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MFPL - 2016 RESEARCH GROUPS

Max F. Perutz Laboratories Research Groups 2016 Research Groups Publications 2016 Publishing Details

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MFPL - 2016 RESEARCH GROUPS

Research Groups Research at the MFPL is curiosity-driven and spans the field of Molecular and Cell Biology. Most groups investigate basic research questions, but a significant number are also active in more applied fields of biology. In 2016, almost 500 people from 40 nations worked at the MFPL. Detailed information about all MFPL research groups, their research focus, list of publications and team can be found on the MFPL website: www.mfpl.ac.at/groups.html

Gustav Ammerer Manuela Baccarini Andreas Bachmair Andrea Barta Dieter Blaas Udo Bläsi Christa Bücker Christopher Campbell Alexander Dammermann Thomas Decker Kristina Djinović-Carugo Gang Dong Silke Dorner Roland Foisner Peter Fuchs Boris Görke Angela Hancock Andreas Hartig Marcela Hermann Joachim Hermisson Reinhold Hofbauer N. Erwin Ivessa Michael Jantsch Verena Jantsch Franz Klein Alwin Köhler Robert Konrat Pavel Kovarik Heinrich Kowalski

Signal transduction and transcriptional regulation in yeast Deciphering the MAPK pathway in vivo Protein modifiers in plants and retrotransposon biology Post-transcriptional regulation of plant gene expression Early interactions of viruses with host cells Post-transcriptional regulation in Bacteria and Archaea Transcriptional regulation during early embryonic development Mechanisms that ensure chromosome segregation fidelity in mitosis Centriole assembly and function Host responses and innate immunity to microbial infection Structural biology of the cytoskeleton Structural studies of ciliogenesis The regulation of gene expression by small ncRNAs Lamins in nuclear organization and human disease Stress response in simple epithelia Signal transduction and post-transcriptional regulation in model bacteria Molecular basis of adaptive evolution Origin and biogenesis of peroxisomes LDL-R gene family, apolipoproteins and lipid transfer Mathematics and BioSciences Group (MaBS) Consequences of carnitine deficiency and CSF-1 inhibition Protein biogenesis and degradation from the ER Mechanisms and consequences of RNA-editing Meiosis in Caenorhabditis elegans Chromosome structure and meiotic recombination Nuclear Pores - Regulators of chromatin and membrane dynamics Computational biology and biomolecular NMR spectroscopy Signalling and gene expression in inflammation Molecular and structural biology of picornaviruses

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MFPL - 2016 RESEARCH GROUPS

Claudine Kraft Karl Kuchler Martin Leeb Thomas Leonard Josef Loidl Sascha Martens Javier Martinez Isabella Moll Ernst Müllner & Ulrich Salzer Johannes Nimpf Egon Ogris Friedrich Propst Florian Raible Johann Rotheneder Matthias Schaefer Peter Schlögelhofer Renée Schroeder Christian Seiser Tim Skern Dea Slade Kristin Tessmar-Raible Gijs Versteeg Arndt von Haeseler Graham Warren Georg Weitzer Gerhard Wiche Angela Witte Ivan Yudushkin Bojan Zagrovic

Regulation and signalling in autophagy Host-pathogen interactions & mechanisms of drug resistance & fungal pathogenesis Molecular control of cell fate decisions Structural biology of lipid-activated signal transduction Meiotic chromosome pairing and recombination Molecular mechanisms of autophagy Molecular mechanisms, biology and diseases linked to mammalian tRNA splicing Bacterial stress response and ribosome heterogeneity Erythrocyte (patho)physiology and storage in transfusion units ApoER2 and VLDL receptor Enzyme biogenesis and monoclonal antibodies The neuronal cytoskeleton in axon guidance Origin and diversification of hormone systems Cell cycle regulation and DNA damage response Methylated Cytosine in RNA: Understanding their impact on RNA stability, gene expression and innate immunity Meiotic recombination Riboregulation of transcription: how RNA shapes the transcriptome Chromatin modifiers in development and disease Interactions between viruses and cells DNA damage response Lunar periodicity and inner brain photoreceptors Ubiquitin-mediated regulation of immune signalling CIBIV - Center for Integrative Bioinformatics Vienna Biogenesis of the Golgi apparatus Stem cells of the heart Cytolinker proteins in signalling and disease φCh1, a model system for gene regulation of haloalkaliphilic Archaea facing two extremes: high pH and salt Functional imaging of signalling networks Laboratory of Molecular Biophysics

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MFPL - 2016 RESEARCH GROUPS

GUSTAV AMMERER

Signal transduction and transcriptional regulation in yeast One of our major aims is to understand the cogs and wheels of phosphorylation-modulated signal transduction machineries in the yeast S. cerevisiae.

Gustav Ammerer

TEAM Jillian Augustine Jessica Ferrari Gabriela Gerecova David Hollenstein Marion Janschitz Aleksandra Jovanovic Alexander Mechtler Wolfgang Ludwig Reiter

cell cycle, it is de-repressed at the START of S-phase and fully induced by a positive feedback mechanism during G2-phase and mitosis. In addition, we have found that genotoxic and replication stress will suppress the activation of CLB2 as well as additional genes that exhibit similar G2/M specific expression patterns. We have therefore addressed the question of how phosphorylation events affect stability and function of the important transcriptional regulators, and how their specific modifications can be correlated with changes in the underlying chromatin structure and chromatin modification patterns.

In this field, important still unresolved questions concern the dynamic interactions between different signalling factors and their effectors - e.g. in what cellular context they might happen, how they are controlled by phosphorylation events and how these interactions change during signalling events. To approach these questions, we have established and optimized a novel enzyme-based protein proximity assay. This assay is based on a mammalian histone methyl-transferase and its highly specific substrate, the N-terminal fragment of Histone 3. Apart from successfully characterizing known protein interactions in well studied signal systems such as the high osmolarity response, we have also started to use this approach for validating protein interactions that have been suggested by quantitative mass spectrometry and/or by genetic data. A second project deals with the problem of how cell cycle dependent signals coordinate the transcriptional regulation of genes. In this case, we have focused on the regulation of the main mitotic cyclin gene CLB2 in yeast. This gene is repressed in the G1-phase of the

Signal system mediating osmotic stress in yeast: cartoon of the important factors and proximity assay between the membrane sensor and adaptor Sho1, tagged with a histoneH3K9 methyl transferase and the protein kinase Ste11, tagged with an H3HA epitope. The Western assays show the methylation pattern obtained in wildtype cells and different signalling mutants before (-) and after stress (+). -Me3K9 depicts signals with methylation specific antibody, -HA provides controls for protein levels of Ste11.

Cartoon of the proposed regulatory factor recruitment and chromatin structures found at the CLB2 locus during different cell cycle stages and arrest conditions in yeast

SELECTED PUBLICATIONS Yelamanchi SK, Veis J, Anrather D, Klug H, Ammerer G. Genotoxic Stress Prevents Ndd1-Dependent Transcriptional Activation of G2/M-Specific Genes in Saccharomyces cerevisiae. Mol Cell Biol. 2014 Feb;34(4):711-24. PMID: 24324010 Reiter W, Klopf E, De Wever V, Anrather D, Petryshyn A, Roetzer A, Niederacher G, Roitinger E, Dohnal I, Görner W, Mechtler K, Brocard C, Schüller C, Ammerer G. Yeast protein phosphatase 2A-Cdc55 regulates the transcriptional response to hyperosmolarity stress by regulating Msn2 and Msn4 chromatin recruitment. Mol Cell Biol. 2013. PMID: 23275436 Zuzuarregui A1, Kupka T, Bhatt B, Dohnal I, Mudrak I, Friedmann C, Schüchner S, Frohner IE, Ammerer G, Ogris E. M-Track: detecting short-lived protein-protein interactions in vivo. Nat Methods. 2012. PMID: 22581371

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MFPL - 2016 RESEARCH GROUPS

MANUELA BACCARINI

Deciphering the MAPK pathway in vivo Constitutive activation of the RAS/RAF/MEK/ERK pathway as a result of mutations is considered a key event in the development of several human malignancies and developmental disorders. Thus, pathway components are attractive therapeutic targets.

Manuela Baccarini

TEAM

Erk signaling and pathway cross-talk mediated by RAF1 and MEK1

The Baccarini lab investigates the essential functions of RAF and MEK in the context of the whole organism, and on determining how the MAPK pathway is wired in vivo. Our work has revealed both kinase-dependent and kinase-independent roles of RAF (1) and MEK (2, 3) in different tissues, most of which are based on

Activation,

Repression

their ability to interact physically with other signal transducers and mediate pathway cross-talk. By showing that their essential in vivo functions are fundamentally different, these results have changed the way we look at RAF and MEK kinases and have opened new possibilities for molecule-targeted therapy.

SELECTED PUBLICATIONS Desideri E, Cavallo AL, Baccarini M. Alike but Different: RAF Paralogs and Their Signaling Outputs. Cell. 2015;161(5):967-970. PMID: 26000477 Raguz J, Jeric I, Niault T, Nowacka JD, Kuzet SE, Rupp C, Fischer I, Biggi S, Borsello T, Baccarini M. Epidermal RAF prevents allergic skin disease. Elife. 2016 Jul 19;5. PMID: 27431613 Zmajkovicova K, Jesenberger V, Catalanotti F, Baumgartner C, Reyes G, Baccarini M. MEK1 Is Required for PTEN Membrane Recruitment, AKT Regulation, and the Maintenance of Peripheral Tolerance. Mol Cell. 2013 Apr 11;50(1):43-55. PMID: 23453810

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Christian Baumgartner Clemens Bogner Tania Brandstรถtter Enrico Desideri Coralie Dorard Mohamed Elgendy Karel Hanak Ines Jeric Stefanie Toifl Andrea Varga Georg Vucak


MFPL - 2016 RESEARCH GROUPS

ANDREAS BACHMAIR

Protein modifiers in plants and retrotransposon biology Many proteins are modified after their synthesis. We are interested in how the small modifier proteins ubiquitin and SUMO are attached to substrate proteins in plants, and how these processes change substrate properties.

Covalent attachment of ubiquitin to substrate proteins is essential for many processes. Best known is its role in protein degradation. Several ubiquitin moieties, linked as a chain to the substrate protein, can serve as a signal for rapid proteolytic destruction of the substrate. An example for this function is the N-end rule pathway, which uses the amino-terminal residue of a protein to select substrates for degradation and is important for a plant´s response to anoxia, for germination and for senescence. We have previously characterized two components (ubiquitin ligases) of this pathway, PRT6 and PRT1. These are essential for degradation of proteins with basic, and with aromatic amino termini, respectively (for review, see Gibbs et

al., 2014, Trends Cell Biol 24, 603-611). Currently, we investigate mutants in a third component of this pathway, which is necessary to degrade proteins with hydrophobic amino termini. Ubiquitylation also has non-proteolytic functions (for review, see Tomanov et al., 2014, Frontiers Plant Sci 15, 5). One ubiquitylation complex of interest to us operates at the cell membrane. The ubiquitin chains formed by this complex have a predominantly regulatory role, and seem to influence the activity state of certain membrane proteins. This process is important for correct establishment of the plant architecture (see Figure). Recently, we discovered enzymes that extend single SUMO residues into chains. Together with previously known ubiquitin ligases that bind to SUMO chains to attach a ubiquitin chain, these enzymes establish a pathway that leads from (mono)sumoylation of substrates via SUMO chain formation to ubiquitin-dependent proteolysis. Mutants in this pathway can better cope with salt stress (NaCl toxicity), but less well with osmotic stress, suggesting that the pathway is important for reaction to environmental inputs. Using quantitative proteomics (collaboration with E. Nukarinen and W. Weckwerth), we have identified proteins with higher abundance in mutants that make less SUMO chains. These proteins are potential targets of the postulated sumoylation-ubiquitylation-degradation pathway. Using purified components, SUMO chain formation can be recapitulated in vitro, resulting either in free SUMO chains, or in chains attached to a model substrate. This system is used to study biochemical aspects of SUMO conjugation.

Andreas Bachmair

TEAM Jolanta AmbrozKumorowski Katarzyna Hanczaryk Lilian Nehlin Konstantin Tomanov

Plants with defect in a membrane-associated ubiquitylation complex have more and smaller shoots, due to decreased apical dominance (right). ©Kalda

SELECTED PUBLICATIONS Martens S, Bachmair A. How cells coordinate waste removal through their major proteolytic pathways. Nat Cell Biol 2015;17(7):841-2. PMID: 26123109 Tomanov K, Zeschmann A, Hermkes R, Eifler K, Ziba I, Grieco M, Novatchkova M, Hofmann K, Hesse H, Bachmair A. Arabidopsis PIAL1 and 2 Promote SUMO Chain Formation as E4-Type SUMO Ligases and Are Involved in Stress Responses and Sulfur Metabolism. Plant Cell 2014;26(11):4547-60. PMID: 25415977 Gibbs DJ, Md Isa N, Movahedi M, Lozano-Juste J, Mendiondo GM, Berckhan S, Marín-de la Rosa N, Vicente Conde J, Sousa Correia C, Pearce SP, Bassel GW, Hamali B, Talloji P, Tomé DF, Coego A, Beynon J, Alabadí D, Bachmair A, León J, Gray JE, Theodoulou FL, Holdsworth MJ. Nitric Oxide Sensing in Plants Is Mediated by Proteolytic Control of Group VII ERF Transcription Factors. Mol Cell 2014;53(3):369-79. PMID: 24462115

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MFPL - 2016 RESEARCH GROUPS

ANDREA BARTA

Post-transcriptional regulation of plant gene expression What determines the complexity of higher organisms? No correlation has been found to DNA content and gene number and therefore studies in the field are now focusing on post-transcriptional processes and the impact of the dynamic transcriptome on the complexity of gene expression.

Alternative splicing is one of the posttranscriptional events to expand the repertoire of gene products, and it has been exploited for various differentiation processes. In plants, the significance of alternative splicing was long underestimated, but we and others have shown that it greatly impacts on development and responses to the environment. As alternative splicing in Arabidopsis is not well characterized, we are using RNAseq to define the rules and targets of alternative splicing. In addition, we are using deep sequencing transcriptome data, several bioinformatics pipelines and evaluation of the results by a PCR panel to optimize the Arabidopsis transcriptome to use in differential splicing analysis.

important for splice site selection and spliceosome assembly. In addition, we have isolated several regulatory proteins which seem to be essential to drive the splicing process, like SRPK kinases, helicases and cyclophilins. To elucidate their mechanisms of action, some of the plant SR proteins and cyclophilins are currently characterized in greater detail in terms of their RNA targets, interacting proteins and their impact on flowering and UV-stress response. Interestingly, some of these factors seem to connect splicing to transcription and are therefore currently investigated in greater detail. As some of the alternative splicing events are regulated by light by signals from the chloroplast, screens to establish the signalling pathways are employed.

SR (Ser/Arg) proteins are important splicing factors and to date we have isolated and partially characterized several Arabidopsis SR proteins, which are

Andrea Barta

TEAM Zahra Ayatollahi Nicola Cavallari Armin Fuchs Yamile Marquez Manali Mishra Ezequiel Petrillo Peter Venhuizen

Plants use chloroplast as light sensors that generate signals able to fine-tune nuclear gene expression. Light perceived in the chloroplast triggers a signal that reaches the nucleus and affects alternative splicing. A light induced signal is able to travel through the plant to the roots affecting alternative splicing in a non-photosynthetic tissue.

SELECTED PUBLICATIONS Marquez Y, Hรถpfler M, Ayatollahi Z, Barta A, Kalyna M. Unmasking alternative splicing inside protein-coding exons defines exitrons and their role in proteome plasticity. Genome Res. 2015. 25: 995-1007. PMID: 25934563 Petrillo E, Godoy Herz MA, Fuchs A, Reifer D, Fuller J, Yanovsky MJ, Simpson C, Brown JW, Barta A, Kalyna M, Kornblihtt AR. A chloroplast retrograde signal regulates nuclear alternative splicing. Science. 2014;344(6182):427-30. PMID: 24763593 Gรถhring A, Jacak J, Barta A. Imaging of endogenous messenger RNA splice variants in living cells reveals nuclear retention of transcripts inaccessible to nonsense-mediated decay in Arabidopsis. Plant Cell. 2014;26(2):754-64. PMID: 24532591

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MFPL - 2016 RESEARCH GROUPS

DIETER BLAAS

Early interactions of viruses with host cells For infection, viruses attach to the cell surface, become engulfed and taken up by the cell along different entry routes. Next, the viral genome has to leave the protective shell and arrive at the appropriate position inside the cell for replication.

Dieter Blaas

TEAM Thomas Dechat Irene Gösler

biological, biophysical, and structural biology techniques, such as selection and characterization of viral mutants, expression library screening, fluorescence correlation spectroscopy, capillary electrophoresis, and cryo-electron microscopy.

Most of the entry pathways converge in endosomes. Depending on whether the virus is covered with a lipid membrane (enveloped) or lacking such a membrane (naked), its genome is then being released into the cytoplasm by different mechanisms. Enveloped viruses usually fuse with cellular membranes, which results in the nucleocapsid arriving in the cytosol. Non-enveloped viruses either disrupt the endosomal membrane or their nucleic acids become threaded through a pore and the remaining empty capsids are further shuttled to lysosomes for degradation. Since single-stranded RNA is usually highly structured with many double-stranded regions, it is unclear by what mechanism it unfolds to pass as a single strand through a small orifice.

In the last few years, we have identified heparan sulphate as an additional receptor for some rhinovirus types and characterized the uptake pathway by this proteoglycan and by the intercellular adhesion molecule 1, the receptor of about 90 different HRV types. We developed a liposomal in vitro system mimicking the transfer of the viral RNA through the endosomal membrane that is currently being used for structural analysis. Within the frame of several international collaborations, we have solved the 3D structure of subviral particles. Contrarily to the assumption that the RNA would exit with the 5’-end first we found that threading starts from the poly-(A) at the 3’-end. One might see it as a flexible probe in search of the hole thereby directing the rest of the RNA towards the exit.

Working with human rhinoviruses (HRVs) that lack a lipid membrane and are the predominant cause of common colds, we aim at identifying the different mechanisms underlying viral uptake, the process of genome release and the structural basis of the transfer of the viral genome through lipid membranes. We address these questions by using biochemical, molecular

Native and subviral common cold virion particles reconstructed from electron microscopic images similar to the one in the background. Some are cut open to allow a view onto the viral genome that became compacted to a rod-like structure upon crosslinking and induction of uncoating.

Cryo-electron micrograph of virus particles attached to liposomal membranes via a derivative of the low-density lipoprotein receptor mimicking viral binding to the host cell.

SELECTED PUBLICATIONS Conzemius R, Ganjian H, Blaas D, Fuchs R. ICAM-1 Binding Rhinoviruses A89 and B14 Uncoat in Different Endosomal Compartments. J Virol 2016;90, 7934-7942. PMID: 27334586 Harutyunyan S, Kowalski H, Blaas D. The Rhinovirus subviral a-particle exposes 3’-terminal sequences of its genomic RNA. J Viro 2014;l 88, 6307-6317. PMID: 24672023 Pickl-Herk A, Luque D, Vives-Adrian L, Querol-Audi J, Garriga D, Trus BL, Verdaguer N, Blaas D Caston JR. Uncoating of common cold virus is preceded by RNA switching as determined by X-ray and cryo-EM analyses of the subviral A-particle. Proc Natl Acad Sci U S A 2013;110, 20063-20068. PMID: 24277846

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MFPL - 2016 RESEARCH GROUPS

UDO BLÄSI

Post-transcriptional regulation in Bacteria and Archaea Prokaryotes in general and microbial human pathogens in particular are constantly challenged by changing environmental conditions. They employ a number of post-transcriptional control mechanisms including proteinaceous translational regulators, small regulatory RNAs as well as features inherent to mRNA structure, which permit a fast adaptation to new environments such as the host. The RNA chaperone Hfq has been recognized as the principle post-transcriptional regulator of catabolite repression in the human pathogen Pseudomonas aeruginosa. Hfq was shown to act as a translational repressor that prevents ribosome loading through binding to A-rich sequences within the ribosome binding site of several mRNAs encoding catabolic enzymes. Furthermore, the non-coding RNA CrcZ was shown to bind to and to sequester Hfq, which in turn abrogates Hfq-mediated translational repression. This novel mechanistic twist on Hfq function not only highlighted the central role of RNA based regulation in PAO1 carbon metabolism, but also broadened the view of Hfq-mediated post-transcriptional mechanisms. In addition, CrcZ-mediated regulation of Hfq was shown to impact on virulence traits such as biofilm formation (Fig. 1) and antibiotic susceptibility. The underlying

molecular events are currently studied. Another research focus is directed towards a better understanding of post-transcriptional regulatory mechanisms in the model crenarchaeon Sulfolobus solfataricus (Sso). Previous studies revealed the sequence of events in archaeal translation initiation as well as unprecedented function(s) of archaeal translation initiation factors. Ongoing studies concentrate on the elucidation of the structure (Fig. 2) and function of archaeal SmAP proteins in RNA turnover and on molecular mechanisms underlying non-coding RNA mediated regulation in Sso. Udo Bläsi

TEAM

Figure 1 Increased biofilm formation of a P. aeruginosa crcZ deletion mutant was visualized by confocal scanning laser microscopy. The life/dead stain distinguishes between live (green) and dead cells (red).

Figure 2 A. X-ray structure of the homo-heptameric SmAP2 protein of S. solfataricus (Märtens et al., 2015). The protomers are color coded. B. RNA binding to SmAP2. UV cross-linking in conjunction with mass spectrometry revealed the binding site of the SmAP2 RNA motif on the proximal site of the heptamer, where it is bound via U residues to Lys25 and to the sub-sequence Leu33-Arg46. The structure and RNA binding site(s) were elucidated in collaboration with the groups of K. Djinović-Carugo (MFPL, Vienna) and H. Urlaub (MPI, Göttingen ), respectively.

SELECTED PUBLICATIONS Tata M, Wolfinger MT, Amman F, Roschanski N, Dötsch A, Sonnleitner E, Häussler S, Bläsi U. RNASeq Based Transcriptional Profiling of Pseudomonas aeruginosa PA14 after Short- and Long-Term Anoxic Cultivation in Synthetic Cystic Fibrosis Sputum Medium. PLoS One 2016;11. PMID: 26821182 Sonnleitner E, Bläsi U. Regulation of Hfq by the RNA CrcZ in Pseudomonas aeruginosa carbon catabolite repression. PLoS Genetics 2014;10(6). PMID: 24945892 Märtens B, Manoharadas S, Hasenöhrl D, Zeichen L ,Bläsi U. Back to translation: removal of aIF2 from the 5´-end of mRNAs by translation recovery factor in the crenarchaeon Sulfolobus solfataricus. Nucl. Acids Res 2014; 42:2505-2511. PMID: 24271401

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Flavia Bassani Isabel Del Pino Gomez Dorothea Heitzinger Astrid Maria Hölzle Birgit Märtens Diego Oxilia Speratti Konstantin Prindl Petra Pusic Linda Rampelt Armin Resch Marlena Rozner Elisabeth Sonnleitner Muralidhar Tata


MFPL - 2016 RESEARCH GROUPS

CHRISTA BÜCKER

Transcriptional regulation during early embryonic development Every mammal starts out as a zygote that carries all the necessary information to form a whole individual. However, at any given developmental time point, environmental cues such as growth factors induce a response in the cell that depends on the cellular identity.

Christa Bücker

TEAM Philipp Fischer Elena Kotova Felix Rosebrock

Every cell state reacts differently to the same growth factor since downstream effectors of growth factors converge with the existing, cell type specific transcription factor landscape to drive cell type specific gene expression patterns. But when and how is a cell able to react to specific cues and how is the differentiation set into motion? In order to study these fundamental questions, we have established and extensively characterized a differentiation strategy based on mouse embryonic stem cells that allows us to closely follow a cell fate transition in vitro. We focus on transcriptional enhancers, short stretches of DNA sequences that drive cell type specific gene expression pattern and that integrate cellular identity with growth factor signalling. We have mapped and identified changes in the enhancer landscape during our differentiation strategy and are now dissecting the contributions of single enhancer elements to the activation of a target gene.

During development, changes in the transcription factor repertoire lead to changes in the active enhancer landscape.

termine the causes for these differences, we employ a combination of single cell methods such as RNA-FISH, life imaging and single cell ATAC-seq. Understanding why heterogeneity arises during differentiation will guide us to develop cleaner and more efficient differentiation strategies for clinical applications in the future.

The expression of a gene is often regulated by multiple single enhancer elements that together form “super enhancers”. However, it is still unclear how each element contributes to the overall expression of the target gene: are all single elements equally important, are they working additively or can loss of a single element be tolerated? In order to further understand the inner workings of super enhancers we are employing CRISPR/Cas9 to delete, mutate and invert single enhancer elements and study the effect of these changes during differentiation on the expression of a target gene. Even though most embryonic stem cells will eventually differentiate, the rate at which differentiation is initiated is not the same throughout a population. To de-

SELECTED PUBLICATIONS Buecker C, Srinivasan R, Wu Z, Calo E, Acampora D, Faial T, Simeone A, Tan M, Swigut T, Wysocka J. Reorganization of enhancer patterns in transition from naive to primed pluripotency. Cell Stem Cell. 2014 Jun 5;14(6):838-53. PMID: 24905168 Buecker C, Wysocka J. Enhancers as information integration hubs in development: lessons from genomics. Trends Genet. 2012 Jun;28(6):276-84. PMID: 22487374 Buecker C, Chen HH, Polo JM, Daheron L, Bu L, Barakat TS, Okwieka P, Porter A, Gribnau J, Hochedlinger K, Geijsen N. A murine ESC-like state facilitates transgenesis and homologous recombination in human pluripotent stem cells. Cell Stem Cell. 2010 Jun 4;6(6):535-46. PMID: 20569691

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MFPL - 2016 RESEARCH GROUPS

CHRISTOPHER CAMPBELL

Mechanisms that ensure chromosome segregation fidelity in mitosis In mitosis, chromosome segregation by the microtubule-based mitotic spindle ensures equal partitioning of the genome between the daughter cells. Cells use multiple mechanisms to ensure that chromosomes are segregated with high fidelity.

chromosomes), and the strength of these attachments is regulated by the kinase Aurora B. Aurora B provides the catalytic activity of the chromosomal passenger complex (CPC). The CPC is a four-subunit complex that detects improper microtubule-kinetochore connections and weakens them via phosphorylation of various targets on the kinetochore. One fundamental question that we wish to address is: how does the CPC specifically destabilize aberrant kinetochore-microtubule connections?

The vast majority of cancer cells are aneuploid (contain the wrong number of chromosomes), indicating that one or more of these segregation fidelity mechanisms has failed. The resulting increase in chromosome segregation errors is termed chromosomal instability (CIN). Despite major recent advances in the genomic characterization of cancer cells, very little is known about how and why cancer cells missegregate their chromosomes. We are interested in understanding the mechanisms that cells use to prevent the missegregation of chromosomes as well as the direct repercussions of chromosome missegregation. Our focus lies in fundamental, conserved processes that identify chromosomes with aberrant attachments to the microtubules and correct those misattachments. To examine these processes, we employ a combination of genetic, biochemical, and microscopy-based techniques using budding yeast as a model organism. Aneuploid cancer cell lines have defects in the strength of attachments between microtubules and the kinetochore (the microtubule-attachment site on

Many cancers have extremely high rates of chromosomal instability (CIN). Some cancers have chromosome segregation errors in every cell division, which would be detrimental to the growth of normal cells. Little is known about how cancers are able to thrive with high levels of CIN. We aim to determine how cells evolve to cope with CIN by creating a model system for persistent chromosomal instability in budding yeast. What types of mutations allow cells to adapt to a constantly shifting genomic content? What are the direct effects of CIN and aneuploidy on the health and viability of cells?

Aberrant attachments between kinetochores and the mitotic spindle must be corrected before anaphase to ensure proper chromosome segregation.

SELECTED PUBLICATIONS Campbell CS, Hombauer H, Srivatsan A, Bowen N, Gries K, Desai A, Putnam CD, Kolodner RD. Mlh2 is an accessory factor for DNA mismatch repair in Saccharomyces cerevisiae. PLoS Genet. 2014 May 8;10(5):e1004327. PMID: 24811092 Campbell CS, Desai A. Tension sensing by Aurora B kinase is independent of survivin-based centromere localization. Nature. 2013 May 2;497(7447):118-21. PMID: 23604256 Hombauer H, Campbell CS, Smith CE, Desai A, Kolodner RD. Visualization of eukaryotic DNA mismatch repair reveals distinct recognition and repair intermediates. Cell. 2011 Nov 23;147(5):1040-53. PMID: 22118461

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Christopher Campbell

TEAM Madhwesh C. Ravichandran Matthew Clarke Sarah Fink Sabine Kral Katrin Loibl Theodor Marsoner


MFPL - 2016 RESEARCH GROUPS

ALEXANDER DAMMERMANN

Centriole assembly and function Centrioles are small cylindrical organelles whose distinguishing feature is an outer wall composed of a nine-fold symmetric array of stabilized microtubules. requirements for centriole assembly. The six-protein molecular pathway we identified has since been found to be conserved from ciliates to vertebrates, and is thought to form the core of the centriole assembly machinery in all eukaryotes. We further identified the hydrolethalus syndrome protein HYLS-1 as a protein that is incorporated into centrioles during their assembly to confer on them the ability to initiate cilia. The single amino acid missense mutation associated with hydrolethalus syndrome impairs HYLS-1 function in ciliogenesis, identifying this disorder as a severe (perinatal lethal) ciliopathy.

Centrioles perform two important functions in eukaryotic cells: 1) they recruit pericentriolar material to form centrosomes that organize the microtubule cytoskeleton and position the mitotic spindle, and 2) they template cilia, cellular projections that perform a variety of critical sensory and motile functions. Centrosome and cilia abnormalities have been linked to aneuploidy and tumorigenesis as well as developmental disorders including ciliopathies and microcephaly. Despite their significance to human physiology and pathology, centrioles have remained poorly understood at the molecular level, largely due to the technical challenges posed by the small size of this organelle. Alexander Dammermann

TEAM Gabriela Cabral Jeroen Dobbelaere Balazs Erdi Triin Laos Max Roessler Cornelia Rumpf-Kienzl Daniel Serwas Tiffany Su

Current research builds on this foundation, seeking to answer three main questions: 1) How do centrioles assemble, and what are the molecular mechanisms that underlie their remarkable stability; 2) how do centrioles recruit pericentriolar material to form centrosomes and what is the molecular nature of this material; and 3) how do centrioles form cilia, focusing on the events immediately downstream of HYLS-1.

