MFPL Research Groups 2013

M A X F . PE R U T Z L A B O R A T O R I ES

2013

Research Groups

A joint venture of

M A X

F .

PE R U T Z

L A B O R A T O R I ES

2013 Research Groups

R e s e arc h

G ro u ps

G u sta v A mm e r e r

Signal transduction and transcriptional regulation in yeast In our fields of interest, important, still unresolved questions concern the dynamic interactions between different signaling factors and their effectors.

Gustav Ammerer team

Jessica Ferrari Rainer Gith Isabella Hansmann Aleksandra Jovanovic Wolfgang Reiter Natalie Romanov Jiri Veis Syam Yelamanchi

To approach these questions we have established and optimized an enzyme-based protein proximity assay based on a mammalian histone methyltransferase and its highly specific substrate, the N-terminal fragment of histone 3. This assay has been successfully applied to osmo-stress and mating specific specific signal transduction problems, to the induction of autophagy and to the validation of protein phosphatase targets.

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. Similar investigations concerned the question of how stress induced signals can cause the dramatic changes in the global gene expression pattern and what factors might control the duration of this response. We specifically investigated the role of protein phosphatase 2A in transcription, an enzyme that has an active role in inducing the so-called environmental stress response genes. Our experiments suggested that PP2A is both necessary for nuclear retention of the relevant transcription factors as well as their correct temporal chromatin recruitment.

Other projects dealt with the problem of how cellular signals coordinate the transcriptional regulation of genes. In one case we have focused on the regulation of the main mitotic cyclin gene CLB2 in yeast. This gene is repressed in the absence of CDK activity, de-repressed at the START of S-phase and finally 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

RNA FISH of the CTT1 gene before and after stress. DNA blue, RNA green. Notice the nuclear RNA signal in the middle panel documenting active transcription of the CTT1 gene.

selected Publications 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 Mar;33(5):105772. n Reiter W, Anrather D, Dohnal I, Pichler P, Veis J, Grøtli M, Posas F, Ammerer G. Validation of regulated protein phosphorylation events in yeast by quantitative mass spectrometry analysis of purified proteins. Proteomics. 2012 Oct;12(19-20):3030-43. n 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.

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R e s e arc h

G ro u ps

M an u e la B accarini

Deciphering the MAPK pathway in vivo The Raf/MEK/ERK pathway represents the oldest paradigm of a cytosolic signal transduction cascade. Its constitutive activation is considered a key event in the development of several human malignancies and developmental disorders. We use conditional knock-out mice and cells to understand the essential roles of Raf and Mek in tissue development, remodeling, and neoplasia.

Focusing specifically on the two best known Raf kinases, B-Raf and C-Raf, we have discovered that B-Raf is the essential ERK activator in all instances in which its ablation results in a phenotype. In contrast, C-Raf mediates pathway cross-talk independently of its kinase activity, by binding to, and inhibiting, the cytoskeleton-based Rok-alpha, which controls cell shape, motility, and in some cell types differentiation.

These non-redundant functions of Raf are best illustrated in a Ras-driven skin carcinogenesis model, in which B-Raf drives MEK/ERK activation and proliferation. In contrast, C-Raf works as an endogenous Rok inhibitor essential for blocking differentiation (Fig. 1). Elimination of B-Raf and

Fig. 1: Before and after: C-Raf ablation results in the regression of Ras-induced epidermal tumors. Once C-Raf is ablated, its binding partner Rok-alpha is hyperactive and induces the differentiation of keratinocytes (K10, brown staining), and the regression of the tumors (Hematoxylin/ Eosin staining), which never relapse.

Manuela Baccarini team

Fig. 2: Nascent cell-cell contacts between endothelial cells. The transmembrane protein VE-Cadherin (green) mediates the formation of cellular junctions and is connected to the intracellular actin cytoskleton (red). The nucleus is stained in blue.

C-Raf from the epidermis enforces abrupt tumor regression, combining delayed proliferation and increased differentiation. These results emphasize that therapies targeting both Raf kinase-dependent and -independent pathways may potentially be more effective and less prone to inducing adverse effects and/or resistance.

Through its interaction with Rok-alpha, C-Raf also regulates the cohesion and the collective migration of endothelial cells, and ultimately sprouting and tumor-induced angiogenesis. Downstream of the small GTPase Rap1, C-Raf is recruited to VE-cadherin containing adherens junctions (Fig. 2), where it is required to anchor Rok-alpha and modulate junctional actomyosin during adherens junction maturation.

We have also established MEK1 as the critical negative regulator of MEK2/ERK signaling. MEK1 mediates the regulation of MEK2 in the context of a MEK1:MEK2 heterodimer that is negatively regulated by ERK-mediated phosphorylation of MEK1.

By showing that their essential in vivo functions are fundamentally different, these results have changed the way we look at Raf and MEK kinases, beginning to reveal how the pathway is wired in vivo.

selected Publications Kern F, Doma E, Rupp C, Niault T, Baccarini M. Essential, non-redundant roles of B-Raf and Raf-1 in Ras-driven skin tumorigenesis. Oncogene. 2012 Jun 25. n Wimmer R, Cseh B, Maier B, Scherrer K, Baccarini M. Angiogenic sprouting requires the fine tuning of endothelial cell cohesion by the Raf-1/Rok-Îą complex. Dev Cell. 2012 Jan 17;22(1):158-71. n Catalanotti F, Reyes G, Jesenberger V, Galabova-Kovacs G, de Matos Simoes R, Carugo O, Baccarini M. A Mek1-Mek2 heterodimer determines the strength and duration of the Erk signal. Nat Struct Mol Biol. 2009 Mar;16(3):294-303.

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Christian Baumgartner Clemens Bogner Stefan Buchleitner Anna Lina Cavallo Botond Cseh Eszter Doma Karin Ehrenreiter Elisabeth Froschauer-Neuhauser Tatjana Hirschmugl Ines Jeric Veronika Jesenberger Thomas Kogler Patrick Lall Joanna Nowacka Josipa Raguz Bettina Riegler Christian Rupp Andrea Varga Bartosz Tarkowski Reiner Wimmer Katarina Zmajkovicova

R e s e arc h

G ro u ps

A ndr e as B ac h mair

Protein modifiers in plants and retrotransposon biology

Andreas Bachmair team

Maria Granilshchikova Bulut Hamali Konstantin Tomanov Andrea Tramontano Eneda Xhikaj

Many proteins are modified after their synthesis. We are interested in how small modifier proteins are linked to substrate proteins in plants, and how this process influences plant responses to adverse conditions.

Covalent attachment of small modifier proteins to substrates is essential for many regulatory processes in plants. Of particular interest to us is the ubiquitin-dependent N-end rule pathway of protein degradation, which regulates seed germination, senescence, and the response to several forms of environmental stress. Mutants in every step of the pathway are available, and we are preparing certain mutant combinations to probe functional redundancy. Collaboration with other labs shall extend the range of known substrates, which is currently still limited. These combined resources are used to obtain a deeper, mechanistic

insight into processes regulated by this pathway.

Another modifier essential for stress responses is the small ubiquitin-related modifier, SUMO, which helps plants to cope with drought, temperature extremes, and nutritional limitations. We study SUMO ligases, key enzymes in the formation of isopeptide bonds between SUMO and substrates.

Both in vitro experiments and mutant analysis help us to elucidate the biochemical basis of stress adaptation.

Our second focus is retrotransposon biology.

Retrotransposons are pieces of DNA that can replicate more often than the normal genomic DNA. They do so by reverse transcribing their mRNA, and by inserting the ensuing DNA copy into the host genome, often at a random position.

Retrotransposon activity is usually triggered by cellular stress, and transposition is a rare event.

In a synthetic biology approach, we have put our element of choice, Tto1, under control of an inducible promoter. In this way, we gained experimental control over the Tto1 life cycle. This novel circuitry is currently tested in the model plant Arabidopsis thaliana and in Arabidopsis plants with mutant genetic background. We also make changes in the Tto1 sequence and observe which consequences the changes have for Tto1 activity. This allows us to learn a lot about the Tto1 life cycle and how Tto1 interacts with host proteins. As an extension of this work, the most successful constructs shall be used for insertion mutagenesis in other plant species, particularly those where current methods of mutant generation are limited. Our work shall thereby help to decrease the knowledge gradient that currently exists between the model plant Arabidopsis thaliana and many plant species used in agriculture.

Arabidopsis thaliana plants with impaired SUMO conjugation (left pot) are smaller than siblings with intact sumoylation system (right pot, plants supported by wood sticks).

selected Publications Tomanov K, Hardtke C, Budhiraja R, Hermkes R, Coupland G, Bachmair A. Small Ubiquitin-Like Modifier Conjugating Enzyme with Active Site Mutation Acts as Dominant Negative Inhibitor of SUMOConjugation in Arabidopsis. J Integr Plant Biol. 2013 Jan;55(1):75-82. n Novatchkova M, Tomanov K, Hofmann K, Stuible HP, Bachmair A. Update on sumoylation: defining core components of the plant SUMO conjugation system by phylogenetic comparison. New Phytol. 2012 Jul;195(1):23-31. n Tramontano A, Donath A, Bernhart SH, Reiche K, BĂśhmdorfer G, Stadler PF, Bachmair A. Deletion analysis of the 3‘ long terminal repeat sequence of plant retrotransposon Tto1 identifies 125 base pairs redundancy as sufficient for first strand transfer. Virology. 2011 Mar 30;412(1):75-82.

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R e s e arc h

G ro u ps

A ndr e a B arta

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 posttranscriptional processes and the impact of the dynamic transcriptome on gene expression.

Alternative splicing is one of the posttranscriptional events to expand the repertoire of genes 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 plant development and responses to the environment.

As alternative splicing in Arabidopsis is not well investigated we are using RNAseq to define the rules and targets of alternative splicing. This analysis showed that more than 60% of intron containing genes are alternatively spliced in Arabidopsis. SR (Ser/Arg) proteins are important splicing factors and to date we have isolated and partially characterized several Arabidopsis

SR proteins, which are important for splice site selection and spliceosome assembly. Currently, we are using deep RNA sequencing to investigate the influence of these splicing factors on alternative splicing. In addition, the impact of the circadian clock on alternative splicing in plants is investigated as well as the effect of alternative splicing on clock genes.

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 using Genomic SELEX, RIP and CLIP methods, as well as their 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 being investigated in greater detail. Molecular Beacons are used to visualize the localization of alternatively spliced products within a cell. Furthermore, a project has been started to investigate the influence of chromatin and DNA modifications on alternative splicing in plants.

Model of how binding of AtCyp59 to the transcript might influence transcription. When the RRM domain (dark blue) of AtCyp59 (light blue) is not engaged in RNA–protein interaction, activity of its PPIase domain is unaffected (green).

However, upon binding of AtCyp59 to the binding motif (purple) on the RNA transcript, the PPIase activity of AtCyp59 decreases (yellow). These changes might serve as a signal to modulate RNA polymerase II activity.

selected Publications Bannikova O, Zywicki M, Marquez Y, Skrahina T, Kalyna M, Barta A. Identification of RNA targets for the nuclear multidomain cyclophilin atCyp59 and their effect on PPIase activity. Nucleic Acids Res. 2013 Feb 1;41(3):1783-96. n Marquez Y, Brown JW, Simpson C, Barta A, Kalyna M. Transcriptome survey reveals increased complexity of the alternative splicing landscape in Arabidopsis. Genome Res. 2012 Jun;22(6):1184-95. n Kalyna M, Simpson CG, Syed NH, Lewandowska D, Marquez Y, Kusenda B, Marshall J, Fuller J, Cardle L, McNicol J, Dinh HQ, Barta A, Brown JW. Alternative splicing and nonsense-mediated decay modulate expression of important regulatory genes in Arabidopsis.

Nucleic Acids Res. 2012 Mar;40(6):2454-69..

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Andrea Barta team

Zahra Ayatollahi Olga Bannikova Armin Fuchs Janett Göhring Markus Höpfner Maria Kalyna Yamile Marquez Manali Mishra

R e s e arc h

G ro u ps

di e t e r blaas

Early interactions of viruses with host cells

Dieter Blaas

For infection, a virus must deliver its genome into a host cell suitable for producing progeny. This involves the mutual recognition of surface structures present on the viral surface and the plasma membrane that finally results in delivery of the virus into the cell.

team

Irene GĂśsler Shushan Harutyunyan Mohit Kumar

To this end, viruses exploit various entry routes that are normally taken by metabolites, hormones, and carriers necessary for maintaining the cell alive.

The viral genome must then leave its protective coat and be delivered into the cytoplasm from where it will be shuttled to a site appropriate for replication.

It is poorly understood how the viral nucleic acid or nucleoprotein that is highly compacted within the limited space of the viral shell, becomes unwound and passes through a small hole supposedly contiguous with a channel in the membrane that appears to form upon interaction with the virus.

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.

For our study object HRV2, one of the many common cold viruses, there is experimental support for the transfer of the RNA genome through a pore in the endosomal membrane. By using cryoelectron microscopy and X-ray crystallography in combination with biophysical and biochemical methods we have recently discovered that the RNA switches its conformation during the structural changes of the virion occurring upon infection. This switch appears to prepare the viral genome for exit through one of the holes at the two-fold axes of the icosahedral protein shell.

Exit must be highly organized to avoid tangling and kinetic traps. This appears to be facilitated by egress starting with the 3’-end of the single stranded RNA genome.

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.

selected Publications Garriga D, Pickl-Herk A, Luque D, Wruss J, CastĂłn JR, Blaas D, Verdaguer N. Insights into minor group rhinovirus uncoating: the X-ray structure of the HRV2 empty capsid. PLoS Pathog. 2012 Jan;8(1):e1002473. n Bilek G, Matscheko NM, Pickl-Herk A, Weiss VU, Subirats X, Kenndler E, Blaas D. Liposomal nanocontainers as models for viral infection: monitoring viral genomic RNA transfer through lipid membranes. J Virol. 2011 Aug;85(16):8368-75. n Konecsni T, Berka U, Pickl-Herk A, Bilek G, Khan AG, Gajdzig L, Fuchs R, Blaas D. Low pH-triggered beta-propeller switch of the low-density lipoprotein receptor assists rhinovirus infection. J Virol. 2009 Nov;83(21):10922-30.

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R e s e arc h

G ro u ps

Udo bl ä si

Post-transcriptional regulation in bacteria and archaea Bacteria are constantly challenged by changing environmental conditions.

They employ a number of posttranscriptional control mechanisms including trans-acting proteins, small regulatory RNAs (sRNAs) as well as features inherent to mRNA structure, which permit a fast adaptation to new environments or to different kinds of stress.

