collegial peer group within the Rudolf Virchow Center. One of the primary goals is to promote interactions and cooperations at the graduate student level.

In 2008, the Virchow Graduate Program comprised 32 students, including 7 recipients of the highly competitive GSLS fellow-ships. A majority of projects aim to identify molecular mecha-nisms of disease and/or attempt to develop tools to monitor, inhibit, or even abrogate pathologic cell behavior. The experi-mental strategies range from the analysis of single molecules to complex in vivo disease models.

The Virchow Graduate Program is fully integrated into the wider biomedical research community in Würzburg. Exchange on a scientific as well as technological level with other laboratories not only in Würzburg but also nationally and internationally is highly encouraged and actively promoted. Furthermore, the pro-gram facilitates individualized hands-on training by organizing practical training units, tutorials and small workshops. Our aim is to provide a training that truly prepares young scientists to a career in biomedical research. Laboratory work is complemented by opportunities to learn about relevant aspects of clinical med-icine and by seminars in which students are taught to critically review the scientific literature, to organize their work-schedules efficiently, and to communicate their science most effectively.

Student activitiesThe graduate students organized their own seminar series, with presentations of ongoing work and discussions of top-ics of broader interest. In September 2008, students also par-ticipated in the Proteomics Workshop and Dynamic Microscopy Workshop organized by members of the Rudolf Virchow Center, where international speakers presented the latest advances in these technologies. These workshops again provided truly state-of-the-art training to participants, reflecting the cutting-edge nature of the Virchow Graduate Program.

All key technological platforms provided by the groups of the Rudolf Virchow Center are fully integrated into the Graduate Program and used by students for their thesis work on a daily basis. These include:

State-of-the-art proteomic approaches to determine pro-tein content and function in cells, including quantita-tive mass spectrometry, phosphoproteomics and interac-tomics.Advanced molecular imaging of molecules and cells in vi-tro and in vivo, including biosensors, single molecule and multiphoton microscopy, as well as optical whole-mouse imaging.Cutting-edge approaches to determine the structures of biological macromolecules at the atomic level, together with biophysical and biochemical methods to analyze their functions.The generation and analysis of transgenic and knockout mouse models of disease, including modulation of gene expression by RNA interference in vivo.

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Public Science CenterE-mail: [email protected]: +49(0)931 201 487 14Fax: +49(0)931 201 487 02http://www.rudolf-virchow-zentrum.de/public/public.html

Sonja Jülich

The overall goal of the Public Science Center is to increase the national and international visibility of the Rudolf Virchow Center and of biomedical re-search in general. The Center provides information about research and teach-

Fig. 1:The award-winning exhibits at the Science Summer Fair in Leipzig 2008: A 3D-projection of a protein structure to illustrate drug design (left) and a large walk-in artery with a 3D-projec-tion of atherosclerotic plaque formation and arterial thrombosis (right) were presented.

ing activities at the Rudolf Virchow Center to the public, politicians and the science community, as well as stimulating debate not only about the achievements, but also about ethical issues in biomedical research. In line with the overall concept of the Rudolf Virchow Center to attract the brightest minds to science early on, one important target group is young people. The Public Science Center’s main instruments of communication are through the media, the Annual Report, as well as organizing public events such as school projects, Science Days and discourses.

Science Summer - Wissenschaft interaktiv 2008In 2008, we participated in the German science festival “Science Summer” in Leipzig, which attracted more than 100 000 people within one week. A team from the Public Science Center together with young scientists from the Rudolf Virchow Center (Sylvia Luckner, Daniela Schneeberger, Frauke May, and Irina Pleines) developed and present-ed two exhibits showing current research in progress (Fig. 1). The team collaborated with experts in the field of designing and building interactive exhibitions. For the first time, “Wissenschaft im Dialog” and the “Stifterverband für die Deutsche Wissenschaft” announced a special audience award (Wissenschaft interaktiv 2008) for exhibits at the Science Summer. The Rudolf Virchow Center exhibits and the presentation Hereinspaziert - Biomedizinische Forschung XXL! were awarded the first prize, which included € 10 000 to be used for future projects.

Science DayIn September 2008, the Rudolf Virchow Center opened its doors to children and high-school students by launching the Vir-chowlab, and celebrated its school projects winning the contest “Deutschland – Land der Ideen (Land of Ideas)”. Guided lab tours and several hands-on experiments demon-strated everyday life in a research center. Several hundred people visited the Center. As a special highlight, the Wissenschaft in-teraktiv exhibits were on display.

Discourse on Animal ResearchIn biomedical sciences, animal research is an issue that raises controversy or even strong opposition. Our aim is to stimu-late constructive dialog between scientists and the public. As a first step, the Public Science Center has organized a discourse on animal research with high-school stu-dents by adopting a BMBF-funded project from the Helmholtz Center for Infectious Diseases (HZI). This provides the op-portunity for a dialog between students and experts with opposing points of view about animal research. In July 2008, a discourse was held at the Deutschhaus Gymnasium (high-school in Würzburg). This was very fruitful and productive, including an intense but extremely fair discussion with positive feedback from all parties.

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General public – Media relations

The development and maintenance of an extensive network with journalists is a main focus of the Public Science Center, since we regard journalists as our most important partners. For example, compared to direct activities with the general pub-lic, our press releases can reach a far wider audience. Information about the Rudolf Virchow Center’s activities is sent out to a network of journalists through press re-leases and the news service Informations-dienst der Wissenschaft. In 2008, 19 press releases resulted in 420 media reports with a reach of more than 30 million. This year, a particular effort was put into increasing research-related media reports and achiev-ing a presence in more nationally distrib-uted media. Therefore, the Public Science Center increased its number of annual press releases concerning the Center’s research from two (2003-2007) to twelve (2008). Reports describing the Center’s research increased from 11 in 2005 to 308 in 2008. These research-related reports have been mainly published in national media. Thus, reports in national media increased consid-erably, especially in online media such as zeit.de, spiegel.de, sueddeutschezeitung.de and focus-online, and TV such as the

Fig. 3:High-school students in the new “Virchowlab”

Selected Media Reports“Was den Lebenssaft zum Stocken bringt”, Spiegel online, February 19, 2008

“Neuer Ansatz bei Chemotherapie-Re-sistenz”, Bayerischer Rundfunk TV, June 11, 2008

“Kleber im Blut“, Mainpost, June 14, 2008

“Wissenschaft interaktiv – Team vom RVZ gewinnt”, Bayerischer Rundfunk 1, July 7, 2008

“Blutgerinnung besser steuern”, Rhein-Neckar-Zeitung, July 25, 2008

“Weniger Nebenwirkungen bei Herzin-farkt”, BR TV /BR Alpha, August 24, 2008

“Gentechniker heilen Herzschwäche”, Handelsblatt & handelsblatt.com, November 30, 2008

“Herzmuskelschwäche bei Maus geheilt”, ARD Videotext, December 01, 2008

“Forscher finden “Schalter für Herz-muskelschwäche”, zeit.de, December 07, 2008

“Ein Schalter lässt das Herz groß und schwach werden”, Dradio Forschung aktuell, December 08, 2008

Politicians and Science Community

In 2008, politicians visited the Rudolf Virchow Center to gain information about the Center’s research and discuss the in-novative structures implemented by the Center within a traditional university set-ting in order to promote scientific excel-lence. Politicians and representatives of the Bavarian State Ministry of Sciences, Research and the Arts, and the Bavarian State Ministry of the Interior visited the Center (see page 12). Since 2006, the Public Science Center has published its Annual Report with an annually circulation of 1500 copies. This report is distributed to politicians and decision makers, and also to collaborators, colleagues, research centers and scientists worldwide.

Fig. 2:Discourse on animal research at the Deutschhaus Gymnasium. Experts were: Horst Spielmann, Freie Universität Berlin, Centre for Documentation and Evalu-ation of Alternatives to Animal Experiments, Berlin; Gabriele Kuesters, Sanofi-Aventis; Winfried Ueckert, veterinary office Würzburg; Eve-Marie Engels, University of Tübin-gen (member of the National Ethics Council); and Martin Lohse, Rudolf Virchow Center.

Attracting young people to science

Right from the start, the Public Science Center set up different school projects with two key aims: to generate interest in bio-medical sciences from childhood on and to attract new generations of students. Four projects, the children‘s lab Rudis Forscher-camp and Rudolf-Virchow-Paten for “Jugend forscht”, the Virchowlab and ForscherRe-porter have been established. Enthusiasm is long-lasting and feedback has been highly positive. We now offer a broad spec-trum of projects for the age range 8 to 18. All school projects have been awarded rec-ognition as one “Ort im Land der Ideen” 2008 in the contest “Deutschland – Land der Ideen”

VirchowlabIn September 2008, the Public Science Center started a new project for students from the age of thirteen to sixteen. The two key aims of this project are to main-tain and generate further interest in sci-ence through a range of research-oriented project activities. The Center opens this lab to whole school classes. Several ex-periments have been developed based on the Bavarian school curriculum and in close collaboration with Ilona Landgraf, a science teacher at the Deutschhaus Gymnasium.

ForscherReporterThe aim of the monthly course is to provide A-level students with the possibility to ori-ent themselves in the occupational field of “research”. At the same time, we motivate them to take on the role of a journalist to gain an insight into how scientists work from a different perspective and also im-prove their ability to communicate this knowledge. So far, eight schools and 70 students have participated, and “Forscher-Reporter” is currently booked until March 2009.

Bayerischer Rundfunk. Two press releases about two publications in Nature and Na-ture Medicine reached over 15 Million read-ers, since they were distributed through the agency dpa (Deutsche Presseagentur).