In our lab, we are using a combination of biochemical, cell biological and genetic approaches in the nematode C. elegans and the fruit fly Drosophila melanogaster to investigate the fundamental and conserved molecular mechanisms underlying centriole assembly and function. In previous work, we have used the C. elegans early embryo to define the molecular

(A) Centriole assembly pathway as delineated in C. elegans. (B) C. elegans early embryo, stained for SAS-4 (centrioles, yellow), Îł-tubulin (pericentriolar material, blue), Aurora-A (peripheral pericentriolar material and astral microtubules, red) and microtubules (black). (C) Depletion of HYLS-1 in Xenopus embryo results in failure of cilia assembly (acetylated tubulin, green). Basal bodies (Îł-tubulin, blue) are disorganized.

SELECTED PUBLICATIONS Laos T, Cabral G, Dammermann A. Isotropic incorporation of SPD-5 underlies centrosome assembly in C. elegans. Curr Biol;25(15):R648-9. PMID: 26241136 Schouteden C, Serwas D, Palfy M, Dammermann A. The ciliary transition zone functions in cell adhesion but is dispensable for axoneme assembly in C. elegans. J CELL BIOL 2015;210(1):35-44. PMID: 26124290 Serwas D, Dammermann A. Ultrastructural analysis of Caenorhabditis elegans cilia. Method Cell Biol 2015;129:341-67. PMID: 26175447

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MFPL - 2016 RESEARCH GROUPS

THOMAS DECKER

Host responses and innate immunity to microbial infection The first line of defence against pathogenic microorganisms is set by the innate immune system, which rapidly limits colonization and spread. regulators of the IFN genes. We have identified an RNA helicase, DDX3X, that enhances the ability of IRF to stimulate IFN transcription. Current activities aim at studying the importance of the DDX3X enzyme for innate immunity in cells and mice. Furthermore, we address the mechanism by which DDX3X contributes to transcriptional regulation of antimicrobial genes. Once infected cells produce IFN, these interact with cell surface receptors and set off cellular signals through Janus protein tyrosine kinases (JAKs) and transcription factors collectively called signal transducers and activators of transcription (STATs). A long-standing interest of the lab is how STATs influence the chromatin of target genes to support transcription. Related to this, we study the coordination of distinct signals in infected cells at chromatin level. Our particular interest is the transcriptional cooperativity of the JAK-STAT and NFkB pathways as a regulatory mechanism impacting on a large group of antimicrobial genes. The overarching aim of our studies is to contribute to a comprehensive view of signalling and transcriptome regulation in infected cells.

To increase protection and produce immunological memory, cells participating in the innate response initiate an adaptive immune response. Protection and immunoregulation by the innate immune system requires that a microbe is detected and physical contact is translated into altered gene expression of the infected cell. Antimicrobial gene products provide protective effector mechanisms. Moreover, secreted cytokines fulfil the task of communicating between cells involved in the antimicrobial response to maximize the common antimicrobial effort. One important group of cytokines is formed by the interferons (IFN), subdivided into three distinct classes (IFN-I, II, III). Collectively, IFN play an indispensable role in the immune system, both as regulators of antimicrobial cell activation and as modulators of the inflammation which accompanies infection. Our research aims at understanding how the synthesis of IFN is initiated when cells or animals are infected with intracellular bacteria, and how IFN give rise to changes in the cellular transcriptome. Microbial infection causes cellular signals that produce activated interferon regulatory factors (IRF), transcriptional

IFNAR  

PRR  

JAK  

IKK   Histone   modifiers  

ISGF3  

NFκB  

Pol  II   kinases  

An=microbial   gene  

Pol  II  

Mediator  

Ini=a=on   complex  

Gene activation in cells infected with Listeria monocytogenes by cooperation of the type I IFN (blue) and NFkB (red) pathways. The type I interferon receptor (IFNAR) activates Janus kinases (JAKs) to phosphorylate transcription factor ISGF3 whereas pattern recognition receptors (PRR) activate the IkB kinase complex (IKK) and NFkB. ISGF3 contacts the initiation and mediator complexes to recruit RNA polymerase II (pol II). NFkB stimulates promoter binding of histone modifying enzymes and it contacts mediator to recruit both pol II and its activating kinases. Together these events render antimicrobial genes competent for transcriptional initiation and elongation.

SELECTED PUBLICATIONS Jamieson A M, Pasman L, Yu S, Gamradt P, Homer R J, Decker T, Medzhitov R. Role of tissue protection in lethal respiratory viral- bacterial coinfection. Science 2013;340(6137) 1230-1234 PMID: 23618765 Rauch I, Rosebrock F, Hainzl E, Heider S, Majoros A, Wienerroither S, Strobl B, Stockinger S, Kenner L, Müller M Decker T. Noncanonical effects of IRF9 in intestinal inflammation: more than type I and type III interferons. Mol. Cell. Biol 2015;35 : 2332-2343. PMID: 25918247 Wienerroither S, Shukla P, Farlik M, Majoros A, Stych B, Vogl C, Cheon H, Stark G R, Strobl B, Müller M, Decker T. Cooperative transcriptional activation of antimicrobial genes by STAT and NFkB pathways through concerted recruitment of the mediator complex. Cell Reports 2015;12: 300-312. PMID: 26146080

13

Thomas Decker

TEAM Duygu Demiröz Katrin Fischer Ekaterini Platanitis Birgit Rapp Ursula Styx Daniel Szappanos Fotima Touraeva Roland Tschismarov


MFPL - 2016 RESEARCH GROUPS

KRISTINA DJINOVIĆ-CARUGO

Structural biology of the cytoskeleton Animal movement is mediated by striated muscles, with the sarcomere being the basal contractile unit within single muscle fibers. Sarcomeric actin and myosin form cross-linked and interdigitated filament bundles whose sliding motion generates force. Antiparallel actin filaments from adjacent sarcomers are anchored at the Z-disk, which plays a central role as the site organizing the molecular machinery that is required for muscle contraction and is regarded as one of the most complex assemblies known to biology (Figure 1A-C).

Kristina Djinović-Carugo

TEAM Oliviero Carugo Julian Ehrmann Irina Grishkovskaya Andreas Hagmüller Tamas Hatfaludi Stefan Hofbauer Julius Kostan Joan Lopez Arolas Moritz Madern Georg Mlynek Miriam Pedron Martin Gerald Puchinger Dominic Pühringer Sara Sajko Claudia Schreiner Julia Schweighofer Antonio Sponga Valeria Stefania Tobias Thöni Karolina Zielinska

We collaborate with several groups at MFPL (Warren, Slade, Skern, Kovarik, Blaesi, Moll), as well as with C. Obinger (University of Natural Resources and Life Sciences, Vienna) (Figure 2). Figure 1. Schematic representation of muscle, its filaments and of the Z-disk. (A) Muscle is composed of a bundle of fibers – muscle cells, which are in turn composed of myofibrils, the building unit of which is a sarcomere. (B) Sarcomere, with its major filaments actin, myosin and titin and the Z-disk anchoring cross linker α-actinin. (C) In the Z-disk actin and titin filaments are cross-linked by its major component α-actinin. (D) A surface representation of the alpha-actinin dimer (Ribeiro Ede et al., Cell, 2014) against a background of muscle sarcomeres viewed under the electron microscope. The sarcomeres display a typical striated pattern, and are connected via joint Z-discs, seen as dark black and grey diagonal stripes. Credit: Julius Kostan, Joan L. Arolas, Tobias Thoeni, Nikos Pinotsis, Kristina Djinović-Carugo, Uni Vienna; Mathias Gautel and Andrea Ghisleni, KCL; Marija Nabernik, Jernec Zupanc, Seyens

The major questions we are addressing are: What is the stoichiometry of the components and the assembly hierarchy of the Z-disk? What is the molecular architecture of both pre- and myofibrillar assemblies? Our long-term goal is to reconstitute a functional Z-disk from individual components, which is the best way to truly understand their disparate functional roles and molecular mechanisms. Apart from its major component α-actinin-2 (Ribeiro Ede et al., Cell, 2014) (Figure 1D), which accounts for ∼20% of the Z-disk mass, the current inventory of proteins in mature Z-disks includes over 40 proteins that form a highly stable and highly ordered unit that can support contractile forces of the muscle. The production of 16 Z-disk proteins is already well established in our laboratory. We use integrative structural biology approaches combining biochemical, biophysical and high resolution structural studies (X-ray diffraction, NMR) with lower resolution approaches that can either yield molecular envelopes (SAXS, SANS, EM) or specific distance information derived from e.g. chemical-cross-linking coupled to mass-spectrometry or NMR. New bioinformatic strategies are being designed to extend our prediction and analysis capabilities.

Figure 2. Examples of collaborative projects. (A) Crystal structure of heptameric SmAP2 from Sulfolobus solfataricus (Märtens et al., Life (Basel), 2015) (B) Overview showing the S2– S1 complex structure assembled from two protomers, with S1 in blue, S2 in yellow. Zn2+ is depicted as a green sphere (Byrgazov et al., Nucleic Acids Res., 2015). (C) Crystal structures of dimeric chlorite dismutase from Nitrobacter winogradskyi (Mlynek et al., J Bacteriol., 2011) and pentameric chlorite dismutase (Kostan et al., J Struct Biol., 2010). (D) Crystal structure of HemQ from Listeria monocytogenes catalyzing the decarboxylation of coproheme to heme b (accepted in FEBS Journal). Credit: Georg Mlynek, Irina Grishkovskaya, Kristina Djinović-Carugo, Uni Vienna

In order to overcome the major bottlenecks in structural and functional studies of proteins, which are availability of milligram amounts of active, chemically and conformationally pure protein and crystallization, an FFG funded Laura Bassi Centre for Optimized Structural Studies (COSS) was established as a joint venture of IMP, Biomin and UNIVIE-MFPL with the goal to set-up an efficient platform to combine the recent advances in protein production and develop customized crystallisation approaches.

SELECTED PUBLICATIONS Ribeiro Ede A Jr, Pinotsis N, Ghisleni A, Salmazo A, Konarev PV, Kostan J, Sjöblom B, Schreiner C, Polyansky AA, Gkougkoulia EA, Holt MR, Aachmann FL, Zagrović B, Bordignon E, Pirker KF, Svergun DI, Gautel M, Djinović-Carugo K. The structure and regulation of human muscle α-actinin. Cell. 2014 Dec 4;159(6):1447-60. PMID: 25433700 Song JG, Kostan J, Drepper F, Knapp B, de Almeida Ribeiro E Jr, Konarev PV, Grishkovskaya I, Wiche G, Gregor M, Svergun DI, Warscheid B, Djinović-Carugo K. Structural insights into Ca2+-calmodulin regulation of Plectin 1a-integrin β4 interaction in hemidesmosomes. Structure. 2015 Mar 3;23(3):558-70. PMID: 25703379 Kostan J, Salzer U, Orlova A, Törö I, Hodnik V, Senju Y, Zou J, Schreiner C, Steiner J, Meriläinen J, Nikki M, Virtanen I, Carugo O, Rappsilber J, Lappalainen P, Lehto VP, Anderluh G, Egelman EH, Djinović-Carugo K. Direct interaction of actin filaments with F-BAR protein pacsin2. EMBO Rep. 2014 Nov;15(11):1154-62. PMID: 25216944

14


MFPL - 2016 RESEARCH GROUPS

GANG DONG

Structural studies of ciliogenesis Eukaryotic cilia and flagella are specialized organelles that are highly conserved from protists to mammals.

vesicle targeting to the ciliary base, flagellar pocket biogenesis and intraflagellar transport. Technically, we mainly use X-ray crystallography to determine the structures of these proteins and their complexes. Other biophysical techniques such as static/dynamic light scattering (SLS/DLS), differential scanning calorimetry, and analytical ultracentrifugation will as well be employed to study the architecture and assembly of the proteins and their complexes. Small proteins and domains are also studied by nuclear magnetic resonance (NMR) spectroscopy, whereas large assemblies are examined by electron microscopy (EM). Our structural studies will be complemented by site-directed mutagenesis, in vitro biochemical experiments, and in vivo assays (mostly with our collaborators) to test our mechanistic hypotheses. The available new structures will enhance our understanding of how these complexes function and provide hints as to how their malfunction leads to human diseases.

These organelles consist of the membrane-sheathed axoneme, which is an extension of the mother centriole, and at least 360 associated proteins. Cilia have attracted much attention in recent years because of their role in the transduction of extracellular signals and their association with an expanding number of human disorders. Such disorders include respiratory distress syndrome, male sterility, polycystic kidney disease, retinal degeneration, and Bardet-Biedl syndrome. Cilium assembly is initiated by the docking and fusion of the mother centriole to the apical membrane of the cell. Cilia are assembled and maintained through intraflagellar transport (IFT). This process is carried out by two distinct protein complexes, IFT complex A and B, which contain at least six and sixteen subunits, respectively. These complexes transport ciliary cargos within cilia and flagella by interactions with the microtubule-associated motor proteins kinesin-II and dynein. We are focusing on a number of aspects in ciliogenesis, including centriole/basal body duplication,

Gang Dong

TEAM Johannes Lesigang Yulia Pivovarova Emma Stepinac Yan Zhang

Research summary

SELECTED PUBLICATIONS Shimanovskaya E, Viscardi V, Lesigang J, Lettman MM, Qiao R, Svergun DI, Round A, Oegema K, Dong G. Structure of the C. elegans ZYG-1 Cryptic Polo Box Suggests a Conserved Mechanism for Centriolar Docking of Plk4 Kinases. Structure. 2014 Aug 5;22(8):1090-104. PMID: 24980795 Vidilaseris K, Shimanovskaya E, Esson HJ, Morriswood B, Dong G. Assembly Mechanism of Trypanosoma brucei BILBO1, a Multidomain Cytoskeletal Protein. J Biol Chem. 2014 Aug 22;289(34):23870-81. PMID: 25031322 Qiao R, Cabral G, Lettman MM, Dammermann A, Dong G. SAS-6 coiled-coil structure and interaction with SAS-5 suggest a regulatory mechanism in C. elegans centriole assembly. EMBO J. 2012 Nov 14;31(22):4334-47. PMID: 23064147

15


MFPL - 2016 RESEARCH GROUPS

SILKE DORNER

The regulation of gene expression by small ncRNAs Post-transcriptional processes such as mRNA splicing, mRNA degradation, mRNA surveillance, RNA editing, translational repression, and RNA-mediated gene silencing, play crucial roles in the regulation of eukaryotic gene expression.

Silke Dorner

TEAM Zahra Ayatollahi Stefanie Hosiner

provides a mechanism to commit mRNAs for degradation. Currently, we investigate the timing and order of recruitment of additional mRNA degradation factors to the miRNA effector complex. The second focus of our work is the tight coupling of the translation and degradation of mRNAs. Since it is not entirely clear where in the cell mRNA degradation occurs, we investigate the possibility of mRNA degradation on ribosome complexes in Drosophila cells. Using polysome profiles and ribosome affinity purification we could demonstrate the co-purification of various deadenylation and decapping factors with ribosome complexes. In addition, AGO1 and GW182, the two key components of the miRNA effector complex, were associated with ribosome complexes. We also isolated decapped mRNA degradation intermediates from ribosome complexes and could demonstrate a high abundance of these intermediates on the ribosome. Thus mRNA degradation not only occurs on ribosome complexes but is also used as a very general site of general and miRNA-mediated - mRNA degradation in cells. In future projects we will investigate the changes of the mRNA degradosome on the ribosome under various stress conditions.

In the past two decades, the finding of small non-coding RNAs has entirely revolutionized the way we think about the regulation of gene expression. The major focus of our research is the RNA-mediated gene silencing by miRNAs (micro RNAs) in Drosophila. miRNAs are small non-coding RNAs that have been well established as key regulators of gene expression, which result in translation repression and/or mRNA destabilization upon binding to their target mRNAs. Key factors for miRNA-mediated mRNA degradation are the components of the miRNA effector complex (AGO1 and GW182) and the general mRNA degradation machinery (deadenylation and decapping enzymes). An important question we address in our projects is how the mRNA decapping machinery gets recruited to mRNAs after being targeted by miRNAs. The decapping step in mRNA degradation is of particular interest because it is an irreversible step and decapped mRNAs are quickly degraded by exonucleolytic digestion. Interestingly, we identified an interaction of GW182 and HPat, a general decapping activator. Thus the decapping step is not only a consequence of deadenylation but decapping activators get recruited to the miRNA effector complex. Furthermore, the recruitment of HPat to the miRNA effector complex

We established an inducible expression system for Drosophila cell culture that allows the measurement of mRNA turnover rates. Left: Northern blot analysis of mRNA levels after a transcriptional pulse. Right: Quantitative analysis of mRNA decay based on the Northern blot experiments shown.

SELECTED PUBLICATIONS Antic S, Wolfinger MT, Skucha A, Hosiner S, Dorner S. General and MicroRNA-Mediated mRNA Degradation Occurs on Ribosome Complexes in Drosophila Cells. Mol Cell Biol. 2015 Jul;35(13):2309-20. PMID: 25918245 Barišić-Jäger E, Kręcioch I, Hosiner S, Antic S, Dorner S. HPat a decapping activator interacting with the miRNA effector complex. PLoS One. 2013 Aug 19;8(8):e71860. PMID: 23977167 Jäger E, Dorner S. The decapping activator HPat a novel factor co-purifying with GW182 from Drosophila cells. RNA Biol. 2010 May-Jun;7(3):381-5. PMID: 20458171

16


MFPL - 2016 RESEARCH GROUPS

ROLAND FOISNER

Lamins in nuclear organization and human disease Lamins form a stable scaffold structure at the nuclear envelope, the lamina, and are also found throughout the nucleoplasm. They determine mechanical properties of the nucleus and are involved in chromatin regulation and gene expression. Lamin mutations cause human diseases ranging from muscular dystrophy to premature-ageing syndromes.

lamin-chromatin interactions, epigenetic profiles and gene expression (See figure). This novel role of LAP2α and lamins in gene regulation may also be involved in the progeria premature ageing disease phenotype. Expression of the progeria-linked lamin mutant, leads to loss of LAP2α and lamins in the nuclear interior and affects expression of extracellular matrix genes, while ectopic LAP2α rescues this phenotype. We currently investigate the potential mechanisms involved.

We aim at understanding molecular mechanisms of lamin functions in nuclear organization and chromatin regulation during differentiation and their role in diseases. In particular, we investigate i) how lamin-binding proteins affect lamin dynamics and functions, ii) how these proteins control lamin-chromatin interactions, and iii) how these processes are affected by lamin disease mutations. We have been studying lamin-interacting proteins of the LEM (LAP-Emerin-MAN1) protein family, which bind chromatin via their LEM motif. While most LEM proteins localize to the inner nuclear membrane, Lamina-associated polypeptide 2α (LAP2α) is in the nucleoplasm and binds lamins in the nuclear interior. In LAP2α-deficient mice we found that LAP2α stabilizes lamins in the nuclear interior and regulates proliferation and differentiation of tissue stem cells, but mechanistic details are unknown and are currently investigated. Chromatin regulation by lamins in health and disease (FWF grant P26492-B20). The lamina interacts with heterochromatic genomic regions, termed lamina-associated domains. By chromatin immunoprecipitation we found that LAP2α and lamins in the nuclear interior associate with open euchromatic regions, and LAP2α loss affects

Endothelial dysfunction and cardiovascular disease in progeria premature aging disease (Progeria research Foundation grant PRF 2016-64). Progeria disease is characterized by severe symptoms resembling features of premature aging, including cardiovascular disease that leads to myocardial infarction. In order to study the molecular basis of progeria-linked cardiovascular disease, we generated a mouse model expressing the lamin mutant in vascular endothelial cells. We find heart left ventricular hypertrophy and pro-atherogenic changes, such as attenuated shear stress response and mechanosignaling by the nucleus. We will investigate these defects at molecular level and aim at identifying new (pro-atherogenic) pathways and components as potential targets for diagnosis and therapy. Lamins interact with heterochromatin at the nuclear envelope and euchromatin in the nuclear interior. Cartoon shows part of the nucleus depicting the nuclear membranes, a nuclear pore complex, and the nuclear lamina underneath the inner nuclear membrane, consisting of B-type lamins (blue) and A-type lamins (yellow). Unlike B-type lamins, A-type lamins also localize within the nucleus in a LAP2α-dependent manner (red). Lamin-associated heterochromatin at the nuclear envelope (LADs, dashed black) and euchromatin (dashed green) in the nuclear interior are shown. The right panel shows Integrative Genomics Viewer (IGV) tracks of genomic DNA bound to peripheral and intra-nucleoplasmic lamin structures shown in the cartoon. Cover art © Kevin Gesson and Roland Foisner

SELECTED PUBLICATIONS Gesson K, Rescheneder P, Skoruppa M, von Haeseler A, Dechat T, Foisner R. A-type lamins bind both hetero- and euchromatin, the latter being regulated by lamina-associated polypeptide 2 alpha. Genome Res 2016;26(4):462-73. PMID: 26798136 Vidak S, Kubben N, Dechat T, Foisner R. Proliferation of progeria cells is enhanced by lamina-associated polypeptide 2α (LAP2α) through expression of extracellular matrix proteins. Gene Dev 2015;29(19):2022-2036. PMID: 26443848 Gruenbaum Y, Foisner R. Lamins: nuclear intermediate filament proteins with fundamental functions in nuclear mechanics and genome regulation. Annual review of biochemistry 2015.;84:131-64. PMID: 25747401

17

Roland Foisner

TEAM Simona Ferraioli Petra Fichtinger Konstantina Georgiou Josef Gotzmann Dragana Mustafic Nana Naetar


MFPL - 2016 RESEARCH GROUPS

PETER FUCHS

Stress response in simple epithelia A major role of the keratin intermediate filaments in simple epithelia is to protect cells from mechanical and non-mechanical stresses.

Peter Fuchs

There is increasing evidence for the involvement of keratin-associated proteins with the modulation of these functions. One of these proteins is epiplakin, a member of the plakin protein family. Compared to the other protein family members, epiplakin has an unusual structure comprising solely 16 (mouse) or 13 (human) plakin repeat domains. Its expression is restricted to epithelial tissues and it binds to intermediate filaments, mainly to keratins, which are the only binding partners identified so far. Epiplakin-deficient (Epi KO) mice generated in our laboratory are viable and show no obvious phenotype. These findings are in clear contrast to other proteins belonging to the plakin protein family like plectin, desmoplakin, and BPAG1, which play an important role in mechanically strengthening the skin as shown by phenotypes of knock-out mice. In order to further elucidate the biological function of epiplakin in simple epithelia, we are performing a combination of experiments using mouse injury models and experiments based on cell culture, biochemistry and video microscopy. In the mouse we use several stress models for simple epithelia in different organ systems which are complemented by experiments with primary cells. In addition, we use biochemical and cell culture based methods to investigate epiplakin interaction with simple epithelial keratins in more detail and to reveal epiplakin functions in keratin filament dynamics and network recovery after stress. Recently we found that epiplakin binds to the hepatic keratins 8 (K8) and K18 via multiple domains and that its filament-associated localization in hepatocytes completely depends on its binding partner keratin. In several liver stress models epiplakin and K8 expression were upregulated in parallel and Epi KO mice suffered from aggravated liver injury. Diseased Epi KO livers contained larger amounts of hepatocellular keratin granules (Figure 1), indicating impaired disease-induced keratin network reorganization. Our data indicate that during situations of cellular stress, associated with strong upregulation and subsequent reorganization of keratins, epiplakin aids in organizing the reassembly of new keratin filaments (Figure 2).

Figure 1. Keratin aggregates formed in stressed livers. Immunofluorescence microscopy displaying hepatocytes comprising keratin aggregates (arrows) formed in stressed livers. Paraffin sections of DDC-treated livers isolated from WT and epiplakin-deficient (Eppk─/─) mice were co-stained with antibodies recognizing K8 and epiplakin.

disease (CBDL, DDC)

_/_

WT

EPPK

healthy cell

healthy cell disease (CBDL, DDC)

N

keratin epiplakin

forced K8 expression

N

forced K8 expression

keratin

keratin reorganization

keratin reorganization cell death?

N

keratin epiplakin

N

cell with keratin granules

N

keratin

N

keratin disorganization liver disease

N

cell with keratin granules

Figure 2. Loss of epiplakin impairs stress-induced keratin reorganization leading to aggravated experimental liver injury. This scheme visualizes a model of epiplakin’s role during disease-induced or forced keratin upregulation and consequent filament reorganization in hepatocytes. In untreated WT cells, epiplakin (blue) colocalizes with keratin filaments (brown). In hepatocytes of both genotypes, keratin network reorganization is triggered by experimental liver disease-induced keratin upregulation or by forced keratin expression. Epiplakin-deficient (Eppk─/─) cells suffer from keratin disorganization which leads to cell death further aggravating liver disease. In contrast to Eppk─/─ hepatocytes, parallel upregulation of epiplakin enables WT cells to tolerate high keratin protein levels by successfully reorganizing their keratin filament networks. N, nucleus.

SELECTED PUBLICATIONS Szabo S, Wögenstein KL, Österreicher CH, Guldiken N, Chen Y, Doler C, Wiche G, Boor P, Haybaeck J, Strnad P, Fuchs P. Epiplakin attenuates experimental mouse liver injury by chaperoning keratin reorganization. J Hepatol. 2015 Jun;62(6):1357-66. PMID: 25617501 Wögenstein KL, Szabo S, Lunova M, Wiche G, Haybaeck J, Strnad P, Boor P, Wagner M, Fuchs P. Epiplakin deficiency aggravates murine caerulein-induced acute pancreatitis and favors the formation of acinar keratin granules. PLoS One. 2014 Sep 18;9(9):e108323. PMID: 25232867 Szabo S, Wögenstein KL, Fuchs P. Functional and Genetic Analysis of Epiplakin in Epithelial Cells. Methods Enzymol. 2016;569:261-85. PMID: 26778563

18


MFPL - 2016 RESEARCH GROUPS

BORIS GÖRKE

Signal transduction and posttranscriptional regulation in bacteria Our research aims to unravel novel principles underlying signal-perception, -transduction and cellular regulation in the model organism Escherichia coli. Mechanistically, we focus on the roles of small regulatory RNAs (sRNAs), mechanisms achieving specificity in RNA turnover and on the functions of protein-protein interaction for regulation.

many more proteins like RapZ explaining how specificity in RNA decay can be achieved. Currently, the identification and characterization of such adaptors is a major task in our laboratory. In addition, we clarify the mechanistic details underlying the GlmY-GlmZRapZ circuit and try to exploit this mechanism for antimicrobial chemotherapy.

In a first project, we investigate the roles of RNase adaptor proteins for regulated turnover of transcripts. We recently identified a novel regulatory circuit, composed of two small RNAs and the RNA binding protein RapZ, which controls synthesis of the cell wall biosynthesis enzyme GlmS. When synthesis of cell wall metabolites such as glucosamine-6-phosphate (GlcN6P) is required, the small RNA GlmZ base-pairs with the glmS mRNA thereby activating translation. The latter process is assisted by the hexameric RNA chaperone Hfq, which promotes base-pairing. In contrast, when dispensable, GlmZ is targeted to degradation by endoribonuclease RNase E. This process requires protein RapZ, which binds GlmZ and recruits RNase E through protein-protein interaction. Degradation of GlmZ is antagonized by a homologous small RNA, GlmY, which acts as decoy and sequesters protein RapZ, if cell wall biosynthesis shall occur. RapZ is the first discovered protein shown to serve as an adaptor to mediate decay of a specific transcript by a globally acting RNase. We speculate that there exist

Furthermore, we investigate the mode of operation of histidine sensor kinases, which represent the primary sensory systems of bacteria. The usually membrane bound kinases sense specific signals in the environment and transduce the gained information into the cell leading to changes in gene expression or of other cellular activities. We previously identified an accessory protein named PtsN, which modulates the activities of histidine kinases from inside the cell through direct interaction. We study how PtsN achieves this task and investigate the physiological meaning of this additional layer of regulation of kinase activities.

Boris Görke

TEAM Svetlana Durica Yvonne Göpel Muna Khan Markus Mörk-Mörkenstein Alexandra Schilder

Regulation of GlmS synthesis by small RNAs GlmY and GlmZ and the RNase adaptor protein RapZ (Göpel et al., 2016).

SELECTED PUBLICATIONS Khan M A, Göpel Y, Milewski S, Görke B. Two Small RNAs Conserved in Enterobacteriaceae Provide Intrinsic Resistance to Antibiotics Targeting the Cell Wall Biosynthesis Enzyme Glucosamine-6-Phosphate Synthase. Front Microbiol 2016;7:908. PMID: 27379045 Göpel Y, Khan M A, Görke B. Domain swapping between homologous bacterial small RNAs dissects processing and Hfq binding determinants and uncovers an aptamer for conditional RNase E cleavage. Nucleic Acids Res 2016;44(2):824-37. PMID: 26531825 Göpel Y, Papenfort K, Reichenbach B, Vogel J, Görke B. Targeted decay of a regulatory small RNA by an adaptor protein for RNase E and counteraction by an anti-adaptor RNA. Gene Dev 2013;27(5):552-64. PMID: 23475961

19


MFPL - 2016 RESEARCH GROUPS

ANGELA HANCOCK

Molecular basis of adaptive evolution Adaptation to different local environments can result in large-scale phenotypic diversity across a species’ range. Determining how this variation is produced and maintained is a central goal of evolutionary biology.