Post-transcriptional regulatory mechanisms are also studied in the model crenarchaeon Sulfolobus solfataricus (Sso). These studies revealed the sequence of events in archaeal translation initiation as well as unprecedented function(s) of archaeal translation initiation factors. We have shown that the g-subunit of translation initiation factor aIF2 exhibits – besides its requirement for initiator-tRNA binding – an additional function with resemblance to the eukaryotic cap-complex.

It binds to the 5´-triphosphate end of mRNAs and counteracts mRNA decay in Sso by a recently identified Sso RNAse with 5´->3´directionality.

In addition, ongoing studies concentrate on the elucidation of the function of archaeal Sm proteins in RNA metabolism and molecular mechanisms underlying non-coding RNA mediated regulation in Sso.

X-ray structure of the Pseudomonas aeruginosa catabolite repression control protein Crc (green) superimposed on the AP endonuclease Nape (magenta) of Neisseria meningitidis.

We are focusing on post-transcriptional control mechanisms exerted by the global regulatory protein Hfq in conjunction with sRNAs in bacteria, with emphasis on the human pathogen Pseudomonas aeruginosa. These studies revealed novel molecular modes of sRNA-mediated regulation as well as Pseudomonas sRNAs contributing to pathogenicity. Other research foci include molecular mechanisms underlying catabolite repression and regulation of antibiotic resistance in P. aeruginosa.

Model of E. coli Hfq RNA chaperone function.

The sRNA DsrA-34 displayed by the full-atom model is shown bound to the proximal face of HfqEc (green surface). Through conformational fluctuations the sRNA can cover a large conformational space (9 representative DsrA34 models are displayed). The mRNA is bound on the distal side (polyA9 orange) to one of the six tripartite binding motifs as shown in the crystal structure pdb3GIB. In the model HfqEc does not only act as a platform for binding and by increasing the local concentration of both ligands, but also serves to promote their flexibility, and consequently successful annealing in a stochastic manner (Ribero et al., 2012).

selected Publications H Hämmerle H, Beich-Frandsen M, Večerek B, Rajkowitsch L, Carugo O, Djinović-Carugo K, Bläsi U. Structural and biochemical studies on ATP binding and hydrolysis by the Escherichia coli RNA chaperone Hfq. PlosOne. 2012;7(11):e50892. n Ribeiro Ede A Jr, Beich-Frandsen M, Konarev PV, Shang W, Vecerek B, Kontaxis G, Hämmerle H, Peterlik H, Svergun DI, Bläsi U, Djinović-Carugo K. Structural flexibility of RNA as molecular basis for Hfq chaperone function. Nucleic Acids Res. 2012 Sep;40,(16):8072-84. n Romeo A, Sonnleitner E, Sorger-Domenigg T, Nakano M, Eisenhaber B, Bläsi U. Transcriptional regulation of nitrate assimilation in Pseudomonas aeruginosa occurs via transcriptional antitermination within the nirBD-PA1779-cobA operon. Microbiology. 2012 Jun;158(Pt 6):1543-52.

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Udo Bläsi team

Hermann Hämmerle Johannes Kassmannhuber Kanstantin Kovalchuk Salim Manoharadas Tetyana Milojevic Petra Pusic Armin Resch Stefan Rindler Alessandra Romeo Elisabeth Sonnleitner Muralidhar Tata

R e s e arc h

G ro u ps

al e xand e r damm e rmann

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.

Alexander Dammermann team

Gabriela Cabral Jeroen Dobbelaere Mate Palfy Max Roessler Clementine Schouteden Veronika Wonesch

Centrioles perform two distinct functions in eukaryotic cells: 1) they recruit pericentriolar material to form centrosomes that organize the microtubule cytoskeleton and position the mitotic spindle, and 2) they template cilia, cellular projections that perform a variety of critical sensory and motile functions. Centrosome and cilia abnormalities have been linked to aneuploidy and tumorigenesis as well as developmental disorders including ciliopathies and microcephaly.

Despite their importance to human physiology and pathology, centrioles have remained poorly understood at the molecular level, largely due to the technical challenges posed by the small size of this organelle.

In our lab we are using a combination of biochemical, cell biological and genetic approaches in the nematode C. elegans and more recently also the fruit fly Drosophila melanogaster to investigate the fundamental and conserved

molecular mechanisms underlying centriole assembly and function. In previous work we have taken advantage of the availability of data from genome-wide RNAi-based screens to define the molecular requirements for centriole assembly. The six-protein molecular pathway we identified has since been found to be conserved from ciliates to vertebrates, and is thought to form the core of the centriole assembly machinery in all eukaryotes.

We further identified the hydrolethalus syndrome protein HYLS-1 as a core centriolar protein that is incorporated into centrioles during their assembly to confer on them the ability to initiate cilia. The single amino acid missense mutation associated with hydrolethalus syndrome impairs HYLS-1 function in ciliogenesis, identifying this disorder as a severe (perinatal lethal) ciliopathy.

Current research builds on this foundation, seeking to answer three main questions: 1) How do centrioles assemble, in particular what are the specific mechanistic contributions of each of the six proteins in the centriole assembly pathway; 2) how do centrioles recruit pericentriolar material to form centrosomes and what is the molecular nature of this material; and 3) how do centrioles form cilia, focusing on the events immediately downstream of HYLS-1.

(A) Centriole assembly pathway as delineated in C. elegans. (B) C. elegans early embryo, stained for SAS-4 (centrioles, yellow), 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 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. n Dammermann A, Pemble H, Mitchell BJ, McLeod I, Yates JR 3rd, Kintner C, Desai AB, Oegema K. The hydrolethalus syndrome protein HYLS-1 links core centriole structure to cilia formation. Genes Dev. 2009 Sep 1;23(17):2046-59. n Dammermann A, MĂźller-Reichert T, Pelletier L, Habermann B, Desai A, Oegema K. Centriole assembly requires both centriolar and pericentriolar material proteins. Dev Cell. 2004 Dec;7(6):815-29.

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R e s e arc h

G ro u ps

t h omas d e ck e r

Host responses and innate immunity to bacteria A large number of pathogenic microbes must be recognized by the immune system and defense mechanisms must be alerted.

The first line of defense is set by the innate immune system which rapidly acts to limit host colonization and the dissemination of microbes.

To increase protection cells participating in the innate response initiate an adaptive immune response. Protection and immunoregulation by the innate immune system requires that a microbe is detected and physical contact is translated into altered gene expression of the infected cell.

Antimicrobial gene products provide protective effector mechanisms. Moreover, secreted cytokines fulfill the task of communicating between cells involved in the antimicrobial response to maximize the common antimicrobial effort. One important group of cytokines is formed by the interferons (IFN), subdivided into three distinct classes (IFN-I, II, III). Collectively IFN play an indispensable role in the immune system as humans or animals with partial or complete losses of responses to IFN are highly immunocompromised. To reprogram gene expression in target cells, IFN employ Jak-Stat signal transduction: after binding to cell surface receptors, receptor-associated Jak tyrosine kinases phosphorylate Stat transcription factors which translocate to the nucleus to stimulate gene expression.

Our research aims to understand how the synthesis of IFN-I is regulated when cells or animals are infected with intracellular bacteria and how the Stats activated upon IFN-I secretion communicate with additional bacteria-derived signals in the process of activating antimicrobial genes (see figure). To this end we infect normal cells and mice and compare them with infected mice that cannot synthesize IFN-I or that cannot respond to them. In addition, mice with reduced or absent responses to IFN are used to study the impact of the cytokines in a mouse model of acute intestinal inflammation. In this situation we test the hypothesis that IFN contribute to inflammation, thus worsening the outcome. These efforts are coordinated with collaborators at the University of Vienna that determine corresponding changes in the composition of the intestinal microbiota.

Infections by viral pathogens such as the influenza virus are frequently followed by superinfection with a bacterial pathogen and severe disease may result from the bacterial rather than the original viral infection. In her project, Amanda Jamieson studies mechanisms causing the immune response to influenza virus to alter and worsen the subsequent infection by a bacterial pathogen. Her recent work emphasizes the importance of the balance between proinflammatory antimicrobial effector mechanisms and the tissue and organ damage inflicted by inflammation. Thus, pathology of infection may result from the innate response rather than pathomechanisms of the infecting microbe.

Signaling in cells infected with Listeria monocytogenes.

Bacteria are recognized by cell surface, endosomal and cytoplasmic pattern recognition receptors that activate NFkB, MAPK and IRF pathways. IRFs stimulate transcription of type I interferon (IFN-I) genes . IFN-I are produced and signal through Jak kinases and Stat transcripton factors. Together NFkB, MAPK –activated transcription factors and Stats shape the gene expression signature of infected cells.

selected Publications Kernbauer E, Maier V, Stoiber D, Strobl B, Schneckenleithner C, Sexl V, Reichart U, Reizis B, Kalinke U, Jamieson A, Müller M, Decker T. Conditional Stat1 ablation reveals the importance of interferon signaling for immunity to Listeria monocytogenes infection. PLoS Pathog. 2012;8(6):e1002763. n Farlik M, Reutterer B, Schindler C, Greten F, Vogl C, Müller M, Decker T. Nonconventional initiation complex assembly by STAT and NF-kappaB transcription factors regulates nitric oxide synthase expression. Immunity. 2010 Jul 23;33(1):25-34. n Jamieson AM, Yu S, Annicelli CH, Medzhitov R. Influenza virus-induced glucocorticoids compromise innate host defense against a secondary bacterial infection. Cell Host Microbe. 2010 Feb 18;7(2):103-14.

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Thomas Decker team

Michael Aichinger Pia Gamradt Sandra Haas Amanda Jamieson Andrea Majoros Birgit Rapp Isabella Rauch Ursula Stix Bernadette Stych Daniel Szappanos Fotima Touraeva Sebastian Wienerroither

R e s e arc h

G ro u ps

K ristina D jino v i Ć - C ar u go

Structural biology of the cytoskeleton Movement is vital to all living organisms, from the transport of cellular organelles to the movement of entire organisms.

Kristina Djinovic´-Carugo team

Euripedes de Almeida Riberio Eduardo Bezerra Oliviero Carugo Ariadna Chamorro Eirini Gkougkoulia Irina Grishkovskaya Andreas Hagmüller Julius Kostan Patrick Lall Anita Lehner Georg Mlynek Adekunle Onipe Nikos Pinotsis Claudia Schreiner Jaegeun Song Bettina Spitzenberger Valeria Stefania

Sarcomeres are the smallest contractile units of striated skeletal and heart muscles. The Z-disk provides the attachment region for neighboring sarcomere units and plays a pivotal role in maintaining muscle architecture. A striking feature of the Z-disk is the high frequency of multiple protein-protein interactions. We aim to generate comprehensive structural information on the protein-protein interaction network in the Z-disk, with focus on macromolecular complexes composed of their basic components – α-actinin and filamin C – and the adaptor and regulatory proteins centered on them.

and displays an ensemble of different spatial arrangements. In the model based on these findings we suggest that the structural flexibility of RNA ligands bound to Hfq stochastically facilitates base pairing and provides the foundation for the RNA chaperone function inherent to Hfq. 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 with the goal to set up a platform for generation of protein targets employing nested constructs design and perform their biophysical characterization to find conditions best suited for structural and functional studies (see page 12).

In studies of selected binary and higher complexes we are adopting an integrative approach, employing biochemical and biophysical characterization combined with high resolution studies (X-ray diffraction, NMR) and lower resolution approaches that can either yield molecular envelopes (SAXS, SANS, EM) or specific distance information (massspectrometry combined with chemical crosslinking, NMR, double electron-electron resonance).

New bioinformatics tools and strategies are being designed to complement our structural analyses and extend our prediction capabilities.

In local collaboration we pursue structural studies of the RNA chaperone Hfq and its interactions with RNA (Bläsi), ribosomal protein S1 and its complex with S2 (Moll) and trypasonomal cytoskeletal protein MORN1 (Warren). In collaboration with M. Wagner (Faculty for Life Sciences, University of Vienna) and C. Obinger (BOKU, Vienna) we study biochemical, structural and phylogenetic aspects of family of bacterial chlorite dismutases.

To study the Hfq:RNA complex we used bioinformatics and biophysical methods combined with molecular and structural biology. We showed that not only C-terminal extensions of Hfq are intrinsically disordered and protrude away from the hexameric core, but also the segment of sRNA DsrA(34) when in complex with Hfq, is extended

Model of E. coli Hfq RNA chaperone function.

The sRNA displayed by the full-atom model is shown bound to the proximal face of Hfq (green solvent accessible surface). The mRNA is bound on the distal side represented by polyA9 orange flat cartoon representation.

The model of a hypothetical mRNA chain is displayed in orange oval cartoon. Hfq acts by restructuring the mRNA, which may be accomplished by the conformationally flexible C-termini. The structural variability of both RNAs in a transient ternary 1:1:1 complex would allow to sample large spaces and Hfq would not only act as a platform for binding and by increasing the local concentration of both ligands, but also serve to promote their flexibility, and consequently successful annealing in a stochastic manner.

selected Publications Ribeiro Ede A Jr, Beich-Frandsen M, Konarev PV, Shang W, Vecerek B, Kontaxis G, Hämmerle H, Peterlik H, Svergun DI, Bläsi U, Djinović-Carugo K. Structural flexibility of RNA as molecular basis for Hfq chaperone function. Nucleic Acids Res. 2012 Sep;40(16):8072-84. n Galkin VE, Orlova A, Salmazo A, Djinović-Carugo K, Egelman EH. Opening of tandem calponin homology domains regulates their affinity for F-actin. Nat Struct Mol Biol. 2010 May;17(5):614-6. n Sjöblom B, Polentarutti M, Djinović-Carugo K. Structural study of X-ray induced activation of carbonic anhydrase. Proc Natl Acad Sci U S A. 2009 Jun 30;106(26):10609-13.

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R e s e arc h

G ro u ps

G ang dong

Structural studies of centriole and cilium biogenesis Built on the centriole-derived basal body, the cilium is a specialized organelle highly conserved from protists to mammals. It consists of a membrane-sheathed axoneme and more than 600 associated proteins.

Both the centriole and the cilium have attracted tremendous attention in recent years because of their association with an expanding number of human disorders. We aim to elucidate the structures of the proteins that are essential for centriole and cilium biogenesis.

Cilia are assembled and maintained through intraflagellar transport (IFT). This process is carried out by two distinct protein complexes, IFT complexes A and B, which contain at least six and sixteen subunits, respectively. Whereas the two complexes have been studied for some years, little is known about their architecture and assembly. We are currently developing protocols for producing soluble and stable protein complexes by reconstituting in vitro or co-expressing all components in bacterial or baculovirus-insect cell expression system. Our goal is to elucidate at the atomic level the assembly mechanisms of the protein complexes for cargo transport to and within the cilium.