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Chairman: Prof. Dr. Martin Lohse, Institute of Pharmacology and ToxicologyVice-Chairs: Prof. Dr. Jörg Hacker, Institute of Molecular Infection Biology (until 2008) Prof. Dr. Manfred Schartl, Theodor-Boveri-Institute, Physiological Chemistry IMembers: Prof. Dr. Caroline Kisker, Rudolf Virchow Center Prof. Dr. Bernhard Nieswandt, Rudolf Virchow Center Prof. Dr. Michael Sendtner, Institute of Clinical Neurobiology

Scientific Advisory Board

Chairman: Prof. Dr. Fritz Melchers, MPI for Infection Biology, BerlinMembers: Prof. Dr. Ueli Aebi, Biocenter, University of Basel, Biocenter Prof. Dr. Volkmar Braun, University of Tübingen Prof. Dr. Sabine Werner, Eidgenössische Technische Hochschule Zürich Prof. Dr. Heiner Westphal, Laboratory of Mammalian Genes and Development, NICHD, Bethesda, MD, USA Prof. Dr. Alfred Wittinghofer, MPI for Molecular Physiology, Dortmund Prof. Dr. Claes Wollheim, University of Geneva

I. Funded Members

Prof. Dr. Dr. Stefan Engelhardt, Rudolf Virchow Center (funded by Sanofi-Aventis, Procorde, Bavarian Ministry of Economic Affairs)

Prof. Dr. Utz Fischer, Theodor-Boveri-Institute, BiochemistryProf. Dr. Peter Friedl, Rudolf Virchow CenterProf. Dr. Manfred Gessler, Theodor-Boveri-Institute, Physiological Chemistry IProf. Dr. Gregory Harms, Rudolf Virchow CenterPD Dr. Heike Hermanns, Rudolf Virchow CenterProf. Dr. Thomas Hünig, Institute of Virology and ImmunobiologyDr. Asparouh Iliev, Emmy Noether-Fellow, Rudolf Virchow Center, Institute of PharmacologyProf. Dr. Caroline Kisker, Rudolf Virchow CenterDr. Stephan Kissler, Rudolf Virchow CenterProf. Dr. Martin Lohse, Institute of Pharmacology and Bio-Imaging CenterProf. Dr. Thomas Müller, Julius von Sachs Institute, Botanic IProf. Dr. Bernhard Nieswandt, Rudolf Virchow CenterProf. Dr. Andreas Rosenwald, Institute of PathologyProf. Dr. Manfred Schartl, Theodor-Boveri-Institute, Physiological Chemistry IProf. Dr. Hermann Schindelin, Rudolf Virchow CenterProf. Dr. Michael P. Schön, Rudolf Virchow CenterProf. Dr. Walter Sebald, Theodor-Boveri-Institute, Physiological Chemistry IIProf. Dr. Albert Sickmann, Rudolf Virchow CenterProf. Dr. Stephan Sigrist, Clinical Neurobiology and Bio-Imaging Center

II. Non-funded Members

Prof. Dr. Gerhard Bringmann, Institute of Organic ChemistryProf. Dr. Matthias Frosch, Institute of Hygiene and MicrobiologyProf. Dr. Werner Goebel, Theodor-Boveri-Institute, MicrobiologyProf. Dr. Jörg Hacker, Institute of Molecular Infection BiologyProf. Dr. Martin Heisenberg, Theodor-Boveri-Institute, Genetic and NeurobiologyProf. Dr. Bert Hölldobler, Theodor-Boveri-Institute, Zoology IIProf. Dr. Peter Jakob, Institute of Physics, BiophysicsProf. Dr. Hans-Konrad Müller-Hermelink, Institute of PathologyProf. Dr. Ulf Rapp, Institute of Medical Radiation and Cell ResearchProf. Dr. Markus Riederer, Theodor-Boveri-Institute, Botany IIProf. Dr. Michael Sendtner, Institute of Clinical NeurobiologyProf. Dr. Klaus V. Toyka, Clinic of NeurobiologyProf. Dr. Ulrich Walter, Institute of Clinical Biochemistry and Pathobiochemistry

Executive Committees and Scientific Members

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Group Stefan Engelhardt

Group leader:Prof. Dr. Dr. Stefan Engelhardt

Postdocs:Dr. Morgan DupuisDr. Xavier LoyerDr. Sabine Merkle

Scientific Staff:Lydia Vlaskin

Graduate Students:Andrea AhlesJayavarshni GanesanPetra GöbelCarina GrossClaudia JentzschDeepak Ramanujam

Bachelor/Master Students:Justus BeckLars KlöcklSimon LeiersederSilke Mühlstedt

Technicians:Isabell FlohrschützNadine Yurdagül-Hemmrich

Group Heike Hermanns

Group leader:PD Dr. Heike Hermanns

Postdoc:Dr. Christine Mais

Graduate Students:Johannes DrechslerChristoph GroßChristoph Hintzen

Technician:Daniela Kraemer

Group Asparouh Iliev

Group leader:Dr. Asparouh Iliev

Graduate Students: Ursula Duda (med.)Christina FörtschSabrina HuppCarolin Wippel

Technician:Alexandra Bohl

Group Stephan Kissler

Group leader:Dr. Stephan Kissler

Graduate Students:Kay FischerJulie JosephPeilin Zheng

Bachelor Student:Fabian Kaiser

Technicians:Nicole GollhoferKatharina Herrmann

Animal care taker:Claudia KühneHeike Rudolf

Core Center

Group Gregory Harms

Group leader:Prof. Dr. Gregory Harms

Postdocs:Dr. Sandra de KeijzerDr. Volodymyr Ermolayev

Graduate Students:Jörg BlachutzikQiang GanKun WangMonika Zelman-Femiak

Bachelor/Master/Diploma Students:Matthias EidelMike FriedrichJohanna KlughammerJan-Hendrik SpilleIsabell Weber

Technicians:Wiebke BuckMarkus HirschbergRevaz Nozadze

Group Caroline Kisker

Group leader:Prof. Dr. Caroline Kisker Postdocs: Dr. Ingrid Teßmer Dr. Jochen Kuper

Graduate Students: Uwe Dietzel Maria Hirschbeck Matthias Leyh (med.)Sylvia Luckner Shambhavi Mishra Vijayan RamachandranFlorian Rohleder Heide Marie Roth Stefanie Wolski

Master/Diploma Students: Christine Betz David Fronczek Dominik Schmitt

Technicians: Nicole BaderSabine ErhardGudrun Michels

Academic Members and Supporting Staff

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Group Hermann Schindelin

Group leader: Prof. Dr. Hermann Schindelin

Postdocs: Dr. Petra Hänzelmann Dr. Daniela Schneeberger

Graduate Students: Carolyn Delto Xaver KoberEun Young Lee Hans Maric Wilko Rauert Bodo Sander Daniel Völler

Diploma Student: Gunnar Knobloch

Technicians: Nicole Bader Silvia Scheuring

Group Albert Sickmann

Group leader:Prof. Dr. Albert Sickmann

Postdocs:Dr. Katrin LokajDr. Urs LewandrowskiDr. René Zahedi

Scientific Staff:Stefanie Wortelkamp

Graduate Students:Julia BurkhartBeate EyrichThomas PremslerStephanie PützJulia Wiesner

Bachelor/Master/Diploma Students:Florian BeckOliver Simon

Technicians:Claudia SchützChristiane Winkler

Research Professorships

Group Peter Friedl

Group leader:Prof. Dr. Peter Friedl

Clinical Associate:Dr. Julian Storim

Graduate Students: Stephanie AlexanderVolker Andresen Antoine KhalilBettina Weigelin

Technicians:Markus HirschbergMonika KuhnEva NaglerMargit Ott

Group Bernhard Nieswandt

Group leader:Prof. Dr. Bernhard Nieswandt

Postdocs:Dr. Attila BraunDr. Margitta ElversDr. Miroslava Pozgajova

Graduate Students:Markus BenderAlejandro Berna ErroShuchi GuptaIna HagedornFrauke MayIrina PleinesRastislav PozgajDavid StegnerJohannes Steinweg (med.)Dávid Varga-SzabóTimo Vögtle

Bachelor/Diploma Students:Ronmy Rivera-GaldosSebastian HofmannTimo Vögtle

Technicians:Stefanie HartmannSylvia HengstJuliana GoldmannBirgit Midloch

Animal care taker:Claudia KühneMario Müller

Group Michael P. Schön

Group leader:Prof. Dr. Michael P. Schön

Research Associate:PD Dr. Margarete Schön

Postdocs:Dr. Sabine GesierichDr. Kai MichaelisDr. Stephanie SchlickumDr. Katrin WallbrechtDr. Gregor Wienrich

Graduate Students:Luise Erpenbeck (med.)Desislava Boyanova (med.)Katharina Amschler (med.)