Our research integrates population genetics, bioinformatics, quantitative genetics and controlled experiments (in Arabidopsis) to clarify how species respond to environmental selection pressures.

this is a daunting task because it requires knowledge of the natural environment, the important adaptive traits, the genetic basis of phenotypic variation, and evidence that differences in this genetic basis equate to fitness differentials in the natural population. Ecologically interesting populations of well-studied model organisms can provide the background knowledge and tools necessary to overcome this challenge. Islands represent powerful systems for unravelling evolutionary histories because in these systems complexity is reduced relative to mainland populations and natural processes can be studied in relative isolation. To this end, we are using populations of the model plant Arabidopsis thaliana from Macaronesian archipelagos to dissect phenotypic variation and reconstruct adaptive histories. Ongoing and planned projects focus on identifying functional genetic variants, modelling their evolutionary histories and testing for fitness differentials in simulated and natural environments using a combination of population genetic analysis, trait mapping, genome editing and field work.

Identifying variation that underlies adaptation to the environment We conduct population genetic analyses on spatially-explicit genomic data sets to understand how adaptation progressed in natural systems. We are particularly interested in clarifying what types of molecular variants underlie adaptation, what mode of selection drove adaptive differentiation and why these factors sometimes differ among species. Angela Hancock

Reconstructing evolutionary histories in island Arabidopsis By comprehensively characterizing the evolutionary process in particular cases, we can uncover general principles of adaptation and evolutionary change. However, in complex organisms from natural populations,

Arabidopsis growing in Cape Verde Islands.

SELECTED PUBLICATIONS Horton MW, Hancock AM, Huang YS, Toomajian C, Atwell A, Auton A, Muliyati NW, Platt A, Sperone FG, WilhjĂĄlmsson BJ, Nordborg M, Borevitz JO, Bergelson J. Genome-wide patterns of genetic variation in worldwide Arabidopsis thaliana accessions from the RegMap panel. Nat. Genet. 2012;44:212-216. PMID: 22231484 Hancock AM, Brachi B, Faure N, Horton MW, Jarymowycz LB, F. Sperone G, Toomajian C, Roux F, Joy Bergelson J. Adaptation to Climate Across the Arabidopsis thaliana Genome. Science 2011;334(6052):83-86. PMID: 21980108 Hancock AM, Witonsky DB, Ehler E, Alkorta-Aranburu G, Beall C, Gebremedhin A, Sukernik R, Utermann G, Pritchard J, Coop G, Di Rienzo A. Colloquium paper: human adaptations to diet, subsistence, and ecoregion are due to subtle shifts in allele frequency. Proc. Natl. Acad. Sci. USA 2010;107 Suppl 2:892430. PMID: 20445095

20


MFPL - 2016 RESEARCH GROUPS

ANDREAS HARTIG

Origin and biogenesis of peroxisomes Eukaryotic cells contain organelles separating metabolic pathways. This spatial separation ensures optimal flux of metabolic intermediates and increases the efficiency of the metabolism.

functional peroxisomes, the transport of peroxisomal matrix proteins across the organellar membrane, and the control of size, shape and number of these compartments. Dispensable peroxisomes are degraded in a process called pexophagy. Employing yeast as model system we aim to elucidate the molecular mechanisms leading to new peroxisomes either through proliferation of already existing ones or via a de novo biogenesis pathway through fission from the ER. Currently, our main interest is focused on the mechanism of the de novo biogenesis initiated at the ER. Proteins exclusively involved in the biogenesis of peroxisomes are called peroxins (Pex-proteins). Among these the Pex11 protein is a membrane elongation factor, and in yeast, we showed that this protein acts only on already existing peroxisomes leading to proliferation. Two distantly related yeast proteins, Pex25p and Pex27p, play similar roles at the peroxisomal membrane and, in addition, participate in the de novo biogenesis. Together with Pex3p the Pex25 protein is essential for the initiation of peroxisome generation at the ER. Distinct vesicles emanating from the ER may slowly mature into peroxisomes or may fuse with each other or already existing peroxisomes to form mature organelles. The priming event at the ER, the proteins involved and the molecular mechanism are so far unknown, and will be the focus of our future work.

Wild type yeast cells accumulate a fluorescent peroxisomal protein in peroxisomes

Peroxisomes are highly versatile organelles and essential for life. They participate in many metabolic processes, most notably the degradation of fatty acids and the glyoxylate cycle. Synthesis of organelles and their degradation has to be tightly regulated in agreement with the metabolic status of the cell. Accordingly, peroxisomes need to be maintained in sufficient number to ensure metabolic homeostasis. A network of interacting proteins guarantees the biogenesis of

SELECTED PUBLICATIONS Kunze M, Hartig A. Permeability of the peroxisomal membrane: lessons from the glyoxylate cycle. Front Physiol 2013;4(204). PMID: 23966945 Huber A, Koch J, Kragler F, Brocard C, Hartig A. A Subtle Interplay between Three Pex11 Proteins Shapes de novo Formation and Fission of Peroxisomes. Traffic 2012;1(13):157-167. PMID: 21951626 Koch J, Pranjic K, Huber A, Ellinger A, Hartig A, Kragler F, Brocard C. PEX11 family members are membrane elongation factors that coordinate peroxisome proliferation and maintenance. J Cell Sci 2010;123:3389-400. PMID: 20826455

21

Andreas Hartig


MFPL - 2016 RESEARCH GROUPS

MARCELA HERMANN

LDL�R gene family, apolipoproteins and lipid transfer Our studies focus on the biology of the growing chicken oocyte and the developing chicken embryo. Specifically, we are interested in unravelling molecular mechanisms involved in the transport of VLDL from the egg yolk to the embryo proper.

Marcela Hermann

TEAM

the modification of LDL may play a key role in early atherogenic events. Modified LDL activates endothelial cells to attract and bind monocytes, and consecutively foam cells are formed, leading to the appearance of the fatty streak lesion. Various diseases such as diabetes, chronic renal insufficiency and obesity come along with elevated levels of blood cholesterol and different modified LDL. We are interested to identify compounds (synthetic, natural) with the potential to act as catalysts or inhibitors of the atherogenic modification of LDL. We also investigate molecular genetic, cell biological, and biochemical details of the role of the recently discovered apolipoprotein O in the etiology of fat liver and pathogenetic interplay between lipid metabolism and intrahepatic lipid accumulation. Non-alcoholic fatty liver is the most common chronic liver disease worldwide, representing the hepatic manifestation of the metabolic syndrome.

In this context, the roles of the LDL receptor gene family members, apolipoproteins and lipid transfer proteins are studied. The developing avian embryo constitutes an excellent system for the study of lipid and lipoprotein transport phenomena. The yolk is the major source of nutrients for the developing embryo, but molecular details of the delivery mechanisms are largely unknown. During the vitellogenic phase of ocyte growth in the chicken, the yolk accumulates via uptake from the circulation of precursor proteins, serves as the sole source of lipid, carbohydrate, and protein. Such uptake, to a large part, occurs via the yolk sac, which utilizes the yolk lipoprotein components, following their degradation or modification, for re-synthesis of lipoproteins which are subsequently secreted and delivered to the embryo through the embryonic circulatory system. We also focus on the role of LDL modification in atherogenesis. The onset of atherosclerosis is a complex process, but there is now some evidence that

Mirjam Giefing Miriam Kamper Brenda Omeze Barbara Schmidinger Anna Weijler

During oogenesis in the chicken, the yolk precursors (e.g., vitellogenin and VLDL) are synthesized by the maternal liver under stringent hormonal control (E2) and taken up into the oocyte via receptor-mediated endocytosis (LRs). After ovulation and fertilization, a major feature of development is the formation of a series of extraembryonic structures including the amnion, chorion, allantois and yolk sac membranes (modified from http://chickscope.beckman.uiuc.edu/). Inset: The yolk sac is a layer of tissue growing over the surface of the yolk containing area vasculosa with blood vessels (bv), endothelial cells (EC), and an inner single layer of endodermal epithelial cells (EEC) with endocytic LRs and basement membrane (bm). A major role of the yolk sac is the uptake of nutrients from the yolk, their degradation and/or modification for re-synthesis and secretion into the embryonic circulation.

SELECTED PUBLICATIONS Schmidinger B, Weijler AM, Schneider W, Hermann M. Hepatosteatosis and estrogen increase apolipoprotein O production in the chicken. Biochimie 2016;127: 37-43. PMID: 27126072 Kamper M, Manns CC, Plieschnig JA, Schneider WJ, Ivessa NE, Hermann M. Estrogen enhances secretion of apolipoprotein B-100 containing lipoproteins by BeWo cells. Biochimie 2015;112:121-128. PMID: 25765953 Eresheim C, Plieschnig J, Ivessa NE, Schneider WJ, Hermann M. Expression of microsomal triglyceride transfer protein in lipoprotein-synthesizing tissues of the developing chicken embryo. Biochimie 2014;101: 67-74. PMID: 24394625

22


MFPL - 2016 RESEARCH GROUPS

JOACHIM HERMISSON

Theoretical population genetics The work of the Mathematics and Biosciences Group (MaBS) is on theoretical population genetics and evolutionary ecology.

Does adaptive evolution typically proceed in many small steps or fewer larger ones? This classical evolutionary question for the “genetic basis of adaptation” has previously been addressed in theoretical models that do not account for the mode of environmental change that causes the selection pressure. Kopp and Hermisson (2009) demonstrate that this ecological information indeed plays a crucial rule: If the environment changes slowly relative to the adaptive potential of a population (small gamma), the step sizes alpha will typically be small. In contrast, large steps are expected for fast changes, when the speed of adaptation is only limited by the mutation rate.

Phenotypic approaches It is widely appreciated (and ever better understood) that the genetic basis of most quantitative traits consists of complex gene networks. However, when and how gene interactions (epistasis) affect evolutionary processes is far less clear. In a series of articles, we have studied the evolutionary role of epistasis in equilibrium and non-equilibrium systems. A special research focus is on the effects on genetic variation and the adaptive process (epistatis and evolvability) and on the evolution of the genotype-phenotype map (robustness, canalization, and modularity).

Evolution is the unifying theory of the biological sciences, and our aim is to design advanced mathematical methods and models that account for the biological complexity involved in most evolutionary processes. Complexity arises on all levels of biological organization: molecular, organismal, and ecological. The key issues of evolutionary research, such as adaptation and speciation, are usually addressed in special sub-disciplines for each of these levels, i.e. molecular population genetics, quantitative genetics, and evolutionary ecology. We work on all three fields with the special goal to create an integrative approach, using a combination of different models, concepts, and methods. Methods include analytical work (stochastic processes, differential equations), extensive computer simulations, and statistical data analysis.

Ecologically motivated approaches The vast majority of population genetic models work under the assumption of a constant fitness landscape. Since fitness depends on variable environments, this is an idealization. Natural fitness landscapes will change over space and time. And because an important aspect of an individual’s environment is the composition of phenotypes in its own population, fitness will also depend on allele frequencies. The aim of this third line of our research is to combine genetic models with ecological factors. Recent studies have focused on conditions for speciation in spatially structured populations with gene-flow (parapatric speciation).

Molecular approaches The availability of DNA polymorphism data on a genome-wide scale (“population genomics”) is arguably the most significant development in evolutionary research today. In this context, the characterization of the adaptive process on the level of the molecular genotype is a primary research focus in our group. Our aim is to extend the population genetic theory of molecular adaptation to a broader range of biological scenarios. Quantities of interest are fixation probabilities and fixation times and the expected footprint of selection on linked neutral variation (so-called selective sweeps).

SELECTED PUBLICATIONS Bank C, Hermisson J, Kirkpatrick M. Can reinforcement complete speciation? Evolution. 2012 Jan;66(1):229-39. PMID: 22220877 Rueffler C, Hermisson J, Wagner GP. Evolution of functional specialization and division of labor. Proc Natl Acad Sci U S A. 2012 Feb 7;109(6):E326-35. PMID: 22308336 Horton MW, Hancock AM, Huang YS, Toomajian C, Atwell S, Auton A, Muliyati NW, Platt A, Sperone FG, Vilhjálms-son BJ, Nordborg M, Borevitz JO, Bergelson J. Genome-wide patterns of genetic variation in worldwide Arabidopsis thaliana accessions from the RegMap panel. Nat Genet. 2012 Jan 8;44(2):212-6. PMID: 22231484

23

Joachim Hermisson

TEAM Andrea Fulgione


MFPL - 2016 RESEARCH GROUPS

REINHOLD HOFBAUER

Consequences of carnitine deficiency and CSF-1 inhibition Although both research areas have a completely different biological background, signalling processes are very important for the transcriptional activation of genes taking place under carnitine deprivation and CSF-1 inhibition.

Reinhold Hofbauer

TEAM

nomics). The results of this research will provide better insight in metabolic aspects of pathologies and their regulation as well as mitochondrial function.

The first research task is dealing with the effects L-carnitine as a nutrigenomical metabolite exerts upon gene expression. We study carnitine deficiency, itself defining a very critical clinical condition, followed by carnitine supplementation in an artificial model system in human liver and fibroblast cells. This cell culture model defines sharp metabolic condition comparable to a patient situation, a precondition to study changes on mRNA expression levels. These promoter specific processes triggered by L-carnitine will be analysed by a variety of molecular techniques, including chip screen analysis, real time RT-PCR, reporter gene and (super) band shift assays. We already have identified genes directly involved in the transcriptional regulation of the “L-carnitine effect”, thus being able to approach clinical pathologies of hyperlipidemia, insulin resistance and type 2 diabetes mellitus, which are often very closely related. We primarily want to reveal so called “candidate or susceptibility” genes, which are associated with these diseases and have an increased sensitivity to diet (= main goal of nutrige-

The second research project is tracing the effects associated with inhibition of the macrophage colony-stimulating factor (CSF-1), which plays a key role in a wide variety of biologic processes. It primarily acts on cells of the mononuclear phagocyte lineage by controlling the differentiation, proliferation and survival of precursor cells as well as the activation of mature macrophages. As the latter are present in many tissues, CSF-1 also has a role in the pathogenesis of several disorders including cancers, because it regulates the production of MMPs and the uPA gene, which are heavily involved in tissue remodelling and tumour invasion. In view of the key role of CSF-1 in tumour progression, we have investigated whether inhibition of CSF-1 expression can serve as a valuable tool to fight tumour growth and decrease the risk of metastasis. Microarray analyses have revealed very promising candidate genes that are re- or induced during CSF-1 inhibition. In theory their inhibition should enhance the inhibitory effect of CSF-1 specific antibodies or RNAi. Additional preclinical animal studies with additional inhibitory agents (monoclonal antibodies, RNAi) delineated from chip screen candidates are the next experimental aims.

Theresa Kleissner Alexander Panzenböck Stefan Sack Eva Steiner Lars Zver

The pivotal role of L-carnitine for the mitochondrial lipid metabolism

SELECTED PUBLICATIONS Blake SM, Strasser V, Andrade N, Duit S, Hofbauer R, Schneider WJ, Nimpf J. Thrombospondin-1 binds to ApoER2 and VLDL receptor and functions in postnatal neuronal migration. EMBO J. 2008 Nov 19;27(22):3069-80PMID: 18946489 Kienesberger K, Pordes AG, Völk TG, Hofbauer R. L-carnitine and PPARα-agonist fenofibrate are involved in the regulation of Carnitine Acetyltransferase (CrAT) mRNA levels in murine liver cells. BMC Genomics. 2014 Jun 24;15:514 PMID: 24962334

24


MFPL - 2016 RESEARCH GROUPS

N. ERWIN IVESSA

Protein biogenesis and degradation from the ER We are interested in the molecular characterization of a quality control system that operates in the endoplasmic reticulum (ER) to ensure that only properly folded proteins will be released.

to prolong their half-lives. Furthermore, the requirement of N-linked glycan trimming for ERAD was shown, and from studies with mutant cell lines with defects in N-glycan assembly the activities of one or more ER Îą1,2-mannosidases could be implicated in ERAD. Interaction partners of ERAD substrate proteins in these mutant cell lines will be identified and further characterized. Another aspect of this project deals with the precise intracellular localization of the ERAD pathway of glycoproteins by indirect immunofluorescence and confocal laser scanning microscopy using appropriate marker proteins.

The ERAD substrate RI332-6HA (B, D) co-localizes with the ER marker calnexin (A, C), but hardly with the marker for the ER-Golgi-intermediate-compartment, ERGIC53 (E, F) in Chinese hamster fibroblast cells.

The role of MTP and PDI in the assembly and secretion of atherogenic lipoprotein particles. Microsomal triglyceride transfer protein (MTP) is a lipid transfer protein required for the assembly and secretion of very low density lipoproteins (VLDL). Active MTP is a heterodimer containing a 58 kDa subunit identified as protein disulfide isomerase (PDI). The MTP complex catalyzes the loading of apolipoprotein B (apoB) with lipids and/or the translocation of apoB into the lumen of the ER. We are studying the effect of estrogen treatment on MTP activity and on the regulation of VLDL secretion that is also determined by lipid availability and apoB degradation. In this context, the consequence of altered intracellular MTP activity on VLDL assembly and secretion is being analyzed. Another aspect of the project is concerned with the mechanism of retention of the MTP complex in the ER.

Misfolded polypeptides are retro-translocated from the ER to the cytosol, where they become poly-ubiquitinated and destroyed by proteasomes. ER-associated degradation (ERAD) is of relevance for a variety of genetically inherited, neurodegenerative, and virally transmitted diseases with protein folding defects. We have previously shown that a truncated form of ribophorin I, a model glycoprotein for ERAD, is degraded by the ubiquitin/proteasome system. The role of N-linked glycans in ERAD was pinpointed as temporary retention devices in the ER. Thus, interaction of N-glycosylated substrates with the calnexin cycle appears

SELECTED PUBLICATIONS Ivessa NE, Rehberg E, Kienzle B, Seif F, Hermann R, Hermann M, Schneider WJ, Gordon DA. Molecular cloning, expression, and hormonal regulation of the chicken microsomal triglyceride transfer protein. Gene. 2013 Jul 1;523(1):1-9. PMID: 23542778 Eresheim C, Plieschnig J, Ivessa NE, Schneider WJ, Hermann M. Expression of microsomal triglyceride transfer protein in lipoprotein-synthesizing tissues of the developing chicken embryo. Biochimie. 2014 Jun;101:67-74. PMID: 24394625 Kamper M, Manns CC, Plieschnig JA, Schneider WJ, Ivessa NE, Hermann M. Estrogen enhances secretion of apolipoprotein B-100 containing lipoproteins by BeWo cells. Biochimie. 2015 May;112:121-8. PMID: 25765953

25

N. Erwin Ivessa


MFPL - 2016 RESEARCH GROUPS

MICHAEL F. JANTSCH

Mechanisms and consequences of RNA-editing RNA-editing by adenosine deaminases acting on RNA (ADARs) converts adenosines to inosines.

Editing as a signalling mark Complete loss of editing leads to elevated interferon signalling and embryonic death. This suggests that the presence of Inosines marks endogenous RNAs as “self�. We are deciphering the types of RNAs that need to be edited to prevent interferon signalling and aim at analysing the underlying pathways.

As inosines are interpreted as guanosines by most cellular processes this type of editing can affect the coding potential of an RNA, its folding, stability, or localization. ADAR mediated editing is widespread in metazoa and affects thousands of transcripts in the human transcriptome. RNA editing is an essential process that regulates processes such as hematopoiesis, smooth muscle contraction, or interferon signalling.

Michael F. Jantsch

Editing in protein coding mRNAs A handful of highly conserved protein coding targets for A to I editing are known today. To understand the impact of editing on these RNAs and their encoded proteins we have generated transgenic mice impaired in editing the Filamin A pre-mRNA. These mice show altered smooth muscle contractions with important consequences for the cardiovascular system.

TEAM Prajakta Bajad Celine Brunner Peter Burg Laura Cimatti Phillip Czermak Mamta Jain Michael Janisiw Utkarsh Kapoor Konstantin Licht Maja Stulic Mansoureh Tajaddod Cornelia Vesely

A

Exon

B

ADAR ExonA I

unedited dsRNA A A A

edited dsRNA I I I

A

I

A

A

MDA5 Ca2+

Glutamate Receptor

pri-miRNA

I Interferon Response

MAVS

pre-miRNA DICER

DROSHA

Ca2+

C

I

mRNA

RISC

RNA editing affects the coding potential, folding, processing but also the recognition of nucleic acids by the innate immune system

The many consequences of RNA editing

SELECTED PUBLICATIONS Licht K, Kapoor U, Mayrhofer E, Jantsch MF. Adenosine to Inosine editing frequency controlled by splicing efficiency. Nucleic Acids Res 2016; 4(13):6398-408. PMID: 27112566 Vesely C, Tauber S, Sedlazeck F, Tajaddod M, von Haeseler A, Jantsch MF. ADAR2 induces re-producible changes in sequence and abundance of mature microRNAs in the mouse brain. Nucleic Acids Res 2014;42(19):12155-68. PMID: 25260591 Barraud P, Banerjee S, Mohamed W, Jantsch MF, Allain, FH. A bimodular nuclear localization signal assembled via an extended double-stranded RNA-binding domain acts as an RNA-sensing signal for transportin 1. P Natl Acad Sci USA 2014;111(18):E1852-61. PMID: 24753571

26


MFPL - 2016 RESEARCH GROUPS

VERENA JANTSCH

Meiosis in Caenorhabditis elegans Meiosis is the specialized cell division that generates haploid germ cells. It not only halves the chromosome content but also ensures genetic diversity by recombination. Errors in meiosis lead to infertility, pregnancy loss and clinical syndromes linked to mental retardation.

Research in my lab is directed towards the identification of genes and processes essential in meiotic prophase in an animal model system. Excellent forward and reverse genetics, transparency and easy cytological observation of all meiotic stages make the nematode Caenorhabditis elegans a powerful system for our studies. During the first meiotic division, faithful chromosome segregation is facilitated by the formation of a physical tether between the parental homologs (mediated by crossover recombination and cohesion). Crossovers require the introduction of DNA double-strand breaks, chromosome pairing, formation of the synaptonemal complex, and double-strand break repair by homologous recombination using the homolog as a repair template. In meiotic prophase I chromosomes are moved by cytoplasmic forces transferred to the nucleus via the SUN/KASH protein module (components of the inner and outer nuclear envelope that connect chromosomes to cytoplasmatic microtubules). Abrogation of chromosome movement, as we demonstrated with the sun-1(jf18) allele, leads to precocious synapsis involving non-homologous chromosomes or self synapsis.

We study the nature of chromosome movement and its regulation. These forces stir chromosomes, helping to bring homologs together and to inhibit undesired interactions. We investigate(d) SUN-1 post-translational modifications and their role in coordination of meiotic events to ensure the formation of a crossover on each chromosome. In addition, we discovered that SUN-1 is an integral part of a meiotic surveillance mechanism that coordinates chromosome synapsis with the obligate crossover on each chromosome. During meiosis programmed double strand break induction leads to formation of about 12 breaks per chromosome in C. elegans. In worms strictly only one of these matures into a crossover product. The rest of these gets resolved or dissolved in a non-crossover manner. We also investigate the role of the RTR complex in meiotic recombination. It is comprised of a RecQ helicase, type 1A topoisomerase, and the structural protein RMI1 (RMH-1 in C. elegans). We revealed that RMH-1 is an essential actor during meiosis, being involved in non-crossover production, crossover designation, reliable crossover resolution and the off-center position of crossovers on chromosomes. Currently we investigate its pro-crossover role in meiosis.

In early C. elegans meiosis one end of each chromosome attaches to the nuclear envelope via meiosis-specific protein complexes (filled blue, yellow and orange circles). Cytoplasmic tubulin (pink bars) provide the driving forces that move chromosomes (blue and brown lines) vigorously along the surface of the inner nuclear envelope. Cytoplasmic driving forces are transmitted to the nucleus via SUN-KASH protein complexes (green and magenta ellipses). Concomitantly the synaptonemal complex forms between homologous chromosomes (pink ladder like lines).

Picture of a nucleus, which will develop into an egg, imaged with structured Illumination Microscopy. It shows the aligned parental chromosomes (cyan). In yellow: the spots where DNA breaks will mature into a connection between the parental chromosomes. Surprisingly, this is where the RMI complex also acts (magenta).

SELECTED PUBLICATIONS Jagut M, Hamminger P, Woglar A, Millonigg S, Paulin L, Mikl M, Dello Stritto MR, Tang L, Habacher C, Tam A, Gallach M, von Haeseler A, Villeneuve AM, Jantsch V. Separable Roles for a Caenorhabditis elegans RMI1 Homolog in Promoting and Antagonizing Meiotic Crossovers Ensure Faithful Chromosome Inheritance. Plos Biology 2016;14(3):e1002412. PMID: 27011106 Woglar A, Daryabeigi A, Adamo A, Habacher C, Machacek T, La Volpe A, Jantsch V. Matefin/SUN-1 Phosphorylation Is Part of a Surveillance Mechanism to Coordinate Chromosome Synapsis and Re-combination with Meiotic Progression and Chromosome Movement. Plos Genet 2013;9(3):e1003335. PMID: 23505384 Penkner AM, Fridkin A, Gloggnitzer J, Baudrimont A, Machacek T, Woglar A, Csaszar E, Pasierbek P, Ammerer G, Gruenbaum Y, Jantsch V. Meiotic chromosome homology search involves modifications of the nuclear envelope protein Matefin/SUN-1. Cell 2009;139 :920-933. PMID: 19913286

27

Verena Jantsch

TEAM Maria Rosaria Dello Stritto NicolĂĄs GarcĂ­a Seyda Angela Graf Eva Janisiw Jana Link Dimitra Paouneskou Nicola Silva Maria Velkova Mona von Harder


MFPL - 2016 RESEARCH GROUPS

FRANZ KLEIN

Chromosome structure and meiotic recombination We study meiotic recombination and chromosome segregation in S. cerevisiae as model organism to understand the interplay between chromosome structure and recombination.

Franz Klein

TEAM Doris Chen Marta Galova Lingzhi Huang Elisa Mayrhofer Kuldepp Nangalia Viktoria Prast Katharina Semrad Prieler Silvia Magdalena Vesely Soma Zoter

During the initiation of meiotic recombination whole double-stranded chromosomal pieces can get excised. Here, we have mapped their ends genome wide by deep sequencing. We show a region around a recombination hotspot on chromosome 4. Each fragment is shown by a half circle. Positions are given in nucleotides. Orange lines represent the strength of individual DNA break sites mapped by Pan et al. 2011.

During meiosis, the genetic content of a diploid cell is reduced to half, a prerequisite for the production of gametes and for sexual reproduction. Our experience shows that the important processes are conserved between yeast and man, so that many of our findings can be generalized. Chromosomes are organized as dynamic structures with distinct micro-domains, such as axis and loop regions, and macro-domains, such as recombination rich – or poor regions, centromeres, telomeres and others. In meiosis, cohesins and axial element proteins

shape the chromosome and mediate recombination as well as correct chromosome segregation. We have established a high-resolution map of protein-chromosome interactions by microarray-analysis, which we currently improve using deep sequencing. We also study in detail the process of chromosome synapsis, a structure that mainly exists to fine-tune recombination pathways. Eventually, these pathways decide over the integrity of the resulting gamete’s genome, and thus over the health of the new individual, that originates from these gametes.

SELECTED PUBLICATIONS Sun X, Huang L, Markowitz TE, Blitzblau HG, Chen D, Klein F, Hochwagen A. Transcription dynamically patterns the meiotic chromosome-axis interface. Elife. 2015 Aug 10;4. PMID: 26258962 Xaver M, Huang L, Chen D, Klein F. Smc5/6-Mms21 prevents and eliminates inappropriate recombination intermediates in meiosis. PLoS Genet. 2013;9(12):e1004067. PMID: 24385936 Panizza S, Mendoza MA, Berlinger M, Huang L, Nicolas A, Shirahige K, Klein F. Spo11-accessory proteins link double-strand break sites to the chromosome axis in early meiotic recombination. Cell. 2011 Aug 5;146(3):372-83. PMID: 21816273

28


MFPL - 2016 RESEARCH GROUPS

ALWIN KÖHLER

Nuclear Pores - Regulators of chromatin and membrane dynamics The cell nucleus is like a fortified city, in which lipid membranes form a defensive wall, and nuclear pore complexes (NPCs) function as gates that control the transport of cargo across these walls. Notably, these city gates not only provide a point of controlled entry and departure, but are zones of molecular communication and trade.

Ubiquitin Signalling on Chromatin Related to our interest in gene expression, we are studying the modification of chromatin by ubiquitin. When appended to histones, ubiquitin functions as an important epigenetic switch to regulate multiple steps of transcription. Histone H2B monoubiquitination is mediated by the E2 and E3 enzymes Rad6 and Bre1 (Gallego et al., PNAS 2016). Their activity is counteracted by a deubiquitinase of the SAGA complex (Köhler et al., Cell 2010). We are dissecting the structure and function of this intricate molecular machinery to understand its biology in health and disease.

My group is interested in three main areas: Adaptors at the Nuclear Pore The function of NPCs in nucleocytoplasmic transport is well studied. However, NPCs carry out additional, unconventional duties. In particular, they impact on chromatin architecture and gene activity by physically interacting with the genome. A key question is how NPCs ‘communicate’ with the gene expression machinery. We recently discovered that the NPC basket employs dedicated Adaptors (Figure 1) such as TREX-2 and Mediator to regulate RNA Polymerase II (Schneider et al., Cell 2015). These findings are stepping stones for further mechanistic analyses on how NPCs participate in decoding the genome.

Shaping the Nuclear Membrane The nuclear envelope encloses and protects the genome. NPCs need to promote the controlled exchange of molecules between nucleus and cytoplasm without disrupting this membrane barrier. Thus, the assembly and insertion of new NPCs is tightly controlled. NPCs are embedded in holes formed by the fusion of the outer and inner nuclear membranes. We have discovered a new function for the NPC basket in remodelling the nuclear membrane to promote NPC and nuclear envelope integrity (Mészáros et al., Dev Cell, 2015). Future studies aim at understanding how various NPC proteins cooperate to sculpt their membrane environment. We use biochemical, structural, genome-wide and cell biological methods to address these questions. Functional reconstitution is central to our approach, as the ability to reconstruct systems from scratch offers key insights into what is minimally needed and how mechanisms emerge from component parts.

The NPC employs Adaptors to contact and regulate the core transcription machinery.