We are also investigating the underlying molecular mechanisms for centriole biogenesis. SAS-5 and SAS-6 are two of the five conserved proteins essential for centriole duplication. It has been reported that SAS-5 and SAS-6 physically interact with each other and are codependent for their targeting to procentrioles. However, it remains unclear how these two proteins interact at the molecular level. We have recently solved the crystal structure of the coiled-coil domain of SAS-6 and further characterized its specific interaction with SAS-5.

BILBO1 is a recently identified 65 kDa cytoskeletal protein located in the flagellar pocket collar (FPC) of parasitic Trypanosomatidae. Due to the lack of structural information, the mechanism of its essential function for cytokinesis remains mysterious.

We have now determined a high resolution structure for the N-terminal domain of T. brucei BILBO1 and have identified a conserved aromatic surface patch on this domain that is essential for BILBO1 function in regulating cell division. Furthermore, we have used EM studies to reveal the assembly mechanism of BILBO1 and identified the domain responsible for its specific localization to the FPC using in vivo immunofluorescence EM analysis.

Technically, we combine X-ray crystallography with other biochemical and biophysical techniques in our studies. Our structural studies are also complemented by site-directed mutagenesis and in vitro/vivo experiments to test the mechanistic hypotheses.

Our long-term goal is to provide a structural view of several aspects of ciliogenesis, including intraflagellar transport,flagellar pocket formation, vesicle targeting and centriole duplication.

selected Publications 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. n Dong G, Wearsch PA, Peaper DR, Cresswell P, Reinisch KM. Insights into MHC class I peptide loading from the structure of the tapasin-ERp57 thiol oxidoreductase heterodimer. Immunity. 2009 Jan 16;30(1):21-32. n Dong G, Medkova M, Novick P, Reinisch KM. A catalytic coiled coil: structural insights into the activation of the Rab GTPase Sec4p by Sec2p. Mol Cell. 2007 Feb 9;25(3):455-62.

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Gang Dong team

Johannes Lesigang Renping Qiao Ekaterina Shimanovskaya Keni Vidilaseris

R e s e arc h

G ro u ps

silk e dorn e r

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

homo­­logous mRNAs for degradation. A genomewide screen for components of the siRNA pathway identified several novel candidate genes in Drosophila cultured cells. Thus, the main focus of this project is the biochemical characterization of these novel candidate genes. This will allow the identification of their role in the siRNA pathway.

In the past decade the discovery of small noncoding RNAs has entirely revolutionized the way we think about the regulation of gene expression.

The major focus of our research is RNA-mediated gene silencing by siRNAs (small interfering RNAs) and miRNAs (micro RNAs) in Drosophila.

MicroRNAs (miRNAs) are an abundant class of small non-coding RNAs (about 22 nt) that are found in a variety of eukaryotic organisms. Over the past decade these small RNAs emerged as crucial factors of gene regulation and play an essential role in developmental and physiological processes.

Generally, animal miRNAs base pair imperfectly to the 3’ untranslated region (3’ UTR) of target mRNAs and have been well established as key regulators of gene expression at the translational level. More recently, evidence accumulated that miRNAs can also accelerate mRNA degradation of some of their targets. It is important to note that miRNA-mediated decay of mRNA does not occur through an endonucleolytic cleavage as in siRNA-mediated gene silencing. However, miRNAs accelerate the mRNA turnover by recruitment of the general mRNA decay machinery. The underlying mechanisms by which miRNAs regulate gene expression are still quite controversial. The aim of this project is to develop kinetic tools to study the mechanism by which miRNAs affect the stability and trans­latability of their target mRNA. Therefore, we will investigate which particular step of mRNA degradation or translation is influenced by miRNAs.

Silke Dorner team

Sanja Antic Stefanie Hosiner Elisabeth Jäger Izabela Krecioch

siRNA mediated gene silencing or RNA interference (RNAi) RNA interference is thought to be a mechanism used to defend viruses and other transposable elements. Once RNAi was discovered it quickly revolutionized reverse-genetic approaches in various systems and has become a broadly powerful tool for the analysis of gene function.

Post-transcriptional silencing by RNAi is initiated by double-stranded RNAs (dsRNAs) that are processed into short interfering RNAs (siRNAs). siRNAs get incorporated into the RNA-induced silencing complex (RISC) which ultimately targets

miRNA-mediated gene silencing

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

selected Publications 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. n Dorner S, Lum L, Kim M, Paro R, Beachy PA, Green R. A genomewide screen for components of the RNAi pathway in Drosophila cultured cells. Proc Natl Acad Sci U S A. 2006 Aug 8;103(32):11880-5. n Dorner S, Brunelle JL, Sharma D, Green R. The hybrid state of tRNA binding is an authentic translation elongation intermediate. Nat Struct Mol Biol. 2006 Mar;13(3):234-41.

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R e s e arc h

G ro u ps

R oland foisn e r

Lamins in chromatin organization and regulation Higher order chromatin organization is an important mechanism to regulate gene expression during development and differentiation.

causes hyperproliferation of early progenitor cells and hyperplasia of regenerating tissues. We propose that lamin-LAP2α complexes regulate adult stem cells in tissue homeostasis and that an impairment of these functions in lamin-linked diseases may contribute to the tissue pathologies.

The nuclear lamina, a scaffold structure at the nuclear envelope, which consists of lamins and several lamin-binding proteins, is involved in “functional chromatin organization” in metazoan nuclei. Mutations in lamins cause a variety of human diseases including muscular dystrophy, lipodystrophy and accelerated aging syndromes.

We study the dynamics and regulation of the assembly and interactions of lamins and their regulatory functions during cell differentiation, as well as the effect of disease causing mutations in lamins. In the past years, we have been focusing on a group of lamin-binding proteins, defined by the presence of a structural motif – the LEM (LAP-Emerin-MAN1) domain – which mediates association of these proteins with chromatin.

While most LEM proteins are integral membrane proteins of the inner nuclear membrane, we studied unique members of this protein family localizing throughout the nucleus: LaminaAssociated Polypeptide 2α (LAP2α) and Ankyrin and LEM domain-containing protein 1 (Ankle1).

LAP2α binds to a dynamic pool of A-type lamins that, unlike the peripheral lamina, are distributed uniformly throughout the nucleoplasm and are involved in overall chromatin organization and gene regulation. Knock-down of LAP2α in mice leads to loss of the nucleoplasmic lamins and

Roland Foisner team

Mirta Boban Andreas Brachner Juliane Braun Ana Catarina Ribeiro Carrão Thomas Dechat Kevin Gesson Christian Knapp Ursula Pilat Sandra Vidak Nikola Woisetschläger Livija Zlopasa Ankle1 expressed in HeLa cells (green) localizes in the cytoplasm. Upon treatment with the nuclear export inhibitor, Leptomycin, it accumulates in the nucleus and causes DNA damage (indecated by marker γH2A.X, red).

DNA is blue. Confocal immunofluorescence images are shown. Bar is 10µm.

Ankle1 is a LEM protein with a C-terminal GIYYIG endonuclease domain that possesses nuclease activity in vitro and in vivo. Ankle1 shuttles between the cytoplasm and the nucleus, and ectopically expressed Ankle1 in the nucleus causes DNA damage. Ankle1 may be a new component of specific DNA repair pathways.

Immunofluorescence analysis of lamin A distribution in cells from LAP2α-deficient transgenic mice expressing wild-type or muscle disease-causing ΔK32-lamin A mutant. Staining intensity profiles along indicated lines in images are shown on right. Wild type lamin is exclusively at the periphery, while mutant lamin localizes throughout the nucleoplasm. selected Publications Brachner A, Braun J, Ghodgaonkar M, Castor D, Zlopasa L, Ehrlich V, Jiricny J, Gotzmann J, Knasmüller S, Foisner R. The endonuclease Ankle1 requires its LEM and GIY-YIG motifs for DNA cleavage in vivo. J Cell Sci. 2012 Feb 15;125(Pt 4):1048-57. n Gotic I, Schmidt WM, Biadasiewicz K, Leschnik M, Spilka R, Braun J, Stewart CL, Foisner R. Loss of LAP2 alpha delays satellite cell differentiation and affects postnatal fiber-type determination. Stem Cells. 2010 Mar 31;28(3):480-8. n Naetar N, Korbei B, Kozlov S, Kerenyi MA, Dorner D, Kral R, Gotic I, Fuchs P, Cohen TV, Bittner R, Stewart CL, Foisner R. Loss of nucleoplasmic LAP2alpha-lamin A complexes causes erythroid and epidermal progenitor hyperproliferation. Nat Cell Biol. 2008 Nov;10(11):1341-8.

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R e s e arc h

G ro u ps

Peter Fuchs

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

Peter Fuchs team

Sandra Szabo Karl Wögenstein

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 (Fig. 1) and it binds to intermediate filaments, mainly to keratins, which are the only binding partners identified so far. Epiplakin-deficient mice generated in our laboratory are viable and show no obvious phenotype. These findings are in clear contrast to other proteins belonging to the plakin protein family like plectin, desmoplakin, and BPAG1, which play an important role in mechanically strengthening the skin as shown by phenotypes of knock-out mice.

Subsequent experiments using primary keratinocytes from epiplakin-deficient mice showed that the biological role of epiplakin seems to be different from these plakins and to be connected with cellular stress response (Fig. 2) rather than with maintenance and regulation of cytoskeletal architecture. This protective function appears to be more prominent in simple epithelial tissues as shown by the knock-down of epiplakin in HeLa cells which led to the disruption of intermediate filament networks, contrasting the situation in keratinocytes.

Fig.1: Immunolocalization of epiplakin in various mouse tissues. Frozen sections prepared from tissues of adult mice, as indicated, were processed for immunolabeling using anti-epiplakin antibodies

However, a comprehensive analysis of the in vivo function of epiplakin in simple epithelia using defined animal models is still missing to date. In order to further elucidate the biological function of epiplakin in simple epithelia, we are performing a combination of experiments using mouse injury models and experiments based on cell culture, biochemistry and video microscopy.

In the mouse we use several stress models for simple epithelia in different organ systems which are complemented by experiments with primary cells. In addition we use biochemical and cell culture based methods to investigate epiplakin interaction with simple epithelial Fig.2: Colocalization and subcellular co-distribution of epiplakin with keratin keratins in more detail and to aggregates after okadaic acid (OA)-induced filament disruption in wild-type reveal epiplakin functions in keratin keratinocytes. Primary mouse keratinocytes, treated with OA for 2, 4 and 6 hours network recovery after stress. were immunolabeled using epiplakin (red) and pan-keratin (green) antibodies. selected Publications Walko G, Vukasinovic N, Gross K, Fischer I, Sibitz S, Fuchs P, Reipert S, Jungwirth U, Berger W, Salzer U, Carugo O, Castañón MJ, Wiche G. Targeted proteolysis of plectin isoform 1a accounts for hemidesmosome dysfunction in mice mimicking the dominant skin blistering disease EBS-Ogna. PLoS Genet. 2011 Dec;7(12):e1002396. n Spazierer D, Raberger J, Gross K, Fuchs P, Wiche G. Stress-induced recruitment of epiplakin to keratin networks increases their resistance to hyperphosphorylation-induced disruption. J Cell Sci. 2008 Mar 15;121(Pt 6):825-33. n Spazierer D, Fuchs P, Reipert S, Fischer I, Schmuth M, Lassmann H, Wiche G. Epiplakin is dispensable for skin barrier function and for integrity of keratin network cytoarchitecture in simple and stratified epithelia. Mol Cell Biol. 2006 Jan;26(2):559-68.

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R e s e arc h

G ro u ps

j u ro gr e gan

Chromosome segregation during mitosis and meiosis How does the cell ensure that during cell division each daughter cell inherits one copy of every chromosome?

Meiosis is a specialized cell division which produces haploid gametes from diploid cells. How is this reduction of chromosome number achieved? We want to understand how cells accurately segregate their chromosomes during mitosis and meiosis.

It is important to understand this process because defects in chromosome segregation (missegregation) during mitosis result in cells with abnormal numbers of chromosomes. Such cells are hallmarks of cancer. More­over, defects during meiosis cause miscarriages, infertility and genetic diseases such as Down’s Syndrome.

Chromosome segregation during meiosis The reduction of chromosome number during meiosis is achieved by two successive rounds of chromosome segregation, called meiosis I and meiosis II. While meiosis II is similar to mitosis in that sister kinetochores are bi-oriented and segregate to opposite poles, recombined homologous chromosomes segregate during the first meiotic division. Formation of chiasmata, mono-orientation of sister kinetochores and protection of centromeric cohesion are three major features of meiosis I

chromosomes which ensure the reductional nature of chromosome segregation. In our studies we use the fission yeast S. pombe, which is an excellent model organism amenable to both genetic and cell biology techniques, to identify new proteins required for proper segregation of chromosomes during meiosis. In order to decipher the molecular functions of identified proteins, we combine biochemical and cell biology techniques. To test the possible functional conservation of identified proteins, we plan to analyze the function of the respective homologs in mammalian cells.

Chromosome segregation during mitosis Accurate chromosome segregation in mitosis depends on the establishment of correct (amphitelic) kinetochore orientation. Merotelic kinetochore orientation is an error which occurs when a single kinetochore is attached to microtubules emanating from opposite spindle poles. Recent studies showing that merotelic kinetochore attachment represents a major mechanism of aneuploidy in mitotic cells and is the primary mechanism of chromosomal instability in cancer cells underline the importance of studying merotely. We focus on fission yeast proteins required to prevent and correct merotelic attachments in order to understand how cells ensure high fidelity of chromosome segregation.

Pcs1/Mde4 complex is a putative clamp which ensures proper microtubule-kinetochore attachment.

selected Publications Gregan J, Polakova S, Zhang L, Tolić-Nørrelykke IM, Cimini D. Merotelic kinetochore attachment: causes and effects.

Trends Cell Biol. 2011 Jun;21(6):374-81 n Rumpf C, Cipak L, Dudas A, Benko Z, Pozgajova M, Riedel CG, Ammerer G, Mechtler K, Gregan J. Casein kinase 1 is required for efficient removal of Rec8 during meiosis I. Cell Cycle. 2010 Jul 1;9(13):2657-62. n Gregan J, Riedel CG, Pidoux AL, Katou Y, Rumpf C, Schleiffer A, Kearsey SE, Shirahige K, Allshire RC, Nasmyth K. The kinetochore proteins Pcs1 and Mde4 and heterochromatin are required to prevent merotelic orientation. Curr Biol. 2007 Jul 17(14):1190-200.