Technician:Helga Sennefelder

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RVZ Network

Group Utz Fischer

Group leader:Prof. Dr. Utz Fischer

Graduate Student:Julia Wiesner

Group Manfred Gessler

Group leader:Prof. Dr. Manfred Gessler

Graduate Student:Julia Heisig

Group Thomas Hünig

Group leader:Prof. Dr. Thomas Hünig

Postdoc:Kirsty McPherson

Group Thomas D. Müller

Group leader:Prof. Dr. Thomas D. Müller

Group Manfred Schartl

Group leader:Prof. Dr. Manfred Schartl

Postdoc:Daniel Liedtke

Scientific Staff:Monika Niklaus-Ruiz

Technician:Isabell Erhard

Group Walter Sebald

Group leader:Prof. Dr. Walter Sebald

Graduate Students:Stefan HarthMarianne Rattel

Bio-Imaging Center

Group Martin Lohse

Group leader:Prof. Dr. Martin LohseStephan Schulz

Postdoc:Dr. Viacheslav NikolaevDr. Davide Calebiro (Humboldt fellow)Dr. Veronika Hlavackova (EMBO fellow)

Group Stephan Sigrist

Group leader:Prof. Dr. Stephan Sigrist Postdocs:Dr. Birgit GreinerDr. Tobias SchwarzDr. Carolin Wichmann

Graduate Students:Frauke Christiansen-EngelhardtWernher FouquetOmid KhorramshahiKaren LiuSara MertelDavid OwaldTill AndlauerRui Tian Diploma student:Harald Depner

Technicians:Christine QuentinFranziska ZeheClaudia Wirth

Central Technologies

Transgene Technologies

Group leader:Dr. Bettina Holtmann

Postdoc:Dr. Eva Schmitteckert

Technicians:Daniela ÖstreichMelanie Maier

Animal care taker:Azer Achmedov

DNA Arrays

Group leader:PD Dr. Andreas Rosenwald

Administration

Administrative Director:Prof. Dr. Karl-Norbert Klotz

Administrative Assistants:Eva AlberoGerhard AntlitzBianca KlotzPetra LütkeMaria Weidner

IT Area Manager:Joachim Baumeister

Teaching and Training

Coordinator:Carmen Dengel

Assistants:Elke DrescherMichaela Reuter

Public Science Center

Group leader:Sonja Jülich

Assistants:Christiane WeberKatja Weichbrodt

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Visiting Scientists 2008 at the Rudolf Virchow Center

Carl A. Machutta, Stony Brook, NY, USA (Jan. - Feb. 2008)

Bennett Van Houten, NIH, Bethesda, MD, USA (several short visits)

Davide Calebiro, Milan, Italy (Jan - July 2008)

Peter Tonge, Stony Brook, NY, USA (two short visits)

Lajos Pinter, Temesvari, Hungary (June - July 2008)

Cory Quammen, Chapel Hill, NC, USA (July 2008)

Sara Field, Cambridge, England (April 2008)

Xavier Loyer, INSERM, Paris, France (Feb. - Dec. 2008)

Marie Vidal, Strasbourg, France (March - Sept. 2008)

Miguel P. Aso, Valencia, Spain (March - April 2008)

Veronica Hlavackova, Praha, Czech Rep. (since Feb. 2008)

Manuela Ambrosio, Milan, Italy (since Oct. 2008)

Johann Heemskerk, Maastricht, Netherlands (one short visit)

Shari Raasi, Konstanz, Germany (August 2008)

Eun Young Lee, Stony Brook, NY, USA (several visits)

Alexander Urban, Potsdam, Germany (April 2008)

Günther Schwarz, Cologne, Germany (May 2008)

Silke Leimkühler, Potsdam, Germany (August 2008)

Takuo Fujisawa, Okayama, Japan (April - May 2008)

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Teaching Committees (Faculties Biology and Medicine)

BSc/MSc Study Committee

Chairman: Prof. Dr. Werner Lutz, Institute of Pharmacology and ToxicologyMembers: Prof. Dr. Manfred Gessler, Theodor-Boveri-Institute, Physiological Chemistry Dr. Ursula Rdest, Theodor-Boveri-Institute, Microbiology Prof. Dr. Wolfgang Rößler, Theodor-Boveri-Institute, Zoology II

BSc/MSc Examination Committee

Chairman: Prof. Dr. Manfred Schartl, Theodor-Boveri-Institute, Physiological ChemistryMembers: Prof. Dr. Thomas Brand, Theodor-Boveri-Institute, Zoology I Prof. Dr. Jürgen Kreft, Theodor-Boveri-Institute, Microbiology Prof. Dr. Georg Nagel, Theodor-Boveri-Institute, Botany I Prof. Dr. Stephan Sigrist, Rudolf Virchow Center, Bio-Imaging Center Prof. Dr. Helga Stopper, Institute of Pharmacology and Toxicology

University of Würzburg Graduate Schools Managing Director: Prof. Dr. Martin Lohse, Institute of Pharmacology and Toxicology, Rudolf Virchow Center

Graduate School of Life Sciences (since 09/2006)

Dean: Prof. Dr. Markus Riederer, Theodor-Boveri-Institute, Botany IIVice Deans: Prof. Dr. Martin Lohse, Institute of Pharmacology and Toxicology, Rudolf Virchow Center Prof. Dr. Heidrun Moll, Institute of Molecular Infection Biology

Section Biomedicine (Graduate School of Life Sciences)

Chairperson: Prof. Dr. Caroline Kisker, Rudolf Virchow CenterVice-Chair: Prof. Dr. Helga Stopper, Institute of Pharmacology and Toxicology

Virchow Graduate Program (Section Biomedicine)

Coordinator: Dr. Stephan Kissler, Rudolf Virchow Center

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Undergraduate program in Biomedicine

Bachelor theses published in 2008

The theses have been written in the context of the under-graduate program in Biomedicine.

“Soluble guanylate cyclase activation with HMR1766 in rats with chronic myocardial infarction”Auinger, Julia

“Molekulare Analyse der SAM-6 induzierten Lipoptose“Baumann, Claudia

“Functional Characterisation of YB-1 Mutants”Bruttel, Valentin

“Charakterisierung der Effekte von Phytoprostanen auf humane Plättchen“Damm, Anna

“Characterisation of the Oncostatin M mediated signal transduction“Erb, Julia

“The biology of the rodent malaria parasite Plasmodium berghei”Fraschka, Sabine

“Analysis of the role of lin9 in development and cell cycle using a lin9 knockout mouse”Frings, Marianne

“Untersuchung der Antikörperantwort gegen “neue“ respiratorische Viren“Godzik, Katharina

“Cloning of a vector for transgenic expression of the signal peptide of the dihydrolipoamide dehydrogenase”Habbaba, Yasmin

“Invasion of HeLa cells by Staphylococcus aureus leads to acidification of the phagosomal compartment”Hellrung, Anke

“Isolation of micronucleated cancer cells and single-cell CGH to detect clonal heterogeneity and migration-associ-ated DNA damage”Imle, Andrea

“Structural and functional analysis of Ena/VASP proteins”Burkhard, Jakob

“Generierung von HT1080-Fibrosarcoma-Zelllinien, in denen Integrin ß1 durch lentivirale RNA-Interferenz gehemmt ist“Kaiser, Fabian Marc Philipp

“The cardiac beta-1-adrenergic receptor as a putative target of miRNAs”Klöckl, Lars-Uwe

“Analysis of Presynaptic Active Zone Proteins in Dro-sophila Melanogaster”Körner, Jörg

“A quantitative histological study of a new genetically engineered mouse model for serotonin deficiency in the central nervous system”Kraft, Stefanie

“Effects of NAD(P)H Oxidase Inhibitors on Thrombocyte Function and Haemostasis”Mühlpfordt, Friederike

“Optimizing methods for time dependent protein expres-sion using a heat shock promoter in larval Drosophila”Nahrendorf, Wiebke

“Alteration of DNA methylation by non-genotoxic car-cinogens“Otto, Mirjam

“Differential responsiveness of Dendritic cells to com-mensal or pathogenic strains of E.coli”Schraut, Karla-Gerlinde

“Einfluss der Oberflächenglykosylierung auf die Funk-tionen Dendritischer Zellen“Strecker, Katrin

“Quantitative analysis of cGMP mediated signaling path-ways in human platelets“Thiessen, Anja

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Master theses published 2008

The theses have been written in the context of the under-graduate program in Biomedicine.

“Signal transduction in skin cancer - the Rb/p16ink4a pathway in Merkel cell carcinoma”Alb, Miriam

“Untersuchungen zur Prozessierung des Signalpeptids der Dihydrolipoamid Dehydrogenase”Althoff, Veit

“The role of the neuronal NO synthase in the myocardi-um: Conditional and cardiac specific nNOS overexpression decreases myocardial contractility”Burkard, Natalie

“Nuclear deformation and DNA damage in non-neoplastic cells during migration through 3D tissues”Burkert, Cornelia

“The Bruchpilot protein and related factors in the pre-sentation of synaptic Ca2+-channels”Depner, Harald

“Structural investigation of the immunodominant staphylococcal antigen A (IsaA)”Drießle, Julia

“Untersuchungen zu Interaktionsproteinen von FANCJ und FANCN”Endt, Daniela

“Behavioural effects of electromagnetic fields on Drosophila melanogaster”Engelmann-Pilger, Kerstin

“The Calcineurin-Calpastatin signaling pathway in the myocardium”Gebhardt, Claudia

“Proteinexpression des Signalpeptides der Dihydrolipoa-mid-Dehydrogenase in E.coli.“Graf, Martin

“Trogocytosis of immunotolerogenic molecules as a mechanism of local immune regulation”Luft, Christin

“Function and Regulation of Stromal Interaction Molecule 1 (STIM1) in Cardiomyocytes”Mühlstedt, Silke

“Construction and characterisation of a foamyviral shRNA-expression vector for the transduction of murine hematopoietic stem cells & Cloning of SIVmac239 Nef into an eukaryotic expression plasmid and analysis of protein functionality”Optiz, Elisa

“The role of histone modifications in the regulation of the Xmrk promoter”Regneri, Janine