SELECTED PUBLICATIONS Gallego LD, Ghodgaonkar Steger M, Polyansky AA, Schubert T, Zagrovic B, Zheng N, Clausen T, Herzog F, Köhler A. Structural mechanism for the recognition and ubiquitination of a single nucleosome residue by Rad6-Bre1. Proc Natl Acad Sci U S A. 2016 Sep 20;113(38):10553-8. PMID: 27601672 Schneider M, Hellerschmied D, Schubert T, Amlacher S, Vinayachandran V, Reja R, Pugh BF, Clausen T, Köhler A. The Nuclear Pore-Associated TREX-2 Complex Employs Mediator to Regulate Gene Expression. Cell. 2015 Aug 27;162(5):1016-28. PMID: 26317468 Mészáros N, Cibulka J, Mendiburo MJ, Romanauska A, Schneider M, Köhler A. Nuclear pore basket proteins are tethered to the nuclear envelope and can regulate membrane curvature. Dev Cell. 2015 May 4;33(3):285-98. PMID: 25942622

29

Alwin Köhler

TEAM Jakub Cibulka Laura Gallego Maria Jose Mendiburo Anete Romanauska Maren Schneider Tobias Schubert


MFPL - 2016 RESEARCH GROUPS

ROBERT KONRAT

Computational Biology and Biomolecular NMR Spectroscopy The sequencing of the human genome has provided a ‘parts list’ of the human inventory comprising potential therapeutic targets for the pharmaceutical and biotechnology industry.

Robert Konrat

TEAM Sven Brüschweiler Nicolas Coudevylle Andrea Flamm Leonhard Geist Tanja Gesell Clemens Kauffmann Georg Kontaxis Karin Ledolter Borja Mateos Lorenz Perschy Andreas Prattinger Tomas Sara Thomas Schwarz Marco Sealey Mate Somlyay Anna Zawadzka-Kazimierczuk

The figure serves as an overview of currently pursued research topics in the group. A central structural biology topic in the group is the structural analysis of the oncogenic transcription factor myc and its differentially regulated target genes. (Top) We have used NMR spectroscopy to analyse the C-terminal (DNA-binding and dimerisation) domain of myc in the individual stages of transcription. Additionally, our structural analysis of myc target genes provided a first glimpse on myc’s cell transforming potential. (Bottom) NMR spectroscopy is a unique tool to identify and analyse high-energy states of proteins.

In order to cope with this huge number of targets, we introduced a new theoretical conception of protein structural biology (meta-structure) that can be used for protein sequence-to-function annotation and drug design. A hallmark of our research is the integrative application of this novel conception and sophisticated NMR spectroscopy directed towards a better understanding of fundamental biological processes.

Finally, as much of protein function is predicated on dynamics, we are developing novel methodological approaches that combine biochemistry, bioorganic chemistry and NMR spectroscopy to unravel the microscopic details of functionally important protein plasticity.

SELECTED PUBLICATIONS Henen MA, Coudevylle N, Geist L, Konrat R. Toward rational fragment-based lead design without 3D structures. J Med Chem. 2012 Sep 13;55(17):7909-19. PMID: 22889313 Brüschweiler S, Konrat R, Tollinger M. Allosteric communication in the KIX domain proceeds through dynamic re-packing of the hydrophobic core. ACS Chem Biol. 2013 Jul 19;8(7):1600-10 PMID: 23651431 Kurzbach D, Schwarz TC, Platzer G, Höfler S, Hinderberger D, Konrat R. Compensatory adaptations of structural dynamics in an intrinsically disordered protein complex. Angew Chem Int Ed Engl. 2014 Apr 7;53(15):3840-3. PMID: 24604825

30


MFPL - 2016 RESEARCH GROUPS

PAVEL KOVARIK

Signalling and gene expression in inflammation Defence against infections requires efficient activation of an inflammatory response and timely reestablishment of immune homeostasis once the pathogen has been eradicated. Unproductive responses result in infectious disease whereas failures in homeostatic processes cause tissue damage and prevent healing. We study the molecular wiring of balanced inflammatory responses in three areas:

2) Regulation of immune homeostasis by mRNA decay The mRNA-destabilizing protein tristetraprolin (TTP) is a key factor regulating the elimination of inflammatory mRNAs (e.g. cytokines). We investigate the function of TTP in the maintenance and reestablishment of immune homeostasis after an insult. Our current study (Sedlyarov et al., Mol Syst Biol 2016) demonstrates that TTP is essential for the initiation of the resolution phase of inflammation. By using PAR-iCLIP, a genome wide high-resolution mapping of TTP binding sites, in the transcriptome of immunostimulated primary mouse macrophages the study establishes a comprehensive map of cis-acting elements controlling mRNA stability during inflammation. The binding sites are functionally annotated and publically accessible via the TTP Atlas webpage.

1) Defence against bacterial pathogens We investigate the mechanisms of efficient activation of the immune system as well as the molecular principles of timely return into immune and tissue homeostasis in models of invasive infection with pathogenic bacteria such as Streptococcus pyogenes. Our recent study (Castiglia et al., Cell Host & Microbe 2016) describes that activation of type I interferon (IFN) signalling in the context of S. pyogenes infection balances the immune response by ensuring that the production of the important pro-inflammatory cytokine IL-1ß reaches protective yet not destructive levels. This function of type I IFNs is essential for preventing lethal systemic hyperinflammation.

3) Regulation of the transcription cycle in cytokine responses Mechanisms which prevent uncontrolled repetition of the transcription cycle once the upstream signal has vanished are not understood. We have recently described a novel way of transcription control in the JAK-STAT signalling pathway: processive transcription initiated by STAT1 feeds back to downregulate STAT1 promoter occupancy and, consequently, limit the transcription output (Wiesauer et al., Mol Cell Biol 2016). Our current data suggest that chromatin-associated signalling acts in cis to allow cross talk between promoter-bound transcription factor(s) and the transcription machinery. In this context, we investigate the role of the CDK8 which we have previously identified as the key factor regulating the STAT transcription factors in a chromatin-associated manner (Bancerek et al., Immunity 2013).

TTP Atlas: Functionally annotated atlas of TTP binding sites in the macrophage transcriptome (Sedlyarov et al., Mol Syst Biol 2016; logo Florian Ebner).

SELECTED PUBLICATIONS Castiglia V, Piersigilli A, Ebner F, Janos M, Goldmann O, Damböck U, Kröger A, Weiss S, Knapp S, Jamieson AM, Kirschning C, Kalinke U, Strobl B, Müller M, Stoiber D, Lienenklaus S, Kovarik P. Type I Interferon Signaling Prevents IL-1β-Driven Lethal Systemic Hyperinflammation during Invasive Bacterial Infection of Soft Tissue. Cell Host Microbe 2016;19(3):375-87. PMID: 26962946 Sedlyarov V, Fallmann J, Ebner F, Huemer J, Sneezum L, Ivin M, Kreiner K, Tanzer A, Vogl C, Hofacker I, Kovarik P. Tristetraprolin binding site atlas in the macrophage transcriptome reveals a switch for inflammation resolution. MOL SYST BIOL 2016;12(5):868. PMID: 27178967 Bancerek J, Poss ZC, Steinparzer I, Sedlyarov V, Pfaffenwimmer T, Mikulic I, Dölken L, Strobl B, Müller M, Taatjes DJ, Kovarik P. CDK8 Kinase Phosphorylates Transcription Factor STAT1 to Selectively Regulate the Interferon Response. Immunity 2013:250-62. PMID: 23352233

31

Pavel Kovarik

TEAM Virginia Castiglia Florian Ebner Kevin Eislmayr Masa Ivin Renata Kleinová Anna Mildner Lucy Sneezum Iris Steinparzer Terezia Vcelkova


MFPL - 2016 RESEARCH GROUPS

HEINRICH KOWALSKI

Molecular and structural biology of picornaviruses Our research is dedicated to unravelling the molecular mechanisms underlying assembly and uncoating of small RNA viruses belonging to the family of picornaviridae, to elucidate the role of host factors required for their efficient replication and to create structural surrogates for the difficult to cultivate C-species rhinoviruses.

Strategy towards structural analysis of non-cultivatable viruses Focusing on C-type rhinoviruses, we have been engaged in the establishment of a baculovirus expression system for production of virus-like particles (VLPs) as surrogates of these difficult to grow respiratory viruses for use as a multivalent immunogen and to facilitate structural analysis.

Infections by picornaviruses present with a wide variety of clinical manifestations, ranging from the mild “common cold” to severe disorders, such as pneumonia, myocarditis, pancreatitis, meningitis and flaccid paralysis. Except for poliovirus no effective vaccines are presently available and despite intense efforts no antiviral small molecule has yet been approved for broad human applications. By molecularly characterizing as yet ill-defined steps in the life-cycle of picornaviruses we also hope to contribute to the discovery of novel drug targets.

Role of N-myristoyltransferases in the picornavirus life-cycle Using HAP1 knock-out cells, siRNA knock-down and small molecule inhibition combined with bioorthogonal “click” chemistry, we have investigated the role of human N-myristoyltransferase isozymes 1 and 2 (HuNMT1 and HuNMT2) in the covalent modification of coxsackievirus B3 (CVB3) capsid proteins VP0/VP4. This study revealed an eminent role of the C14:0 saturated fatty acid moiety in the assembly of infectious virions. Strikingly, we find that this co-translationally acquired modification also governs efficient penetration of the RNA genome from endocytic compartments following virus entry into the host cell.

We are presently studying three crucial aspects related to picornavirus biology:

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Effect of NMT isozyme knock-out on CVB3 production. (A) Western blot analysis of NMT1 and NMT2 protein levels in wild type (wt), NMT1 single and NMT2 single knockout Hap1 cells. Tubulin was used as loading control. (B) NMT1 but not NMT2 knock-out significantly reduces production of progeny CVB3 compared to wt HAP1 as measured by TCID50/ml.

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Gregor Augustin Tanja Gumpenberger Irena Čorbić Ramljak Julia Stanger

log TCID50/ml

TEAM

Higher structure organization of the rhinoviral RNA genome The aim of the project is to determine the conformation of the viral RNA in- and outside the virion of representative rhinoviruses using SHAPE-MaP, a chemical RNA modification method combined with massively parallel sequencing. We also plan to produce pseudovirus particles of rhinovirus type 2 featuring progressively truncated genomes to elucidate organizational principles of the encapsidated RNA through cryo-EM analysis and 3D-image reconstruction. The results shall considerably advance our understanding of the rules governing the assembly and disassembly (uncoating) of rhinovirus particles.

Ha

Heinrich Kowalski

SELECTED PUBLICATIONS Weiss VU, Subirats X, Kumar M, Harutyunyan S, Gösler I, Kowalski H, Blaas D. Capillary electrophoresis, gas-phase electrophoretic mobility molecular analysis, and electron microscopy: effective tools for quality assessment and basic rhinovirus research. Methods Mol Biol. 2015;1221:101-28. PMID: 25261310 Harutyunyan S, Kowalski H, Blaas D. The Rhinovirus subviral a-particle exposes 3’-terminal sequences of its genomic RNA. J Virol. 2014 Jun;88(11):6307-17. PMID: 24672023 Harutyunyan S, Kumar M, Sedivy A, Subirats X, Kowalski H, Köhler G, Blaas D. Viral uncoating is directional: exit of the genomic RNA in a common cold virus starts with the poly-(A) tail at the 3’-end. PLoS Pathog. 2013;9(4):e1003270. PMID: 23592991

32


MFPL - 2016 RESEARCH GROUPS

CLAUDINE KRAFT

Regulation and signalling in autophagy Our group aims at understanding how the molecular machinery of the cellular waste disposal system works.

is absolutely essential for autophagy function, yet how Atg1/ULK1 regulates autophagosome formation at different stages during the process and the underlying signalling and mechanistic events remain unclear. We want to dissect the mechanisms by which Atg1/ ULK1 kinase activity is regulated and how Atg1/ULK1 itself regulates downstream events in autophagy. We employ a diverse range of classical in vitro and in vivo methods. Recently we have established a complementary synthetic in vivo approach. This approach allows us to achieve higher spatial and temporal resolution when studying autophagy in living cells and to define what is necessary and sufficient in vivo at specific steps in autophagy. Most of our in vivo work is performed in budding yeast, which is an excellent model organism for studying the mechanisms of autophagy due to the highly conserved nature of the process up to mammals. Additionally, we also use mammalian cell culture systems to test our findings for conservation in higher eukaryotes. Deciphering the mechanisms governing autophagy will help to better understand the molecular basis of diseases associated with autophagy dysfunction, including cancer and neurodegenerative disorders like Alzheimer’s disease.

One of the major cellular responses when nutrients are scarce is the activation of a degradation pathway termed autophagy, in which the cell digests its own components. This mechanism provides the cell with nutrients to maintain vital cellular functions during times of starvation. It also serves a house-keeping function by eliminating superfluous or damaged organelles, misfolded proteins, and invading microorganisms, thereby acting as the cellular waste disposal and recycling system. Even though autophagy has been extensively studied from yeast to mammals, the molecular events of starvation sensing and autophagy induction remain elusive. One of the key regulators of autophagy is the target of rapamycin (TOR) kinase, which shuts off autophagy in the presence of nutrients and growth factors. Nutrient-limiting conditions lead to TOR kinase inactivation and autophagy induction, which results in the sequestration of cytosol and organelles into a double-membrane organelle followed by their subsequent delivery to the vacuole/lysosome for breakdown and recycling (Figure 1-3). A major conserved upstream regulator of autophagy is the Atg1 (yeast) / ULK1 (mammals) kinase complex, which is thought to be a direct target of nutrient signalling by TOR. The activity of the Atg1/ULK1 complex

Figure 1. The Atg1/ULK1 kinase complex receives the autophagy-inducing signal, resulting in the initiation of autophagy. An isolation membrane expands and engulfs cytoplasmic material to form a double-membraned autophagosome. After fusion with the lysosome/vacuole the contents of the autophagosome are degraded and recycled for further use.

Figure 2. An oversized autophagic cargo (magenta) is engulfed by an isolation membrane (green) in a yeast cell, monitored by live cell fluorescence imaging.

Figure 3. Native cargo (magenta) in an autophagosome (green) visualized by immuno-electron microscopy.

SELECTED PUBLICATIONS Torggler R, Papinski D, Brach T, Bas L, Schuschnig M., Pfaffenwimmer T., Rohringer S, Matzhold T, Schweida D, Brezovich A and Kraft C. Two Independent Pathways within Selective Autophagy Converge to Activate Atg1 Kinase at the Vacuole. Mol Cell. 2016 Oct 20;64(2):221-235. PMID: 27768871 Pfaffenwimmer T, Reiter W, Brach T, Nogellova V, Papinski D, Schuschnig M, Abert C, Ammerer G, Martens S, Kraft C. Hrr25 kinase promotes selective autophagy by phosphorylating the cargo receptor Atg19. Embo Rep 2014;15(8):862-70. PMID: 24968893 Pfaffenwimmer T, Kijanska M, Stoffel I, Lee SS, Brezovich A, Lou JH, Turk BE, Aebersold R, Ammerer G, Peter M, Kraft C. Early Steps in Autophagy Depend on Direct Phosphorylation of Atg9 by the Atg1 Kinase. Mol Cell 2014;53(3):471-83. PMID: 24440502

33

Claudine Kraft

TEAM Levent Bas Akif Ciftci Mariya Licheva Daniel Papinski Sabrina Rohringer Martina Schuschnig Raffaela Torggler


MFPL - 2016 RESEARCH GROUPS

KARL KUCHLER

Host-Pathogen interactions & mechanisms of drug resistance & fungal pathogenesis We study the molecular mechanisms of fungal pathogenicity and fundamental problems in infection biology, using a combination of molecular, as well as genome-wide and systems biology approaches.

Candida albicans cells forming colonies of markedly different phenotypes on agar plates due to distinct chromatin modifications, which modulate transcriptional regulatory networks controlling morphogenesis.

Karl Kuchler

TEAM Aybala Eker Christa Gregori-Schüller Fabian Istel Sabrina Jenull Narakorn Khunweeraphong Filomena Nogueira Leonel Pereira Andriy Petryshyn Michael Riedelberger Bernhard Scheidl Raju Shivarathri Nathalie Uwamahoro Florian Zwolanek

Finally, we study structure-function relationships of fungal ABC multidrug transporters, and we pursue systems biology approaches to answer how the molecular cross-talk of stress response signalling pathways impact cellular growth control and ion homeostasis in simple model organisms such as baker’s yeast.

First, we use reverse genetics approaches to identify virulence and antifungal drug resistance genes in the most prevalent human fungal pathogens such as Candida glabrata and C. albicans. For instance, we have generated a genome-scale gene deletion collection of C. glabrata currently comprising some 700 single gene deletions. Further, we decipher the role of histone modification genes in morphogenetic switching, cell fate determination and virulence. We would like to define the genetic networks and signalling pathways facilitating immune evasion and driving invasion of host cells. We also study the genomic and genetic adaptations occurring in pathogen genomes during host niche or organ colonization and systemic dissemination. On the host side, we are studying the mechanisms of host-pathogen interaction and cytokine signalling response in primary dendritic cells / macrophages, as well as in mouse infection models, to define their contribution to virulence. Along this line, we delineate the interplay of adaptive and innate immunity in immune surveillance, and, particularly the role of Tec kinases and type I interferons in virulence and dissemination in host tissues and organs.

SELECTED PUBLICATIONS Hnisz D, Bardet AF, Nobile CJ, Petryshyn A, Glaser W, Schöck U, Stark A, Kuchler K. A histone deacetylase adjusts transcription kinetics at coding sequences during Candida albicans morphogenesis. PLoS Genet. 2012;8(12):e1003118. PMID: 23236295 Majer O, Bourgeois C, Zwolanek F, Lassnig C, Kerjaschki D, Mack M, Müller M, Kuchler K. Type I interferons promote fatal immunopathology by regulating inflammatory monocytes and neutrophils during Candida infections. PLoS Pathog. 2012;8(7):e1002811. PMID: 22911155 Tierney L, Linde J, Müller S, Brunke S, Molina JC, Hube B, Schöck U, Guthke R, Kuchler K. An Interspecies Regulatory Network Inferred from Simultaneous RNA-seq of Candida albicans Invading Innate Immune Cells. Front Microbiol. 2012 Mar 12;3:85. PMID: 22416242

34


MFPL - 2016 RESEARCH GROUPS

MARTIN LEEB

Molecular control of cell fate decisions Proper cell identity control is essential for normal development. My group investigates the molecular mechanisms of cell fate determination in mammals using high throughput screening platforms combined with defined cell culture systems.

Embryonic stem cells hold great potential for use in regenerative medicine and could be employed to treat diseases such as Parkinson’s and diabetes. This assumption is based on the fact that ES cells can differentiate into cell types of all three germ layers in vitro and can contribute to normal development in chimeric mice. However, whereas developmental progression and changes in cellular identity in the embryo unfold in a deterministic manner, in vitro differentiation is asynchronous and disorganized. As a consequence, robust protocols to control primary lineage decision experimentally do not exist and efficient differentiation into authentic cell types of clinical relevance remains largely elusive. Work in my lab aims to bridge this gap in knowledge by contributing to a broader understanding of the molecular mechanisms that determine cellular identity during the course of embryonic development. The discovery of haploid ES cells as a potent discovery platform for unbiased random mutagenesis-based screens in mammalian cells has enabled us to address this question in a high throughput approach. In screens performed over the last few years we have already identified a cohort of novel players involved in the exit from ES cell self-renewal. Future efforts in my laboratory will focus on the in depth functional analysis of selected candidate genes and pathways. For this we will use refined ES cell culture techniques together with defined differentiation protocols in order to dissect the regulatory cascade that controls differentiation in high molecular resolution. We will further seek to design strategies and experimental platforms that will allow us to dissect the genetics of cell identity decisions downstream of the exit from ES cell self-renewal.

A haploid karyotype allows detection of recessive mutations in genetic screens

Schematic overview of a screen to identify key players involved in ES cell differentiation.

SELECTED PUBLICATIONS Leeb M, Dietmann S, Paramor M, Niwa H, Smith A. Genetic exploration of the exit from self-renewal using haploid embryonic stem cells. Cell Stem Cell. 2014;14(3):385-93. PMID: 24412312 Leeb M, Wutz A. Derivation of haploid embryonic stem cells from mouse embryos. Nature. 2011;479(7371):131-4. PMID: 21900896 Leeb M, Pasini D, Novatchkova M, Jaritz M, Helin K, Wutz A. Polycomb complexes act redundantly to repress genomic repeats and genes. Genes Dev. 2010;24(3):265-76. PMID: 20123906

35

Martin Leeb

TEAM Elena Galimberti Andreas Lackner Christina Manakanatas Julia Ramesmayer Wanhui You


MFPL - 2016 RESEARCH GROUPS

THOMAS LEONARD

Structural biology of lipid-activated signal transduction Membranes are sites of intense signalling activity in eukaryotic cells. Essential processes such as autophagy, cytokinesis, exo- and endocytosis, and cytoskeletal remodelling depend on signal propagation at cellular membranes.

Thomas Leonard

TEAM Daniel Elsner Iva Lucic Linda Trübestein Freia von Raußendorf

second messengers can turn on signalling pathways at the membrane. Many of the lipid responsive human protein kinases belong to the AGC family of kinases, of which the paradigmatic lipid-regulated kinase is protein kinase C (PKC). We would like to understand how lipid-engagement by these protein kinases is coupled to their activation at the molecular level. Ultimately, we hope to elucidate common principles of the molecular mechanisms that govern lipid-mediated signal transduction.

Dysregulation of signal transduction at these sites is the cause of a number of hereditary and non-hereditary diseases, including Coffin-Lowry syndrome, spinocerebellar ataxia, myotonic dystrophy, and various cancers. Over 500 kinases and 130 phosphatases regulate signal transduction by phosphorylating or dephosphorylating their target proteins. Of the more than 500 kinases, 54 contain known lipid-binding or membrane-interacting domains, and whilst much is known about how these proteins are targeted to cellular membranes, very little is known about how membrane engagement is coupled to signal transduction. We are using a spectrum of biophysical (including X-ray crystallography), biochemical, and cell biological techniques to address two questions central to signal transduction at membranes. One of the most important consequences of the activation of cell surface receptors is the generation of small molecule second messengers. In addition to the freely diffusible second messengers such as cAMP and inositol triphosphate (IP3), a number of cellular second messengers are lipids. Despite being of fundamental importance to the exquisite spatial and temporal regulation of many cellular processes, the molecular mechanisms of lipid-mediated signal transduction are not well understood. Our goal is to understand how lipid

The second question relates to how signal transduction pathways are organized. Scaffolding of signalling proteins in the same pathway enhances specificity, promotes signal amplification by reducing noise, and, ultimately, improves signal propagation through the pathway. Membranes act as the scaffolds for many signalling reactions, including those involved in visual signalling in Drosophila and those involved in regulating cellular growth processes. Our studies are aimed at understanding how diverse signals are integrated, how substrate specificity is encoded not just at the kinase level, and the influence of the membrane environment on multi-component signalling hubs. This is an exciting area of research with frontiers in ageing, cancer, metabolic diseases such as diabetes, and obesity. Dimeric Rho-kinase (ROCK) comprises N-terminal kinase domains and C-terminal membrane-binding domains joined by a long, parallel coiled-coil of 107 nm. For comparison, the 80S eukaryotic ribosome is drawn to scale alongside. Removal of the membrane-binding domains or truncation of the coiled-coil has no effect on ROCK activity in vitro, but truncation of the coiled-coil blocks stress fiber formation in cells. We propose that the coiled-coil of ROCK functions like a molecular ruler, bridging the membrane to ROCK substrates in the actin cytoskeleton. (Truebestein et al., Nat Commun., 2015).

A dual-colour membrane translocation kinetics as a proxy for the stability of the autoinhibited state of protein kinase C. Mutations in conserved intramolecular, inter-domain interfaces are manifested in accelerated membrane translocation kinetics as a consequence of the increased exposure of the DAG-binding C1 domains. Small perturbations in the accessibility of these domains can be accurately quantitated by monitoring the translocation of wild type and mutant proteins in the same cell. (Lučić et al., J Mol Biol., 2016).

SELECTED PUBLICATIONS Truebestein L, Elsner DJ, Fuchs E, Leonard TA. A molecular ruler regulates cytoskeletal remodelling by the Rho kinases. Nat Commun. 2015 Dec 1;6:10029PMID: 26620183 Lučić I, Truebestein L, Leonard TA. Novel Features of DAG-Activated PKC Isozymes Reveal a Conserved 3-D Architecture. J Mol Biol. 2016 Jan 16;428(1):121-41. PMID: 26582574 Leonard TA, Różycki B, Saidi LF, Hummer G, Hurley JH. Crystal structure and allosteric activation of protein kinase C βII. Cell. 2011 Jan 7;144(1):55-66PMID: 21215369

36


MFPL - 2016 RESEARCH GROUPS

JOSEF LOIDL

Meiotic chromosome pairing and recombination Meiosis is a pivotal process in the sexual reproduction cycle: it compensates for the doubling of the chromosome number at fertilization and it provides the progeny with newly assorted sets of alleles, which is the basis of their genetic heterogeneity. Failures in meiosis may lead to gametes with aberrant chromosome numbers and thus to progeny with congenital defects.

Josef Loidl

A pair of conjugating Tetrahymena cells, each with a spindle-shaped generative micronucleus in meiotic prophase. (The large round structures are the somatic nuclei, which do not undergo meiosis). Shown in green are patches of a chromatin-associated protein, which is probably involved in the spatial separation of recombination and transcription.

the structural integrity of chromosomes. Compared to other eukaryotes, endowment with cohesins and their auxiliary proteins is poor; in particular, a single set of cohesins is active in mitosis and meiosis. In contrast, Tetrahymena has extra versions of condensin proteins that seem to have adopted a range of new functions in genome elimination. Another study is devoted to novel meiosis-specific members of the transcription initiation complex, of which we speculate that they may coordinate the spatial regulation of concurrent recombination and transcription activities.

We are studying meiotic chromosome organisation and behaviour in the protist Tetrahymena thermophila, which is an evolutionarily very distant relative of the common model organisms. This divergent system allows us to learn which adaptations and changes to the meiotic process have occurred in evolution. Ultimately, our studies will help to understand the origin and function of conserved meiotic features such as the SCs, the chromosomal bouquet, and the regulation of meiotic recombination. We are knocking out genes that are expressed in sexually reproducing cells and determine their function via mutant analysis. We have discovered numerous genes with a function in meiosis, which have no homologs in other organisms.

Finally, our group is hosting a project of the University of Vienna´s INDICAR programme. It investigates the role of chromatin remodelling in postmeiotic cells during the dedifferentiation of gamete nuclei into vegetative nuclei.

Other projects in our group investigate cohesins and condensins, protein complexes that are required for

SELECTED PUBLICATIONS Loidl J. Conservation and variability of meiosis across the eukaryotes. Annu. Rev. Genet 2016;50. PMID: 27686280 Loidl J, Lorenz A. DNA double-strand break formation and repair in Tetrahymena meiosis. Semin Cell Dev Biol 2016;54:126-34. PMID: 26899715 Lukaszewicz A, Shodhan A, Loidl J. Exo1 and Mre11 execute meiotic DSB end resection in the protist Tetrahymena. DNA repair 2015;35:137-43. PMID: 26519827

37

TEAM Takahiko Akematsu Emine Ali Rachel A. Howard-Till Anura Shodhan Miao Tian


MFPL - 2016 RESEARCH GROUPS

SASCHA MARTENS

Molecular mechanisms of autophagy Autophagy is an evolutionarily conserved and important process during which our cells digest or cannibalize small parts of themselves.

sosome within which the content is degraded. The degraded content can subsequently be used for the synthesis of factors that are essential for the survival of the cell.

Autophagy plays an essential role during starvation, the defence against pathogenic microorganisms, the removal of protein aggregates and the degradation of damaged organelles. Misregulated or defective autophagy can result in neurodegeneration and premature aging and is thus highly relevant to a plethora of human diseases.

Sascha Martens

TEAM

Although many genes that are important for autophagy have been identified we have only a very limited understanding of how this important and fascinating process is regulated and executed. Thus, the challenge now is to assign functions to these genes in order to gain a better understanding of the mechanisms that orchestrate autophagy.

Autophagy is induced by an upstream signal such as starvation, the detection of pathogenic microorganisms in the cytosol or by damaged mitochondria. This signal triggers the most enigmatic and fascinating step of autophagy, the de novo formation of autophagosomes. Initially a small double membrane bound structure is formed, which grows and adopts the shape of a cup. This cup-shaped structure eventually fuses at its rims to form a double membrane bound organelle enclosing a part of the cell’s cytoplasm. The autophagosome then fuses with components of the classical endosomal system thereby maturing to an autoly-

We are a multidisciplinary team employing cell biology, biochemistry, light- and electron microscopy as well as structural biology approaches. Our findings will give important insights into mechanisms of autophagy.

Christine Abert Dorotea Fracchiolla Annamaria Gamper Veronika Nogellova Julia Romanov Kathrin Runggatscher Justyna Sawa-Makarska Martin Sztacho Eleonora Turco Gabriele Zaffagnini Bettina Zens

Scheme showing the generation of autophagosomes. Initially a small double membrane-bound structure called isolation membrane is formed. This structure expands to adopt a cuplike shape thereby gradually enclosing cytoplasmic cargo. This structure fuses at its rims giving rise to the mature autophagosome. Subsequently, autophagosomes fuse with lysosomes. Within these so-called autolysosomes the inner membrane and the cargo are degraded.

(A) A picture taken by confocal microscopy showing giant unilamellar vesicles (GUVs). The membrane of the GUVs was labelled by incorporation of a fluorescent lipid. (B) A picture showing human cells which express green and red labelled proteins that are targeted to autophagosomes.