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Juro Gregan team

Zsigmond Benko Lubos Cipak Ines Kovacikova Swastika Sanyal Lijuan Zhang

R e s e arc h

G ro u ps

A l e xand e r v on G abain

R & D Programs at Intercell AG, a spin off of the MFPL and the IMP

Alexander von Gabain Professor at the MFPL, Foreign Adjunct Professor at the Karolinska Institute in Stockholm, Chairman elect of the European Institute of Innovation and Technology, EIT, and Strategic advisor and Chairman of the SAB at Intercell.

Vaccination is arguably the most successful medical intervention which has become during the last century a mandatory part of most countries’ health care programs and shown to be an effective instrument in the control of infectious diseases worldwide.

development of novel infectious disease vaccines.

The company has worldwide launched a novel prophylactic vaccine against Japanese encephalitis virus which is based on an attenuated, inactivated, cell culture produced and highly purified vaccine antigen. In the clinical pipeline are protein subunit vaccines against bacterial pathogens, such as Mycobacterium tuberculosis, causing TB, and Pseudomas aeruginosa, Clostridium difficile, both causing hospital acquired infections, but also a However, development and launch of novel vaccines therapeutic vaccine against the Hepatitis C virus. has not seen a turn around before the late 1980ies.

The Intercell team works also on the development of Dramatic progress made in the scientific fields of anti-infective monoclonal antibodies that are derived immunology, molecular biology, genomics and hostfrom humans exposed to the pathogens. The vaccine parasite interaction, but also in the arena of novel development is supported by technology platforms manufacturing technologies has facilitated the that help to dissect the protective human immune development of novel vaccines. Intercell, a spin response, to identify vaccine antigens, structures off of the Campus Vienna Biocenter and researchmediating protection against the pathogen, and driven Biotech Company takes advantage of to design vaccine adjuvants, substances inducing this trend and devotes its R&D programs to the and facilitating the proper type of immunity in the vaccinated subjects. Many of Intercell’s vaccine technologies and R & D projects are partnered with pharmaceutical industries, including Merck & co, sanofi and Novartis. Additionally, the company is actively collaborating with many academic and public institutions, including the Centre of disease Control, CDC, Max Plank Institutes, Karolinska Institute and MFPL. Vaccine development at Intercell is largely financed by private investments and revenues. However, the company is also grateful for the generous support of Austrian, Viennese and US funding agencies, but also indebted to Vaccine development at Intercell is aiming to reduce the formulation to a PATH and AERAS foundations minimal number of antigens and to an adjuvant. Consequently an adaptive and largely carried by the Melinda and protective immune response is induced in the vaccinated subjects. Our antigens Bill Gates Foundation. are identified by using the immune system of pathogen-exposed individuals as read out. We are optimizing our adjuvants by analysing their effect on the innate immune system. Our needle-free delivery system is delivering antigens and adjuvant to the first layer of immune defence where macrophages are prevalent.

For more information: www.intercell.com

selected Publications Senn BM, Visram Z, Meinke AL, Neubauer C, Gelbmann D, Sinzinger J, Hanner M, Lundberg U, Boisvert H, Reinscheid D, von Gabain A, Nagy E. Monoclonal antibodies targeting different cell wall antigens of group B streptococcus mediate protection in both Fc-dependent and independent manner. Vaccine. 2011 May 31;29(24):4116-24. n Aichinger MC, Ginzler M, Weghuber J, Zimmermann L, Riedl K, SchĂźtz G, Nagy E, von Gabain A, Schweyen R, Henics T. Adjuvating the adjuvant: facilitated delivery of an immunomodulatory oligonucleotide to TLR9 by a cationic antimicrobial peptide in dendritic cells. Vaccine. 2011 Jan 10;29(3):426-36. n Fritzer A, Senn BM, Minh DB, Hanner M, Gelbmann D, Noiges B, Henics T, Schulze K, Guzman CA, Goodacre J, von Gabain A, Nagy E, Meinke AL. Novel conserved group A streptococcal proteins identified by the antigenome technology as vaccine candidates for a non-M protein-based vaccine. Infect Immun. 2010 Sep;78(9):4051-67.

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R e s e arc h

G ro u ps

B oris G ö rk e

Signal transduction and post-transcriptional regulation in model bacteria Mechanisms acting at the posttranscriptional level allow bacteria to adapt instantly to changes in the environment in a precise manner.

Our research deals with the regulatory circuits underlying signal perception and transduction, and cellular regulation in the model bacteria Escherichia coli and Bacillus subtilis. Mechanistically, we focus on the roles of small regulatory RNAs (sRNAs) and the functions of protein phosphorylation and protein-protein interaction for signal transduction and cellular regulation.

In a first project, we are investigating the regulation of the cell wall biosynthesis pathway in E. coli, which involves a network of two sRNAs and the novel RNA-binding protein RapZ. This network controls synthesis of enzyme GlmS, which catalyzes formation of glucosamine-6-phosphate (GlcN6P), a key metabolite required for cell envelope biosynthesis. Both sRNAs are highly similar, but only sRNA GlmZ is able to directly activate the glmS mRNA through a base-pairing process. The homologous sRNA GlmY, however, activates glmS

indirectly by protecting GlmZ from degradation.

Protein RapZ binds GlmZ and targets it to cleavage by RNase E. GlmY counteracts this process by sequestration of RapZ. GlmY accumulates when the concentration of GlcN6P decreases in the cell.

As a result, synthesis of GlmS is adjusted to the level of its enzymatic product, thereby mediating GlcN6P homeostasis.

Secondly, we focus on two-component systems (TCSs), which are the primary sensory systems of bacteria. A TCS consists of a sensor histidine kinase, which perceives a signal and transduces this information via phosphorylation of a response regulator, leading to changes in gene expression.

We recently identified the phosphorelay system PTSNtr as an accessory system, which modulates the activities of two central histidine kinases, the K+ sensing kinase KdpD and the phosphate sensing kinase PhoR in E. coli. KdpD and PhoR regulate genes required for uptake of potassium and phosphate sources, respectively. Notably, the nonphosphorylated form of protein EIIANtr, the output domain of PTSNtr, stimulates the activities of both kinases through protein-protein interaction.

Regulation of GlmS expression by sRNAs GlmY and GlmZ and the RNase adaptor protein RapZ.

selected Publications 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. Genes Dev. 2013 Mar 1;27(5):552-64. n Lüttmann D, Göpel Y, Görke B. The phosphotransferase protein EIIA(Ntr) modulates the phosphate starvation response through interaction with histidine kinase PhoR in Escherichia coli. Mol Microbiol. 2012 Oct;86(1):96-110. n Göpel Y, Görke B. Rewiring two-component signal transduction with small RNAs. Curr Opin Microbiol. 2012 Apr;15(2):132-9.

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Boris Görke team

Yvonne Göpel Muna Ayesha Khan

R e s e arc h

G ro u ps

R e s e arc h

G ro u ps

A rndt v on h a e s e l e r

Bioinformatics The Center for Integrative Bioinformatics Vienna (CIBIV, www. cibiv.at) serves as a central facility to coordinate the Bioinformatics activities at the MFPL. Moreover, it is involved in providing infrastructure and bioinformatics expertise for the various research groups at MFPL and on campus.

More recently we have expanded our research interests to address mathematically and computationally tractable problems that may help to assist in conservation decisions. We have employed the integer linear programming paradigm to explore conservation scenarios in the presence of external constraints.

Finally, we have started to develop tools to efficiently analyse deep sequencing data that pose a new challenge to Bioinformatics. To this end we have developed an efficient optimal local alignment tool, which maps millions of reads to a reference genome in a few seconds. The mapping of reads to a reference genome is the first, and possibly crucial step for any further analysis. To understand the performance of different mapping strategies we suggest a new evaluation tool that allows a graphical view of the mapping accuracy.

We have also developed tools to analyse bisulfite deep sequencing data and could show that one can improve the results considerably by employing a specifically tailored algorithm.

Besides this data analysis part, the CIBIV pursues its own research agenda. The group’s main effort is to understand the evolutionary processes that have shaped the genomes of contemporary species.

To this end, the CIBIV applies methods from statistics, computer sciences, and mathematics to detect the traces ancient evolutionary events have left in modern genomes. The CIBIV is involved in several international projects, like the Deep Metazoan Phylogeny project, where it coordinates the Bioinformatics aspects (www.deep-phylogeny. org). The figure below shows the results of a large scale evolutionary analysis for all available sequence data from fungi.

The development of efficient algorithms and further statistical tools to analyse the data will be a major research focus of the group during the next years.

Supplementary Fig. S9 CAPSASPORA OWCZARZAKI (1,1) Amoebidium parasiticum (1,1)

Glomeromycota )

Mucoromycotina

4,4

) ) (4,3 (4,4 ) es es lla (1,1 cet cet re my my o tie llata o y i r n tr o i bo cci M rtic Pu cro ve Mi Agaricomycetes (14,11)

s(

M

Microsporidia (4,3)

uc or ale

Metazoa (5,12)

Nuclearia (2,2*)

Glomus intraradices (1,-)

Neocallimastigomycota (2,2*) Chytridiomycota (2,2)

Ustilaginomycotina (2,2) Schizosaccharomycetes (4,4)

Blastocladiomycota (2,2*)

Pneumocystis carini (1,1*) Taphrina deformans (1,1*) Saitoella complicata (1,1*)

Entomophthoromycotina (2,2*) Monacrosporium haptotylum (1,1*)

Orbiliomycetes Hypocreales (25,11) Phyllachorales (2,2)

Taphrinomycotina

YARROWIA_LIPOLYTICA (1,1)

Leotiomycetes (5,5)

CRYPHONECTRIA PARASITICA (1,1) Sordariales (8,7)

Basidiomycota

Tremellomycetes (3,3)

PICHIA PASTORIS (1,1) Pezizomycetes (2,2*)

Pleosporales (5,3) MAGNAPORTHE Ophiostoma piliferum (1,1) GRISEA (1,1)

Saccharomycetes WGD/Protoploid clade (14,14)

Onygenales (10,10) Eurotiales (12,12)

Aureobasidium Capnodiales (3,3) pullulans (1,1) Sordariomycetes

Eurotiomycetes

Saccharomycetes CTG clade (9,9)

Saccharomycotina

Supernetwork summarizing the eight fungal backbone trees inferred from two data sets.

Colors highlight major systematic groups (Ascomycota: red; Basidiomycota: blue; Mucoromycotina: magenta; Glomeromycota: purple; Entomophthoromycotina: yellow; Blastocladiomycota: marine; and Chytriodiomycota/Neocallimastigomycota: green).

Dothideomycetes Ascomycota

selected Publications Ebersberger I, de Matos Simoes R, Kupczok A, Gube M, Kothe E, Voigt K, von Haeseler A. A consistent phylogenetic backbone for the fungi. Mol Biol Evol. 2012 May;29(5):1319-34. n Dinh HQ, Dubin M, Sedlazeck FJ, Lettner N, Mittelsten Scheid O, von Haeseler A. Advanced methylome analysis after bisulfite deep sequencing: an example in Arabidopsis.

PLoS One. 2012;7(7):e41528. n Thi Nguyen MA, Gesell T, von Haeseler A. ImOSM: intermittent evolution and robustness of phylogenetic methods. Mol Biol Evol. 2012 Feb;29(2):663-73.

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Arndt von Haeseler team

Quang Minh Bui Olga Chernomor Huy Quang Dinh Ingo Ebersberger Mareike Fischer Wolfgang Fischl Miguel Gallach Tanja Gesell Manuel Gil Iris Gruber Tina Köstler Milica Krunic Steven Müller Minh Anh Thi Nguyen Tung Lam Nguyen Jovana Nolic Mikhail Okun Susanne Pfeifer Martin Plochberger Niko Popitsch Celine Prakash Philipp Rescheneder Heiko Schmidt Fritz Sedlazeck Alexander Seitinger David Szkiba Stefanie Tauber

R e s e arc h

G ro u ps

A ndr e as h artig

Origin and biogenesis of peroxisomes Synthesis and degradation of organelles are tightly regulated in agreement with the metabolic status of the cell.

Andreas Hartig team

Gisela Dechat

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. Accordingly, peroxisomes need to be maintained in sufficient number to ensure metabolic homeostasis. A network of interacting proteins guarantees the biogenesis of functional peroxisomes, the transport of peroxisomal matrix proteins across the organellar membrane, and the control of size, shape and number of these compartments.

Dispensable peroxisomes are degraded in a process called pexophagy. Employing yeast as a 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 endoplasmic reticulum (ER). Currently, our main interest is focused on the mechanism of the de novo biogenesis initiated at the ER.

Peroxisomes are either transported to the newly born bud or are generated de novo from the ER. Yeast cells carrying a mutation in the inheritance process depend exclusively on the de novo biogenesis.

Proteins exclusively involved in the biogenesis of peroxisomes are called peroxins (Pex-proteins).

Among these the Pex11 protein is a membrane elongation factor, and we showed that this protein promotes the proliferation of peroxisomes already present in the cell. Two distantly related yeast proteins, Pex25p and Pex27p, participate in the de novo biogenesis. Together with Pex3p the Pex25 protein is essential for the initiation of the de novo peroxisome biogenesis from 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 employing genetic and biochemical screening procedures.

Wild type yeast cells accumulate a fluorescent peroxisomal protein in peroxisomes.

selected Publications 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 Jan;13(1):157-67. n 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 Oct 1;123(Pt 19):3389-400. n Kunze M, Pracharoenwattana I, Smith SM, Hartig A. A central role for the peroxisomal membrane in glyoxylate cycle function. Biochim Biophys Acta. 2006 Dec;1763(12):1441-52.

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R e s e arc h

G ro u ps

e rwin h e b e rl e - bors

Biotechnological use of plant reproductive development Previous and yet unpublished work in my laboratory has involved the plant DCN1 gene, which encodes for an E3 ligase involved in neddylation.

I am lecturing at the University of Vienna on Molecular Genetics for students of the Humanities and non-biological Science, and I give courses at the FH Campus Vienna in General Biology, Molecular Biology and Genetics, as well as Bioethics.

We have shown that DCN1 is involved in the reprogramming of microspores towards totipotency (microspore embryogenesis). Other work of my group, prior to my retirement, focused on a MAP kinase involved in auxin signal transduction/ transport.