“Isolation and Characteristation of Melanoma Stem Cells”Synwoldt, Peggy

“Analysis of pluripotency and induced differentiation of stem cells“Thoma, Eva

“Bestimmung ausgewählter Parameter des oxidativen Stresses im Rahmen eines Estrogenersatz-Modells”Troppens, Stefan

“Circadian and Metabolic Control of Gene Expression in Mouse Liver”Vollmers, Christopher Stephan

“Learning and the presynapse in Drosophila melanogas-ter Investigating protein-protein interactions and larval learning in fruitflies”Wegener, Stephanie

“Flow Cytometric and Functional Analyses of T-cells in STIM1-deficient Mice”Vögtle, Timo

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“Pore formation and small GTPase activation by pneumo-lysin”Förtsch, Christina

“In vivo assembly of synapses“Fouquet, Wernher

“In vivo visualization of Smad signaling by dynamic high resolution microscopy“Gan, Qiang

“Translational control in cardiomyocytes”Ganesan, Jayavarshni

“Identification of the cardiac ß-adrenergic phosphopro-teome”Göbel, Petra

“Analysis of the oncogenic potential of ∆Np73”Griesmann, Heidi

“The role of miRNAs in cardiac disease”Gross, Carina

“Studies on cytoskeletal regulation of platelet spreading, granule release and coagulant activity”Gupta, Shuchi

“In vitro and in vivo analysis of thrombus formation under flow conditions”Hagedorn, Ina

“Hey bHLH transcription factors and their target genes”Heisig, Julia

“Immunoregulatory functions of the Interleukin-6-type cytokine oncostatin M”Hintzen, Christoph

“Structure based drug design”Hirschbeck, Maria

“Function, regulation and biological role of Chronophin”Hoffmann, Axel

“Role of sub-cellular and sub-compartmental distribution of regulatory GEFs and GAPs in mediating activation of small GTPases by pneumolysin”Hupp, Sabrina

“Signaling mechanisms in cardiac failure”Jentzsch, Claudia

“Analysis of G protein-coupled receptor activation by optical methods”Ahles, Andrea

“Collective cancer cell invasion along blood vessels”Alexander, Stephanie

“Structure, development and plasticity of mammalian synapses”Andlauer, Till

“New approaches in nonlinear microscopy: applications in biomedicine”Andresen, Volker

“Cellular regulation of platelet adhesion receptors”Bender, Markus

“Function of the stromal interaction molecule 2 (STIM2) in hemostasis and thrombosis “Berna Erro, Alejandro

“Biological and biophysical characterization of proteins and lipids in plant rafts”Blachutzik, Jörg

“Phosphorylation-specific interaction and regulation of Vasodilator-stimulated phosphoprotein in platelets”Burkhardt, Julia

“Synaptic organization and synaptic plasticity in the olfactory system of Drosophila“Christiansen-Engelhardt, Frauke

“Architecture of gephyrin”Delto, Carolyn

“X-ray crystallographic studies on Rhodesain and SARS-PLpro, two papain-like proteases in complex with new inhibitors”Dietzel, Uwe

“Impact of Interleukin-6-type cytokines on cardiovascu-lar diseases”Drechsler, Johannes

“Characterization of AUM, a novel HAD-type phosphatise”Duraphe, Prashant

“Quantitative analysis of proteins enriched in lipid rafts”Eyrich, Beate

“Role and function of the human susceptibility gene KIAA0350 in the NOD mouse model of type 1 diabetes”Fischer, Kay

PhD theses at the Virchow Graduate Program (2008)

77

“Evaluating the therapeutic potential of Men1 modula-tion in the NOD model of type 1 diabetes”Joseph, Julie

“Role of connexins in collective cell invasion”Antoine Khalil

“The role of glutamate receptors in plasticity processes at the Drosophila NMJ”Khorramshahi, Omid

“Structural and functional characterization of protein disulfide isomerase”Kober, Xaver

“Oncogene-induced senescence in melanocytes”Leikam, Claudia

“Genetic dissection of active zone assembly”Liu, Karen

“Towards the development of high affinity InhA/KasA inhibitors with activity against drug-resistant strains of Mycobacterium tuberculosis”Luckner, Sylvia

“Anchoring of GABA(A) receptors”Maric, Hans

“The La-related protein LARP7 is a component of the 7SK ribonucleoprotein and affects transcription of cellular and viral polymerase II genes”Markert, Andreas

“Signaling in platelets via ITAM-coupled receptors”May, Frauke

“Molecular and functional analysis of active zone pro-teins in Drosophila”Mertel, Sara

“Structure based drug design on proteins from Mycobac-terium tuberculosis”Mishra, Shambhavi

“Studies on kallikrein-kinin system-driven edema forma-tion and infection in vivo”Oschatz, Chris Tina

“Cell cycle regulation by ΔN-p73”Oswald Claudia

“Active zone organization in control of pre-synaptic vesicle release at the Drosophila NMJ”Owald, David

“Pathological activation of the contact-phase-systems in vivo”Pleines, Irina

“Phosphorylation-specific interaction and regulation of vasodilator-stimulated phosphoprotein in endothelial cells”Premsler, Thomas

“Differential and quantitative proteome analysis of a cell culture model for malignant transformation”Pütz, Stephanie

“Structural and biochemical investigation of ubiquitin activation”Rauert, Wilko

“Structural analysis of eukaryotic DNA repair mechanism”Rohleder, Florian

“New insights into nucleotide excision repair from recog-nition to incision”Roth, Heide-Marie

“Profiling the gephyrin-neuroligin2 interaction”Sander, Bodo

“Functional analysis of p53 via fluorescence microscopy”Sauer, Markus

“Transcriptional regulation by the p53 family of tumor suppressors”Schlereth, Katharina

“Evaluation of activating antibodies by means of fluores-cence resonance energy transfer microscopy”Schlipp, Angela

“Characterisation of posttranslational modifications of guanylyl cyclase A (GC-A)”Schröter, Juliane

“Generation and characterization of mice deficient in gi24”Stegner, David

“Analysis of gene functions related to mental retardation and synapse architecture”Tian, Rui

“Store-operated calcium entry in platelets”Varga-Szabó, Dávid

“Store-operated calcium entry in immune cell activation and signalling”Vögtle, Timo


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Concluded in 2008

“A role for Caspase-1 in heart failure”Merkle, Sabine

“Studies on formation and stabilization of pathological thrombi in vivo”Pozgajová, Miroslava

“Visualization and characterization of the TGF-β activa-tion response with disease causing mutations via fluo-rescence and FRET based Biosensors”Wang, Kun

“Quantification and characterization of multi-protein entities using the example of the human SMN complex”Wiesner, Julia

“Effects of pneumolysin on dendritic spine function and synapse formation via small GTPases”Wippel, Carolin

“Structural and functional characterization of Nucleotid-Excision-Repair proteins”Wolski, Stefanie

“Tracking and Signaling of Interleukin Receptors”Zelman-Femiak, Monika

“Role of the susceptibility gene Ptpn22 in the selec-tion and function of T-cell in the NOD model of type 1 diabetes”Zheng, Peilin


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Publications


Group Stefan Engelhardt

Cook, A.R., Bardswell, S.C., Pretheshan, S., Dighe, K., Kanaganayagam, G.S., Jabr, R.I., Merkle, S., Marber, M.S., Engelhardt, S., and Avkiran, M. (2008) Paradoxical resistance to myocardial ischemia and age-related cardiomyopathy in NHE1 transgenic mice: A role for ER stress? J Mol Cell Cardiol, in press.

Lewin, G., Matus, M., Basu, A., Frebel., K., Rohsbach, S.P., Safronenko, A., Seidl, M.D., Stümpel, F., Buchwalow, I., König, S., Engelhardt, S., Lohse, M.J., Schmitz, W., and Müller, F.U. (2009) Critical role of transcription factor CREM in beta1-adre-noceptor-mediated cardiac dysfunction. Circulation, 119, 79-88.

Thum, T., Gross, C., Fiedler, J., Fischer, T., Kissler, S., Bussen, M., Galuppo, P., Just, S., Rottbauer, W., Frantz, S., Casttoldi, P., Soutschek, J., Koteliansky, V., Rosenwald, A., Basson, M.A., Licht, J.D., Pena, J.T.R., Muckenthaler, M., Tuschl, T., Martin, G.R., Bauersachs, J., and Engelhardt., S. (2008) miR-21 derepresses fibroblast MAPkinase signalling and contributes to myocardial disease. Nature, 456, 980-4.

Vilardaga, J.-P., Bünemann, M., Feinstein, T.M., Lambert, N., Nikolaev, V.O., Engel-hardt, S, Lohse, M.J., and Hoffmann, C. (2008) GPCR and G proteins: drug efficacy and activation in live cells. Mol Endo, in press.

Group Heike Hermanns

Hintzen, C., Evers, C., Lippok, B.E., Volkmer, R., Heinrich, P.C., Radtke, S.*, and Hermanns, H.M.* (2008) Box 2 region of the oncostatin M receptor determines specificity for recruitment of Janus kinases and STAT5 activation. J Biol Chem, 283, 19465-19477.(* authors contributed equally)

Hintzen, C., Haan, C., Tuckermann, J.P., Heinrich, P.C., and Hermanns, H.M. (2008) Oncostatin M-induced chemokine expres-sion promotes monocyte and T-lymphocyte migration and is in part negatively regu-lated through STAT5-induced CIS expres-sion. J Immunol, 181, 7341-9.