SELECTED PUBLICATIONS Wurzer B, Zaffagnini G, Fracchiolla D, Turco E, Abert C, Romanov J, Martens S. Oligomerization of p62 allows for selection of ubiquitinated cargo and isolation membrane during selective autophagy. Elife. 2015 Sep 28;4:e08941. PMID: 26413874 Sawa-Makarska J, Abert C, Romanov J, Zens B, Ibiricu I, Martens S. Cargo binding to Atg19 unmasks additional Atg8 binding sites to mediate membrane-cargo apposition during selective autophagy. Nat Cell Biol. 2014 May;16(5):425-33. PMID: 24705553 Romanov J, Walczak M, Ibiricu I, SchĂźchner S, Ogris E, Kraft C, Martens S. Mechanism and functions of membrane binding by the Atg5-Atg12/Atg16 complex during autophagosome formation. EMBO J. 2012 Nov 14;31(22):4304-17. PMID: 23064152

38


MFPL - 2016 RESEARCH GROUPS

JAVIER MARTINEZ

Molecular mechanisms, biology and diseases linked to mammalian tRNA splicing

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Transfer RNAs (tRNA) are encoded in the genome as precursor molecules that must undergo processing in order to generate mature, functional tRNAs for the translation of mRNAs.

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Partial purification of a human cyclic-phosphodiesterase/phosphatase that converts a 2’, 3’-cyclic-phosphate at the end of an RNA molecule into a 2’OH, 3’OH terminal nucleotide. Starting from cytoplasmic extracts of HeLa cells, the novel enzymatic activity has been purified over several chromatographic steps. Here, active fractions from a Butyl-Sepharose column have been pooled and loaded onto a Heparin column. The cyclic-phosphodiesterase and phosphatase activity peaks in fractions 7-9 (in red).

Current projects deal with mutations in TSEN leading to pontocerebellar hypoplasia, the unexpected role of the tRNA ligase complex during oxidative stress and redox control, and with RNA recognition aspects of innate immunity by investigating the in vivo functions of the RNA 3’ Terminal Phosphate Cyclase RTCD1, an enzyme described back in 1983 with a still elusive in vivo function. Digging into the yet obscure topic of RNA repair, we are purifying a novel RNA processing factor in HeLa cells entailing a dual 2’, 3’-cyclic phosphodiesterase and phosphatase activity.

Processing entails a vast number of chemical modifications, as well as removal of 5’-leader, 3’-trailer and intronic sequences. The removal of introns during pre-tRNA splicing requires two enzymatic activities: an endonuclease and a ligase. Over the last years, our laboratory revealed the mammalian pre-tRNA splicing machinery by identifying and characterizing: a) CLP1, a subunit of the tRNA splicing endonuclease (TSEN) complex as the first 5’ RNA phosphorylating activity described in mammalian cells; b) the long sought mammalian tRNA ligase complex, joining tRNA exon halves; and c) Archease, a conserved protein that provides multiple turnover activity to the tRNA ligase.

Taken together, we combine biochemistry and mouse genetics to explore RNA metabolism in mammals. This research contributes to a renewed interest in the so-called “old” tRNA molecules and the enzymatic machinery devoted to their synthesis and processing.

We have stepped into in vivo biology by generating mouse models and analysing fibroblasts from patients with mutations in tRNA splicing factors. As a result, we uncovered the function of CLP1 in motor neuron diseases and assigned the tRNA ligase and archease to the cytoplasmic splicing of the Xbp1-mRNA. This is a critical event during the unfolded protein response which in turn is essential for plasma cells to produce immunoglobulins and for the survival of cancer cells.

SELECTED PUBLICATIONS Jurkin J, Henkel T, Nielsen A, Minnich M, Popow J, Kaufmann T, Heindl K, Hoffmann T, Busslinger M, Martinez J. The mammalian tRNA ligase complex mediates splicing of XBP1 mRNA and controls antibody secretion in plasma cells. EMBO J. 2014; 33(24):2922-36. PMID: 25378478 Popow J, Englert M, Weitzer S, Schleiffer A, Mierzwa B, Mechtler K, Trowitzsch S, Will C L, Lührmann R, Söll D, Martinez J. HSPC117 is the essential subunit of a human tRNA splicing ligase complex. Science 2001;331(6018):760-4. PMID: 21311021 Weitzer S, Martinez J. The human RNA kinase hClp1 is active on 3’ transfer RNA exons and short interfering RNAs. Nature 2007;447(7141):222-6. PMID: 17495927

39

Javier Martinez

TEAM Igor Asanovic Jutta Dammann Devon Germain Marion Godeck Theresa Henkel Jennifer Jurkin Dhaarsini Koneswarakantha Silvia Panizza Paola Hentges Pinto Stefan Weitzer


MFPL - 2016 RESEARCH GROUPS

ISABELLA MOLL

Bacterial stress response and ribosome heterogeneity The universal process of protein synthesis is performed by the ribosome, a complex assembly composed of RNA and protein elements.

manding transcriptional stress response we provided evidence that ribosome heterogeneity opens the possibility to instantaneously adapt protein synthesis to sudden alterations of environmental cues (Vesper et al, 2011). The hallmark of this post-transcriptional stress response is the modification of ribosomes by the endoribonuclease MazF, the toxin component of the toxin-antitoxin (TA) module mazEF. By removing the functionally important 3´-terminus of the 16S rRNA, MazF generates specialized stress-ribosomes that selectively translate mRNAs likewise processed by MazF within their 5´-UTR (Figure 1, i and ii) (Sauert et al, 2014; Sauert, 2016; Vesper et al, 2011). Moreover, we recently uncovered that this ‘one-step ribosome specialization’ mediated by MazF is reversible. The RNA ligase RtcB re-ligates the removed 3´-terminus to the truncated 16SΔ43 rRNA present in stress-ribosomes. Thereby, the ability of the ribosome to translate mRNAs harbouring a canonical 5´-UTR is fully restored (Figure 1, iii and iv; Temmel et al., 2016, in press).

Although ribosome heterogeneity was observed already more than 40 years ago, the ribosome is traditionally viewed as an unchangeable entity that has to be equipped with all ribosomal components and translation factors in order to precisely accomplish all steps in protein synthesis. In the recent years this concept was challenged by several studies highlighting a broad variation in the composition of the translational machinery in response to environmental signals, which leads to its adaptation and functional specialization.

Isabella Moll

Our research is centered on bacterial stress response mechanisms at the post-transcriptional level with a major focus on ribosome heterogeneity. In general, bacteria that encounter stress adapt to the given conditions by the alteration of the transcriptome, which requires the expression of alternative sigma factors to specify the RNA polymerase to distinct promoters. In contrast to this time- and energy-de-

TEAM

Our data suggest that these antagonizing factors provide a flexible and energy-efficient link between the number of specialized ribosomes and changing environmental conditions, which underscores the physiological importance of reversible ribosome heterogeneity as a key mechanism of the bacterial stress response network and raises the translational apparatus from a cellular factory required to make proteins to a control unit with an immense regulatory capacity.

Tanino Giuseppe Albanese Folke Ebert Christian Müller Mykola Roiuk Katharina Otto Martina Sauert

The reversible stress adaptation of the translational machinery in E. coli. During stress (red) the endoribonuclease MazF generates stress-ribosomes (i), which selectively translate MazF-processed mRNAs to adapt protein synthesis to the adverse conditions (ii). Upon stress relief the specialized ribosomes are regenerated by the RNA ligase RtcB (iii), thereby restoring the translational proficiency of 70S ribosomes to ensure canonical translation during relaxed conditions (green) (iv). (Taken from Temmel et al., 2016, in press)

SELECTED PUBLICATIONS Vesper O, Amitai S, Belitsky M, Byrgazov K, Kaberdina AC, Engelberg-Kulka H, Moll I. Selective translation of leaderless mRNAs by specialized ribosomes generated by MazF in Escherichia coli. Cell. 2011 Sep 30;147(1):147-57. PMID: 21944167 Sauert M, Wolfinger MT, Vesper O, Müller C, Byrgazov K, Moll I. The MazF-regulon: a toolbox for the post-transcriptional stress response in Escherichia coli. Nucleic Acids Res. 2016 Aug 19;44(14):6660-75. PMID: 2690865 Temmel, H., Müller C., Sauert, M., Vesper, O., Reiss, A., Popow, J., Martinez, J., and Moll, I. The RNA ligase RtcB reverses MazF-induced ribosome heterogeneity in Escherichia coli. Nucleic Acids Res. 2016, in press.

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MFPL - 2016 RESEARCH GROUPS

ERNST MÜLLNER & ULRICH SALZER

Erythrocyte (patho)physiology and storage in transfusion units In contrast to long-held views, erythrocytes are not merely bags full of haemoglobin but equipped with a large set of signalling components, currently emerging as important players in various physiologic processes.

Assessing the quality of erythrocytes in transfusion units Blood transfusion is routinely applied in many clinical settings. Apart from blood typing and a storage time limit of 42 days, however, standards regarding quality of transfusion units are still lacking. We investigate molecular changes that occur during the storage of transfusion units. This aims at the assessment of inter-donor and -recipient variability and at the identification of novel molecular markers indicating the degree of senescence within stored erythrocytes.

We, Ernst Müllner and Ulrich Salzer, teamed up with clinical cooperators from Vienna, Munich and Dresden to explore the intricate regulatory properties, diagnostic potential and therapeutic possibilities/limitations of these most abundant blood cells. Basic research on erythrocyte physiology • Erythrocytes actively engage in blood clotting and thrombus formation. Lysophosphatidic acid (LPA) from activated platelets triggers rapid exposure of phophatidylserine (PS) at the cell surface, resulting in an amplification of the clotting cascade. We currently explore the intracellular signalling and the molecular players of this process. • Due to their function as oxygen carriers, erythrocytes are loaded with an arsenal of molecules preventing oxidative stress. In an ongoing project, we study the complex regulation of the response to intracellular oxidative stress and the involvement of erythrocytes in the defense against oxidative stressors in blood plasma.

Ernst Müllner

Research on erythrocytes to identify molecular mechanisms underlying neurodegeneration The rare congenital neurodegenerative disorders chorea acanthocytosis (ChAC), McLeod syndrome and pantothenate kinase-associated neurodegeneration(PKAN) are classified as neuroacanthocytoses (NA) due to the occurrence of mis-shaped erythrocytes aka acanthoytes. We found that LPA signalling is deregulated in NA erythrocytes resulting in aberrant PS exposure and calcium uptake. Moreover, we found that drug-induced endovesiculation is impaired in NA erythrocytes (Siegl et al, 2013). • A current study on ChAc erythrocytes indicates that the altered endovesiculation properties are due to reduced dynamics and reversibility in the interaction between plasma membrane and cytoskeleton (Figure 1). • PKAN is often caused by point mutations in the PANK2 gene (Schiessl-Weyer et al, 2015). We use erythrocytes from PKAN patients to assess stability and activity of Pank2 mutants and study the impact on coenzyme A level and regulatory properties within these cells.

Figure 1. The ability of erythrocytes for induced endovesicle formation depends on interaction dynamics between membrane and the underlying cytoskeleton. Neuro-acanthocytosis erythrocytes show reduced ability to form endovesicles.

Ulrich Salzer

Figure 2. Storage time-dependent markers of erythrocyte senescence (PS exposure and hemolysis) show large inter-donor differences as seen by the differential development of these markers in individual transfusion units at longer storage times.

SELECTED PUBLICATIONS Kostan J, Salzer U, Orlova A, Törö I, Hodnik V, Senju Y, Zou J, Schreiner C, Steiner J, Meriläinen J, Nikki M, Virtanen I, Carugo O, Rappsilber J, Lappalainen P, Lehto VP, Anderluh G, Egelman EH, Djinović-Carugo K. Direct interaction of actin filaments with F-BAR protein pacsin2. EMBO Rep. 2014 Nov;15(11):1154-62. PMID: 2521694 Schiessl-Weyer J, Roa P, Laccone F, Kluge B, Tichy A, De Almeida Ribeiro E, Prohaska R, Stoeter P, Siegl C, Salzer U. Acanthocytosis and the c.680 A>G Mutation in the PANK2 Gene: A Study Enrolling a Cohort of PKAN Patients from the Dominican Republic. PLoS One. 2015 Apr 27;10(4):e0125861. PMID: 25915509 Siegl C, Hamminger P, Jank H, Ahting U, Bader B, Danek A, Gregory A, Hartig M, Hayflick S, Hermann A, Prokisch H, Sammler EM, Yapici Z, Prohaska R, Salzer U. Alterations of red cell membrane properties in neuroacanthocytosis. PLoS One. 2013 Oct 3;8(10):e76715. PMID: 24098554

41

TEAM Magdalena Bürkle Nina Küntzel Thomas Öhlinger Maike Werning


MFPL - 2016 RESEARCH GROUPS

JOHANNES NIMPF

ApoER2 and VLDL receptor We study the biology of LDL receptor related proteins (VLDL receptor and ApoER2), a group of cell surface receptors which mediate transport of macromolecules across cell membranes and play important roles in signal transduction.

Johannes Nimpf

The biological systems we are working with are the chicken oocyte and the mammalian brain. These two systems reflect the functional dichotomy of the receptors which function in endocytosis (follicles) and signal transduction (brain development). The best characterized function of VLDLR in follicles of egg laying species is endocytosis of yolk precursors into the growing oocyte. These yolk precursors (VLDL and Vitellogenin) are synthesized in the liver and rapidly taken up by the growing oocyte. Recently we have started to elucidate cell signalling functions of VLDLR and ApoER2 in granulosa cells, which support the maturation of oocytes within the follicle. In respect to brain development both receptors act as Reelin-signal transducers. The Reelin signal orchestrates the correct positioning of newly generated neurons within laminated structures of the

brain. In the development of the olfactory system in rodents, the structure of the olfactory bulb depends on neurons generated throughout life in the subventricular zone. These neurons migrate via the rostral migratory stream towards the olfactory bulb. This migration also depends on the presence of ApoER2 and VLDLR but seems to be independent on Reelin. To this end we have characterized thrombospondin-1 and clusterin as novel ligands for ApoER2 and VLDLR. Currently, we are focusing on a potential involvement of the receptors in the development of Alzheimer’s disease. A key feature of this disease is an imbalance of production and clearance of the Aβ-peptide which results in amyloid deposits in the brain. Since clusterin forms complexes with the Aβ-peptide, these complexes might get removed from the extracellular space by the action of ApoER2 and VLDLR.

Model of the intracellular fates of ApoER2 and VLDLR upon Reelin stimulation. Upon binding of Reelin, both ApoER2 and VLDLR mediate phosphorylation of Dab1(1). VLDLR internalizes Reelin rapidly via Clathrin-mediated endocytosis (2) and is separated from Reelin in the compartment of uncoupling of receptor and ligand (3). VLDLR then recycles back to the plasma membrane (4) while Reelin is delivered to the lysosome for degradation (5). ApoER2 internalizes Reelin via the same pathway although the receptor originally resides in lipid rafts and endocytoses its ligand with a much slower rate. In contrast to VLDLR, ApoER2 is not recycled but ends up in the lysosome together with Reelin (6). As an additional feedback mechanism, Reelin stimulation induces secretase-mediated cleavage of ApoER2, thereby generating a soluble extracellular fragment (8). This fragment can, together with another N-terminal fragment produced from an ApoER2 isoform by furin cleavage (9), inhibit the Reelin signal by sequestering free Reelin in the cell’s surrounding. The function of the soluble intracellular domain of ApoER2 is not well understood yet.

TEAM Paula Dlugosz Tobias Nimpf Harald Rumpler

SELECTED PUBLICATIONS Riegler B, Besenboeck C, Bauer R, Nimpf J, Schneider WJ. Enzymes involved in hepatic acylglycerol metabolism in the chicken. Biochem Bioph Res Co 2011. PMID: 21316342 Duit S, Mayer H, Blake SM, Schneider WJ, Nimpf J. Differential functions of ApoER2 and VLDL receptor in Reelin signaling depend on differential sorting of the receptors. J Biol Chem 2010; 285(7):4896-908. PMID: 19948739 Hong C, Duit S, Jalonen P, Out R, Scheer L, Sorrentino V, Boyadjian R, Rodenburg KW, Foley E, Korhonen L, Lindholm D, Nimpf J, van Berkel TJ, Tontonoz P, Zelcer N. The E3 ubiquitin ligase IDOL induces the degradation of the low density lipoprotein receptor family members VLDLR and ApoER2. J Biol Chem 2010; 285(26):19720-6. PMID: 20427281

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MFPL - 2016 RESEARCH GROUPS

EGON OGRIS

Enzyme biogenesis and monoclonal antibodies Cells employ reversible protein phosphorylation to regulate the functional state of their proteins. The enzymes catalysing these reactions, the protein kinases and phosphatases, are important regulators of almost all aspects of life.

Protein phosphatase 2A (PP2A), an essential phosphoserine/threonine phosphatase, is a tumour suppressor and target of cancer causing viruses. PP2A comprises a family of many different multisubunit holoenzymes, each of which possesses - dependent on its subunit composition - presumably unique substrate specificity. Decreased PP2A activity is associated with the development of human diseases indicating that proper PP2A function is required for cellular and organismal homeostasis. Our aim is to understand the molecular mechanisms of PP2A biogenesis and substrate specificity. A second translational research focus is the improvement of the monoclonal antibody technology and the development of novel antibodies specific for human disease-linked proteins and posttranslational modifications. The interactions between PP2A and its substrates are short-lived and thus difficult to detect with standard biochemical methods. By using a detection method that by itself is based on a short-lived enzyme-substrate reaction we succeeded in capturing PP2A “in flagrante” with substrates and are using this method termed M-Track (for Methyl-Tracking) for PP2A substrate validation/identification (Figure 1). Our study of PP2A biogenesis in yeast led to a model, in which a chaperone-dependent activation step is coupled to methylation-dependent holoenzyme assembly and is under the surveillance of the PP2A methylesterase PPE1. Despite the high degree of conservation not much is known about PP2A biogenesis in higher eukaryotes. To study PP2A biogenesis in mammals, we generated a conditional knockout mouse of the PP2A chaperone PTPA. Deletion of PTPA in the epiblast reveals an essential function of PTPA during embryonic development. Mouse embryonic fibroblasts lacking PTPA show severe phenotypic alterations, and these correlate with changes in PP2A complex composition suggesting a key role of PTPA in holoenzyme maturation. Within the scope of the translational research focus, we generate high quality monoclonal antibodies for the PP2A as well as for the larger research community,

e.g. the worldwide first monoclonal antibodies against the CRISPR/Cas9 endonuclease. To allow the simultaneous detection of coloured molecular weight markers in the chemiluminescent Western blot analysis we developed the anti-BLUE and anti-RAINBOW monoclonals (Figure 2), which render obsolete the manual charting of the marker bands on film. In addition, we are providing a monoclonal antibody service (please refer to the “At a Glance booklet” for details on this service).

Egon Ogris

Figure 1. Cartoon of an M-Track assay: Bait protein “Y”, prey protein “X”, histone lysine methyltransferase (HKMT), N terminus of histone H3 (H3K9). Upon interaction with the bait, the prey is stably marked by methylation (M, methyl group).

Figure 2. Anti-dye-specific antibodies for the visualization of prestained protein molecular weight markers in the chemiluminescent Western blot analysis.

SELECTED PUBLICATIONS Zuzuarregui A, Kupka T, Bhatt B, Dohnal I, Mudrak I, Friedmann C, Schüchner S, Frohner IE, Ammerer G, Ogris E. M-Track: detecting short-lived protein-protein interactions in vivo. Nat Methods. 2012 Jun;9(6):594-6. PMID: 22581371 Schüchner S, Andorfer P, Mudrak I, Ogris E. Anti-RAINBOW dye-specific antibodies as universal tools for the visualization of prestained protein molecular weight markers in Western blot analysis. Sci Rep. 2016 Aug 17;6:31363. PMID: 27531616 Yabe R, Miura A, Usui T, Mudrak I, Ogris E, Ohama T, Sato K. Protein Phosphatase Methyl-Esterase PME-1 Protects Protein Phosphatase 2A from Ubiquitin/Proteasome Degradation. PLoS One. 2015 Dec 17;10(12):e0145226. PMID: 26678046

43

TEAM Ingrid Frohner Stephanie Kronlachner Florian Martys Martina Mitterhuber Ingrid Mudrak Stefan Schüchner Jiri Veis


MFPL - 2016 RESEARCH GROUPS

FRIEDRICH PROPST

The neuronal cytoskeleton in axon guidance Axon extension, branching, and retraction are morphological changes that neurons have to execute to accomplish correct wiring of the nervous system during development and regeneration. These transformations are guided by extracellular signals which ultimately need to be translated into rearrangement of the neuronal cytoskeleton.

Friedrich Propst

TEAM Irmgard Fischer Bernhard Kaiser Petra-Franziska Kalman Alexa Kiss

disordered. In collaboration with Robert Konrat (MFPL) we use nuclear magnetic resonance techniques to reveal structural changes in its interaction with microtubules. Recently, we also engaged in a systems biological multi-scale, multi-parametric image analysis of microtubule dynamics in axonal growth cones of live primary neurons of wild-type and mutant mice. This study is aimed at determining whether growth cone behaviour (migration, pausing, collapse and retraction) is associated with distinct sets of microtubule dynamic parameters (Figure 2). We measure about 80 parameters of microtubule dynamics and growth cone behaviour in hundreds of migrating growth cones yielding tens of thousands of observations. We apply advanced statistical methods to determine if and how the behaviour of individual growth cones (minutes time scale) is linked to microtubule dynamic parameters (seconds time scale).

We study mechanisms involved in the orchestrated reorganization of neuronal microtubules and actin filaments. Our approach combines gene ablation in the mouse with cell biological and molecular analyses in cultured neurons as well as biochemical and ultrastructural analysis. One focus of our research is the role of microtubule-associated proteins of the MAP1 family. In the past, we have shown that MAP1B is a component of a pathway that links calcium influx and activation of neuronal nitric oxide synthase to reconfiguration of axonal microtubules, and thus might contribute to physiological and pathological effects of nitric oxide in the brain. We have since demonstrated that MAP1B is essential for signal transduction of several other repulsive axon guidance cues as well. These include semaphorin 3A and draxin which critically depend on MAP1B which in response to these extracellular cues is phosphorylated through a PI3 kinase-Akt-GSK-3beta pathway (Figure 1). To complement our work in mice and primary neurons, we investigate the ultrastructural details of the microtubule binding domain of MAP1 proteins. This domain is about 125 amino acids in length and intrinsically

Figure 2. A. YFP-EB3 comets (top) and their segmentation (red outlines, bottom) in a DRG growth cone. Based on their unique IDs (yellow numbers), single EB3-comets are traced throughout their lifetime. The segmented growth cone outline is displayed in blue. B. Imaging setup using multiple temporal scales (seconds‐minutes). Growth cones are repeatedly imaged (red arrow) in every 10 minutes, with a 2‐second time resolution. C. Spatial and temporal hierarchy of the dataset. Multiple parameters are simultaneously recorded across all of the scales (comets, growth cones). The dataset may be dis/re-aggregated according to these inter‐connected levels. D. Putative multivariate separation of growth cone behavioural categories based on their microtubule dynamic properties.

Figure 1. MAP1B is required for draxin- and semaphorin 3A-induced growth cone collapse. Growth cones of cortical neurons from newborn wildtype or MAP1B-/- mice, treated for 1 h with 100 nM draxin or 30 min with 100 ng/ml semaphorin 3A (Sema3A), fixed and stained for F-actin. Scale bar = 10 μm.

SELECTED PUBLICATIONS Stroissnigg H, Trancíková A, Descovich L, Fuhrmann J, Kutschera W, Kostan J, Meixner A, Nothias F, Propst F. S-nitrosylation of microtubule-associated protein 1B mediates nitric-oxide-induced axon retraction. Nat Cell Biol 2007; 9(9):1035-45. PMID: 17704770 Meli R, Weisová P, Propst F. Repulsive axon guidance by Draxin is mediated by protein Kinase B (Akt), glycogen synthase kinase-3β (GSK-3β) and microtubule-associated protein 1B. Plos One 2015;10(3):e0119524. PMID: 25775433 Cheng L, Desai J, Miranda CJ, Duncan JS, Qiu W, Nugent AA, Kolpak AL, Wu CC, Drokhlyansky E, Delisle MM, Chan WM, Wei Y, Propst F, Reck-Peterson SL, Fritzsch B, Engle EC. Human CFEOM1 mutations attenuate KIF21A autoinhibition and cause oculomotor axon stalling. Neuron 2014;82(2):334-49. PMID: 24656932

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MFPL - 2016 RESEARCH GROUPS

FLORIAN RAIBLE

Origin and diversification of hormone systems We are interested in the origin and evolution of hormone systems. Central to our work is the exploration of a novel invertebrate model system, Platynereis dumerilii.

University of Vienna Research Platform (“Rhythms of Life”) that allows us to interact with colleagues from the Analytical Chemistry and Neurobiology. Together, we have recently gained critical insight into one of the enigmatic hormones involved in the synchronized reproduction of the animal.

Our past work has shown that this marine worm exhibits a unique combination of ancestral-type genomic characteristics not found in insect and nematode model species. Moreover, we have identified numerous components of ancestral-type hormone pathways in Platynereis. Therefore, Platynereis is highly interesting for comparison with other animal hormone systems - including the ones of vertebrates - and for our understanding of marine life.

Exploring a new marine model system Over the past years, Platynereis has emerged as a very promising “next-generation” model system. We have pioneered transgenic technology in Platynereis that allows us to mark and interrogate cell types with unprecedented precision. Likewise, we have helped to establish targeted mutagenesis in the worm, allowing us to test if a given gene is required for regeneration or reproductive timing. Finally, we make use of the remarkable transparency of Platynereis to observe neurons and stem cells in the living animal. These approaches provide entry points into the fascinating biology of a new marine model species. Besides the action of hormones, we are actively investigating the evolution of gene-regulatory logic and the orchestration of cellular processes involved in the sculpting of bristles. Our vision is to firmly establish Platynereis as a reference species for marine biology.

The hormonal control of reproduction and regeneration What could be the function of ancestral-type hormones in Platynereis? One of the systems that we dissect is the hormonal machinery orchestrating reproduction and regeneration. Platynereis is an excellent object for this analysis, as it has been a central model for the link between chronobiology and reproduction. Our bioinformatic analyses, as well as transcriptomic, proteomic and targeted biochemical analyses, have revealed a spectrum of hormones present in Platynereis. Thanks to the establishment of new molecular tools, we are now able to systematically assess the impact of these candidates on the development and maturation of the animals. These experiments are supported by an ERC starting grant (HOR.MOON), as well as a

Hormonal orchestration of regeneration and reproduction by the medial Platynereis brain. (A) Classical transplantation studies revealed that immature Platynereis heads are the source for an endocrine brain hormone inhibiting maturation and supporting regeneration. (B) Implantations of small brain fragments (blue) map the source of the brain hormone to the medial Platynereis brain. Recent work has allowed us to gain first insight into the molecular identity of the brain hormone.

SELECTED PUBLICATIONS Bannister S, Antonova O, Polo A, Lohs C, Hallay N, Valinciute A, Raible F, Tessmar-Raible K. TALENs Mediate Efficient and Heritable Mutation of Endogenous Genes in the Marine Annelid Platynereis dumerilii. Genetics 2014;197(1):77-89. PMID: 24653002 Backfisch B, Veedin Rajan VB, Fischer RM, Lohs C, Arboleda E, Tessmar-Raible K, Raible F. Stable transgenesis in the marine annelid Platynereis dumerilii sheds new light on photoreceptor evolution. P Natl Acad Sci USA 2013;110(1):193-8. PMID: 23284166 Tessmar-Raible K, Raible F, Arboleda E. Another place, another timer: Marine species and the rhythms of life. BIOESSAYS 2011;33(3):165-72. PMID: 21254149

45

Florian Raible

TEAM Stephanie Bannister Andrej Belokurov Caroline Broyart Carola Jaunecker Roger Revilla-i-Domingo Sven Schenk Clara Schmidt Karim Pyarali Vadiwala Martin Zurl

Ancestral-type hormones in a simple invertebrate. Individual hormone-producing cells are visualized (green colour) in an adult Platynereis brain. Whereas cell bodies (“mc”) localize to the medial brain, neuronal projections (np) project into the region of the infracerebral gland, an annelid neurohemal organ.


MFPL - 2016 RESEARCH GROUPS

JOHANN ROTHENEDER

Cell cycle regulation and DNA damage response My laboratory is focused on the mechanisms controlling growth and cell cycle of the mammalian cell. They respond to perturbations like replication errors or DNA damage by inducing cell cycle arrest, senescence, or apoptosis. knock down of p21 reduces the anti-apoptotic activities of overexpressed EAPP. This suggests that p21 at least in part mediates this activity of EAPP. EAPP stimulates p21 expression by binding to its promoter and seems to be required for the assembly of the transcription initiation. The knock down of EAPP facilitates apoptosis and goes along with reduced p21.

Dysfunction of these mechanisms often results in the malignant transformation of a cell and the development of cancer. E2F is a family of transcription factors that integrate cell-cycle progression with transcription through cyclical interactions with important cell cycle regulators.

Johann Rotheneder

TEAM

We have recently identified and characterized a protein that we called EAPP (E2F Associated PhosphoProtein). EAPP interacts with E2F1-3, comprising the activator group of E2F proteins, and modulates E2F-dependent transcription. Tumour cells often overexpress EAPP, indicating that it confers a selective advantage to these cells. EAPP levels increase upon DNA damage and higher EAPP levels seem to protect cells from apoptosis. This protection can also be achieved by ectopic expression of EAPP and correlates with an increased number of cells in G1 phase and an upregulation of p21. Increased p21 inhibits cyclin/cdk activity which is required for cell cycle progression, but also interferes with apoptosis. The RNAi-mediated

Our findings suggest that EAPP is indispensable for the survival of a cell. The required amount of EAPP seems to depend on the environmental conditions. Preliminary evidence suggests that the role of EAPP in transcription is not limited to the p21 promoter. Active promoters are occupied by multiple types of complexes and EAPP seems to be an important component of at least some of them. Lowering EAPP levels influences the expression of some of the genes examined including important cell-cycle regulators. In the future, we will examine which genes are influenced by EAPP and scrutinize the biochemical details of its activity.