Erwin Heberle-Bors

As a consultant for the Bill & Melinda Gates Foundation, I am involved in a project to produce for the first time doubled haploids in Cassava (Manihot esculenta), a staple food in tropical Africa. I am also writing a book on the role of Yersiana pestis-related plague in human history, and am preparing a manuscript entitled “Plague and nomads: an ironic connection�.

selected Publications Ribarits A, Mamun AN, Li S, Resch T, Fiers M, Heberle-Bors E, Liu CM, Touraev A. Combination of reversible male sterility and doubled haploid production by targeted inactivation of cytoplasmic glutamine synthetase in developing anthers and pollen. Plant Biotechnol J. 2007 Jul;5(4):483-94. n Ribarits A, Abdullaev A, Tashpulatov A, Richter A, Heberle-Bors E, Touraev A. Two tobacco proline dehydrogenases are differentially regulated and play a role in early plant development. Planta. 2007 Apr;225(5):1313-24.

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M arc e la h e rmann

LDL-R gene family, apolipoproteins and lipid transfer

Marcela Hermann team

Karin Eigner Dominik Habrina Patricia Mamesa Julia Plieschnig Max Strauss Desire Ĺ ubik Katarina Winter

Our studies focus on the biology of the growing chicken oocyte and the developing chicken embryo.

Specifically, we are interested in unraveling molecular mechanisms involved in the transport from the egg yolk to the embryo proper.

In this context, the roles of the LDL receptor gene family members, apolipoproteins and lipid transfer proteins, are studied. The developing avian embryo constitutes an excellent system for the study of lipid and lipoprotein transport phenomena. The yolk is the major source of nutrients for the developing embryo, but molecular details of the delivery mechanisms are largely unknown. During the vitellogenic phase of oocyte growth in the chicken, the yolk accumulates via uptake from the circulation of precursor proteins and serves as the sole source of lipid, carbohydrate, and protein. Only 350 mg of the 5-6 g of lipid in the yolk are mobilized during the first two weeks of embryogenesis; the major portion is transported during the final week. Such uptake, to a large part, occurs via the yolk sac, which utilizes the yolk lipoprotein components, following their degradation or modification, for resynthesis of lipoproteins,

which are subsequently secreted and delivered to the embryo through the embryonic circulatory system. The chick yolk sac is characterized by an outer layer of loosely associated mesenchymal tissue containing fetal blood islands and an inner single layer of endodermal cells, which line the lumen of the yolk sac cavity. The yolk-sac derived lipoproteins, mainly VLDL, contain much higher proportions of cholesteryl esters than yolk VLDL and harbor the intact form of apoB-100 rather than proteolytic fragments thereof. Furthermore, they lack apoVLDL-II, which is synthesized by laying hens and is present in yolk VLDL. These findings suggest that processing of yolk components inside the yolk sac proceeds in controlled fashion, initially involving degradation of their constituents.

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 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. We are interested in identifying compounds (synthetic, natural) with the potential to act as catalysts or inhibitors of the atherogenic modification of LDL.

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 resynthesis and secretion into the embryonic circulation. selected Publications Bauer R, Plieschnig JA, Finkes T, Riegler B, Hermann M, Schneider WJ. The developing chicken yolk sac acquires nutrient transport competence by an orchestrated differentiation process of itsendodermal epithelial cells. J Biol Chem. 2013 Jan 11;288(2):1088-98. n Plieschnig JA, Gensberger ET, Bajari TM, Schneider WJ, Hermann M. Renal LRP2 expression in man and chicken is estrogen-responsive. Gene. 2012 Oct 15;508(1):49-59. n Schreier SM, Steinkellner H, Jirovetz L, Hermann M, Exner M, Gmeiner BM, Kapiotis S, Laggner H. S-carbamoylation impairs the oxidant scavenging activity of cysteine: its possible impact on increased LDL modification inuraemia. Biochimie. 2011 Apr;93(4):772-7.

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J oac h im h e rmisson

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

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 subdisciplines 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 of creating an integrative approach, using a combination of different models, concepts, and methods. Methods include analytical work (stochastic processes, differential equations), extensive computer simulations, and statistical data analysis.

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

Phenotypic approaches It is widely appreciated (and ever better understood) that the

genetic basis of most quantitative traits consists of complex gene networks. However, how and when 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 genotypephenotype map (robustness, canalization, and modularity).

Ecologically motivated approaches The vast majority of population genetic models work under the assumption of a constant fitness landscape. Since fitness depends on variable environments, this is an idealization. Natural fitness landscapes will change over space and time.

And because an important aspect of an individual‘s environment is the composition of phenotypes in its own population, fitness will also depend on allele frequencies. The aim of this third line of our research is to combine genetic models with ecological factors. Recent studies have focused on conditions for speciation in spatially structured populations with gene-flow (parapatric speciation).

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

In contrast, large steps are expected for fast changes, when the speed of adaptation is only limited by the mutation rate.

selected Publications Bank C, Bürger R, Hermisson J. The limits to parapatric speciation: Dobzhansky-Muller incompatibilities in a continent-island model. Genetics. 2012 Jul;191(3):845-63. n Hancock AM, Brachi B, Faure N, Horton MW, Jarymowycz LB, Sperone FG, Toomajian C, Roux F, Bergelson J. Adaptation to climate across the Arabidopsis thaliana genome. Science.

2011 Oct 7;334(6052):83-6. n Kopp M, Hermisson J. The genetic basis of phenotypic adaptation II: the distribution of adaptive substitutions in the moving optimum model. Genetics. 2009 Dec;183(4):1453-76.

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Joachim Hermisson team

Claudia Bank Gregory Ewing Andrea Fulgione Angela Hancock Ines Hellmann Christian Huber Derek Setter MaBS members at Mathematics Department: Alexandre Blanckaert Ilse Höllinger Sebastian Matuszewski Agnes Rettelbach Claus Rüffler Hannes Svardal Hildegard Uecker

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r e in h old h ofba u e r

Consequences of carnitine deficiency and CSF-1 inhibition

Reinhold Hofbauer team

Klemens Kienesberger Christian Schäfer Eva Steiner

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

The first research task is dealing with the effects that 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 a sharp metabolic condition comparable to a patient situation, a precondition to study changes on mRNA expression levels. These promoter specific processes triggered by L-carnitine will be analyzed by a variety of molecular techniques, including chip screen analysis, real time RT-PCR, reporter gene and (super) band shift assays. We have identified factors 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 (the main goal of nutrigenomics).

The second research project is tracing the effects associated with inhibition of the macrophage colony-stimulating factor (CSF-1), which plays a key role in a wide variety of biologic processes.

It primarily acts on cells of the mononuclear phagocyte lineage by controlling the differentiation, proliferation and survival of precursor cells as well as the activation of mature macrophages.

As the latter are present in many tissues, CSF-1 also has a role in the pathogenesis of several disorders including cancers, because it regulates the production of MMPs and the uPA gene, which are heavily involved in tissue remodeling and tumor invasion. In view of the key role of CSF-1 in tumor progression, we detected that inhibition of CSF-1 expression can serve as a valuable tool to fight tumor growth and decrease the risk of metastasis.

Microarray analyses have revealed very promising candidate genes that are re- or induced during CSF-1 inhibition. Preclinical animal studies with inhibitory agents (monoclonal antibodies, RNAi) delineated from chip screen candidates are the next experimental aims.

Recently we characterized potentially cytotoxic genes (e.g.

Tmem66, superactive thymidine kinase 1), that were identified or generated in my group in previous research projects. These genes were cloned into an inducible vector system (pUHD-Hygr) and then transfected into model tumor cells (MCF-7 pretransfected with a puromycin resistance gene carrying a silencer construct). The final aim is to develop a genetic approach to attack tumor cells in a mammalian organisms.

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):3069-80. n Godárová A, Litzlbauer E, Brunner S, Agu AC, Lohninger A, Hofbauer R. L-Carnitine regulates mRNA expression levels of the carnitine acyltransferases CPT I, CPT II and CRAT. Chem. Monthly 2005 136:1349-1363. n Hofbauer R, Godárová A, Litzlbauer E, Agu AC, Kletzmayr J, Lohninger A. Chronic hemodialysis and pregnancy- L-carnitine supplementation to human sera in vitro restoring normal expression levels of carnitine acyltransferases. Chem. Monthly 2005 136: 1509-1521.

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N . e rwin i v e ssa

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.

Misfolded polypeptides are retro-translocated from the ER to the cytosol, where they become polyubiquitinated and destructed by proteasomes. ERassociated degradation (ERAD) is of relevance for a variety of genetically inherited, neurodegenerative, and virally transmitted diseases with protein folding defects.

with mutant cell lines with defects in N-glycan assembly the activities of one or more ER α1,2mannosidases could be implicated in ERAD. Interaction partners of ERAD substrate proteins in these mutant cell lines will be determined in immunoprecipitation experiments with cell lysates from cells grown in the presence of proteasome and/ or glycan processing inhibitors. Positive candidates will be identified using antibodies to known ERassociated proteins, and/or by mass spectro­scopy, and then further characterized. Another aspect of this project deals with the precise intracellular localization of the ERAD pathway of glyco­proteins by indirect immunofluorescence and confocal laser scanning microscopy using appropriate marker proteins.

The role of MTP und PDI in the assembly and secretion of atherogenic lipoprotein particles Microsomal triglyceride transfer protein (MTP) is a lipid transfer protein required for the assembly and secretion of very low density lipoproteins (VLDL).

Active MTP is a heterodimer containing a 97 kDa catalytic subunit and a 58 kDa subunit identified as protein disulfide isomerase (PDI). The MTP complex Furthermore, the requirement of N-linked glycan catalyzes the loading of apolipoprotein B (apoB) trimming for ERAD was shown, and from studies with lipids and/or the translocation of apoB into the lumen of the endoplasmic reticulum (ER). In avians, the synthesis of VLDL is inducible by estrogen. We are studying the effect of estrogen treatment on MTP activity and on the regulation of VLDL secretion that is also determined by lipid availability and apoB degradation. In this context, the consequence of altered intracellular MTP activity on VLDL assembly and secretion is being analyzed. Another aspect of the project is concerned with the mechanism of retention of The ERAD substrate RI332-6HA (B, D) co-localizes with the ER marker calnexin the MTP complex in the ER. We have previously shown that a truncated form of ribophorin I, a model glycoprotein for ERAD, is degraded by the ubiquitin/proteasome system. The role of N-linked glycans in ERAD was pinpointed as temporary retention devices in the ER. Thus, interaction of N-glycosylated substrates with the calnexin cycle prolongs their half lives.

(A, C), but hardly with the marker for the ER-Golgi-intermediate-compartment, ERGIC53 (E, F) in Chinese hamster fibroblast cells.

selected Publications Kitzmüller C, Caprini A, Moore SE, Frénoy JP, Schwaiger E, Kellermann O, Ivessa NE, Ermonval M. Processing of N-linked glycans during endoplasmic-reticulum-associated degradation of a short-lived variant of ribophorin I. Biochem J. 2003 Dec 15;376(Pt 3):687-96. n Hermann M, Foisner R, Schneider WJ, Ivessa NE. Regulation by estrogen of synthesis and secretion of apolipoprotein A-I in the chicken hepatoma cell line, LMH-2A. Biochim Biophys Acta. 2003 Jun 17;1641(1):25-33.

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N. Erwin Ivessa team

Fikret Rifatbegović

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MICHAEL F. JANTSCH

Transcriptome diversification through RNA editing The number of genes found in different organismic groups does not reflect their biological complexity.

Instead, transcriptome diversification can increase biological complexity.

Michael F. Jantsch team

Robert Aufreiter Silpi Banerjee Jacki Haraud Mamta Jain Konstantin Licht Maja Stulic Mansoureh Tajaddod Cornelia Vesely

One mechanism to achieve this, is RNA editing by adenosine deaminases that act on RNA (ADARs).

ADARs convert adenosines to inosines in structured and double-stranded RNAs. As inosines are interpreted as guanosines by most cellular processes, this type of editing can affect the coding potential, but also the folding, stability, and localization of RNAs. ADAR-mediated editing is widespread in metazoa and affects thousands of transcripts. Our research is focused on topics related to this type of RNA editing and aims at understanding the biochemical, cellular, and organismic consequences of A to I conversion.

Editing of repetitive elements in mRNAs. Repetitive elements (such as Alu elements) can basepair if inserted in opposite orientation. The basepaired regions are recognized by ADAR enzymes and can thus provide a substrate for RNA editing.

Editing in protein coding mRNAs

Regulators of RNA editing

A handful of protein coding targets for A to I editing are known today. The impact of editing on these RNAs and their encoded proteins is studied using transgenic mice. Our studies show that lack of editing of the mRNAs encoding the actin crosslinking protein filamin A leads to physiological defects at the organismic level but also to cellular defects such as cell migration and outgrowth.

The extent of RNA editing changes throughout development and differs between specific substrates despite a rather uniform expression of ADARs. We have therefore screened for factors that can stimulate and repress editing. Apparently, RNA-binding proteins that bind substrates specifically can act as inhibitors of RNA-editing.

Understanding the network of these RNA-binding proteins and their interaction with ADARs and their substrates is a current focus of our research.

Repetitive elements as modulators of gene expression Massive editing can be found in highly structured 3’ ends of mRNAs formed by inverted repetitive elements of the SINE family. Analysis of these untranslated regions in reporter gene assays demonstrates that inverted SINES can regulate gene expression. Deciphering the mechanisms by which these 3’ ends control gene expression is another research goal of our group.

selected Publications Tariq A, Garncarz W, Handl C, Balik A, Pusch O, Jantsch MF. RNA-interacting proteins act as site-specific repressors of ADAR2-mediated RNA editing and fluctuate upon neuronal stimulation. Nucleic Acids Res. 2012 Dec 28. n Vesely C, Tauber S, Sedlazeck FJ, von Haeseler A, Jantsch MF. Adenosine deaminases that act on RNA induce reproducible changes in abundance and sequence of embryonic miRNAs. Genome Res. 2012 Aug;22(8):1468-76. n Fritz J, Strehblow A, Taschner A, Schopoff S, Pasierbek P, Jantsch MF. RNA-regulated interaction of transportin-1 and exportin-5 with the double-stranded RNA-binding domain regulates nucleocytoplasmic shuttling of ADAR1. Mol Cell Biol.

2009 Mar;29(6):1487-97.

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v e r e na jantsc h

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 therefore 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.

chromosome movement. This checkpoint system monitors establishment of the obligate crossover, inducible only in leptotene/zygotene. Unrepaired DSBs and unsynapsed chromosomes maintain this checkpoint, but a crossover intermediate is necessary to shut it down.

Finally, we study a component of a putative double holiday junction dissolvase complex. During meiosis programmed double strand break induction leads to formation of about 12 breaks per chromosome in C. elegans. In worms only one of these matures into a crossover product. The rest of these gets resolved or dissolved in a noncrossover manner. To understand the interplay between these pathways will be essential to understand how a balanced number of crossovers will be established, since too few or too many lead to unfaithful chromosome partitioning into gametes.