Group Asparouh Iliev

Iliev, A., Djannatian, J.R., Opazo, F., Gerber, R., Nau, R., Mitchell, T.J., and Wouters, F.S. (2008) Rapid microtubule bundling and stabilization by the Strepto-coccus pneumoniae neurotoxin pneumoly-sin in a cholesterol-dependent, non-lytic and Src-kinase dependent manner inhibits intracellular trafficking. Mol Microbiol, in press.

Group Stephan Kissler

Thum, T., Gross, C., Fiedler, J., Fischer, T., Kissler, S., Bussen, M., Galuppo, P., Just, S., Rottbauer, W., Frantz, S., Casttoldi, P., Soutschek, J., Koteliansky, V., Rosenwald, A., Basson, M.A., Licht, J.D., Pena, J.T.R., Muckenthaler, M., Tuschl, T., Martin, G.R., Bauersachs, J., and Engelhardt., S. (2008) miR-21 derepresses fibroblast MAPkinase signalling and contributes to myocardial disease. Nature, 456, 980-4.

80

Core Center

Group Gregory Harms

Dogan, T., Harms, G.S., Hekman, M., Karre-man, C., Oberoi, T.K., Alnemri, E.S., Rapp, U.R., and Rajalingam, K. (2008) X-linked and cellular IAPs modulate the stability of C-RAF kinase and cell motility. Nature Cell Biol, in press.

Sauer, M., Bretz, A.C., Beinoraviciute- Kellner, R., Beitzinger, M., Burek, C., Rosenwald, A., Harms, G.S., and Stiewe, T. (2008) C-terminal diversity within the p53 family accounts for differences in DNA binding and transcriptional activity. Nucleic Acids Res, 36, 1900-12.


Steinmeyer, R., and Harms, G.S. (2008) Fluorescence resonance energy transfer and anisotropy reveals both hetero- and homo-energy transfer in the pleckstrin homology-domain and the parathyroid hormone-receptor. Microsc Res Tech, in press.

Group Hermann Schindelin

Daniels, J.N., Wuebbens, M.M., Rajagopal-an, K.V., and Schindelin, H. (2008) Crystal structure of a molybdopterin synthase-precursor Z complex: insight into its sulfur transfer mechanism and its role in molyb-denum cofactor deficiency. Biochemistry, 47, 615-26.

Kadurin, I., Golubovic, A., Leisle, L., Schindelin, H., and Gründer, S. (2008) Differential effects of N-glycans on surface expression suggest structural differences between the acid-sensing ion channel (ASIC) 1a and ASIC1b. Biochem J, 412, 469-75.

Lee, I., and Schindelin, H. (2008) Struc-tural insights into E1-catalyzed ubiquitin activation and transfer to conjugating enzymes. Cell, 134, 268-78.

Li, G., Zhao, G., Schindelin, H., and Len-narz, W.J. (2008) Tyrosine phosphorylation of ATPase p97 regulates its activity during ERAD. Biochem Biophys Res Commun, 375, 247-51.

Group Caroline Kisker

Dietzel, U., Kuper, J., Doebbler, J.A., Schulte, A., Truglio, J.J., Leimkühler, S., and Kisker, C. (2008) Mechanism of Sub-strate and Inhibitor Binding of Rhodo-bacter capsulatus Xanthine Dehydrogenase. J Biol Chem, in press.

Lu, H., England, K., amEnde, C., Truglio, J.J., Luckner, S., Marlenee, N., Knudson, S.E., Knudson, D.L., Bowen, R.A. Kisker, C., Slayden, R.A., and Tonge, P.J (2008) Slow-Onset Inhibition of the FabI Enoyl Reductase from Francisella Tularensis:Residence Time and In vivo Activity. ACS Chem Biol, in press.

Li, H., Chavan, M., Schindelin, H., and Lennarz, W.J. (2008) Structure of the oligosaccharyl transferase complex at 12 Å resolution. Structure, 16, 432-40.

Paukert, M., Chen, X., Polleichtner, G., Schindelin, H., and Gründer, S. (2008) Candidate amino acids involved in H+ gat-ing of acid-sensing ion channel 1a. J Biol Chem, 283, 572-81.

Schmitz, J., Chowdhury, M.M., Hänzel-mann, P., Nimtz, M., Lee, E.Y., Schinde-lin, H., and Leimkühler, S. (2008) The sulfurtransferase activity of Uba4 presents a link between ubiquitin-like protein con-jugation and activation of sulfur carrier proteins. Biochemistry, 47, 6479-89.

Tian, G., Kober, F.X., Lewandrowski, U., Sickmann, A., Lennarz, W.J., and Schin-delin, H. (2008) The catalytic activity of protein disulfide isomerase requires a conformationally flexible molecule. J Biol Chem, 283, 33630-40.

Zhao, G., Li, G., Zhou, X., Matsuo, I., Ito, Y., Suzuki, T., Lennarz, W.J., and Schinde-lin, H. (2008) Structural and mutational studies on the importance of oligosac-charide binding for the activity of yeast PNGase. Glycobiology, in press.

Respicio, L., Nair, P.A., Huang, Q., Anil, B., Tracz, S., Truglio, J.J., Kisker, C., Raleigh, D.P., Ojima, I., Knudson, D.L., Tonge, P.J., and Slayden, R.A. (2008) Characterizing septum inhibition in Myco-bacterium tuberculosis for novel drug dis-covery. Tuberculosis (Edinb), 88, 420-9.

Wolski, S.C., Kuper, J., Hänzelmann, P., Truglio, J.J., Croteau, D.L., Van Houten, B., and Kisker, C. (2008) Crystal structure of the FeS cluster-containing nucleotide excision repair helicase XPD. PLoS Biol, 6, e149.

81

Kroiss, M., Schultz, J., Wiesner, J., Chari, A., Sickmann, A., and Fischer, U. (2008) Evolution of an RNP assembly system: a minimal SMN complex facilitates formation of UsnRNPs in Drosophila melanogaster. Proc Natl Acad Sci USA, 105, 10045-50.

Lewandrowski, U., Sickmann, A., Cesaro, L., Brunati, A.M., Toninello, A., and Salvi, M. (2008) Identification of new tyrosine phosphorylated proteins in rat brain mito-chondria. FEBS Lett, 582, 1104-10.

Lewandrowski, U., Lohrig, K., Zahedi, R.P., Wolters, D., and Sickman, A. (2008) Glycosylation site analysis of human platelets by electrostatic repulsion hydro-philic interaction chromatography. Clin Proteomics, 4, 25-36.

Mack, C., Sickmann, A., Lembo, D., and Brune, W. (2008) Inhibition of proinflam-matory and innate immune signaling pathways by a cytomegalovirus RIP1-interacting protein. Proc Natl Acad Sci USA, 105, 3094-9.

Markert, A., Grimm, M., Martinez, J., Wiesner, J., Meyerhans, A., Meyuhas, O., Sickmann, A., and Fischer, U. (2008) The La-related protein LARP7 is a component of the 7SK ribonucleoprotein and affects transcription of cellular and viral poly-merase II genes. EMBO Rep, 9, 569-75.

Meisinger, C., Sickmann, A., and Pfanner, N. (2008) The mitochondrial proteome: from inventory to function. Cell, 134, 22-4.

Peisker, K., Braun, D., Wolfle, T., Hentschel, J., Funfschilling, U., Fischer, G., Sickmann, A., and Rospert, S. (2008) Ribosome-associated complex binds to ribosomes in close proximity of Rpl31 at the exit of the polypeptide tunnel in yeast. Molecular biology of the cell, 19, 5279-5288.

Group Albert Sickmann

Baljuls, A., Schmitz, W., Mueller, T.D, Zahedi, R.P., Sickmann, A., Hekman, M., and Rapp, U.R. (2008) Positive regulation of A-RAF by phosphorylation of isoform-specific hinge segment and identification of novel phosphorylation sites. J Biol Chem, 283, 27239-54.

Benz, P.M., Blume, C., Moebius, J., Oschatz, C., Schuh, K., Sickmann, A., Walter, U., Feller, S.M., and Renne, T. (2008) Cytoskeleton assembly at endo-thelial cell-cell contacts is regulated by αII-spectrin-VASP complexes. J Cell Biol, 180, 205-19.

Benz, P.M., Feller, S.M., Sickmann, A., Walter, U., and Renne, T. (2008) Pros-taglandin-induced VASP phosphoryla-tion controls αII-spectrin breakdown in apoptotic cells. Int Immunopharmacol, 8, 319-24.

Bolender, N., Sickmann, A., Wagner, R., Meisinger, C., and Pfanner, N. (2008) Mul-tiple pathways for sorting mitochondrial precursor proteins. EMBO Rep, 9, 42-9.

Chari, A., Golas, M.M., Klingenhäger, M., Neuenkirchen, N., Sander, B., Englbrecht, C., Sickmann, A., Stark, H., and Fischer, U. (2008) An assembly chaperone collabo-rates with the SMN-complex to generate spliceosomal snRNPs. Cell, 135, 497-509.

Dittrich, M., Birschmann, I., Mietner, S., Sickmann, A., Walter, U., and Dandekar, T. (2008) Platelet protein interactions: map, signaling components, and phosphoryla-tion groundstate. Arterioscler Thromb Vasc Biol, 28, 1326-31.

Hofbauer, A., Ebel, T., Waltenspiel, B., Oswald, P., Chen, Y.C., Halder, P., Biskup, S., Lewandrowski, U., Winkler, C., Sick-mann, A., et al. (2009) The Wuerzburg Hy-bridoma Library against Drosophila Brain. Journal of neurogenetics, 1-14.