Sarah Gobetzky Christoph Penz Cheng Zhang

A model showing three different scenarios with elevated, normal and reduced levels of EAPP

SELECTED PUBLICATIONS Andorfer P, Rotheneder H. EAPP: gatekeeper at the crossroad of apoptosis and p21-mediated cell-cycle arrest. Oncogene. 2011 Jun 9;30(23):2679-90. PMID: 21258403 Schwarzmayr L, Andorfer P, Novy M, Rotheneder H. Regulation of the E2F-associated phosphoprotein promoter by GC-box binding proteins. Int J Biochem Cell Biol. 2008;40(12):2845-53. PMID: 18588995 Andorfer P, Schwarzmayr L, Rotheneder H. EAPP modulates the activity of p21 and Chk2. Cell Cycle. 2011 Jul 1;10(13):2077-82. PMID: 21572256

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MFPL - 2016 RESEARCH GROUPS

MATTHIAS SCHAEFER

Methylated cytosine in RNA: Unterstanding their impact on RNA stability, gene expression and innate immunity RNAs carry more than 130 distinct post-transcriptional modifications, but their biological functions remain mostly unexplored.

In contrast to the limited number of known DNA modifications, RNAs are “decorated” with more than 100 distinct post-transcriptional modifications. The chemical nature of these modifications, especially in non-coding RNAs, can be diverse and very complex. However, the majority involves methylation reactions indicating the importance of methylated nucleotides for the function of RNA or DNA. 5-methylcytosine (m5C) is a simple modification that is present both in RNA and DNA. While m5C in DNA plays various roles in the epigenetic regulation of gene expression the function of m5C in RNAs remains still unclear.

melanogaster to understand how m5C in RNA • Impacts on RNA stability and function • Controls stress-induced RNA processing • Affects the interaction with RNA-binding proteins • Contributes to the regulation of gene expression Our long-term goal is to contribute to the characterization of the “epitranscriptome” and to our understanding as to how RNA modifications impact on gene regulation during development and under changing environmental conditions.

My group applies genetic and biochemical tools, human cell culture and the model organism Drosophila

Matthias Schaefer

TEAM Aleksej Drino Bianca Genenncher Daniela Zinkl

m5C affects RNA stability, especially during stress conditions and m5C RNA methyltransferase mutants show various pleiotropic phenotypes. It is presently unclear how exactly m5C-methylated RNAs impact on processes such as protein translation, mobile element control and innate immunity.

SELECTED PUBLICATIONS Durdevic Z, Mobin MB, Hanna K, Lyko F, Schaefer M. The RNA methyltransferase Dnmt2 is required for efficient Dicer-2-dependent siRNA pathway activity in Drosophila. Cell Rep. 2013;4(5):931-7. PMID: 24012760 Durdevic Z, Hanna K, Gold B, Pollex T, Cherry S, Lyko F, Schaefer M. Efficient RNA virus control in Drosophila requires the RNA methyltransferase Dnmt2. Embo Rep. 2013;14(3):269-75. PMID: 23370384 Schaefer, M. RNA 5-Methylcytosine Analysis by Bisulfite Sequencing. Methods in Enzymology. 2015; 560(14): 297-329. PMID: 26253976

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MFPL - 2016 RESEARCH GROUPS

PETER SCHLÖGELHOFER

Meiotic recombination We focus our research on meiotic recombination, mainly working with the model plant Arabidopsis thaliana and to some extent with the yeast Saccharomyces cerevisiae.

Peter Schlögelhofer

Our research efforts are well embedded in the Department of Chromosome Biology with five other groups performing meiosis research in various organisms. Meiosis is a specialised, two-step cell division that ensures the reduction of the genome prior to the formation of generative cells. During meiosis, homologous centromeres are segregated during the first, and sister centromeres during the second division. As there is no intervening DNA replication between the two meiotic divisions, each of the final division products contains only half of the initial DNA content. For a given diploid organism, the developing generative cells are then haploid. It is important to note, that during meiosis, genetic information between maternal and paternal chromosomes is mutually exchanged, leading to novel combinations of genetic traits in the follow-

ing generation. Two genetically diverse generative cells fuse during the process of fertilization, re-establish the species-specific original genome content and constitute an individual with a unique genetic set-up. Novel combinations between parts of paternal and maternal chromosomes are generated through the process of homologous recombination (HR). A pre-requisite for HR are DNA double strand breaks (DSBs), generated by a protein complex with the conserved protein SPO11 being its catalytically active subunit. DSBs are formed at non-random sites throughout the genome, known as hot spots of meiotic recombination. We are interested in 1) cis and trans acting factors that mediate meiotic DSB formation, 2) mechanisms of meiotic DSB processing, 3) the biochemical details of subsequent DSB repair and 4) the coordination of all these events. We use a broad range of techniques (molecular biology, cytology, biochemistry and genetics) and take advantage of the on-site facilities (Bio-optics, deep-sequencing, mass-spectrometry, bioinformatics).

TEAM Nele Aigner Christian Gruber Marie-Therese Kurzbauer Ignacio Prusén Carla Schachner Katja Schneider Katharina Schropp Jason Sims Nguyen Tan-Trung Faye Wheeler Teresa Wöhrer

The panel shows a preparation of meiotic chromosomes isolated from meiocytes of a mutant Arabidopsis plant. The depicted stage of meiosis is called “pachytene” with all five chromosome pairs in close alignment, stabilized by a protein complex known as the “synaptonemal complex” (SC). To visualise the DNA and associated proteins a specific DNA dye (DAPI) and antibodies (coupled to fluorescent molecules) specifically detecting certain meiotic proteins have been applied. The DNA is stained in blue, a protein of the SC is stained in green, and a DNA repair protein is stained in red.

SELECTED PUBLICATIONS Roitinger E, Hofer M, Köcher T, Pichler P, Novatchkova M, Yang J, Schlögelhofer P, Mechtler K. Quantitative phosphoproteomics of the ataxia telangiectasia-mutated (ATM) and ataxia telangiectasia-mutated and rad3-related (ATR) dependent DNA damage response in Arabidopsis thaliana. Mol Cell Proteomics. 2015 Mar;14(3):556-71. PMID: 25561503 Cabral G, Marques A, Schubert V, Pedrosa-Harand A, Schlögelhofer P. Chiasmatic and achiasmatic inverted meiosis of plants with holocentric chromosomes. Nat Commun. 2014 Oct 8;5:5070. PMID: 25295686 Uanschou C, Ronceret A, Von Harder M, De Muyt A, Vezon D, Pereira L, Chelysheva L, Kobayashi W, Kurumizaka H, Schlögelhofer P, Grelon M. Sufficient amounts of functional HOP2/MND1 complex promote interhomolog DNA repair but are dispensable for intersister DNA repair during meiosis in Arabidopsis. Plant Cell. 2013 Dec;25(12):4924-40. PMID: 24363313

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MFPL - 2016 RESEARCH GROUPS

RENÉE SCHROEDER

Riboregulation of transcription: how RNA shapes the transcriptome Life is a self-organized process, that needs to control growth and establish homeostasis in response to very diverse environmental cues.

If RNA is to play the role of the molecule that enabled life to emerge, it should be able to control its own synthesis. Our laboratory is interested in identifying RNAs that interact with RNA polymerases to control transcription. We are currently studying RNA polymerase-binding RNA aptamers (RAPs) that are able to cross talk to RNA polymerases in cis and modulate the transcription of nascent RNAs hosting these RAPs. By performing genomic SELEX using RNA libraries derived from E. coli, yeast and human genomic DNA and highly purified RNA polymerases as bait, we isolated thousands

of RAPs. RAP-mediated control of transcription elongation and termination is a novel level of regulation that is conserved from bacteria to humans. RNAs can control gene expression at all steps. We are especially interested in antisense RNA mediated regulation of the transcriptome via double-stranded RNA formation and transcription interference. The human transcriptome is modulated by unconventional splicing events like nested and recursive splicing. We are currently addressing the role of intronic splicing on the efficiency of formation of mature RNA. We recently revealed a novel function for small RNAs in the regulation of transcription in E. coli. A large number of E. coli genes have long 5’ UTRs whose transcription is turned off prematurely by the RNA helicase Rho under exponential growth. Under stress conditions small RNAs are synthesized that bind to these 5’ UTRs competing with rho for interaction with the RNA. By doing this they induce antitermination of transcription leading to full length gene expression. Thus sRNAs not only regulate gene expression post-transcriptionally but already co-transcriptionally.

A novel role for sRNAs in bacteria: sRNAs compete with the termination factor Rho to induce transcription antitermination under stress conditions. This represents a novel level of transcription regulation by inducing antitermination.

SELECTED PUBLICATIONS Sedlyarova N, Shamovsky I, Bharati BK, Epshtein V, Chen J, Gottesman S, Schroeder R, Nudler E. sRNA-Mediated Control of Transcription Termination in E. coli. Cell. 2016 Sep 22;167(1):111-121.e13. PMID: 27662085 Aronica L, Kasparek T, Ruchman D, Marquez Y, Cipak L, Cipakova I, Anrather D, Mikolaskova B, Radtke M, Sarkar S, Pai CC, Blaikley E, Walker C, Shen KF, Schroeder R, Barta A, Forsburg SL, Humphrey TC. The spliceosome-associated protein Nrl1 suppresses homologous recombination-dependent R-loop formation in fission yeast. Nucleic Acids Res. 2016 Feb 29;44(4):1703-17. PMID: 26682798 Weiss A. Lamarckian Illusions. Trends Ecol Evol. 2015 Oct;30(10):566-8. PMID: 26411613

49

Renée Schroeder

TEAM Eva Klopf Elzbieta Kowalska Andres Magan Garcia Murielle Moes Maximilian Radtke Natascha Rziha Nadezda Sedlyarova Ismet Srndic


MFPL - 2016 RESEARCH GROUPS

CHRISTIAN SEISER

Chromatin modifiers in development and disease Dynamic acetylation of histone proteins induces local changes in the chromatin structure and thereby controls important biological processes such as transcription, replication and DNA repair. recently revealed distinct but overlapping functions of HDAC1 and HDAC2 enzymes during epidermal development and tumorigenesis (Winter et al., 2013), in neurogenesis (Hagelkruys et al., 2014) and in collaboration with Wilfried Ellmeier during T cell development (Boucheron et al.,2014).

Histone deacetylases (HDACs) remove acetyl groups from histones and other proteins and act as transcriptional co-regulators. Small molecule inhibitors of HDACs are used in anti-tumour therapy and for treatment of neurological disorders, parasitic and inflammatory diseases. Our research focuses on the class I deacetylases HDAC1 and HDAC2.

Christian Seiser

TEAM Brigitte Gundacker Astrid Hagelkruys Christian Humer Mirjam Moser Verena Moser Stephanie Schneider

To dissect catalytic and non-catalytic functions of HDAC1 and HDAC2 we have now generated novel knock-in mice expressing catalytically inactive HDAC1 or HDAC2 versions instead of the wildtype protein. Strikingly, heterozygous expression of inactive HDAC2 but not HDAC1 results in perinatal lethality due to defects in brain development. Thus, the catalytically inactive HDAC enzymes can act as dominant negative mutants and might mimic isoform specific inhibitors. Corresponding conditional knock-in mouse lines will be therefore excellent tools for studying HDAC1 and HDAC2 as potentially relevant targets for anti-tumour drugs.

We have originally identified mouse HDAC1 as growth factor inducible gene in T cells (Bartl et al., 1997). HDAC1 gene disruption leads to reduced proliferation and severe developmental problems resulting in embryonic lethality of HDAC1 knockout mice (Lagger et al., 2002). One crucial function of HDAC1 in the context of proliferation control is the repression of the CDK inhibitor p21/WAF1 suggesting a potential role of HDAC1 in tumorigenesis (Zupkovitz et al., 2010). Surprisingly, absence or reduced expression of HDAC1 in murine or human teratomas leads to increased proliferation and reduced differentiation and is linked with a more malignant phenotype (Lagger et al., 2010). By using conditional HDAC knockout mice we have

Changes in murine brain architecture upon heterozygous expression of catalytically inactive HDAC2. Brains of mutant mice are smaller and more fragile and show reduced sizes of the cortex and cerebellum and a diminished foliation of the cerebellum. The mutant mice die few hours after birth (Hagelkruys et al., 2015).

SELECTED PUBLICATIONS Hagelkruys A, Mattes K, Moos V, Rennmayr M, Ringbauer M, Sawicka A, Seiser C. Essential Nonredundant Function of the Catalytic Activity of Histone Deacetylase 2 in Mouse Development. Mol Cell Biol. 2015 Nov 23;36(3):462-74. PMID: 26598605 Hagelkruys A, Lagger S, Krahmer J, Leopoldi A, Artaker M, Pusch O, Zezula J, Weissmann S, Xie Y, Schöfer C, Schlederer M, Brosch G, Matthias P, Selfridge J, Lassmann H, Knoblich JA, Seiser C. A single allele of Hdac2 but not Hdac1 is sufficient for normal mouse brain development in the absence of its paralog. Development. 2014 Feb;141(3):604-16PMID: 24449838 Boucheron N, Tschismarov R, Göschl L, Moser MA, Lagger S, Sakaguchi S, Winter M, Lenz F, Vitko D, Breitwieser FP, Müller L, Hassan H, Bennett KL, Colinge J, Schreiner W, Egawa T, Taniuchi I, Matthias P, Seiser C, Ellmeier W. CD4(+) T cell lineage integrity is controlled by the histone deacetylases HDAC1 and HDAC2. Nat Immunol. 2014 May;15(5):439-48. PMID: 24681565

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MFPL - 2016 RESEARCH GROUPS

TIM SKERN

Interactions between viruses and cells Most viruses interfere with or modulate host systems to ensure successful replication. My group looks at interactions between the leader proteinase of foot-and-mouth disease virus and the 2A proteinase of the common cold virus with the cellular protein called eukaryotic initiation factor 4G (eIF4G) as well as investigating the immunemodulator protein A46 of vaccinia virus.

Vaccinia virus is the viral vaccine that was used to eradicate smallpox virus. Both vaccinia virus and smallpox virus encode many proteins that reduce the affectivity of the host immune system. One such protein is vaccinia virus A46, a protein that counteracts several immune regulators of the infected cell to prevent the inflammation response. We have determined the three-dimensional structure of residues 87to 229 of A46 and investigated the interaction of this protein with purified host cell proteins. The structure suggests how the A46 specifically interacts with its targets in the host cell and why its spectrum of interaction partners is different to those of closely related vaccinia virus proteins. Further investigation should help to illuminate how the cellular immune regulators interact with each other.

eIF4G is involved in recruiting capped cellular mRNA to the ribosomes for protein synthesis. Cleavage of this molecule during replication of the above mentioned viruses thus prevents capped cellular mRNA from being translated. Viral protein synthesis is unaffected as it initiates internally downstream of the 5’ end of its RNA. We have determined the molecular structures of three of these proteinases and investigated the sites at which they interact with eIF4G. The leader proteinase is a relative of the plant cysteine proteinase papain. However, in contrast to papain, the leader proteinase is very specific, with only three target proteins identified at present. Nevertheless, a consensus sequence representing the cleavage site has been difficult to define as the three known cleavage sites show considerable differences. We have determined the structure of this proteinase in complex with an inhibitor to show the reasons why this proteinase is specific. In addition, we have also used nuclear magnetic resonance to see how a short fragment of eIF4G is recognized by the proteinases.

Tim Skern

TEAM Gustavo Arruda Bezerra Martina Aumayr Daniel Azar Nina Bobik Sofiya Fedosyuk Amelie Schoenenwald Öykü Üzülmez

The foot-and-mouth disease leader proteinase.

Structure of A46 protein from vaccinia virus.

SELECTED PUBLICATIONS Fedosyuk S, Grishkovskaya I, De Almeida Ribeiro E, Skern T. Characterisation and structure of the vaccinia virus NF- κB antagonist A46. J Biol Chem. 2014;289(6):3749-62. PMID: 24356965 Steinberger J, Kontaxis G, Rancan C, Skern T. Comparison of self-processing of footand-mouth disease virus leader proteinase and porcine reproductive and respiratory syndrome virus leader proteinase nsp1α. Virology 2013;443(2):271-7. PMID: 23756127 Steinberger J, Grishkovskaya I, Cencic R, Juliano L, Juliano MA, Skern T. Foot-and-mouth disease virus leader proteinase: Structural insights into the mechanism of intermolecular cleavage. Virology. 2014;468-470:397-408. PMID: 25240326

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MFPL - 2016 RESEARCH GROUPS

DEA SLADE

DNA damage response DNA damage response (DDR) is a complex regulatory network that involves DNA damage sensing, signalling and repair. These processes are carried out by diverse enzymatic activities that must be precisely co-ordinated as to ensure the efficient, accurate and timely repair of DNA damage and the preservation of genomic integrity.

Dea Slade

TEAM Lisa Appel Melania Bruno Joachim Garbrecht Sebastien Herbert Tanja Kaufmann Eva Kukolj Karin Olek Eva Scheuringer

and inflammatory stress, which alter NAD levels. They regulate cellular response to stress through changes in chromatin structure, gene expression, DNA repair, cell cycle, metabolism and cell fate. Among 17 PARP family members and seven sirtuins in mammals, PARP1, PARP2, PARP3, SIRT1, and SIRT6 are hitherto known to affect DDR. PARP1/2 and SIRT1 deficiencies sensitize the cells to DNA-damaging agents and result in embryonic lethality due to genomic instability. However, little is known about DNA repair processes regulated by sirtuins and the relationship between sirtuin- and PARP-mediated modifications of DNA repair proteins. The observations that PARPs and sirtuins regulate each other’s levels and activities and have opposite effects on the same pathway such as cell death suggest a functional interplay between these NAD-consuming enzyme families. Our aim is to identify unknown DDR protein targets regulated by PARPs and sirtuins and characterize their functional interplay by using biochemical and cell biological techniques in mammalian systems.

The dynamics of the DDR protein network are governed by post-translational modifications including phosphorylation, methylation, acetylation, ubiquitination, sumoylation and poly(ADP-ribosyl)ation (PARylation). They regulate the recruitment of DNA repair factors to the sites of DNA damage, their enzymatic activity, interactions and the choice of the DNA repair pathway. Our research focuses on the regulation of DDR by reversible PARylation and acetylation modifications. Poly(ADP-ribose) (PAR) is rapidly produced in response to DNA damage by PARP polymerases and elicits the recruitment of different DDR factors to DNA damage sites. PAR is rapidly degraded by the PARG glycosylase to ensure a transient effect. While PAR is the largest post-translational protein modification, acetylation is the most prevalent. Among numerous deacetylases, in recent years sirtuins (SIRTs) have emerged as crucial regulators of gene expression, metabolism and genome integrity that interconnect with the PAR processes. As NAD-consuming enzymes, PARPs and sirtuins are activated in conditions of genotoxic, oxidative, metabolic

PARPs and sirtuins regulate stress response after DNA damage.

SELECTED PUBLICATIONS Slade D, Dunstan MS, Barkauskaite E, Weston R, Lafite P, Dixon N, Ahel M, Leys D, Ahel I. The structure and catalytic mechanism of a poly(ADP-ribose) glycohydrolase. Nature. 2011 Sep 4;477(7366):616-20. PMID: 21892188 Chen D, Vollmar M, Rossi MN, Phillips C, Kraehenbuehl R, Slade D, Mehrotra PV, von Delft F, Crosthwaite SK, Gileadi O, Denu JM, Ahel I. Identification of macrodomain proteins as novel O-acetyl-ADP-ribose deacetylases. J Biol Chem. 2011 Apr 15;286(15):13261-71. PMID: 21257746 Slade D, Radman M. Oxidative stress resistance in Deinococcus radiodurans. Microbiol Mol Biol Rev. 2011 Mar;75(1):133-91. PMID: 21372322

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MFPL - 2016 RESEARCH GROUPS

KRISTIN TESSMAR-RAIBLE

Lunar periodicity and inner brain photoreceptors The main interest of my lab is to investigate how solar and lunar light are sensed by the nervous system and how this light information impacts on the animals‘ information processing and endogenous clocks.

The function of vertebrate inner brain opsins Nature’s own optogenetics? Starting with experiments at the beginning of the 20th century it has become apparent that light can be perceived by cells in the inner brain of vertebrates, independent of eyes and pineal organs. Subsequent studies established that measurable amounts of light can penetrate deep inside the brain of vertebrates, including mammals. Our study of non-visual photoreceptors in fishes recently revealed that vertebrate brains harbor directly light-sensory motor- and interneurons. This led us to hypothesize that environmental light impacts on the information processing and output in the vertebrate

brain by modulating the membrane potential of Opsin-expressing inter- and motorneurons, and hence leads to light-dependent behavioural alterations. We now test this hypothesis. Molecular neurobiology of a moonlight entrained circalunar clock While the function of vertebrate non-visual opsins is likely connected to solar light perception, lunar light is also a strong environmental stimulus for animals. The lunar cycle synchronizes reproductive behaviour and sexual maturation of animals as diverse as corals, midges, polychaetes and fishes. In animals such as the annelid Platynereis dumerilii or the midge Clunio marinus, dim nocturnal light serves as entrainment cue for an endogenous oscillator – a circalunar clock – that orchestrates reproductive and behavioral cycles. As circalunar clocks run with a (semi-)monthly period, they represent fundamental biological phenomena clearly distinct from the widely studied, solar light-entrained circadian (24h) clocks. Jointly with Florian Raible’s group we established several critical functional tools for Platynereis dumerilii, including stable transgenesis and TALEN-mediated targeted genome mutagenesis. Using Platynereis dumerilii, my group has started to obtain first insights into the circalunar clock and its interactions with the circadian clock. Differing from Platynereis dumerilii (whose worm ancestors never left the oceans), the midge Clunio marinus likely acquired its circalunar clock de novo during the past 20.000 years. Strains of different geographic origins exhibit differences in their circalunar and circadian timing (“time races”). Clunio marinus thus represents an ideal model to understand the evolution of circalunar clocks across species (in comparison to Platynereis dumerilii), but also across the population of its own species (“time races”).

Lunar reproductive periodicity of Platynereis dumerilii is synchronized by light and controlled by a clock mechanism.

SELECTED PUBLICATIONS Zantke J, Ishikawa-Fujiwara T, Arboleda E, Lohs C, Schipany K, Hallay N, Straw AD, Todo T, Tessmar-Raible K. Circadian and circalunar clock interactions in a marine annelid. Cell Rep. 2013 Oct 17;5(1):99-113. PMID: 24075994 Fischer RM, Fontinha BM, Kirchmaier S, Steger J, Bloch S, Inoue D, Panda S, Rumpel S, Tessmar-Raible K. Co-expression of VAL- and TMT-opsins uncovers ancient photosensory interneurons and motorneurons in the vertebrate brain. PLoS Biol. 2013;11(6):e1001585. PMID: 23776409 Backfisch B, Veedin Rajan VB, Fischer RM, Lohs C, Arboleda E, Tessmar-Raible K, Raible F. Stable transgenesis in the marine annelid Platynereis dumerilii sheds new light on photoreceptor evolution. Proc Natl Acad Sci U S A. 2013 Jan 2;110(1):193-8. PMID: 23284166

53

Kristin Tessmar-Raible

TEAM Thomas Ayers Margaryta Borysova Marcus Dekens Sarfaraz Farooqui Bruno Fontinha Maximilian Hofbauer Lukas Orel Andreas Poehlmann Birgit Poehn Barbara Rodin Hiroki Takekata Vinoth Babu Veedin Rajan


MFPL - 2016 RESEARCH GROUPS

GIJS VERSTEEG

Ubiquitin-mediated regulation of immune signalling We are interested in understanding how the immune system is regulated during infection and inflammatory disease.

Precise regulation and fine-tuning of immune signalling pathways is critical to strike the right balance between conferring sufficient antimicrobial activity during infection and preventing hyper-immune activation resulting in auto-immunity. The molecular mechanisms regulating these signalling molecules in different cell types during the innate immune response remain relatively poorly defined.

Gijs Versteeg

regulates innate immune cytokine expression (Versteeg & Rajsbaum et al. Immunity 2013). In addition, preliminary data implicate several other TRIM proteins as negative regulators. Our data show that individual TRIMs act at various different stages of immune signalling, suggesting that many of them act on different target molecules. My lab focuses on identifying the molecular mechanisms through which TRIM E3 ligases act to balance innate immune cytokine responses using biochemical, proteomic and cell biology approaches. This will be done at a TRIM family-wide scale, after which the significance of individual molecules will be assessed in reconstituted in vitro models, relevant primary human cells (e.g. macrophages, dendritic cells) and ultimately small animal models.

The post-translational modifier ubiquitin is essential for both positive and negative immune regulation. Ubiquitin can be covalently attached to target molecules by so-called E3 ligases, after which the properties of these targets are dramatically changed. An important family of E3 ligases is formed by the 75-member tri-partite motif (TRIM) proteins. We recently demonstrated that half of the 75-member family of human TRIM ubiquitin E3 ligases positively

TEAM Benedikt Agerer Stefan Benke Richard Wallner

TRIM protein domain architecture. TRIM proteins have a conserved N-terminal tri-partite constellation and variable C-terminal domains. The N-terminal RING domain has putative E3 ubiquitin ligase activity.

SELECTED PUBLICATIONS Rajsbaum R, Versteeg GA, Schmid S, Maestre AM, Belicha-Villanueva A, Martínez-Romero C, Patel JR, Morrison J, Pisanelli G, Miorin L, Laurent-Rolle M, Moulton HM, Stein DA, Fernandez-Sesma A, tenOever BR, García-Sastre A. Unanchored K48-linked polyubiquitin synthesized by the E3-ubiquitin ligase TRIM6 stimulates the interferon-IKKε kinase-mediated antiviral response. Immunity. 2014 Jun 19;40(6):880-95. PMID: 24882218 Versteeg GA, Rajsbaum R, Sánchez-Aparicio MT, Maestre AM, Valdiviezo J, Shi M, Inn KS, Fernandez-Sesma A, Jung J, García-Sastre A. The E3-ligase TRIM family of proteins regulates signaling pathways triggered by innate immune pattern-recognition receptors. Immunity. 2013 Feb 21;38(2):384-98. PMID: 23438823 Mata MA, Satterly N, Versteeg GA, Frantz D, Wei S, Williams N, Schmolke M, Peña-Llopis S, Brugarolas J, Forst CV, White MA, García-Sastre A, Roth MG, Fontoura BM. Chemical inhibition of RNA viruses reveals REDD1 as a host defense factor. Nat Chem Biol. 2011 Sep 11;7(10):712-9. PMID: 21909097

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MFPL - 2016 RESEARCH GROUPS

ARNDT VON HAESELER

CIBIV-Center for Integrative Bioinformatics Vienna The CIBIV serves as a central facility to coordinate the Bioinformatic activities at the MFPL. It also provides infrastructure and expertise for the various research groups at MFPL and elsewhere.

Besides this data analysis part, the S.picudilla G.cuvier CIBIV pursues its own research S.barracuda Squids Octopuses agenda. The group’s main effort is to C.leucas Chitons understand the evolutionary processBivalves es that have shaped the genomes of E.evelynae contemporary species. To this end, the Sponges H.plumierii CIBIV applies methods from statistics, computer sciences, and mathematics S.planifrons Crabs to detect the traces ancient evoluShrimps tionary events have left in modern Amphipods genomes. The CIBIV is involved in several international projects, like the Pycnogonids Deep Metazoan Phylogeny project, Ophiuroids O.atlanticus where it coordinates the BioinforHermit crabs matics aspects (www.deep-phylogeny. Spiny lobsters org). The figure shows the results of H.ciliaris a large scale evolutionary analysis Holothurians Isopods for all available sequence data from Zooplankton fungi. Phytoplankton Microfauna More recently we have expanded Symbiotic algae Organic matter Benthic autotrophs our research interests to address mathematically and computationally tractable problems that may help to assist in conservation decisions. We have employed Food web restricted to only those taxa present in S1 or S2 the integer linear programming paradigm to explore (see main text). Red, green and blue nodes depict the taxa present exclusively in S1, exclusively in S2, and in both sets, conservation scenarios in the presence of external respectively. Light blue nodes correspond to aggregated groups. constraints. Arrows connect from predators to their preys with thickness Finally, we have started to develop tools to efficiently reflecting the prey proportion in the predator diet. Arrows analyse deep sequencing data that pose a new chalpointing to or from green and red nodes are coloured green lenge to bioinformatics. To this end we have developed and red respectively. Arrows between blue nodes are coloured an efficient optimal local alignment tool, which maps blue. Note that the arrows between green and red nodes are millions of reads to a reference genome in a few ignored. (From selected publications 2) seconds. The mapping of reads to a reference genome is the first, and possibly crucial, step for any further the results considerably by employing a specifically analysis. To understand the performance of different tailored algorithm. The development of efficient algomapping strategies we suggest a new evaluation tool rithms and further statistical tools to analyse the data that allows a graphical view of the mapping accurawill be a major research focus of the group during the cy. We also developed tools to analyse bisulfite deep next years. sequencing data and could show that one can improve

SELECTED PUBLICATIONS Nguyen LT, Schmidt HA, von Haeseler A, Minh BQ. IQ-TREE: a fast and effective stochastic algorithm for estimating maximum-likelihood phylogenies. Mol Biol Evol. 2015 Jan;32(1):268-74. PMID: 25371430 Chernomor O, Minh BQ, Forest F, Klaere S, Ingram T, Henzinger M, von Haeseler A. Split diversity in constrained conservation prioritization using integer linear programming. Methods Ecol Evol. 2015 Jan;6(1):83-91. PMID: 25893087 Smolka M, Rescheneder P, Schatz MC, von Haeseler A, Sedlazeck FJ. Teaser: Individualized benchmarking and optimization of read mapping results for NGS data. Genome Biol. 2015 Oct 22;16:235. PMID: 26494581

55

Arndt von Haeseler

TEAM Quang Minh Bui Christina Buhl Olga Chernomor Stephen Crotty Maurits Evers Miguel Gallach Iris Gruber Robert Happel Milica Krunic Konstantina Kyriakouli Luis Felipe Paulin Paz Susanne Pfeifer Florian Pflug Celine Prakash Philipp Rescheneder Heiko Schmidt Moritz Smolka David Szkiba Jana Trifinopoulos Anna Zappe


MFPL - 2016 RESEARCH GROUPS

GRAHAM WARREN

Biogenesis of the Golgi apparatus During normal growth and division, cells double in mass and divide into two equally-sized daughters. All cell constituents must be duplicated and then segregated equally during mitosis.