During the first meiotic division, faithful segregation is facilitated by the formation between the parental homologs of a physical tether called crossover. 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 outer and inner nuclear envelope that connect chromosomes to cytoplasmatic microtubules).

Abrogation of chromosome movement, as we demonstrated with the sun-1(jf18) allele, leads to precocious synapsis involving non-homologous chromosomes. We study the nature of chromosome movement and its regulation. These forces stir chromosomes, helping to bring homologs together and to inhibit undesired interactions.

In addition we discovered that SUN-1 is an integral part of a meiotic surveillance mechanism that coordinates chromosome synapsis and recombination with meiotic progression and

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).

selected Publications Labella S, Woglar A, Jantsch V, Zetka M. Polo kinases establish links between meiotic chromosomes and cytoskeletal forces essential for homolog pairing. Dev Cell. 2011 Nov 15;21(5):948-58. n Baudrimont A, Penkner A, Woglar A, Machacek T, Wegrostek C, Gloggnitzer J, Fridkin A, Klein F, Gruenbaum Y, Pasierbek P, Jantsch V. Leptotene/zygotene chromosome movement via the SUN/KASH protein bridge in Caenorhabditis elegans. PLoS Genet. 2010 Nov 24;6(11):e1001219. n 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 Nov 25;139(5):920-33.

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Verena Jantsch team

Anahita Daryabeigi Cornelia Habacher Bence Hajdusits Patricia Hamminger Marlene Jagut Maria Kalldremxhiu Thomas Machacek Manuel Rauter Judith Windisch Alexander Woglar

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F ranz kl e in

Chromosome structure and meiotic recombination A “magic” interplay between structure and enzymology governs the function of chromosomes.

Franz Klein team

Lingzhi Huang Kuldeep Nangalia Feng Peng Silvia Prieler Viktoria Prast Martin Xaver Susanne Zich

During generation of gametes for sexual reproduction (meiosis), maternal and paternal chromosomes exchange fragments by recombination following the repair of meiotic DNAbreaks (DSBs), ensuring that each gamete receives a unique mix of parental properties. Chromosomes are made of DNA and dynamically attached proteins and are organized in distinct domains.

This organization requires structural differentiation into axis (or cohesin) sites, telomeres, kinetochores etc. Meiotic recombination is embedded in this chromosomal landscape, as only loop sequences receive DSBs and recombine, while axis-sequences don’t. We have recently published a breakthrough in correlating structure and function for the meiotic chromosome: 1) The machinery Recombination-Initiation is based on the chromosome axis.

Because initiation of meiotic recombination occurs at special sites in DNA loop regions, it was not understood why chromosome axis components play an important role for it. Using highresolution microarray technology, we were able to map components of the DSB machinery to axis associated DNA and to show the dependence of this localization on axis components. This finding has many significant implications.

2) Sumoylation of the yeast SUMO E2 converts it into a functional E3 and is essential for chromosome synapsis.

Synapsis is the alignment of chromosome axes during meiotic prophase at 100nm distance, which affects the fate of meiotic breaks and The machine for DSB formation, (three colored balls symbolize three located components) was localized to the base of DNA loops (Panizza et al., Cell, 2011). In contrast, DSBs are made on chromosome loops (Blatt et al., Cell, 2002).

Glimpse into the nano-world – section of a chromosome with “DNA-break machines“. Loops of sister chromatids (blue and turquoise) are linked and held in shape by ring-molecules. The machines are anchored between the DNA-loops at the axis of the chromosome. The model is illustrative, but postulates unknown details, such as the way the ring-molecules hold DNA-loops.

prevents chromosome entangling. Our collaborator Andrea Pichler (MPI, Freiburg) found that upon Sumoylation of its K153K157, Ubc9*SUMO acquires E3 competence and stimulates SUMO-chain by Ubc9 formation in vitro. We discovered that the defect of unsumoylatable Ubc9-KR mutants is the lack of synapsis in meiosis. This result strongly points to a role of SUMO chains in the genesis of synapsis and to Ubc9 as its key regulator.

3) A key role for the Smc5/6 complex in meiotic recombination.

Like the other structural maintenance of chromosomes (Smc) complexes, cohesin and condensin, the Smc5/6 complex is essential, but its role, particularly in meiosis, remains largely unknown. We have shown that this complex prevents the accumulation of Spo11 dependent recombination intermediates, called doubleHolliday Junctions (DHJ). The Smc5/6 associated SUMO ligase activity Mms21 is defective in preventing aberrant intermediates, which require Mus81/Mms4 for resolution. In the complete absence of Smc6, DHJs arise which can’t be resolved before anaphase, leading to catastrophic meiosis.

selected Publications Panizza S, Mendoza MA, Berlinger M, Huang L, Nicolas A, Shirahige K, Klein F. Spo11-accessory proteins link doublestrand break sites to the chromosome axis in early meiotic recombination. Cell. 2011 Aug 5;146(3):372-83. n Uanschou C, Siwiec T, Pedrosa-Harand A, Kerzendorfer C, Sanchez-Moran E, Novatchkova M, Akimcheva S, Woglar A, Klein F, Schlögelhofer P. A novel plant gene essential for meiosis is related to the human CtIP and the yeast COM1/SAE2 gene. EMBO J. 2007 Dec 12;26(24):5061-70. n Penkner AM, Prinz S, Ferscha S, Klein F. Mnd2, an essential antagonist of the anaphase-promoting complex during meiotic prophase. Cell. 2005 Mar 25;120(6):789-801.

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A lwin K ö h l e r

Gene expression and chromosome dynamics My group is broadly interested in genome organization and the mechanisms of gene expression.

We focus on two areas: First, we explore the role of nuclear pore complexes (NPCs) in genome regulation. Interphase chromosomes are not randomly spread throughout the nucleus but are fairly well organized, with different gene loci found in different regions of the nucleus. At the same time, chromatin can undergo extensive motion. In fact, some inducible genes dramatically change nuclear positions depending on whether they are active or not.

A fascinating new line of research suggests that activated genes can become hooked to nuclear pores – large transport channels, which protrude into the nuclear interior with a basket-like structure. According to this view, NPCs serve as anchors for the gene expression machineries and play a role in tuning gene activities. We would like to understand which factors mediate chromatinNPC interactions, how these links are formed and broken and how they contribute mechanistically to transcription, RNA processing and export.

Ultimately, our goal is to unravel basic principles of how nuclear architecture determines cellular function.

NPCs and proteins of the inner nuclear membrane partition the genome into areas of silent (yellow) and active chromatin (green). Gene-NPC interactions require various adaptors including the SAGA histone acetyltransferase (HAT) (Köhler & Hurt, Mol Cell, 2010).

Secondly, we investigate how ubiquitin signaling controls gene expression. While ubiquitin is wellknown for tagging proteins for destruction by the proteasome, its role in regulating chromatin is far less understood. We are particularly interested in the enzymatic toolkit for histone ubiquitination (ligases & deubiquitinases). When appended to histones, ubiquitin can function as a reversible molecular switch to regulate transcription, gene silencing and DNA repair. Recently, we have determined the structure of a histone deubiquitinase together with our collaborators and uncovered its sophisticated activation mechanism. Intriguingly, the Ubp8 deubiquitinase forms a protein module with three co-factors, which act in concert to assemble the module, shape the catalytic center and recognize the substrate.

The deubiquitinase module is part of SAGA, a multifunctional transcription co-activator. Our studies serve as a paradigm to explain how a deubiquitinase is switched on at the right time and place inside the cell. In addition, we aim to discover novel ubiquitin functions related to RNA and chromatin biology.

Multi-step activation and structure of a Ubiquitin Pac-Man (Köhler et al., Cell, 2010). selected Publications Köhler A*, Zimmerman E, Schneider M, Hurt E, Zheng N*. Structural basis for assembly and activation of the heterotetrameric SAGA histone H2B deubiquitinase module. Cell. 2010 May 14;141(4):606-17. *corresponding authors n Köhler A, Hurt E. Gene regulation by nucleoporins and links to cancer. Mol Cell. 2010 Apr 9;38(1):6-15. n Köhler A, Schneider M, Cabal GG, Nehrbass U, Hurt E. Yeast Ataxin-7 links histone deubiquitination with gene gating and mRNA export. Nat Cell Biol. 2008 Jun;10(6):707-15.

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Alwin Köhler team

Stefan Amlacher Gustavo Arruda Bezerra Laura Gallego Valle Ana Krolo Maria Jose Mendiburo Noemi Meszaros Maren Schneider Tobias Schubert Eleonora Turco

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G ottfri e d K ö h l e r

Biomolecular optical spectroscopy

Gottried Köhler team

Erwin Gaubitzer Gottfried Grabner Martin Knapp Christoph Miksch Karin Müller Martin Puchinger Julia Schindelar Arthur Sedivy Aamir Shazad

Biophysical characterisation of biomolecules and of their interactions in solution as well as on a live cell level represents the main object of our research.

Methods include single molecule fluorescence and time resolved Fig. 2: Time dependence of ACTH concentrations in response to the external signal and techniques performed the reconstruction by the model. over a wide range of time resolution. Studies by optical spectroscopy of endocytosis and transport in single living are complemented by biocalorimetry (DSC). cells. These measurements provide the basis for Quantitative studies on molecular dynamics on a mathematical modeling of complex dynamic single molecule level are performed using advanced behavior in biosystems, implemented in close cofluorescence correlation techniques (FCS). operation with other research groups.

Among others, these methods are applied on studies of ligand-receptor interactions relevant for hormone regulation and of the mechanisms

As an example we show the mathematical model for ACTH release from pituitary gland cells in dependence on the external cortisol or CRH concentration (see Fig. 1).

This model reproduces the time dependence of measured ACTH concentrations only in the case fast non-genomic feedback reactions, which are indicated in red or blue respectively, are taken into account (see Fig. 2). This result shows the importance of fast non-genomic reactions in stress response.

Fig. 1: Model for ACTH release from pituitary gland cells in response to external cortisol or CRH signals. The green signal cascade is the genomic pathway whereas the fast response to cortisol or CRH are marked in red or blue, respectively.

selected Publications Hiroi N, Lu J, Iba K, Tabira A, Yamashita S, Okada Y, Flamm C, Oka K, Köhler G, Funahashi A. Physiological environment induces quick response - slow exhaustion reactions. Front Physiol. 2011;2:50. n Shahzad A, Knapp M, Lang I, Köhler G. The use of fluorescence correlation spectroscopy (FCS) as an alternative biomarker detection technique: a preliminary study. J Cell Mol Med. 2011 Dec;15(12):2706-11. n Smetana W, Balluch B, Atassi I, Kügler P, Gaubitzer E, Edetsberger M, Köhler G. A Ceramic Microfluidic Device for Monitoring Complex Biochemical reactive Systems. Biomedical Engineering Systems and Technologies, Communications in Computer and Information Science. 2010;52 (2): 110-123.

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R ob e rt konrat

Physico-chemical biology and biomolecular NMR 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.

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. Secondly, 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.

The historical understanding that a protein’s function is encoded in the three dimensional structure is seriously being questioned by the observation that proteins in general and intrinsically disordered proteins (IDPs) in particular do not exist in single conformations but exchange between many different conformational substates.

IDPs have attracted substantial attention in recent years, in particular because of the discovery of their importance in eukaryotic life and of their central role in protein interaction networks. The efficient sampling of a vast and heterogeneous conformational space endows them with enormous potential to simultaneously interact with and control multiple binding partners. This structural polymorphism is intriguing from a biophysical point of view and thus calls for new approaches, combining appropriate theoretical concepts and experimental technologies. In our research we develop both novel theoretical concepts for protein sequence analysis and combine, for example, electron paramagnetic resonance (EPR) and nuclear magnetic resonance (NMR) to provide unique information about the vast conformational space accessible to IDPs and how this structural plasticity encodes biological function.

The individual stages of fragment-based lead drug design (FBLD). Starting from a suitable chosen small molecule fragment library, biophysical techniques are used to identify weak binders. (A) Structure-based FBLD exploits 3D structural information about ligand binding modes to rationally evolve starting fragments in iterative rounds of optimizations. (B) Fragment evolution is performed by either merging individual fragments binding to different interaction sites or by ligand extension using medicinal chemistry substitution. (C,D) Meta-structure based fragment-based lead drug design strategies for ligand merging (C) and extension (D). (C) Meta-structure homologies are used to discern putative binding modes based on available 3D structure information of the homologue. (D) Suitable sites for ligand derivatization are identified using ligand-based NMR spectroscopy (AFP-NOESY). Protons exposed to the solvent exhibit a sign inversion with increasing spin lock power (red). In contrast protons embedded in hydrophobic clusters display a markedly different behavior (blue) due to spin diffusion. This differential behavior can be used to identify suitable sites for ligand derivatization. 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. n Platzer G, Schedlbauer A, Chemelli A, Ozdowy P, Coudevylle N, Auer R, Kontaxis G, Hartl M, Miles AJ, Wallace BA, Glatter O, Bister K, Konrat R. The metastasis-associated extracellular matrix protein osteopontin forms transient structure in ligand interaction sites. Biochemistry. 2011 Jul 12;50(27):6113-24. n Konrat R. The meandering of disordered proteins in conformational space. Structure. 2010 Mar 14;18(4):416-9.

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Robert Konrat team

Nicolas Coudevylle Andrea Flamm Leonhard Geist Morkos Henen Gönül Kisilzavas Karin Ledolter Gerald Platzer Thomas Schwarz

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G ro u ps

P a v e l K o v arik

Signaling and gene expression in inflammation The innate immune system dynamically responds to infecting pathogens by initiation of protective host responses and by a rapid termination of these responses once the infection agent is no longer present.

Pavel Kovarik team

Marion Aberle Joanna Bancerek Virginia Castiglia Florian Ebner Christina Fieber Ivana Mikulic Bastian Oppl Vitaly Sedlyarov Iris Steinparzer

Inefficient or uncontrolled responses may result in infectious or inflammatory diseases. The molecular mechanisms that allow a robust yet temporally precisely restricted inflammatory reaction are studied in our laboratory at the level of transcription, mRNA stability and pathogen recognition.

Chromatin-associated signaling in interferoninduced transcription The STAT transcription factors play a central role in the immune system. STATs are phosphorylated within their transactivation domain (TAD). The precise function of the TAD phosphorylation and the TAD kinase have been elusive for years.