Schindler, J., Lewandrowski, U., Sickmann, A., and Friauf, E. (2008) Aqueous polymer two-phase systems for the proteomic analysis of plasma membranes from minute brain samples. J Proteome Res 7, 432-42.

Stumpf, T., Zhang, Q., Hirnet, D., Lewandrowski, U., Sickmann, A., Wissen-bach, U., Dörr, J., Lohr, C., Deitmer, J.W., and Fecher-Trost, C. (2008) The human TRPV6 channel protein is associated with cyclophilin B in human placenta. J Biol Chem, 283, 18086-98.

Tafelmeyer, P., Laurent, C., Lenormand, P., Rousselle, J.C., Marsollier, L., Reysset, G., Zhang, R., Sickmann, A., Stinear, T.P., Namane, A., and Cole, S.T. (2008) Compre-hensive proteome analysis of Mycobacte-rium ulcerans and quantitative comparison of mycolactone biosynthesis. Proteomics, 8, 3124-38.

Tian, G., Kober, F.X., Lewandrowski, U., Sickmann, A., Lennarz, W.J., and Schin-delin, H., (2008) The catalytic activity of protein disulfide isomerase requires a conformationally flexible molecule. J Biol Chem, in press.

Wegierski, T., Lewandrowski, U., Muller, B., Sickmann, A., and Walz, G. (2008) Tyrosine phosphorylation modulates the activity of TRPV4 in response to defined stimuli. J Biol Chem, in press.

Wiesner, J., Premsler, T., and Sickmann, A. (2008) Application of electron transfer dissociation (ETD) for the analysis of post-translational modifications. Proteomics, 8, 4466-83.

Zahedi, R.P., Lewandrowski, U., Wiesner, J., Wortelkamp, S., Moebius, J., Schütz, C., Walter, U., Gambaryan, S., and Sick-mann, A. (2008) Phosphoproteome of resting human platelets. J Proteome Res, 7, 526-34.

Core Center

82

Research Professors

Group Peter Friedl

Alexander, S., Koehl, G.E., Hirschberg, M., Geissler, E.K., and Friedl, P. (2008) Dynamic imaging of cancer growth and invasion: a modified skin-fold chamber model. Histochem Cell Biol, in press.

Andresen, V., Alexander, S., Heupel, W.M., Hirschberg, M., and Friedl, P. (2008) Infrared multiphoton microscopy: subcel-lular-resolved deep tissue imaging. Curr Opin Biotechnol, in press.

Belletti, B., Nicoloso, M.S., Schiappacassi, M., Berton, S., Lovat, F., Wolf, K., Canzon-ieri, V., D‘Andrea, S., Zucchetto, A., Friedl, P., Colombatti, A., and Baldassarre, G. (2008) Stathmin activity influences sar-coma cell shape, motility, and metastatic potential. Mol Biol Cell, 19, 2003-13.

Friedl, P., and Weigelin, B. (2008) Inter-stitial leukocyte migration and immune function. Nature Immunol, 9, 960-9.

Friedl, P., and Wolf, K. (2008) Proteolytic interstitial cell migration: a five-step process. Cancer Metast Rev, in press.

Friedl, P., and Wolf, K. (2008) Tube travel: protease functions in individual and col-lective cancer invasion. Cancer Res, 68, 7247-7249.

Hartmann, A., Quist, J., Hamm, H., Broecker, E.B., and Friedl, P. (2008) Transplantation of autologous keratinocyte suspension in fibrin matrix to chronic venous leg ulcers: improved long-term healing after removal of the fibrin carrier. Dermatol Surg, 34, 922-9.

Muessel, M.J., Scott, K.S., Friedl, P., Bradding, P., and Wardlaw, A.J. (2008) CCL11 and GM-CSF differentially use the Rho GTPase pathway to regulate motility of human eosinophils in a three-dimen-sional microenvironment. J Immunol,180, 8354-60.

Wolf, K., and Friedl, P. (2008) Mapping proteolytic cancer cell-extracellular matrix interfaces. Clin Exp Metastasis. 10.1007/s10585-008-9190-2 (AOP, published on July 4, 2008).

Group Bernhard Nieswandt

Braun, A., Gessner, J.E., Varga-Szabo, D., Syed, S.N., Konrad, S., Stegner, D., Vögtle, T., Schmidt, R.E., and Nieswandt, B. (2008) STIM1 is essential for Fcγ receptor activation and autoimmune inflammation. Blood, in press.

Braun, A., Varga-Szabo, D., Kleinschnitz, C., Pleines, I., Bender, M., Austinat, M., Bosl, M., Stoll, G., and Nieswandt, B. (2008) Orai1 (CRACM1) is the platelet SOC channel and essential for pathological thrombus formation. Blood, in press.

Elzey, B.D., Schmidt, N.W., Crist, S.A., Kre-sowik, T.P., Harty, J.T., Nieswandt, B., and Ratliff, T.L. (2008) Platelet-derived CD154 enables T-cell priming and protection against Listeria monocytogenes challenge. Blood, 111, 3684-91.

Geng, H., Zhang, H., Zhang, W., Nieswandt, B., Bray, P.F., and Leng, X. (2008) Transdermal 17-β estradiol replace-ment therapy reduces megakaryocyte GPVI expression. Thromb Res, 123, 93-9.

Kleinschnitz, C., Braeuninger, S., Pham, M., Austinat, M., Nolte, I., Renne, T., Nieswandt, B., Bendszus, M., and Stoll, G. (2008) Blocking of platelets or intrinsic coagulation pathway-driven thrombosis does not prevent cerebral infarctions induced by photothrombosis. Stroke, 39, 1262-8.

Kuijpers, M.J., Gilio, K., Reitsma, S., Nergiz-Unal, R., Prinzen, L., Heeneman, S., Lutgens, E., van Zandvoort, M.A., Nieswandt, B., Oude Egbrink, M.G., and Heemskerk, J.W. (2008) Complementary roles of platelets and coagulation in thrombus formation on plaques acutely ruptured by targeted ultrasound treat-ment: a novel intravital model. J Thromb Haemost, in press.

Moser, M., Nieswandt, B., Ussar, S., Pozga-jova, M., and Fässler, R. (2008) Kindlin-3 is essential for integrin activation and platelet aggregation. Nature Med, 14, 325-30.

Nieswandt, B. (2008) How do platelets prevent bleeding? Blood, 111, 4835.

Pleines, I., Elvers, M., Strehl, A., Pozga-jova, M., Varga-Szabo, D., May, F., Chrostek-Grashoff, A., Brakebusch, C., and Nieswandt, B. (2008) Rac1 is essential for phospholipase C-γ2 activation in platelets. Pflügers Arch, in press.

Stoll, G., Kleinschnitz, C., and Nieswandt, B. (2008) Molecular mechanisms of throm-bus formation in ischemic stroke: novel insights and targets for treatment. Blood, 112, 3555-62.

Varga-Szabo, D., Authi, K.S., Braun, A., Bender, M., Ambily, A., Hassock, S.R., Gudermann, T., Dietrich, A., and Nieswandt, B. (2008) Store-operated Ca2+ entry in platelets occurs independently of transient receptor potential (TRP) C1. Pflügers Arch, 457, 377-87.

Varga-Szabo, D., Braun, A., Kleinschnitz, C., Bender, M., Pleines, I., Pham, M., Renne, T., Stoll, G., and Nieswandt, B. (2008) The calcium sensor STIM1 is an essential mediator of arterial thrombosis and ischemic brain infarction. J Exp Med, 205, 1583-91.

Varga-Szabo, D., Pleines, I., and Nieswandt, B. (2008) Cell adhesion mechanisms in platelets. Arterioscler Thromb Vasc Biol, 28, 403-12.

Wong, C., Liu, Y., Yip, J., Chand, R., Wee, J.L., Oates, L., Nieswandt, B., Reheman, A., Ni, H., Beauchemin, N., and Jackson, D. (2008) CEACAM1 negatively regulates platelet-collagen interactions and throm-bus growth in vitro and in vivo. Blood, in press

83

Group Michael P. Schön

Alban, S., Ludwig, R.J., Bendas, G., Schön, M.P., Oostingh, G.J., Radeke, H.H., Fritzsche, J., Pfeilschifter, J., Kaufmann, R., and Boehncke, W.H. (2008) PS3, a semisynthetic b-1,3-glucan sulfate, dimin-ishes contact hypersensitivity responses through inhibition of L- and P-selectin functions. J Invest Dermatol, in press.

Dörrie, J., Schaft, N., Müller, I., Wellner, V., Schunder, T., Hänig, J., Oostingh, G.J., Schön, M.P., Robert, C., Kämpgen, E., and Schuler, G. (2008) Introduction of functional chimeric E/L-selectin by RNA electroporation to target dendritic cells from blood to lymph nodes. Cancer Im-munol Immunother, 57, 467-77.

Geserick, P., Drewniok, C., Hupe, M., Haas, T.L., Diessenbacher, P., Sprick, M.R., Schön, M.P., Henkler, F., Gollnick, H., Walczak, H., and Leverkus, M. (2008) Suppression of cFLIP is sufficient to sensi-tize human melanoma cells to TRAIL- and CD95L-mediated apoptosis. Oncogene, 27, 3211-20.

Gesierich, A., Stoevesandt, J., Kneitz, C., Bröcker, E.B., and Schön, M.P. (2008) Adult-onset Still‘s disease: an uncom-mon differential diagnosis of urticaria and treatment with anakinra. J Eur Acad Dermatol Venereol, in press.