Graham Warren

TEAM Emine Sevil Davidson Isabelle Walters

For some constituents, such as the chromosomes, the underlying principles and the mechanistic details are relatively clear. For membrane-bound organelles, such as the Golgi, the principles and mechanism have been controversial. The primary aim of our research work is to understand how the cell creates another copy of the Golgi during the cell cycle and partitions them equally between the two daughter cells, thereby ensuring that this organelle is propagated through successive generations. The Golgi lies at the heart of the secretory pathway receiving the entire output of newly-synthesized cargo proteins from the endoplasmic reticulum, modifying any bound oligosaccharides, and then sorting them to their final destinations. Typically comprising a stack of closely-apposed and flattened cisternae, the Golgi presents a complex architecture that needs to be duplicated and partitioned every cell cycle. Most studies have focused on the partitioning of the Golgi during mitosis in mammalian cells and most studies suggest that the Golgi undergoes a dramatic conversion to thousands of small vesicles that can then be stochastically distributed between daughter cells. This conversion is triggered by mitotic kinases acting on structural proteins such as GRASPs and golgins. Golgi duplication has been more difficult to study since most mammalian cells have several hundred copies, subsumed into a ribbon-like structure next to the centrosomes and often the nucleus. This precludes facile observation of the duplication process. We have solved this problem by focusing on organisms that have only a single Golgi that undergoes duplication during the cell cycle and partitioning during mitosis.

Protozoan parasites are the best model systems since many have had their genomes sequenced and a variety of molecular biological techniques are available to manipulate protein levels. Trypanosoma brucei is the causative agent of sleeping sickness in sub-Saharan Africa, and provides a highly-simplified and organized secretory system that is ideal for studying the process of Golgi biogenesis. The duplication of the single Golgi can be observed using GFP-tagged Golgi proteins, and video fluorescence microscopy shows that the old Golgi is involved in the construction of the new. Furthermore, both are located on a novel bilobe structure that appears to act as a template, determining both the size of the Golgi and its inheritance. The composition and duplication of this bilobe are presently under investigation as is the molecular mechanism that generates the new Golgi.

Early in the cell cycle (left panel) the old Golgi (G; red) in T. brucei is located near to one lobe of a bilobe structure (green, closed arrowheads). Later in the cell cycle (right panel) the new Golgi is found associated with the other lobe suggesting that the bilobe has a role to play in the duplication process. N=nucleus; K=kinetoplast (mitDNA); open arrowheads=basal bodies.

SELECTED PUBLICATIONS Demmel L, Schmidt K, Lucast L, Havlicek K, Zankel A, Koestler T, Reithofer V, de Camilli P, Warren G. The endocytic activity of the flagellar pocket in Trypanosoma brucei is regulated by an adjacent phosphatidylinositol phosphate kinase. J Cell Sci. 2014 May 15;127(Pt 10):2351-64. PMID: 24639465 Sealey-Cardona M, Schmidt K, Demmel L, Hirschmugl T, Gesell T, Dong G, Warren G. Sec16 determines the size and functioning of the Golgi in the protist parasite, Trypanosoma brucei. Traffic. 2014 Jun;15(6):613-29 PMID: 24612401 Morriswood B, Havlicek K, Demmel L, Yavuz S, Sealey-Cardona M, Vidilaseris K, Anrather D, Kostan J, Djinovic-Carugo K, Roux KJ, Warren G. Novel bilobe components in Trypanosoma brucei identified using proximity-dependent biotinylation. Eukaryot Cell. 2013 Feb;12(2):356-67 PMID: 23264645

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MFPL - 2016 RESEARCH GROUPS

GEORG WEITZER

Stem cells of the heart In recent years, numerous groups provided compelling evidence for the existence of somatic stem cells in the heart of different mammalian species.

hearts and could maintain these cells as monoclonal self-renewing cells lines expressing Oct4, Sox2 and Nanog for several years. These cells obviously committed to the mesodermal lineage and expressing early myocardial transcription factors Brachyury, Nkx2.5, GATA4, and Isl1 exclusively differentiate to cardiomyocytes, smooth muscle cells, and vascular endothelial cells and thus were named cardiovascular progenitor cells (CVPCs). Cardiomyogenic progenitors further differentiate to equal numbers of functional pacemakers, atrial and ventricular cardiomyocytes with a near-adult action potential. Stimulation of CVPCs with Activin A and Retinoic Acid did not yield any cell types of the endodermal and ectodermal lineage, respectively. Addition of BMP2 and SPARC promoted cardiomyogenesis and led to the upregulation of genes for the mesoderm specific transcription factor Brachyury and the early myocardial transcription factor Nkx2.5. In our ongoing research, we try to reveal the molecular pathways which allow SPARC, BMP2 and Nodal to activate Brachyury and Nkx2.5 expression in CVPCs and how Brachyury, Nanog and Nkx2.5 interact on the transcriptional level in undifferentiated and differentiating CVPCs. Our long term scientific goal is to understand early cardiomyogenesis and how somatic stem cells may contribute to homeostasis of the heart. Understanding the molecular and cellular interplay regulating stem cell self-renewal and differentiation may contribute to future targeted therapies utilizing growth factors or small molecules for the temporal endogenous activation of the stem cell pool.

Localisation of Oct4 protein during cell division of CVPCs. Immunofluorescence microscopy of CVPCs with Oct4 antibodies (green), and DAPI (blue). Bar: 15 Îźm. Arrows, top, Metaphase; middle, Anaphase, and bottom, Telophase. Asterisks, Oct4 negative nucleus of a SNL76/7 feeder cell.

Stem cells and progenitor cells are supposed to exist in niches, where they remain in an undifferentiated and quasi-dormant state until external signals stimulate commitment and differentiation to specific somatic cells which may contribute to the repair or maintenance of homeostasis of an organ. Mimicking a stem cell niche of the heart in vitro, we succeeded in the isolation of somatic stem cells from postnatal murine

SELECTED PUBLICATIONS Hoebaus J, Heher P, Gottschamel T, Scheinast M, Auner H, Walder D, Wiedner M, Taubenschmid J, Miksch M, Sauer T, Schultheis M, Kuzmenkin A, Seiser C, Hescheler J, Weitzer G. Embryonic Stem Cells Facilitate the Isolation of Persistent Clonal Cardiovascular Progenitor Cell Lines and Leukemia Inhibitor Factor Maintains Their Self-Renewal and Myocardial Differentiation Potential in vitro. Cells Tissues Organs 2013;197(4):249-68. PMID: 23343517 Fuchs C, Scheinast M, Pasteiner W, Lagger S, Hofner M, Hoellrigl A, Schultheis M, Weitzer G. Self-Organization Phenomena in Embryonic Stem Cell-Derived Embryoid Bodies: Axis Formation and Breaking of Symmetry during Cardiomyogenesis. Cells Tissues Organs 2012;195(5):377-9. PMID: 21860211 Taubenschmid J, Weitzer G. Mechanisms of cardiogenesis in cardiovascular progenitor cells. Int Rev Cel Mol Bio 2012;293:195-267. PMID: 22251563

57

Georg Weitzer

TEAM Laura Ablasser Sabrina Bail Kerstin Fiedler Melanie Hobik Mario Ivankovic Valeria Kizner


MFPL - 2016 RESEARCH GROUPS

GERHARD WICHE

Cytolinker proteins in signalling and disease The cytoskeleton provides the structural basis for physical robustness, shape, movement, and intracellular dynamics of eukaryotic cells.

Gerhard Wiche

TEAM Maria J. Castañón Lilli Winter

Cancer: mechanisms of plectin-mediated metastasis in pancreatic and urinary bladder cancer

We are interested in cytoskeletal linker proteins (cytolinkers), a family of multi-modular, highly versatile proteins of exceptional size, that by networking and anchoring intermediate (10 nm) filaments (IFs) regulate the dynamics and architecture of the cytoskeleton. We are studying the role of cytolinkers in normal development, cellular stress response and disease, combining mouse genetics with cell and structural biology. Several years ago we discovered plectin, a ubiquitous cytolinker that became the prototype of what meanwhile is a whole family of similar proteins. Plectin has key functions in shaping cell architecture, mechanical stabilization, polarization, and migration of cells, positioning of organelles, signal transduction, nerve conduction, and others. Thus, loss or dysfunction of plectin leads to diseases affecting a variety of cell types and tissues. Plectin’s versatility is based on an unusual diversity of isoforms distinguished by short sequences that determine the differential targeting of IFs within cells. We have generated a panel of transgenic mouse lines, including full knockout (KO), and over a dozen single isoform and conditional/tissue-restricted KOs, and knock-in lines. This unique repertoire of genetically distinct mouse lines serves us and our collaborators around the world as animal models for a variety of plectin-related human diseases and source of cell systems for basic research topics with focus on:

Gene therapy, disease treatment: utilization of mini-genes in mouse models, application of chemical chaperons in clinical trials

Composite image of four (laterally aligned) teased skeletal muscle fibers isolated from (left to right): wild-type, skeletal muscle-restricted (MCK-Cre) conditional KO, isoform P1b-specific KO, and isoform P1d-specific KO mice. Specimens were co-immunolabeled using antibodies to cytochrome c (red) and α-actinin (green) to visualize mitochondria and Z-disks, respectively. (Cover image of Hum Mol Genet 2015, 240, issue Aug 15)

Myofibrillar myopathies: the hallmarks of which are formation of protein aggregates in skeletal muscle, mitochondrial dysfunction, and disturbed heart function Skin blistering disease (EBS): due to lack of hemidesmosome stabilization Neuropathies and memory loss: caused by deregulated microtubule dynamics, reduced nerve cell branching and nerve conduction, as well as unbalanced metabolism

Primary lung endothelial (mouse) cells deficient in the cytolinker protein plectin at an early stage of adherens junction formation. Cells were subjected to triple immunofluorescence microscopy using antibodies to vascular-endothelial cadherin (green), actin (red) and vimentin (blue). Adherens junctions (yellow) have lost their straight and in-line arrangement typical of wild-type cells. (Cover image of J Cell Sci 2015, 128, issue Nov 15)

Mechanosensing and -transduction: with fibroblasts, keratinocytes, endothelia, cardiac/skeletal muscle used as model systems

SELECTED PUBLICATIONS Osmanagic-Myers S, Rus S, Wolfram M, Brunner D, Goldmann WH, Bonakdar N, Fischer I, Reipert S, Zuzuarregui A, Walko G, Wiche G. Plectin reinforces vascular integrity by mediating crosstalk between the vimentin and the actin networks. J Cell Sci. 2015; 128:4138-50. PMID: 26519478 Wiche G, Osmanagic-Myers S, Castañón MJ. Networking and anchoring through plectin: a key to IF functionality and mechanotransduction. Curr Opin Cell Biol. 2015;32:21-29. PMID: 25460778 Winter L, Staszewska I, Mihailovska E, Fischer I, Goldmann WH, Schröder R, Wiche G. Chemical chaperone ameliorates pathological protein aggregation in plectin-deficient muscle. J Clin Invest. 2014;124: 1144-1157. PMID: 24487589

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MFPL - 2016 RESEARCH GROUPS

ANGELA WITTE

φCh1, a model system for gene regulation of haloalkali-

philic Archaea facing two extremes: high pH and salt The virus φCh1 was found by spontaneous lysis of a culture of the haloalkaliphilic, archaeon, Natrialba magadii, an isolate from the soda lake, Lake Magadii in Kenya.

This organism has an optimal growth at 3.5M NaCl and at a pH of 9.5. The virus itself is used as a model system to analyse gene expression in haloalkaliphilic organisms, facing with two extremes: a high pH and high concentrations of salt. The sequence of φCh1, infecting the haloalkaliphilic archaeon Natrialba magadii, contains an open reading frame (int1) in the central part of its genome that belongs to the λ integrase family of site-specific recombinases. The flanking sequences of int1 contain several direct repeats of 30 bp in length (IR-L and IR-R), which are orientated in an inverted direction. The invertible region encodes two structural proteins (gp34 and gp36, encoded by ORF34 and ORF36) expected to represent the viral tail fibre proteins.

Angela Witte

In vitro experiments using purified protein variants gp341 and gp3452 (containing the C-terminus of gp36) revealed exclusive binding of gp3452 but not of gp341 to cells of the cured strain N. magadii L13. This specific binding could be inhibited by the addition of α-D-galactose. α-D-galactose also significantly reduced the infectivity of φCh1. The C-terminus contains a domain with similarities to the super-family of “galactose-like binding” proteins. In summary, the experiments gave evidence that gp3452 represents the anti-receptor of φCh1 that binds to specific carbohydrate ligands located on the cell surface of N. magadii. Currently the work concentrates on the identification and function of repressor and activator molecules encoded by the virus, gene regulation due to a recombination event, identification of the receptor for the virus on the cell surface of N. magadii and the transformation/shuttle vector system developed by the group. In addition, the method is used to construct different mutants.

Electron micrograph of φCh1 particle negatively stained with uranyl acetate.

SELECTED PUBLICATIONS Klein R, Rössler N, Iro M, Scholz H, Witte, A. Haloarchaeal myovirus φCh1 harbours a phase variation system for the production of protein variants with distinct cell surface adhesion specificities. Mol Microbiol 2012;83:137-50. PMID: 22111759 Mayrhofer-Iro M, Ladurner A, Meissner C, Derntl C, Reiter M, Haider F, Dimmel K, Rössler N, Klein R, Baranyi U, Scholz H, Witte A. Utilization of virus φCh1 elements to establish a shuttle vector system for halo(alkali)philic Archaea via transformation of Natrialba magadii. Appl Environ Microbiol 2013;79:2741-2748. PMID: 23416999 Derntl C, Selb R, Klein R, Alte B, Witte A. Genomic manipulations in alkaliphilic haloarchaea demonstrated by a gene disruption in Natrialba magadii. FEMS Microbiol Lett 2015;362(21). PMID: 26424765

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TEAM Yan Gillen Martin Kaufmann Michael Tschurtschenthaler


MFPL - 2016 RESEARCH GROUPS

IVAN YUDUSHKIN

Functional imaging of signalling networks Adaptive character of responses to signals from the environment is a fundamental property of all living organisms. At the cellular level, it is brought about by a highly integrated process of transmembrane and intracellular signal transduction. Although many signalling molecules have been identified, how exactly the dynamics of their interactions ensure the ultimate specificity and adaptive character of cellular responses remains poorly understood. How changes in flux of substrates via an enzyme affects signalling specificity? How intracellular localization of a protein is coupled to its signalling role? What is the function of a given protein in network regulation?

Ivan Yudushkin

TEAM Michael Ebner Volodymyr Shubchynskyy

To address these questions, we employ a combination of biochemical and advanced imaging techniques. We are developing tools to visualize the functional state of signalling molecules in live cells to determine how spatial distribution, regulation of specific activity and the corresponding changes in the flux of substrates via individual enzymes determine their signalling function. Furthermore, to examine the physiological relevance of individual enzymes in signalling, we will be developing new tools to selectively manipulate their localization and activity in live cells.

(A) T cell receptor complex is phosphorylated on multiple tyrosine residues upon binding of an antigen. This phosphorylation can be detected in live T cells using translocation- (A) or FRET-based (B) fluorescent reporters. Our data demonstrate that in T cells, active T cell receptor (C, green) localizes on endosomal vesicles (C, red) and may recruit and activate the essential downstream kinase ZAP-70.

The ultimate specialization of T lymphocytes provides a cell biologist with a unique model system for functional studies. Specifically, we will be addressing the functional role of protein compartmentalization in T cell signalling, probe the flux of substrates via the Src family kinases in T cells and develop methods to acutely destabilize select signalling proteins to elucidate their physiological functions in T cells. The outlined approaches should also be broadly applicable in other model systems. Our findings will address a spectrum of fundamental questions of cellular physiology as well as provide insight into the molecular mechanisms of activation of T cells. In the lab we are seeking to create a friendly, collaborative environment that promotes dynamic exchange of ideas and expertise between colleagues in and outside the lab and values initiative, originality and scientific rigor. The group is looking for PhD students and postdocs to pursue challenging, technology-oriented research projects.

SELECTED PUBLICATIONS Yudushkin IA, Vale RD. Imaging T-cell receptor activation reveals accumulation of tyrosine-phosphorylated CD3Îś in the endosomal compartment. Proc Natl Acad Sci U S A. 2010 Dec 21;107(51):22128-33 PMID: 21135224 de Graffenried CL, Anrather D, Von RauĂ&#x;endorf F, Warren G. Polo-like kinase phosphorylation of bilobe-resident TbCentrin2 facilitates flagellar inheritance in Trypanosoma brucei. Mol Biol Cell. 2013 Jun;24(12):1947-63. PMID: 23615446 Yudushkin IA, Schleifenbaum A, Kinkhabwala A, Neel BG, Schultz C, Bastiaens PI. Live-cell imaging of enzyme-substrate interaction reveals spatial regulation of PTP1B. Science. 2007 Jan 5;315(5808):115-9 PMID: 17204654

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MFPL - 2016 RESEARCH GROUPS

BOJAN ZAGROVIC

Laboratory of Molecular Biophysics The function of biomolecules arises from the interplay between their structure, dynamics and interactions with the environment.

protein folding or post-translational modifications of proteins. In particular, we are interested in studying how crowding affects the co-localization of binding partners and employ MD and Brownian dynamics simulations and structural bioinformatics methods to address this question. Finally, we have recently discovered a remarkably robust correspondence between the nucleobase density profiles of mRNAs and the nucleobase affinity profiles of their cognate protein sequences. For example, the purine-density profile of a typical mRNA coding sequence in H. sapiens matches the guanine-affinity profile of its cognate protein with an absolute value of the Pearson correlation of 0.8 on average. We believe this finding supports and extends the stereochemical hypothesis concerning the origin of the genetic code and suggests that cognate mRNAs and proteins may be physico-chemically complementary to each other and bind, especially if unstructured. Moreover, these findings suggest a novel principle of RNA-protein interactions beyond the cognate context, with potential implications in different areas of RNA-protein biology. We use different biophysical methods including MD simulations, structural bioinformatics techniques, free energy calculations and in vitro experiments to further explore this hypothesis.

We explore this fundamental principle through the use of computational and theoretical methods, including molecular dynamics (MD) simulations and structural bioinformatics techniques, in close collaboration with experimentalists. The work in our group is organized around three principal directions. First, we are interested in the role of dynamics and conformational entropy in non-covalent biomolecular interactions. We develop new methods for calculating conformational entropy of biomolecules from computer simulations and for measuring it experimentally. In addition to function, dynamics also affects the very process of biomolecular structure determination. We use MD simulations to help interpret time- and ensemble-averaged X-ray and NMR experiments and analyse the impact of conformational averaging on the derived structures. Second, all biomolecular processes occur in crowded, dynamic, constantly changing environments. We study how crowding affects biomolecular interactions and other basic processes such as

A) Matching of the mRNA purine-density profile and the guanine-affinity profile of the cognate protein of a typical, average human mRNA/protein pair; B) The Complementarity Hypothesis: mRNAs and their cognate proteins are complementary to each other and bind in a co-aligned manner, especially if unstructured. The degree of complementarity is negatively regulated by the mRNA adenine content.

SELECTED PUBLICATIONS Polyansky AA, Zagrovic B. Evidence of direct complementary interactions between messenger RNAs and their cognate proteins. Nucleic Acids Res. 2013 Oct;41(18):8434-43. PMID: 23868089 de Ruiter A, Zagrovic B. Absolute binding-free energies between standard RNA/DNA nucleobases and amino-acid sidechain analogs in different environments. Nucleic Acids Res. 2015 Jan;43(2):708-18. PMID: 25550435 Fleck M, Polyansky AA, Zagrovic B. PARENT: A Parallel Software Suite for the Calculation of Configurational Entropy in Biomolecular Systems. J Chem Theory Comput. 2016 Apr 12;12(4):2055-65. PMID: 26989950

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Bojan Zagrovic

TEAM Lukas Bartonek Markus Fleck Mathias Kreuter Anton A. Polyansky David Weichselbaum


MFPL - 2016 RESEARCH GROUPS

Publications 2016 As of October 31st, 2016 Temmel H, Müller C, Sauert M, Vesper O, Reiss A, Popow J, Martinez J, Moll I. The RNA ligase RtcB reverses MazF-induced ribosome heterogeneity in Escherichia coli. Nucleic Acids Res. 2016 Oct 26. pii: gkw1018. PMID: 27789694

Furtmüller PG, Oostenbrink C, Obinger C. Chemistry and Molecular Dynamics Simulations of Heme b-HemQ and Coproheme-HemQ. Biochemistry. 2016 Sep 27;55(38):5398412. PMID: 27599156.

Tajaddod M, Tanzer A, Licht K, Wolfinger MT, Badelt S, Huber F, Pusch O, Schopoff S, Janisiw M, Hofacker I, Jantsch MF. Transcriptome-wide effects of inverted SINEs on gene expression and their impact on RNA polymerase II activity. Genome Biol. 2016 Oct 25;17(1):220. PMID: 27782844

Pantazopoulou M, Boban M, Foisner R, Ljungdahl PO. Cdc48 and Ubx1 participate in an inner nuclear membrane associated degradation pathway that governs the turnover of Asi1. J Cell Sci. 2016 Aug 26. pii: jcs.189332. [Epub ahead of print] PMID: 27566164.

Torggler R, Papinski D, Brach T, Bas L, Schuschnig M, Pfaffenwimmer T, Rohringer S, Matzhold T, Schweida D, Brezovich A, Kraft C. Two Independent Pathways within Selective Autophagy Converge to Activate Atg1 Kinase at the Vacuole. Mol Cell. 2016 Oct 20;64(2):221-235. PMID: 27768871.

Martinez J, Zagrovic B. A code within a code: how codons influence mRNA stability. EMBO J. 2016 Oct 4;35(19):20642065. PMID: 27562506.

Hagelkruys A, Moser MA, Seiser C. Generation of Tissue-Specific Mouse Models to Analyze HDAC Functions. Methods Mol Biol. 2017;1510:169-192. PMID: 27761821. Hofbauer S, Mlynek G, Milazzo L, Pühringer D, Maresch D, Schaffner I, Furtmüller PG, Smulevich G, Djinović-Carugo K, Obinger C. Hydrogen peroxide-mediated conversion of coproheme to heme b by HemQ - Lessons from the first crystal structure and kinetic studies. FEBS J. 2016 Oct 18. PMID: 27758026. Putz EM, Majoros A, Gotthardt D, Prchal-Murphy M, Zebedin-Brandl EM, Fux DA, Schlattl A, Schreiber RD, Carotta S, Müller M, Gerner C, Decker T, Sexl V. Novel non-canonical role of STAT1 in Natural Killer cell cytotoxicity. Oncoimmunology. 2016 May 19;5(9):e1186314. PMID: 27757297. Nowacka JD, Baumgartner C, Pelorosso C, Roth M, Zuber J, Baccarini M. MEK1 is required for the development of NRAS-driven leukemia. Oncotarget. 2016 Oct 10. PMID: 27741509. Machovina TS, Mainpal R, Daryabeigi A, McGovern O, Paouneskou D, Labella S, Zetka M, Jantsch V, Yanowitz JL. A Surveillance System Ensures Crossover Formation in C. elegans. Curr Biol. 2016 Oct 1. pii: S0960-9822(16)310569. PMID: 27720619. Molodtsov MI, Mieck C, Dobbelaere J, Dammermann A, Westermann S, Vaziri A. A Force-Induced Directional Switch of a Molecular Motor Enables Parallel Microtubule Bundle Formation. Cell. 2016 Oct 6;167(2):539-552.e14. PMID: 27716509. Berk KA, Vongpromek R, Jiang M, Schneider WJ, Timman R, Verhoeven AJ, Bujo H, Sijbrands EJ, Mulder MT. Levels of the soluble LDL receptor-relative LR11 decrease in overweight individuals with type 2 diabetes upon diet-induced weight loss. Atherosclerosis. 2016 Sep 22;254:67-72. PMID: 27697674.

Wei Q, Zhang Y, Schouteden C, Zhang Y, Zhang Q, Dong J, Wonesch V, Ling K, Dammermann A, Hu J. The hydrolethalus syndrome protein HYLS-1 regulates formation of the ciliary gate. Nat Commun. 2016 Aug 18;7:12437. PMID: 27534274. Juen MA, Wunderlich CH, Nußbaumer F, Tollinger M, Kontaxis G, Konrat R, Hansen DF, Kreutz C. Excited States of Nucleic Acids Probed by Proton Relaxation Dispersion NMR Spectroscopy. Angew Chem Int Ed Engl. 2016 Sep 19;55(39):12008-12. Epub 2016 Aug 17. PMID: 27533469. Cuchillo-Ibañez I, Mata-Balaguer T, Balmaceda V, Arranz JJ, Nimpf J, Sáez-Valero J. The β-amyloid peptide compromises Reelin signaling in Alzheimer’s disease. Sci Rep. 2016 Aug 17;6:31646. PMID: 27531658 Schüchner S, Andorfer P, Mudrak I, Ogris E. Anti-RAINBOW dye-specific antibodies as universal tools for the visualization of prestained protein molecular weight markers in Western blot analysis. Sci Rep. 2016 Aug 17;6:31363. PMID: 27531616. Truebestein L, Leonard TA. Coiled-coils: The long and short of it. Bioessays. 2016 Sep;38(9):903-16. doi: 10.1002/ bies.201600062. PMID: 27492088. Tosato V, Sims J, West N, Colombin M, Bruschi CV. Post-translocational adaptation drives evolution through genetic selection and transcriptional shift in Saccharomyces cerevisiae. Curr Genet. 2016 Aug 4. [Epub ahead of print] PMID: 27491680. Yoon SW, Lee MS, Xaver M, Zhang L, Hong SG, Kong YJ, Cho HR, Kleckner N, Kim KP. Meiotic prophase roles of Rec8 in crossover recombination and chromosome structure. Nucleic Acids Res. 2016 Aug 2. pii: gkw682. [Epub ahead of print] PMID: 27484478. Schrempf D, Minh BQ, De Maio N, von Haeseler A, Kosiol C. Reversible polymorphism-aware phylogenetic models and their application to tree inference. J Theor Biol. 2016 Oct 21;407:362-70. PMID: 27480613.

Sedlyarova N, Shamovsky I, Bharati BK, Epshtein V, Chen J, Gottesman S, Schroeder R, Nudler E. sRNA-Mediated Control of Transcription Termination in E. coli. Cell. 2016 Sep 22;167(1):111-121.e13. PMID: 27662085.

Ruivo EF, Gonçalves LM, Carvalho LA, Guedes RC, Hofbauer S, Brito JA, Archer M, Moreira R, Lucas SD. Clickable 4-Oxoβ-lactam-Based Selective Probing for Human Neutrophil Elastase Related Proteomes. ChemMedChem. 2016 Sep 20;11(18):2037-42. PMID: 27465595.

Prakash C, Von Haeseler A. An Enumerative Combinatorics Model for Fragmentation Patterns in RNA Sequencing Provides Insights into Nonuniformity of the Expected Fragment Starting-Point and Coverage Profile. J Comput Biol. 2016 Sep 23. PMID: 27661099.

Loponte S, Segré CV, Senese S, Miccolo C, Santaguida S, Deflorian G, Citro S, Mattoscio D, Pisati F, Moser MA, Visintin R, Seiser C, Chiocca S. Dynamic phosphorylation of Histone Deacetylase 1 by Aurora kinases during mitosis regulates zebrafish embryos development. Sci Rep. 2016 Jul 26;6:30213. PMID: 27458029.

Gallego LD, Ghodgaonkar Steger M, Polyansky AA, Schubert T, Zagrovic B, Zheng N, Clausen T, Herzog F, Köhler A. Structural mechanism for the recognition and ubiquitination of a single nucleosome residue by Rad6-Bre1. Proc Natl Acad Sci U S A. 2016 Sep 20;113(38):10553-8. PMID: 27601672.

Tinsley JN, Molodtsov MI, Prevedel R, Wartmann D, Espigulé-Pons J, Lauwers M, Vaziri A. Direct detection of a single photon by humans. Nat Commun. 2016 Jul 19;7:12172. PMID: 27434854.