We have recently discovered that the CDK8 kinase, a submodule of the Mediator complex, phosphorylates TADs of chromatin-bound STATs. In the interferon (IFN) pathway, CDK8 is recruited

to the promoters of interferon-regulated genes in a STAT1-dependent manner. CDK8 subsequently phosphorylates STAT1 TAD to properly adjust STAT1-regulated transcription and to establish antiviral response. We are currently investigating the precise role for CDK8 in cytokine responses.

Control of immune homeostasis by mRNA stability A considerable proportion of genes induced during the acute phase of inflammation are strongly regulated at the level of mRNA stability.

Many proinflammatory mRNAs contain in their 3´ untranslated regions cis-acting AU-rich regulatory elements (AREs) that are targeted by RNA-stabilizing and -destabilizing proteins. The RNA-destabilizing protein tristetraprolin (TTP) plays a fundamental role in the attenuation of inflammation. In a systems biology approach we recently determined that TTP is the driving force of a regulatory circuit controlling the timing and extent of elimination of one third of inflammatory mRNAs. We are currently studying the molecular signatures that define a particular mRNA as TTP target. Further, we investigate the role of TTP in inflammatory diseases using conditional TTP deletion.

Responses of innate Streptococcus pyogenes

CDK8 occupancy at IFN-γ-regulated genes is STAT1dependent. CDK8 controls IFN-stimulated transcription in two ways: (i) CDK8 signals upstream by phosphorylating STAT1 in the transactivation domain (S727) resulting in gene expression changes. (ii) CDK8 directly regulates the transcription machinery. Together, these regulatory steps are required for antiviral response

immune

cells

to

S. pyogenes is a Gram-positive human pathogen causing mild (e.g. tonsillitis) as well as severe (e.g. toxic shock) diseases. It is still not known how this bacterium is recognized by the innate immune system. We have recently shown that, surprisingly, S. pyogenes is recognized by a receptor that is distinct from any so far described receptors for bacterial pathogens. The identification of the receptor for S. pyogenes and the elucidation of inflammatory signaling cascades in the host cells are currently the major goals of the project.

selected Publications 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 Jan 23. n Kratochvill F, Machacek C, Vogl C, Ebner F, Sedlyarov V, Gruber AR, Hartweger H, Vielnascher R, Karaghiosoff M, Rülicke T, Müller M, Hofacker I, Lang R, Kovarik P. Tristetraprolin-driven regulatory circuit controls quality and timing of mRNA decay in inflammation. Mol Syst Biol. 2011 Dec 20;7:560. n Gratz N, Hartweger H, Matt U, Kratochvill F, Janos M, Sigel S, Drobits B, Li XD, Knapp S, Kovarik P. Type I interferon production induced by Streptococcus pyogenes-derived nucleic acids is required for host protection. PLoS Pathog. 2011 May;7(5):e1001345.

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cla u din e kraft

Regulation and signaling in autophagy Fasting has been part of health practices from ancient times to the present. This tradition may be partially rooted in a cellular process we are now beginning to understand in modern scientific terms.

One of the major cellular responses during fasting is the activation of the lysosomal degradation pathway of autophagy, a process in which the cell digests its own components. This mechanism provides the cell with nutrients to maintain vital cellular functions during fasting and eliminates superfluous or damaged organelles, misfolded proteins, and invading microorganisms. Autophagy is now emerging as a central biological pathway, which promotes health and longevity. Accurate autophagy is pivotal for cell survival under stress conditions such as nutrient limitation. Autophagy has been found to play an important role in several human diseases including neurodegeneration and cancer.

The level of autophagy in a cell must be carefully balanced, as too little can be deleterious, but excessive levels of autophagy may also be harmful. Even though autophagy has been extensively studied from yeast to mammals, the molecular events of starvation sensing and autophagy induction remain largely elusive.

Nutrient-limiting conditions lead to autophagy induction, which results in the sequestration of cytosolic components into a double-membrane organelle followed by their subsequent delivery to the vacuole/lysosome for breakdown and recycling (Fig. 1). A major upstream regulator of autophagy is the Atg1 kinase complex, which is thought to sense the nutrient signal and subsequently transduce it to the autophagic machinery. This complex is part of the phagosome assembly site (PAS), yet how its location in the cell is organized and regulated remains unclear.

To understand autophagy regulation, we aim to identify the molecular events governing PAS assembly and formation, as well as to understand the function of Atg1 kinase in this process. We are studying the regulation of autophagy in budding yeast, using phospho-proteomic approaches, genetic screening techniques as well as modern biochemical and cell biological methods. We are mainly focusing on two aspects: • Composition and dynamics of the PAS. • Regulation of the Atg1 kinase and identification of its substrates.

These approaches aim to understand the mechanistics of a central player in autophagy regulation.

Since the PAS components and Atg1 kinase are highly conserved in other eukaryotes, this work will also help us to better understand diseases associated with autophagy misregulation.

Fig. 1: Schematic of the autophagy pathway. The nutrient signal is sensed by the PAS. Autophagy is then induced, and membranes are formed at the PAS. Membranes grow and engulf cytoplasmic components, which can include entire organelles. Membranes fuse to form a double-membraned autophagosome. The outer membrane fuses with the vacuolar membrane, releasing the inner autophagic vesicle into the lumen of the vacuole, where it is degraded by the vacuolar proteases. The Atg1 kinase is part of the PAS core and regulates the shuttling of membrane vesicles between the PAS and peripheral membrane structures. selected Publications Kraft C, Kijanska M, Kalie E, Siergiejuk E, Lee SS, Semplicio G, Stoffel I, Brezovich A, Verma M, Hansmann I, Ammerer G, Hofmann K, Tooze S, Peter M. Binding of the Atg1/ULK1 kinase to the ubiquitin-like protein Atg8 regulates autophagy. EMBO J. 2012 Sep 12;31(18):3691-703. n Cebollero E, Reggiori F, Kraft C. Reticulophagy and ribophagy: regulated degradation of protein production factories. Int J Cell Biol. 2012;2012:182834. n Kraft C, Martens S. Mechanisms and regulation of autophagosome formation. Curr Opin Cell Biol. 2012 Aug;24(4):496-501.

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Claudine Kraft team

Thorsten Bach Andrea Brezovich Daniel Papinski Thaddäus Pfaffenwimmer Martina Schuschnig Larissa Wilhelm

R e s e arc h

G ro u ps

K arl K u c h l e r

Host-pathogen interactions & mechanisms of fungal virulence We study fundamental problems in infection biology and the mechanisms governing host-fungus interactions, using a combination of molecular, as well as genome-wide and systems biology approaches.

Karl Kuchler team

Christelle Bourgeois Christa Gregori Denes Hnisz Fabian Istel Regina Klaus Cornelia Klein Olivia Majer Christina Rashkova Saren Tasciyan Lanay Tierney Michael Tscherner Philipp Wittmann Florian Zwolanek

Most if not all host-pathogen interactions are characterized by highly dynamic and interdependent relationships due to alternating attack and defense mechanisms exerted by each partner. Thus, we have set out to decipher fungal virulence by studying the molecular signaling processes occuring both in the pathogen and in its host. Iterative systems biology approaches enable us to study the dynamics of reciprocal attack and defense events during fungal infections. Our work stretches from functional and mechanistic studies on genes and signaling pathways to genomewide approaches through quantitative RNA-Seq transcriptomics, functional genomics and/or reverse-genetics. Finally, we use interdisciplinary theoretical approaches to better understand and infer interdependent genetic regulatory networks that control the molecular cross-talk of signaling during host-pathogen interactions. The major goal is therefore to unravel the complexity of molecular signaling processes that determine fungal virulence and shape the host immune response.

We are particularly interested in delineating adaptive mechanisms, which enable microbial pathogens to evade immune surveillance, thereby leading to dissemination and invasive diseases, as well as mechanisms that promote tolerance during host colonization.

On the pathogen side, we use reverse-genetic approaches as well as forward-genetics to: i) identify fungal virulence genes and those

implicated in antifungal resistance, ii) decipher the role of histone-modification enzymes such as histone deacetylases / histone acetyl transferases (HDAC/HAT) in impact gene regulation in morphogenesis, cell fate determination and stress response, iii) study the genetic and genomic adaptation processes occurring in fungal pathogens during host niche colonization, and iv) decipher the structure-function relationships of ABC transporters conferring antifungal drug resistance in fungal species.

On the host immune response aspect, we are delineating: i) the signaling response in primary ex vivo innate immune cells, as well as in mouse models infected with fungal pathogens, including the function of novel microbial pathogen receptors, ii) the signaling cross-talk of adaptive and innate immunity during immune surveillance, iii) the role of type I interferons (i.e. IFN-β in fungal virulence and host dissemination, and which mechanism IFN-β signaling exploits to stimulate migration of inflammatory phagocytes), iv) the signaling mechanisms by which innate phagocytes provide instructions for the adaptive immune response, but also how signaling drives the recruitment of inflammatory phagocytes and controls the outcome of fungal infections.

Our work has been supported by grants from the Christian Doppler Research Society, the 7th European framework programme, the Austrian Science Foundation FWF, the transnational ERANet Pathogenomics scheme, the SysMO projects through the Austrian GenAU research programme, the Austrian Academic Exchange Service OeAD and the FFG.

Candida albicans cells forming colonies of markedly different phenotypes on agar plates due to distinct chromatin modifications, which modulate transcriptional regulatory networks controlling morphogenesis. 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 Dec;8(12):e1003118. n 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. n Hnisz D, Majer O, Frohner IE, Komnenovic V, Kuchler K. The Set3/Hos2 histone deacetylase complex attenuates cAMP/PKA signaling to regulate morphogenesis and virulence of Candida albicans. PLoS Pathog. 2010 May 13;6(5):e1000889.

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T h omas L e onard

How lipids turn on protein kinases Membranes are sites of intense signaling activity in eukaryotic cells. Essential processes such as autophagy, cytokinesis, exo- and endocytosis, and cytoskeletal remodeling depend on signal propagation at cellular membranes.

complexes in quiescent cells (Fig. 1). We are simultaneously investigating the roles of posttranslational phosphorylation, nucleotide binding, catalytic activity, conformational flexibility, and inter-domain interfaces in maintaining the autoinhibited states of these proteins. The results of these experiments will be used to guide the rational design of constructs for crystallization and structure determination.

Dysregulation of signal transduction at membranes is the cause of a number of hereditary and non-hereditary diseases. 54 of 518 human protein kinases 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 elucidate the mechanisms of membrane-mediated kinase activation.

In parallel, we are investigating the regulation of the C-terminal tail of these kinases by mTORC2 in an effort to better understand the mechanisms by which they are activated in the cell. mTORC2 has been shown to be responsible for the phosphorylation of key regulatory sites in the C-tail of a number of these kinases, but it is unclear how mTORC2, which contains a lipid-kinase domain, achieves this directly. Using a combination of biochemical, cell-biological, and biophysical tools, we are addressing (a) the identity of the kinase responsible for phosphorylation of the turn motif of Akt and PKC, and (b) the structure and catalytic activity of the mTOR kinase itself. Preliminary studies have shown that we can recapitulate the reported inhibition of hydrophobic motif phosphorylation of Akt by mTORC2 knockdown using a specific MAPK inhibitor. Furthermore, turn motif phosphorylation is also attenuated in these experiments. We are now trying to identify whether this is a direct or indirect effect of MAPK inhibition.

We are currently focusing our attention on members of the Akt, Rock, and PKC families of AGC kinases that respond to the lipid second messengers PIP 3 and DAG respectively. We are monitoring membrane translocation of these proteins in live cells using fluorescence microscopy, and have recently determined important features of the regulatory domains that regulate the autoinhibited, intramolecular

Fig. 1: Mutation of conserved surface-exposed residues in the C1A domain of PKCs (α, β, δ, η, and θ) results in accelerated translocation to the plasma membrane in response to cell stimulation with phorbol esters. Since the same effect is qualitatively observed in both conventional and novel PKCs (which differ in the order of their regulatory domains in their primary sequences), we can conclude that the topological arrangement of the domains in 3 dimensions is the same. This implies conservation of the mechanism by which they are activated by membrane binding.

selected Publications Leonard TA, Hurley JH. Regulation of protein kinases by lipids. Curr Opin Struct Biol. 2011 Dec;21(6):785-91. n 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):55-66. n Leonard TA, Hurley JH. Two kinase family dramas. Cell. 2007 Jun 15;129(6):1037-8.

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Thomas Leonard team

Teresa Cvetkov Iva Lučić Linda Trübestein

R e s e arc h

G ro u ps

jos e f loidl

Meiotic chromosome pairing and recombination

Josef Loidl

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.

team

Rachel Howard-Till Agnieszka Lukaszewicz Anura Shodhan

During meiosis, homologous chromosomes of paternal and maternal origin pair, exchange parts in a process called crossing over, and segregate to different daughter nuclei. Errors in these processes in humans can lead to congenital defects in the progeny, stillbirths or miscarriages. We are studying various aspects of meiotic chromosome organization and behaviour in the protist Tetrahymena, an unconventional, evolutionarily distant model system, to identify conserved and derived features of meiosis. This will help to understand the evolutionary origin of conserved meiotic structures such as the synaptonemal complex and the chromosomal bouquet, and provide insight into many of the still mysterious meiotic processes such as homology recognition and recombination interference.

Crossing over is initiated by programmed DNA double-strand breaks (DSBs). The broken ends invade and ligate with intact homologous DNA strands. This creates joint molecules, four-stranded branched DNA structures, which are central intermediates of meiotic recombinational repair.

DNA synthesis that accompanies recombinational repair can be detected in situ by the incorporation and (immuno)labeling of base analogs. Using this technique, we found that in Tetrahymena there is a 90 min lag between DSB formation and repair (Fig. 1A-B). This suggests the existence of a mechanism that prevents immediate repair using the sister chromatid as a template. Suppression of repair via the sister (which, because of its proximity, is more accessible) is necessary to promote repair via the homologue. Only homologous repair ensures bivalent formation and the orderly segregation of homologous chromosomes. Hence, we hypothesize that repair is postponed to a stage when homologous chromosomes are aligned. We also apply meiotic BrdU incorporation to study recombination in various mutants. We found that in the absence of the DNA damage sensor kinase ATR, recombination does not occur (Fig. 1C). Currently, we are developing tools to immunoprecipitate BrdU-containing DNA to recover and sequence DNA regions which are hotspots for DSB formation.

Fig. 1A-C: Immunodetection of BrdU incorporated during meiotic recombination (red). Each picture shows a pair of conjugating Tetrahymena cells, each with a somatic macronucleus (MAC) and a generative micronucleus (MIC) which is undergoing meiosis.