Giegold, O., Ludwig, R.J., Hardt, K., Will, J., Schön, M.P., Oostingh, G.J., Pfeilschifter, J.M., Boehncke, W.H., and Radeke, H.H. (2008) Computer-aided analysis of cell interactions under dynamic flow conditions. Exp Dermatol, in press.

Goodman, R.S., Kirton, C.M., Oostingh, G.J., Schön, M.P., Clark, M.R., Bradley, J.A., and Taylor, C.J. (2008) PECAM-1 polymorphism affects monocyte adhesion to endothelial cells. Transplantation, 85, 471-7.

Haenssle, H.A., Kaune, K.M., Buhl, T., Thoms, K.M., Padeken, M., Emmert, S., and Schön, M.P. (2008) Melanoma arising in segmental nevus spilus: Early detection with sequential digital dermoscopy.J Am Acad Dermatol, in press.

Kaune, K., Haas, E., Emmert, S., Schön, M.P., and Zutt, M. (2008) Successful treatment of severe keratosis pilaris rubra with a 595 nm pulsed dye laser. Dermatol Surg, in press.

Li, Y.Y., Zollner, T.M., and Schön, M.P. (2008) Targeting leukocyte recruitment in the treatment of psoriasis. Clin Dermatol, 26, 527-38.

Mössner, R., Schön, M.P., and Reich, K. (2008) Tumor necrosis factor antagonists in the therapy of psoriasis. Clin Dermatol, 26, 486-502.

Papavassilis, C., de Souza, E.M., Bröcker, E.B., and Schön, M.P. (2008) Rapidly growing nodule on the ear lobe. J Dtsch Dermatol Ges, 6, 63-64.

Schlickum, S., Sennefelder, H., Friedrich, M., Harms, G., Lohse, M.J., Kilshaw,P., and Schön, M.P. (2008) Integrin αE(CD103)β7 influences cellular shape and motility in a ligand-dependent fashion. Blood, 112, 619-625.

Schön, M.P. (2008) Animal models of pso-riasis: a critical appraisal. Exp Dermatol, 17, 703-12.

Schön, M.P. (2008) Efalizumab in the treatment of psoriasis: mode of action, clinical indications, efficacy, and safety. Clin Dermatol, 26, 509-14.

Schön, M.P. (2008) Treatment of psoriasis: a journey from empiricism to evidence. Clin Dermatol, 26, 417-418.

Schön, M.P., and Schön, M. (2008) TLR7 and TLR8 as targets in cancer therapy. Oncogene, 27, 190-9.

Schön, M.P., and Schön, M. (2009) To die or not to die, that´s the question - and the answer may depend on netrin-1. J Natl Cancer Inst, in press.

Schön, M., Wienrich, B.G., Kneitz, S., Sennefelder, H., Amschler, K., Vöhringer, V., Weber, O., Stiewe, T., Ziegelbauer, K., and Schön, M.P. (2008) KINK-1, a novel small-molecule inhibitor of IKKβ, and the susceptibility of melanoma cells to antitu-moral treatment. J Natl Cancer Inst, 100, 862-75.

Wienrich, B.G., Schuppe, H.C., and Schön, M.P. (2008) Expression and putative func-tion of lymphocyte endothelial epithelial-cell adhesion molecule in human testis. Andrologia, 40, 252-8.

Research Professors

84

RVZ Network

Group Utz Fischer

Chari, A., Golas, M.M., Klingenhäger, M., Neuenkirchen, N., Sander, B., Englbrecht, C., Sickmann, A., Stark, H., and Fischer, U. (2008) An assembly chaperone collaborates with the SMN-complex to generate spliceo-somal SnRNPs. Cell, 135, 497-509.

Kroiss, M., Schultz, J., Wiesner, J., Chari, A., Sickmann, A., and Fischer, U. (2008) Evolution of an RNP assembly system: a minimal SMN complex facilitates formation of UsnRNPs in Drosophila melanogaster. Proc Natl Acad Sci U S A, 105, 10045-10050.

Markert, A., Grimm, M., Martinez, J., Wi-esner, J., Meyerhans, A., Meyuhas, O., Sick-mann, A., and Fischer, U. (2008) The La-related protein LARP7 is a component of the 7SK ribonucleoprotein and affects tran-scription of cellular and viral polymerase II genes. EMBO Rep, 9, 569-575.

Group Manfred Gessler

Doetzlhofer, A., Basch, M.L., Ohyama, T., Gessler, M., Groves, A.K., and Segil, N. (2008) Hey2 regulation by FGF provides a Notch-independent mechanism for maintaining pillar cell fate in the organ of Corti. Developmental Cell, in press.

Hu, X., Chung, A.Y., Wu, I., Foldi, J., Chen, J., Ji, J.D., Tateya, T., Kang, Y.J., Han, J., Gessler, M., Kageyama, R., and Ivashkiv, L.B. (2008) Integrated Regula-tion of Toll-like Receptor Responses by Notch and Interferon-gamma Pathways. Immunity, 29, 691-703.

Group Thomas Hünig

Azuma, H., Isaka, Y., Li, X., Hunig, T., Sakamoto, T., Nohmi, H., Takabatake, Y., Mizui, M., Kitazawa, Y., Ichimaru, N., et al. (2008) Superagonistic CD28 antibody induces donor-specific tolerance in rat renal allografts. Am J Transplant,8, 2004-2014.

Beyersdorf, N., Ding, X., Blank, G., Den-nehy, K.M., Kerkau, T., and Hunig, T. (2008) Protection from graft-versus-host disease with a novel B7 binding site-spe-cific mouse anti-mouse CD28 monoclonal antibody. Blood, 112, 4328-4336.

Gogishvili, T., Elias, F., Emery, J.L., McPherson, K., Okkenhaug, K., Hunig, T., and Dennehy, K.M. (2008) Proliferative signals mediated by CD28 superagonists require the exchange factor Vav1 but not phosphoinositide 3-kinase in primary peripheral T-cells. Eur J Immunol, 38, 2528-2533.

Guilliams, M., Bosschaerts, T., Herin, M., Hunig, T., Loi, P., Flamand, V., De Baetselier, P., and Beschin, A. (2008) Experimental expansion of the regulatory T-cell population increases resistance to African trypanosomiasis. J Infect Dis, 198, 781-791.

Kitazawa, Y., Fujino, M., Sakai, T., Azuma, H., Kimura, H., Isaka, Y., Takahara, S., Hunig, T., Abe, R., and Li, X.K. (2008) Foxp3-expressing regulatory T-cells ex-panded with CD28 superagonist antibody can prevent rat cardiac allograft rejection. J Heart Lung Transplant, 27, 362-371.

Na, S.Y., Cao, Y., Toben, C., Nitschke, L., Stadelmann, C., Gold, R., Schimpl, A., and Hunig, T. (2008b) Naive CD8 T-cells initiate spontaneous autoimmunity to a sequestered model antigen of the central nervous system. Brain, 131, 2353-2365.

85

Group Walter Sebald

Klages, J., Kotzsch, A., Coles, M., Sebald, W., Nickel, J., Muller, T., and Kessler, H. (2008) The solution structure of BMPR-IA reveals a local disorder-to-order transition upon BMP-2 binding. Biochemistry, 47, 11930-11939.

Kotzsch, A., Nickel, J., Seher, A., Hei-necke, K., van Geersdaele, L., Herrmann, T., Sebald, W., and Mueller, T.D. (2008) Structure analysis of bone morphoge-netic protein-2 type I receptor complexes reveals a mechanism of receptor inactiva-tion in juvenile polyposis syndrome. J Biol Chem, 283, 876-87.

Saremba, S., Nickel, J., Seher, A., Kotzsch, A., Sebald, W., and Mueller, T.D. (2008) Type I receptor binding of bone mor-phogenetic protein 6 is dependent on N-glycosylation of the ligand. Febs J, 275, 172-183.

Zhang, J.L., Qiu, L.Y., Kotzsch, A., Wei-dauer, S., Patterson, L., Hammerschmidt, M., Sebald, W., and Mueller, T.D. (2008) Crystal structure analysis reveals how the Chordin family member crossveinless 2 blocks BMP-2 receptor binding. Dev Cell, 14, 739-50.

Group Thomas D. Müller

Klages, J., Kotzsch, A., Coles, M., Sebald, W., Nickel, J., Mueller, T., and Kessler, H. (2008) The solution structure of BMPR-IA reveals a local disorder-to-order transition upon BMP-2 binding. Biochemistry, 47, 11930-11939.

Kotzsch, A., Nickel, J., Seher, A., Heinecke, K., van Geersdaele, L., Herrmann, T., Sebald, W., and Mueller, T.D. (2008) Structure analy-sis of bone morphogenetic protein-2 type I receptor complexes reveals a mechanism of receptor inactivation in juvenile polyposis syndrome. J Biol Chem, 283, 5876-87.


Zhang, J.L., Qiu, L.Y., Kotzsch, A., Weidauer, S., Patterson, L., Hammerschmidt, M., Sebald, W., and Mueller, T.D. (2008) Crystal structure analysis reveals how the Chordin family member crossveinless 2 blocks BMP-2 receptor binding. Dev Cell, 14, 739-50.

RVZ Network

Group Manfred Schartl

Herpin, A., Fischer, P., Liedtke, D., Klüver, N., Neuner, C., Raz, E., and Schartl, M. (2008) Active Sdf1a and b-induced mobil-ity guides Medaka PGC migration. Dev Biol, 320, 319-27.

Klüver, N., Herpin, A., Braasch, I., Drießle, J., and Schartl, M. (2009) Regulatory back-up circuit of medaka wt1 co-ortho-logs ensures PGC maintenance. Dev Biol, 325, 179-88.