Hofbauer S, Dalla Sega M, Scheiblbrandner S, Jandova Z, Schaffner I, Mlynek G, Djinović-Carugo K, Battistuzzi G,

Raguz J, Jeric I, Niault T, Nowacka JD, Kuzet SE, Rupp C, Fischer I, Biggi S, Borsello T, Baccarini M. Epidermal RAF

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prevents allergic skin disease. Elife. 2016 Jul 19;5. pii: e14012. PMID: 27431613. Żerko S, Byrski P, Włodarczyk-Pruszyński P, Górka M, Ledolter K, Masliah E, Konrat R, Koźmiński W. Five and four dimensional experiments for robust backbone resonance assignment of large intrinsically disordered proteins: application to Tau3x protein. J Biomol NMR. 2016 Aug;65(3-4):193-203. PMID: 27430223. Wirnsberger G, Zwolanek F, Asaoka T, Kozieradzki I, Tortola L, Wimmer RA, Kavirayani A, Fresser F, Baier G, Langdon WY, Ikeda F, Kuchler K, Penninger JM. Inhibition of CBLB protects from lethal Candida albicans sepsis. Nat Med. 2016 Aug;22(8):915-23. PMID: 27428901. Tomanov K, Ziba I, Bachmair A. SUMO Chain Formation by Plant Enzymes. Methods Mol Biol. 2016;1450:97-105. PMID: 27424748. Yan GX, Zhang J, Shodhan A, Tian M, Miao W. Cdk3, a conjugation-specific cyclin-dependent kinase, is essential for the initiation of meiosis in Tetrahymena thermophila. Cell Cycle. 2016 Sep 16;15(18):2506-14. PMID: 27420775. Abert C, Kontaxis G, Martens S. Accessory Interaction Motifs in the Atg19 Cargo Receptor Enable Strong Binding to the Clustered Ubiquitin-related Atg8 Protein. J Biol Chem. 2016 Sep 2;291(36):18799-808 PMID: 27402840. Summers JA, Harper AR, Feasley CL, Van-Der-Wel H, Byrum JN, Hermann M, West CM. Identification of Apolipoprotein A-I as a Retinoic Acid-binding Protein in the Eye. J Biol Chem. 2016 Sep 2;291(36):18991-9005. PMID: 27402828. Khan MA, Göpel Y, Milewski S, Görke B. Two Small RNAs Conserved in Enterobacteriaceae Provide Intrinsic Resistance to Antibiotics Targeting the Cell Wall Biosynthesis Enzyme Glucosamine-6-Phosphate Synthase. Front Microbiol. 2016 Jun 15;7:908. PMID: 27379045. Grumaz S, Stevens P, Grumaz C, Decker SO, Weigand MA, Hofer S, Brenner T, von Haeseler A, Sohn K. Next-generation sequencing diagnostics of bacteremia in septic patients. Genome Med. 2016 Jul 1;8(1):73. PMID: 27368373. Conzemius R, Ganjian H, Blaas D, Fuchs R. ICAM-1 Binding Rhinoviruses A89 and B14 Uncoat in Different Endosomal Compartments. J Virol. 2016 Aug 12;90(17):793442. PMID: 27334586. Polakova S, Molnarova L, Hyppa RW, Benko Z, Misova I, Schleiffer A, Smith GR, Gregan J. Dbl2 Regulates Rad51 and DNA Joint Molecule Metabolism to Ensure Proper Meiotic Chromosome Segregation. PLoS Genet. 2016 Jun 15;12(6):e1006102. PMID: 27304859. Agustinho DP, de Oliveira MA, Tavares AH, Derengowski L, Stolz V, Guilhelmelli F, Mortari MR, Kuchler K, Silva-Pereira I. Dectin-1 is required for miR155 upregulation in murine macrophages in response to Candida albicans. Virulence. 2016 Jun 13:1-12. [Epub ahead of print] PubMed PMID: 27294852. Drmota Prebil S, Slapšak U, Pavšič M, Ilc G, Puž V, de Almeida Ribeiro E, Anrather D, Hartl M, Backman L, Plavec J, Lenarčič B, Djinović-Carugo K. Structure and calcium-binding studies of calmodulin-like domain of human non-muscle α-actinin-1. Sci Rep. 2016 Jun 7;6:27383. PMID: 27272015. Zhang W, Thieme CJ, Kollwig G, Apelt F, Yang L, Winter N, Andresen N, Walther D, Kragler F. tRNA-Related Sequences Trigger Systemic mRNA Transport in Plants. Plant Cell. 2016 Jun;28(6):1237-49. PMID: 27268430.


MFPL - 2016 RESEARCH GROUPS Müller C, Sokol L, Vesper O, Sauert M, Moll I. Insights into the Stress Response Triggered by Kasugamycin in Escherichia coli. Antibiotics (Basel). 2016 Jun 1;5(2). pii: E19. PMID: 27258317.

Carugo O, Djinović-Carugo K. Criteria to Extract High-Quality Protein Data Bank Subsets for Structure Users. Methods Mol Biol. 2016;1415:139-52. PMID: 27115631.

Turco E, Martens S. Insights into autophagosome biogenesis from in vitro reconstitutions. J Struct Biol. 2016 Oct;196(1):29-36. PMID: 27251905.

Licht K, Kapoor U, Mayrhofer E, Jantsch MF. Adenosine to Inosine editing frequency controlled by splicing efficiency. Nucleic Acids Res. 2016 Jul 27;44(13):6398-408. PMID: 27112566.

Blaas D, Fuchs R. Mechanism of human rhinovirus infections. Mol Cell Pediatr. 2016 Dec;3(1):21. PMID: 27251607. Lenz DA, Mladek BM, Likos CN, Blaak R. Thermodynamic stability and structural properties of cluster crystals formed by amphiphilic dendrimers. J Chem Phys. 2016 May 28;144(20):204901. PMID: 27250325. Keller AC, Badani H, McClatchey PM, Baird NL, Bowlin JL, Bouchard R, Perng GC, Reusch JE, Kaufer BB, Gilden D, Shahzad A, Kennedy PG, Cohrs RJ. Varicella zoster virus infection of human fetal lung cells alters mitochondrial morphology. J Neurovirol. 2016 Oct;22(5):674-682. PMID: 27245593. Zlopasa L, Brachner A, Foisner R. Nucleo-cytoplasmic shuttling of the endonuclease ankyrin repeats and LEM domain-containing protein 1 (Ankle1) is mediated by canonical nuclear export- and nuclear import signals. BMC Cell Biol. 2016 Jun 1;17(1):23. PMID: 27245214. Poretska O, Yang S, Pitorre D, Rozhon W, Zwerger K, Uribe MC, May S, McCourt P, Poppenberger B, Sieberer T. The Small Molecule Hyperphyllin Enhances Leaf Formation Rate and Mimics Shoot Meristem Integrity Defects Associated with AMP1 Deficiency. Plant Physiol. 2016 Jun;171(2):1277-90. PMID: 27208298.

Daryabeigi A, Woglar A, Baudrimont A, Silva N, Paouneskou D, Vesely C, Rauter M, Penkner A, Jantsch M, Jantsch V. Nuclear Envelope Retention of LINC Complexes Is Promoted by SUN-1 Oligomerization in the Caenorhabditis elegans Germ Line. Genetics. 2016 Jun;203(2):733-48. PMID: 27098914. Maric-Biresev J, Hunn JP, Krut O, Helms JB, Martens S, Howard JC. Loss of the interferon-γ-inducible regulatory immunity-related GTPase (IRG), Irgm1, causes activation of effector IRG proteins on lysosomes, damaging lysosomal function and predicting the dramatic susceptibility of Irgm1-deficient mice to infection. BMC Biol. 2016 Apr 20;14:33. PMID: 27098192. Jin W, Jiang M, Han X, Han X, Murano T, Hiruta N, Ebinuma H, Piao L, Schneider WJ, Bujo H. Circulating soluble form of LR11, a regulator of smooth muscle cell migration, is a novel marker for intima-media thickness of carotid arteries in type 2 diabetes. Clin Chim Acta. 2016 Jun 1;457:137-41. PMID: 27095609. Hajnic M, Ruiter Ad, Polyansky AA, Zagrovic B. Inosine Nucleobase Acts as Guanine in Interactions with Protein Side Chains. J Am Chem Soc. 2016 May 4;138(17):551922. PMID: 27093234.

Yan GX, Dang H, Tian M, Zhang J, Shodhan A, Ning YZ, Xiong J, Miao W. Cyc17, a meiosis-specific cyclin, is essential for anaphase initiation and chromosome segregation in Tetrahymena thermophila. Cell Cycle. 2016 Jul 17;15(14):1855-64. PMID: 27192402.

Alqaisi KM, Lamare MD, Grattan DR, Damsteegt EL, Schneider WJ, Lokman PM. A comparative study of vitellogenesis in Echinodermata: Lessons from the sea star. Comp Biochem Physiol A Mol Integr Physiol. 2016 Aug;198:72-86. PMID: 27085373.

Sedlyarov V, Fallmann J, Ebner F, Huemer J, Sneezum L, Ivin M, Kreiner K, Tanzer A, Vogl C, Hofacker I, Kovarik P. Tristetraprolin binding site atlas in the macrophage transcriptome reveals a switch for inflammation resolution. Mol Syst Biol. 2016 May 13;12(5):868. PMID: 27178967.

Trifinopoulos J, Nguyen LT, von Haeseler A, Minh BQ. W-IQ-TREE: a fast online phylogenetic tool for maximum likelihood analysis. Nucleic Acids Res. 2016 Jul 8;44(W1):W232-5. PMID: 27084950.

Blaas D. Viral entry pathways: the example of common cold viruses. Wien Med Wochenschr. 2016 May;166(78):211-26. PMID: 27174165. Tortola L, Nitsch R, Bertrand MJ, Kogler M, Redouane Y, Kozieradzki I, Uribesalgo I, Fennell LM, Daugaard M, Klug H, Wirnsberger G, Wimmer R, Perlot T, Sarao R, Rao S, Hanada T, Takahashi N, Kernbauer E, Demiröz D, Superti-Furga G, Decker T, Pichler A, Ikeda F, Kroemer G, Vandenabeele P, Sorensen PH, Penninger JM. The Tumor Suppressor Hace1 Is a Critical Regulator of TNFR1-Mediated Cell Fate. Cell Rep. 2016 May 17;15(7):1481-92. PMID: 27160902. Link J, Benavente R, Alsheimer M. Analysis of Meiotic Telomere Behavior in the Mouse. Methods Mol Biol. 2016;1411:195-208. PMID: 27147043. Martens S. Necessary, but also Sufficient? Trends Cell Biol. 2016 Jul;26(7):467-9. PMID: 27142894. Schaupper M, Jeltsch M, Rohringer S, Redl H, Holnthoner W. Lymphatic Vessels in Regenerative Medicine and Tissue Engineering. Tissue Eng Part B Rev. 2016 Oct;22(5):395-407. PMID: 27142568. Schmidinger B, Weijler AM, Schneider WJ, Hermann M. Hepatosteatosis and estrogen increase apolipoprotein O production in the chicken. Biochimie. 2016 Aug;127:3743. PMID: 27126072. Chernomor O, von Haeseler A, Minh BQ. Terrace Aware Data Structure for Phylogenomic Inference from Supermatrices. Syst Biol. 2016 Apr 26. pii: syw037. [Epub ahead of print] PMID: 27121966.

Terai K, Jiang M, Tokuyama W, Murano T, Takada N, Fujimura K, Ebinuma H, Kishimoto T, Hiruta N, Schneider WJ, Bujo H. Levels of soluble LR11/SorLA are highly increased in the bile of patients with biliary tract and pancreatic cancers. Clin Chim Acta. 2016 Jun 1;457:130-6. PMID: 27079357. Petrov D, Daura X, Zagrovic B. Effect of Oxidative Damage on the Stability and Dimerization of Superoxide Dismutase 1. Biophys J. 2016 Apr 12;110(7):1499-509. PMID: 27074676. Truebestein L, Elsner DJ, Leonard TA. Made to measure keeping Rho kinase at a distance. Small GTPases. 2016 Apr 2;7(2):82-92. PMID: 27070834. Papinski D, Kraft C. Regulation of Autophagy By Signaling Through the Atg1/ULK1 Complex. J Mol Biol. 2016 May 8;428(9 Pt A):1725-41. PMID: 27059781. Gallach M, Betrán E. Dosage Compensation and the Distribution of Sex-Biased Gene Expression in Drosophila: Considerations and Genomic Constraints. J Mol Evol. 2016 May;82(4-5):199-206. PMID: 27059220. Licht K, Jantsch MF. Rapid and dynamic transcriptome regulation by RNA editing and RNA modifications. J Cell Biol. 2016 Apr 11;213(1):15-22. PMID: 27044895. Richter B, Sliter DA, Herhaus L, Stolz A, Wang C, Beli P, Zaffagnini G, Wild P, Martens S, Wagner SA, Youle RJ, Dikic I. Phosphorylation of OPTN by TBK1 enhances its binding to Ub chains and promotes selective autophagy of damaged mitochondria. Proc Natl Acad Sci U S A. 2016 Apr 12;113(15):4039-44. PMID: 27035970. Schubert T, Köhler A. Mediator and TREX-2: Emerging links between transcription initiation and mRNA export. Nucleus. 2016 Apr 25;7(2):126-31. PMID: 27028218.

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Baccarini M, Dikic I. Editorial overview: Cell regulation. Curr Opin Cell Biol. 2016 Apr;39:iv-vi. PMID: 27017261. Jagut M, Hamminger P, Woglar A, Millonigg S, Paulin L, Mikl M, Dello Stritto MR, Tang L, Habacher C, Tam A, Gallach M, von Haeseler A, Villeneuve AM, Jantsch V. Separable Roles for a Caenorhabditis elegans RMI1 Homolog in Promoting and Antagonizing Meiotic Crossovers Ensure Faithful Chromosome Inheritance. PLoS Biol. 2016 Mar 24;14(3):e1002412. PMID: 27011106. Braun J, Meixner A, Brachner A, Foisner R. The GIYYIG Type Endonuclease Ankyrin Repeat and LEM Domain-Containing Protein 1 (ANKLE1) Is Dispensable for Mouse Hematopoiesis. PLoS One. 2016 Mar 24;11(3):e0152278. PMID: 27010503 Cojoc G, Roscioli E, Zhang L, García-Ulloa A, Shah JV, Berns MW, Pavin N, Cimini D, Tolić IM, Gregan J. Laser microsurgery reveals conserved viscoelastic behavior of the kinetochore. J Cell Biol. 2016 Mar 28;212(7):767-76. PMID: 27002163. Fleck M, Polyansky AA, Zagrovic B. PARENT: A Parallel Software Suite for the Calculation of Configurational Entropy in Biomolecular Systems. J Chem Theory Comput. 2016 Apr 12;12(4):2055-65. PMID: 26989950. Schwarz T, Montanari F, Cseke A, Wlcek K, Visvader L, Palme S, Chiba P, Kuchler K, Urban E, Ecker GF. Subtle Structural Differences Trigger Inhibitory Activity of Propafenone Analogues at the Two Polyspecific ABC Transporters: P-Glycoprotein (P-gp) and Breast Cancer Resistance Protein (BCRP). ChemMedChem. 2016 Jun 20;11(12):1380-94. PMID: 26970257. Grutsch S, Brüschweiler S, Tollinger M. NMR Methods to Study Dynamic Allostery. PLoS Comput Biol. 2016 Mar 10;12(3):e1004620. PMID: 26964042. Castiglia V, Piersigilli A, Ebner F, Janos M, Goldmann O, Damböck U, Kröger A, Weiss S, Knapp S, Jamieson AM, Kirschning C, Kalinke U, Strobl B, Müller M, Stoiber D, Lienenklaus S, Kovarik P. Type I Interferon Signaling Prevents IL-1β-Driven Lethal Systemic Hyperinflammation during Invasive Bacterial Infection of Soft Tissue. Cell Host Microbe. 2016 Mar 9;19(3):375-87. PMID: 26962946. Rettelbach A, Servedio MR, Hermisson J. Speciation in peripheral populations: effects of drift load and mating systems. J Evol Biol. 2016 May;29(5):1073-90. PMID: 26929184. Sauert M, Wolfinger MT, Vesper O, Müller C, Byrgazov K, Moll I. The MazF-regulon: a toolbox for the post-transcriptional stress response in Escherichia coli. Nucleic Acids Res. 2016 Aug 19;44(14):6660-75. PMID: 26908653. Decker T. Emancipation from transcriptional latency: unphosphorylated STAT5 as guardian of hematopoietic differentiation. EMBO J. 2016 Mar 15;35(6):555-7. PMID: 26893391. Majoros A, Platanitis E, Szappanos D, Cheon H, Vogl C, Shukla P, Stark GR, Sexl V, Schreiber R, Schindler C, Müller M, Decker T. Response to interferons and antibacterial innate immunity in the absence of tyrosine-phosphorylated STAT1. EMBO Rep. 2016 Mar;17(3):367-82. PMID: 26882544. Murphy AC, Lindsay AJ, McCaffrey MW, Djinović-Carugo K, Young PW. Congenital macrothrombocytopenia-linked mutations in the actin-binding domain of α-actinin-1 enhance F-actin association. FEBS Lett. 2016 Mar;590(6):685-95. PMID: 26879394. Zaffagnini G, Martens S. Mechanisms of Selective Autophagy. J Mol Biol. 2016 May 8;428(9 Pt A):1714-24. PMID: 26876603. Maronova M, Kalyna M. Generating Targeted Gene Knockout Lines in Physcomitrella patens to Study Evolution of Stress-Responsive Mechanisms. Methods Mol Biol. 2016;1398:221-34. PMID: 26867627.


MFPL - 2016 RESEARCH GROUPS

Hofbauer S, Howes BD, Flego N, Pirker KF, Schaffner I, Mlynek G, Djinović-Carugo K, Furtmüller PG, Smulevich G, Obinger C. From chlorite dismutase towards HemQ the role of the proximal H-bonding network in haeme binding. Biosci Rep. 2016 Feb 8;36(2). pii: e00312. PMID: 26858461.

Nemoto TK, Ohara-Nemoto Y, Bezerra GA, Shimoyama Y, Kimura S. A Porphyromonas gingivalis Periplasmic Novel Exopeptidase, Acylpeptidyl Oligopeptidase, Releases N-Acylated Di- and Tripeptides from Oligopeptides. J Biol Chem. 2016 Mar 11;291(11):5913-25. PMID: 26733202.

Lučić I, Truebestein L, Leonard TA. Novel Features of DAG-Activated PKC Isozymes Reveal a Conserved 3-D Architecture. J Mol Biol. 2016 Jan 16;428(1):121-41. PMID: 26582574.

Vidak S, Foisner R. Molecular insights into the premature aging disease progeria. Histochem Cell Biol. 2016 Apr;145(4):401-17. PMID: 26847180.

Negishi T, Veis J, Hollenstein D, Sekiya M, Ammerer G, Ohya Y. The Late S-Phase Transcription Factor Hcm1 Is Regulated through Phosphorylation by the Cell Wall Integrity Checkpoint. Mol Cell Biol. 2016 Jan 4;36(6):941-53. PMID: 26729465.

Tata M, Wolfinger MT, Amman F, Roschanski N, Dötsch A, Sonnleitner E, Häussler S, Bläsi U. RNASeq Based Transcriptional Profiling of Pseudomonas aeruginosa PA14 after Short- and Long-Term Anoxic Cultivation in Synthetic Cystic Fibrosis Sputum Medium. PLoS One. 2016 Jan 28;11(1):e0147811. PMID: 26821182.

Yerlikaya S, Meusburger M, Kumari R, Huber A, Anrather D, Costanzo M, Boone C, Ammerer G, Baranov PV, Loewith R. TORC1 and TORC2 work together to regulate ribosomal protein S6 phosphorylation in Saccharomyces cerevisiae. Mol Biol Cell. 2016 Jan 15;27(2):397-409. PMID: 26582391.

Loi M, Cenni V, Duchi S, Squarzoni S, Lopez-Otin C, Foisner R, Lattanzi G, Capanni C. Barrier-to-autointegration factor (BAF) involvement in prelamin A-related chromatin organization changes. Oncotarget. 2016 Mar 29;7(13):15662-77. PMID: 26701887.

Le Rhun A, Beer YY, Reimegård J, Chylinski K, Charpentier E. RNA sequencing uncovers antisense RNAs and novel small RNAs in Streptococcus pyogenes. RNA Biol. 2016;13(2):177-95. PMID: 26580233.

Burki F, Kaplan M, Tikhonenkov DV, Zlatogursky V, Minh BQ, Radaykina LV, Smirnov A, Mylnikov AP, Keeling PJ. Untangling the early diversification of eukaryotes: a phylogenomic study of the evolutionary origins of Centrohelida, Haptophyta and Cryptista. Proc Biol Sci. 2016 Jan 27;283(1823). pii: 20152802. PMID: 26817772.

Aronica L, Kasparek T, Ruchman D, Marquez Y, Cipak L, Cipakova I, Anrather D, Mikolaskova B, Radtke M, Sarkar S, Pai CC, Blaikley E, Walker C, Shen KF, Schroeder R, Barta A, Forsburg SL, Humphrey TC. The spliceosome-associated protein Nrl1 suppresses homologous recombination-dependent R-loop formation in fission yeast. Nucleic Acids Res. 2016 Feb 29;44(4):1703-17. PMID: 26682798

Kumar S, Kolodkin-Gal I, Vesper O, Alam N, Schueler-Furman O, Moll I, Engelberg-Kulka H. Escherichia coli Quorum-Sensing EDF, A Peptide Generated by Novel Multiple Distinct Mechanisms and Regulated by trans-Translation. MBio. 2016 Jan 26;7(1):e02034-15. PMID: 26814184. Gesson K, Rescheneder P, Skoruppa MP, von Haeseler A, Dechat T, Foisner R. A-type lamins bind both hetero- and euchromatin, the latter being regulated by lamina-associated polypeptide 2 alpha. Genome Res. 2016 Apr;26(4):462-73. PMID: 26798136. Gautel M, Djinović-Carugo K. The sarcomeric cytoskeleton: from molecules to motion. J Exp Biol. 2016 Jan;219(Pt 2):135-45. PMID: 26792323. Fuchs C, Gawlas S, Heher P, Nikouli S, Paar H, Ivankovic M, Schultheis M, Klammer J, Gottschamel T, Capetanaki Y, Weitzer G. Desmin enters the nucleus of cardiac stem cells and modulates Nkx2.5 expression by participating in transcription factor complexes that interact with the nkx2.5 gene. Biol Open. 2016 Jan 19;5(2):140-53. PMID: 26787680. Szabo S, Wögenstein KL, Fuchs P. Functional and Genetic Analysis of Epiplakin in Epithelial Cells. Methods Enzymol. 2016;569:261-85.PMID: 26778563. Rezniczek GA, Winter L, Walko G, Wiche G. Functional and Genetic Analysis of Plectin in Skin and Muscle. Methods Enzymol. 2016;569:235-59. PMID: 26778562. Barnat M, Benassy MN, Vincensini L, Soares S, Fassier C, Propst F, Andrieux A, von Boxberg Y, Nothias F. The GSK3-MAP1B pathway controls neurite branching and microtubule dynamics. Mol Cell Neurosci. 2016 Apr;72:921. PMID: 26773468. Boban M, Foisner R. Degradation-mediated protein quality control at the inner nuclear membrane. Nucleus. 2016;7(1):41-9. PMID: 26760377. Sousa BL, Silva-Filho JC, Kumar P, Graewert MA, Pereira RI, Cunha RM, Nascimento KS, Bezerra GA, Delatorre P, Djinovic-Carugo K, Nagano CS, Gruber K, Cavada BS. Structural characterization of a Vatairea macrocarpa lectin in complex with a tumor-associated antigen: A new tool for cancer research. Int J Biochem Cell Biol. 2016 Mar;72:27-39. PMID: 26751394. Flamm AG, Żerko S, Zawadzka-Kazimierczuk A, Koźmiński W, Konrat R, Coudevylle N. 1H, 15N, 13C resonance assignment of human GAP-43. Biomol NMR Assign. 2016 Apr;10(1):171-4. PMID: 26748655. Pevala V, Truban D, Bauer JA, Košťan J, Kunová N, Bellová J, Brandstetter M, Marini V, Krejčí L, Tomáška Ľ, Nosek J, Kutejová E. The structure and DNA-binding properties of Mgm101 from a yeast with a linear mitochondrial genome. Nucleic Acids Res. 2016 Mar 18;44(5):2227-39. PMID: 26743001.

Varga A, Baccarini M. Knock-in(g) RAF for a loop. EMBO J. 2016 Jan 18;35(2):118-20. PMID: 26671982. Dose A, Sindlinger J, Bierlmeier J, Bakirbas A, Schulze-Osthoff K, Einsele-Scholz S, Hartl M, Essmann F, Finkemeier I, Schwarzer D. Interrogating Substrate Selectivity and Composition of Endogenous Histone Deacetylase Complexes with Chemical Probes. Angew Chem Int Ed Engl. 2016 Jan 18;55(3):1192-5. PMID: 26662792. Crozet P, Margalha L, Butowt R, Fernandes N, Elias CA, Orosa B, Tomanov K, Teige M, Bachmair A, Sadanandom A, Baena-González E. SUMOylation represses SnRK1 signaling in Arabidopsis. Plant J. 2016 Jan;85(1):120-33. PMID: 26662259. Liu ES, Raimann A, Chae BT, Martins JS, Baccarini M, Demay MB. c-Raf promotes angiogenesis during normal growth plate maturation. Development. 2016 Jan 15;143(2):348-55. PMID: 26657770. Montanari F, Pinto M, Khunweeraphong N, Wlcek K, Sohail MI, Noeske T, Boyer S, Chiba P, Stieger B, Kuchler K, Ecker GF. Flagging Drugs That Inhibit the Bile Salt Export Pump. Mol Pharm. 2016 Jan 4;13(1):163-71. PMID: 26642869. Cabedo Martinez AI, Weinhäupl K, Lee WK, Wolff NA, Storch B, Żerko S, Konrat R, Koźmiński W, Breuker K, Thévenod F, Coudevylle N. Biochemical and Structural Characterization of the Interaction between the Siderocalin NGAL/LCN2 (Neutrophil Gelatinase-associated Lipocalin/ Lipocalin 2) and the N-terminal Domain of Its Endocytic Receptor SLC22A17. J Biol Chem. 2016 Feb 5;291(6):291730. PMID: 26635366. Uecker H, Hermisson J. The Role of Recombination in Evolutionary Rescue. Genetics. 2016 Feb;202(2):721-32. PMID: 26627842. Murano T, Yamaguchi T, Tatsuno I, Suzuki M, Noike H, Takanami T, Yoshida T, Suzuki M, Hashimoto R, Maeno T, Terai K, Tokuyama W, Hiruta N, Schneider WJ, Bujo H. Subfraction analysis of circulating lipoproteins in a patient with Tangier disease due to a novel ABCA1 mutation. Clin Chim Acta. 2016 Jan 15;452:167-72. PMID: 26616730. Fallmann J, Sedlyarov V, Tanzer A, Kovarik P, Hofacker IL. AREsite2: an enhanced database for the comprehensive investigation of AU/GU/U-rich elements. Nucleic Acids Res. 2016 Jan 4;44(D1):D90-5. PMID: 26602692. Holthaus KB, Strasser B, Sipos W, Schmidt HA, Mlitz V, Sukseree S, Weissenbacher A, Tschachler E, Alibardi L, Eckhart L. Comparative Genomics Identifies Epidermal Proteins Associated with the Evolution of the Turtle Shell. Mol Biol Evol. 2016 Mar;33(3):726-37. PMID: 26601937. Schneider WJ. Lipid transport to avian oocytes and to the developing embryo. J Biomed Res. 2016 May;30(3):174-80. PMID: 26585559.

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Göpel Y, Khan MA, Görke B. Domain swapping between homologous bacterial small RNAs dissects processing and Hfq binding determinants and uncovers an aptamer for conditional RNase E cleavage. Nucleic Acids Res. 2016 Jan 29;44(2):824-37. PMID: 26531825. Flamm AG, Le Roux AL, Mateos B, Díaz-Lobo M, Storch B, Breuker K, Konrat R, Pons M, Coudevylle N. N-Lauroylation during the Expression of Recombinant N-Myristoylated Proteins: Implications and Solutions. Chembiochem. 2016 Jan 1;17(1):82-9. PMID: 26522884. Lesigang J, Dong G. Analysis of Three-Dimensional Structures of Exocyst Components. Methods Mol Biol. 2016;1369:191-204. PMID: 26519314. Zemora G, Handl S, Waldsich C. Human telomerase reverse transcriptase binds to a pre-organized hTR in vivo exposing its template. Nucleic Acids Res. 2016 Jan 8;44(1):41325. PMID: 26481359. Segré CV, Senese S, Loponte S, Santaguida S, Soffientini P, Grigorean G, Cinquanta M, Ossolengo G, Seiser C, Chiocca S. A monoclonal antibody specific for prophase phosphorylation of histone deacetylase 1: a readout for early mitotic cells. MAbs. 2016;8(1):37-42. PMID: 26467746. Sidonskaya E, Schweighofer A, Shubchynskyy V, Kammerhofer N, Hofmann J, Wieczorek K, Meskiene I. Plant resistance against the parasitic nematode Heterodera schachtii is mediated by MPK3 and MPK6 kinases, which are controlled by the MAPK phosphatase AP2C1 in Arabidopsis. J Exp Bot. 2016 Jan;67(1):107-18. PMID: 26438412. Sims J, Bruschi CV, Bertin C, West N, Breitenbach M, Schroeder S, Eisenberg T, Rinnerthaler M, Raspor P, Tosato V. High reactive oxygen species levels are detected at the end of the chronological life span of translocant yeast cells. Mol Genet Genomics. 2016 Feb;291(1):423-35. PMID: 26423068. Kurzbach D, Vanas A, Flamm AG, Tarnoczi N, Kontaxis G, Maltar-Strmečki N, Widder K, Hinderberger D, Konrat R. Detection of correlated conformational fluctuations in intrinsically disordered proteins through paramagnetic relaxation interference. Phys Chem Chem Phys. 2016 Feb 17;18(8):5753-8. PMID: 26411860. Carugo O. Statistical survey of the buried waters in the Protein Data Bank. Amino Acids. 2016 Jan;48(1):193-202. PMID: 26315961. Dichlberger A, Schlager S, Kovanen PT, Schneider WJ. Lipid droplets in activated mast cells - a significant source of triglyceride-derived arachidonic acid for eicosanoid production. Eur J Pharmacol. 2016 Aug 15;785:59-69. PMID: 26164793. Tajaddod M, Jantsch MF, Licht K. The dynamic epitranscriptome: A to I editing modulates genetic information. Chromosoma. 2016 Mar;125(1):51-63. PMID: 26148686


MFPL - 2016 RESEARCH GROUPS

MFPL wishes to thank the following institutions for financial support of research projects: Parent Institutions

University of Vienna

Medical University of Vienna

Publishing Details Published by Max F. Perutz Laboratories Support GmbH Editors Caterina Purini contributions from MFPL researchers Pictures MFPL staff and scientists Daniel Hinterramskogler Point of View Barbara Mair IMP/IMBA Graphics Department / Michael Sazel Layout & Design XeroGrafiX GmbH, Vienna Print druck.at

Funding Organizations and Programs

Abcam

Active Motif

BMWFW – Federal Ministry of Science, Research and Economy

Boehringer Ingelheim

City of Vienna

EMBO – European Molecular Biology Organization

ERC – European Research Council

FFG – Austrian Research Promotion Agency

FWF – Austrian Science Fund

HFSP – Human Frontier Science Program

Millipore

Neuropore Therapeutics

NIH

ÖAW – Austrian Academy of Sciences

Origimm Biotechnology GmbH

Research Executive Agency (REA, EU)

Santa Cruz Biotechnology

Wellcome Trust

Wings for Life

WWTF – Vienna Science and Technology Fund

Herzfelder Stiftung

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MFPL Research Groups 2016