Leikam, C., Hufnagel, A., Schartl, M., and Meierjohann, S. (2008) Oncogene activation in melanocytes links reactive oxygen to multinucleated phenotype and senescence. Oncogene, in press.

Schartl, M. (2008) Evolution of Xmrk: an oncogene, but also a speciation gene? Bioessays, 30, 822-32.

Schartl, M., and Herpin, A. (2008) Regulatory putsches create new ways of determining sexual development. EMBO Rep, 9, 966-8.

86

Bio-Imaging Center

Group Martin Lohse/ Stefan Schulz

Herget, S., Lohse, M.J., and Nikolaev, V.O. (2008) Real-time monitoring of phospho-diesterase inhibition in intact cells. Cell Signal, 20, 1423-31.

Hoffmann, C., Ziegler, N., Reiner, S., Kra-sel, C., and Lohse, M.J. (2008) Agonist-selective, receptor-specific interaction of human P2Y receptors with β-arrestin-1 and -2. J Biol Chem, 283, 30933-41.

Hoffmann, C., Zürn, A., Buenemann, M., and Lohse, M.J. (2008) Conformational changes in G-protein-coupled recep-tors-the quest for functionally selective conformations is open. Br J Pharmacol, 153 S1, S358-66.

Iancu, R.V., Ramamurthy, G., Warrier, S., Nikolaev, V.O., Lohse, M.J., Jones, S.W., and Harvey, R.D. (2008) Cytoplasmic cAMP concentrations in intact cardiac myocytes. Am J Physiol Cell Physiol, 295, C414-22.

Jacobs, S., and Schulz, S. (2008) Intracel-lular trafficking of somatostatin receptors. Mol Cell Endocrinol, 286, 58-62.

Jahns, R., Boivin, V., Schwarzbach, V., Ertl, G., and Lohse, M.J. (2008) Pathologi-cal autoantibodies in cardiomyopathy. Autoimmunity, 41, 454-61.

Krasel, C., Zabel, U., Lorenz, K., Reiner, S., Al-Sabah, S., and Lohse, M.J. (2008) Dual role of the β2-adrenergic receptor C-terminus for the binding of β-arrestin and receptor internalization. J Biol Chem, 283, 31840-8.

Lesche, S., Lehmann, D., Nagel, F., Schmid, H.A., and Schulz, S. (2008) Dif-ferential effects of octreotide and pasire-otide on somatostatin receptor internal-ization and trafficking in vitro. J Clin Endocrinol Metab, in press.

Lewin, G., Matus, M., Basu, A., Frebel, K., Rohsbach, S.P., Safronenko, A., Seidl, M., Stümpel, F., Buchwalow, I., König, S., Engelhardt, S., Lohse, M.J., Schmitz, W., and Müller, F.U. (2008) Critical role of transcription factor CREM in β1-adrenocep-tor-mediated cardiac dysfunction. Circulation, in press.

Lohse, M.J., and Klenk, C. (2008) Block-ing them all: β-arrestins inhibit cellular signaling. Mol Cell, 31, 619-21.

Lohse, M.J., Hein, P., Hoffmann, C., Niko-laev, V.O., Vilardaga, J.P., and Buenemann, M. (2008) Kinetics of G-protein-coupled receptor signals in intact cells. Br J Phar-macol, 153 S1, S125-32.

Lohse, M.J., Nikolaev, V.O., Hein, P., Hoffmann, C., Vilardaga, J.P., and Buen-emann, M. (2008) Optical techniques to analyze real-time activation and signaling of G-protein-coupled receptors. Trends Pharmacol Sci, 29, 159-65.

Lorenz, K., Schmitt, J.P., Schmitteckert, E.M., and Lohse, M.J. (2008) A new type of ERK1/2-autophosphorylation causes cardiac hypertrophy. Nature Med, in press.


Schmitt, J.P., Ahmad, F., Lorenz, K., Hein, L., Schulz, S., Asahi, M., MacLennan, D.H., Seidman, C.E., Seidman, J.G., and Lohse, M.J. (2008) Alterations of phospholamban function can exhibit cardiotoxic effects independent of excessive SERCA2a inhibi-tion. Circulation, in press.

Shafer, O.T., Kim, D.J., Dunbar-Yaffe, R., Nikolaev, V.O., Lohse, M.J., and Taghert, P.H. (2008) Widespread receptivity to neuropeptide PDF throughout the neuronal circadian clock network of Drosophila revealed by real-time cyclic AMP imaging. Neuron, 58, 223-37.

Vilardaga, J.P., Nikolaev, V.O., Lorenz, K., Ferrandon, S., Zhuang, Z., and Lohse, M.J. (2008) Conformational cross-talk between α2A-adrenergic and µ-opioid receptors controls cell signaling. Nature Chemical Biology, 4, 126-31

Group Stephan Sigrist

Ataman, B., Ashley, J., Gorczyca, M., Ramachandran, P., Fouquet, W., Sigrist, S.J., and Budnik, V. (2008) Rapid activity-dependent modifications in synaptic struc-ture and function require bidirectional Wnt signaling. Neuron, 57, 705-18.

Bogdanik, L., Framery, B., Frölich, A., Franco, B., Mornet, D., Bockaert, J., Sigrist, S.J., Grau, Y., and Parmentier, M.L. (2008) Muscle dystroglycan organizes the postsynapse and regulates presynaptic neurotransmitter release at the Drosophila neuromuscular junction. PLoS ONE, 3, e2084.

Schmid, A., and Sigrist, S.J. (2008) Analy-sis of neuromuscular junctions: histology and in vivo imaging. Methods Mol Biol, 420, 239-51.

Schmid, A., Hallermann, S., Kittel, R.J., Khorramshahi, O., Frölich, A.M., Quentin, C., Rasse, T.M., Mertel, S., Heckmann, M., and Sigrist, S.J. (2008) Activity-depen-dent site-specific changes of glutamate receptor composition in vivo. Nature Neurosci, 11, 659-66.

Imp

rin

tThe Annual Report 2008 is a magazine providing information about the activities in Research, Teaching and Public Relations at the Rudolf Virchow Center/DFG Research Center for Experimental Biomedicine of the University of Würzburg.

Editor:Rudolf Virchow Center/DFG Research Center for Experimental Biomedicine of the University of Würzburg

Editor in chief:Sonja Jülich

Editorial:Avril Arthur-Göttig

Design, Layout & Prepress:Sascha KregerE-mail: [email protected]://www.sk-grafik.com

Print:Laub GmbH & Cohttp://www.laub.de

Notice:Neither the Rudolf Virchow Center, nor any person acting on its behalf may be held responsible for the use to which information contained in this publication may be put, or for any errors which, despite careful preparation and checking may appear.

©RVZ, 2009

Non-commercial reproduction authorized, subject to acknowledgement of source.

Circulation: 1500

Editor`s office:Rudolf Virchow CenterDFG Research Center for Experimental Biomedicine of the University of WürzburgPublic Science CenterVersbacher Str. 997078 WürzburgPhone: +49(0)931 201 487 14Fax: +49(0)931 201 487 02E-mail: [email protected]://www.rudolf-virchow-zentrum.de

Images:(from top) Science: Antje Gohla/Hermann Schindelin/Peter Friedl/Martin Lohse (U1), Archiv Pathologie Universität Würzburg (U4), Sascha Kreger (p.4), Public Science Center (PSC)/PSC (p.5), Ilja C. Hendel für Wissenschaft im Dialog (p.6), PSC (p.7) Stefan Engelhardt/Hermann Schindelin (p.8), Caroline Kisker/ Peter Friedl (p.9), Bernhard Nieswandt (p.10), PSC (p.11), Universitätsbauamt Würzburg/Beatrice Döge/PSC (p.12-13), Gerhard Antlitz/Horst Pfrang (p.14), Sascha Kreger (p.15) (from top) Stefan Engelhardt/Gregory Harms/Manfred Gessler/Stephan Sigrist (p.16-17), Stefan Engelhardt (p.18-19), Heike Hermanns (p.20-21), Asparouh Iliev (22-23), Stephan Kissler (p.24-25), Antje Gohla (p.26), Alma Zernecke (p.27), Gregory Harms (p.28-29), Caroline Kisker (p.30-31), Herrmann Schindelin (p.32-33), Albert Sickmann (p.34-35), Peter Friedl (p.36-37), Bernhard Nieswandt (p.38-39), Michael P. Schön (p.40-41), Utz Fischer (p.42-43), Manfred Gessler (p.44-45), Thomas Hünig (p.46-47), Thomas D. Müller (p.48-49), Manfred Schartl (p.50-51), Walter Sebald (p.52-53), Martin Eilers (p.54), Roland Jahns (p.55), Martin Lohse (p.56-57), Stephan Sigrist (p.58-59), Christiane Weber (p.60), Sascha Kreger (p.63), Beatrice Döge (p.64), Christiane Weber/Sonja Jülich (p.66-67)

Front Image:Drawing of the new RVZ building (Architecture: Gerber Architekten) (picture modified by Sascha Kreger); Scientific pictures of (from top) a cell undergoing cytokinesis (Antje Gohla), a crystal structure of the Dlc-gephyrin interaction (Hermann Schindelin), of migrating tumor cells (Peter Friedl), and imaging of a cAMP-signal to local receptor stimulation (Martin Lohse).

Back:Excerpt from the section book of Rudolf Virchow at the Institute of Pathology, University of Würzburg.

annual report - rudolf virchow center · welcome to the 2008 annual report of the rudolf virchow...

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