A. DSBs trigger the extreme elongation of MICs early in meiotic prophase; at this stage BrdU is not incorporated.

B. At a later stage, when MICs exit from the elongated state, BrdU is incorporated at ~100 sites, indicating recombinational repair synthesis at DSBs. C. No meiotic DNA synthesis takes place if ATR is inhibited.

selected Publications Loidl J, Lukaszewicz A, Howard-Till RA, Koestler T. The Tetrahymena meiotic chromosome bouquet is organized by centromeres and promotes interhomolog recombination. J Cell Sci. 2012 Dec 1;125(Pt 23):5873-80. n Estreicher A, Lorenz A, Loidl J. Mug20, a novel protein associated with linear elements in fission yeast meiosis. Curr Genet. 2012 Apr;58(2):119-27.

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S asc h a M A rt e ns

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

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

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

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

Sascha Martens team

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

autophagosome then fuses with components of the classical endosomal system thereby maturing to an autolysosome within which the content is degraded.

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

We employ biochemistry, light- and electron microscopy to investigate these mechanisms. We are particularly interested in the mechanisms that sculpt cellular membranes into autophagosomes.

Our ultimate goal is to reconstitute crucial steps of auto­ phagosome formation in vitro and to translate our findings back to in vivo models.

(A) A picture taken by confocal microscopy showing giant unilamellar vesicles (GUVs).

The membrane of the GUVs was labelled by incorporation of a fluorescent lipid.

(B) A picture showing human cells which express green and red labelled proteins that are targeted to autophagosomes.

Our findings will give important insights into the generation of membrane curvature, the formation of specialized membrane domains and organelle formation in general.

selected Publications Romanov J, Walczak M, Ibiricu I, Schüchner 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):430417. n Kraft, C, Martens, S. Mechanisms and regulation of autophagosome formation. Curr Opin Cell Biol. 2012 Aug;24(4):496-501. n Groffen AJ*, Martens S*, DÍez Arazola R, Cornelisse LN, Lozovaya N, de Jong AP, Goriounova NA, Habets RL, Takai Y, Borst JG, Brose N, McMahon HT, Verhage M. Doc2b Is a High-Affinity Ca2+ Sensor for Spontaneous Neurotransmitter Release. Science 2010 Mar 26;327(5973):1614-8. * first and corresponding authors 39

Christine Aberts Iosune Ibiricu Justyna Sawa-Makarska Julia Romanov Marta Walczak Bettina Wurzer Gabriele Zaffagnini

R e s e arc h

G ro u ps

I r u t e M e ski e n e

Cell signaling control by MAPK phosphatases

Irute Meskiene

Environmental and developmental signals are transmitted inside the cell to induce corresponding responses.

Reversible protein phosphorylation is a major mechanism of intracellular signaling, where protein phosphatases act as important regulators.

team

Zahra Ayatollahi Justyna Boniecka Alois Schweighofer Volodymyr Shubchynskyy

The model plant Arabidopsis provides excellent possibilities to study stress-induced or developmental signaling on cellular and whole plant level.

We study the roles of Arabidopsis protein phosphatases of PP2C-type (AP2Cs 1-4) in regulation of MAPK (mitogen-activated protein kinase) signaling. After exposure to stress, MAPKs MPK3, MPK4 and MPK6 are activated within minutes by phosphorylation of conserved Thr and Tyr in their activation loop. AP2C1-4 dephosphorylate the phospho-Thr of the MAPKs and thus inhibit their kinase activity. Inactivation of the MAPKs by AP2C leads to signaling “switch off“. Using BiFC we have shown that AP2Cs are interacting with these MAPKs in vivo.

plant immunity. Our findings reveal that gene expression profiles, different plant hormones and plant phenotypes are strongly affected by these protein phosphatases. Currently, we are studying how MAPK phosphatases channel signaling pathways towards specific responses under biotic stress conditions.

Study of protein phosphatase mutant plants enabled us to identify specific genes regulated by these phosphatases. We suggest that AP2C protein phosphatases may help to channel MAPKmediated signals to specific targets. Our results are linking MAPK signaling to transcriptional reprogramming of plant cells and demonstrate importance of protein phosphatases in regulation of plant growth, development and survival under stress conditions.

A

B

Interaction of MAPK phosphatase AP2C3 with MAPK MPK6 in Arabidopsis protoplasts visualized by BiFC.

We have demonstrated that in plants AP2Cs control MAPKs during stress and in stomata development. Stomata are cells that regulate plant gas and water exchange with environment. MAPK phosphatase AP2C3 modulates cell cycle and stomata development, whereas AP2C1 controls

AP2C3 affects epidermal cell fate inducing differentiation of multiple stomata: FLPpro:GUS-GFP in leaves ofAP2C3oe (A) and WT (B) seedlings.

selected Publications Umbrasaite J, Schweighofer A, Kazanaviciute V, Magyar Z, Ayatollahi Z, Unterwurzacher V, Choopayak C, Boniecka J, Murray JA, Bogre L, Meskiene I. MAPK phosphatase AP2C3 induces ectopic proliferation of epidermal cells leading to stomata development in Arabidopsis. PLoS One. 2010 Dec 23;5(12):e15357. n Michniewicz M, Zago MK, Abas L, Weijers D, Schweighofer A, Meskiene I, Heisler MG, Ohno C, Zhang J, Huang F, Schwab R, Weigel D, Meyerowitz EM, Luschnig C, Offringa R, Friml J. Antagonistic regulation of PIN phosphorylation by PP2A and PINOID directs auxin flux.

Cell. 2007 Sep 21;130(6):1044-56. n Schweighofer A, Kazanaviciute V, Scheikl E, Teige M, Doczi R, Hirt H, Schwanninger M, Kant M, Schuurink R, Mauch F, Buchala A, Cardinale F, Meskiene I. The PP2C-type phosphatase AP2C1, which negatively regulates MPK4 and MPK6, modulates innate immunity, jasmonic acid, andethylene levels in Arabidopsis.

Plant Cell. 2007 Jul;19(7):2213-24.

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G ro u ps

I sab e lla M oll

Bacterial stress response and ribosome heterogeneity We are interested in the regulation of protein synthesis at different levels in the model organism Escherichia coli.

Aside from studying translation regulation mediated by sRNAs as well as protein factors, the focus of my group extended towards the intriguing phenomenon of translation regulation by ribosome heterogeneity. Traditionally, the bacterial ribosome is viewed as highly conserved machinery with an invariable rRNA and protein complement, a conception that was supported by the high resolution crystal structures of the ribosome. Thus, they were not considered to have an intrinsic regulatory capacity. In contrast, studying the phenomenon of sustained translation of leaderless mRNAs in E. coli in the presence of the antibiotic kasugamycin (Ksg), we unexpectedly observed the formation of protein-deficient 61S ribosomal particles formed in the presence of Ksg in vivo, which selectively translate leaderless mRNAs. Notwithstanding the artificial conditions, which trigger the release of more than six proteins of the small ribosomal subunit, this study presented the first evidence for the functionality of ribosomes devoid of multiple proteins, and puts forward the idea that the protein complement of the ribosome can vary in response to changed physiological conditions. Later on, we elucidated a novel posttranscriptional stress adaptation mechanism and successfully identified the molecular pathway for the selective translation in response to mazF induction. This observation represents the first report on the generation of functionally heterogeneous ribosomes when bacteria encounter adverse conditions. Thus, the stress-responsive modulation of protein synthesis by processing of the 16S rRNA from already active 70S ribosomes by a toxin completely changed the perception of the bacterial ribosome as unchangeable translational machinery and introduced an additional level of complexity to the regulation of gene expression.

bacteria. Despite being essential, the structure of S1 is lacking on the high resolution structures available for the ribosome. Hence, based on the observation that S1 requires protein S2 for binding to the 30S ribosomal subunit, we verified that the N-terminal domain (NTD) of protein S1 is absolutely required for the interaction with the ribosome.

Currently, we aim to determine the structure of the S1-NTD when bound to the ribosome.

Isabella Moll team

Konstantin Byrgazov Paul Kollmann Martina Sauert Hannes Temmel Oliver Vesper

Molecular pathway for the selective translation in response to mazF induction by stress conditions.

The mazEF module can be triggered by stressful conditions (indicated by an arrow), which results in degradation of the antidote MazE by the ClpAP protease . The activity of released MazF leads to degradation of the majority of transcripts. In addition, we have shown that it removes the 5´-UTR of specific mRNAs thus rendering them leaderless, and moreover, specifically removes the 3´-terminus of the 16S rRNA comprising helix 45 as well as the aSDsequence, which is essential for translation initiation on canonical ribosome binding sites. Consequently, MazF activity leads to selective translation of a novel “leaderless mRNA regulon”.

Aside from these studies, we focus on the structural and functional characterization of protein S1, the largest ribosomal protein in Gram-negative

selected Publications Byrgazov K, Manoharadas S, Kaberdina AC, Vesper O, Moll I. Direct interaction of the N-terminal domain of ribosomal protein S1 with protein S2 in Escherichia coli. PLoS One. 2012;7(3):e32702. n Moll I, Engelberg-Kulka H. Selective translation during stress in Escherichia coli. Trends Biochem Sci. 2012 Nov;37(11):493-8. n 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.

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Ernst W . M ü lln e r

Signal transduction and hematopoiesis/erythropoiesis Hematopoiesis starts from stem cells in the bone marrow which gradually differentiate towards the well-known specific lineages of blood cells.

Ernst W. Müllner team

Matthias Artaker Katrin Fischhuber Manfred Schifrer

The process ends with late-stage committed progenitors undergoing terminal maturation. Our group focused on molecules critically involved in reaching the balance between sustained proliferation versus terminal differentiation of progenitors, with particular emphasis on erythropoiesis.

As systems, we used fetal liver- or bone marrowderived mouse erythroblasts as well as myeloid progenitors from various genetically modified mouse strains. Moreover, we employed cells from cordor peripheral blood of healthy or diseased human donors. These cell types can be kept immature in culture for extended periods and terminally differentiated in a highly synchronous manner.

These cellular tools were mainly used to study: 1 – Signaling pathways emanating from extracellular ligands like growth factors (stem cell factor, Wnt, erythropoietin) or steroid hormones (thyroid hormone T3, glucocorticoid, androgen)

ses. Most complete Stat5-/- embryos developed to term but suffered from severe microcytic anemia resulting in perinatal lethality. We could attribute this phenotype to multiple defects in erythropoiesis, mainly a massive increase in apoptosis in the fetal liver. ad 2 – We also uncovered an additional unexpected defect on cellular iron metabolism due to Stat5 deficiency, involving a significant down regulation of TfR-1 expression in vivo, resulting in massively reduced iron uptake. In particular, levels of the erythroid TfR-1 regulator IRP-2 were strongly decreased. IRP-2 itself were shown to be a direct target gene of Stat5 (see Figure). ad 3 – Experiments with e.g. erythroblasts and fibroblasts synchronized in the cell cycle by centrifugal elutriation provided evidence that vertebrate cells respond to cell size alterations (induced through different growth factor signaling or DNA synthesis inhibitors) by compensatory shortening of the subsequent G1 phase. This suggests an active size-threshold mechanism in G1 to re-adjust cell-cycle duration in the next cycle, ensuring maintenance of a proper balance between growth and proliferation rates.

2 – Cell-type specific features in the regulation of iron metabolism during erythropoiesis 3 – Cell size control - this topic was originally triggered by the decrease in cell volume accompanying erythroid maturation ad 1 – Erythropoiesis depends on signaling through the EpoRJak2-Stat5 axis, regulating proliferation, differentiation and survival. While Jak2 and EpoR deficiency are embryonically lethal, the exact role of Stat5 had remained puzzling, since the original Stat5-ko mice had carried a hypomorphic Stat5 allele (Stat5DN/DN), impeding full analy-

Reduced iron uptake into Stat5-/- erythroblasts. Cells display decreased Irp-2 expression (left; Western blot and quantification) due to functional Stat5 binding sites in the Irp-2 promoter (right; promoter architecture and reporter assay) which in turn down-modulates levels of TfR-1 protein and thus iron import.

selected Publications Friedbichler K, Kerenyi MA, Kovacic B, Li G, Hoelbl A, Yahiaoui S, Sexl V, Müllner EW, Fajmann S, Cerny-Reiterer S, Valent P, Beug H, Gouilleux F, Bunting KD, Moriggl R. Stat5a serine 725 and 779 phosphorylation is a prerequisite for hematopoietic transformation. Blood. 2010 Sep 2;116(9):1548-58. ”Doc-Award 2011” from the University of Vienna n Kerenyi M, Müllner EW. Muscle iron in stress erythropoiesis? Blood. 2009 Jun 25;113(26):6507-8. n Kerenyi MA, Grebien F, Gehart H, Schifrer M, Artaker M, Kovacic B, Beug H, Moriggl R, Müllner EW. Stat5 regulates cellular iron uptake of erythroid cells via IRP-2 and TfR-1. Blood. 2008 Nov 1;112(9):3878-88. ”Doc-Award 2009” from the University of Vienna; “Promotion Award of the City of Vienna 2009” (Förderungspreis der Stadt Wien) 42

R e s e arc h

G ro u ps

J o h ann e s N impf

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 the transport of macromolecules across cell membranes and play important roles in signal transduction.

The biological systems we are working with are the chicken oocyte and the mammalian brain. These

two systems reflect the functional dichotomy of the receptors which function in endocytosis (follicles) and signal transduction (brain development). The best characterized function of VLDLR in follicles of egg laying species is endocytosis of yolk precursors into the growing oocyte.

These yolk precursors (VLDL and Vitellogenin) are synthesized in the liver and rapidly taken up by the growing oocyte. Recently we have started to elucidate cell signaling functions of VLDLR and ApoER2 in granulosa cells which support the maturation of oocytes within the follicle. With respect to brain development both receptors act as Reelinsignal transducers. The Reelin signal orchestrates the correct positioning of newly generated neurons within laminated structures of the brain. In the development of the olfactory system in rodents the structure of olfactory bulb depends on neurons generated throughout life in the subventricular zone.

Model of the intracellular fates of ApoER2 and VLDLR upon Reelin stimulation.

Upon binding of Reelin, both ApoER2 and VLDLR mediate phosphorylation of Dab1 (1). VLDLR internalizes Reelin rapidly via Clathrin-mediated endocytosis (2) and is separated from Reelin in the compartment of uncoupling of receptor and ligand (3).

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

These neurons migrate via the rostral migratory stream towards the olfactory bulb.

This migration also depends on the presence of ApoER2 and VLDLR but seems to be independent on Reelin. To this end we have characterized thrombospondin-1 as a novel ligand for ApoEr2 and VLDLR present in the rostral migratory stream.

Currently, we are focusing on the search for other alternative ligands for the receptors which are involved in orchestrating the migration of neuroblasts and/or which are involved in maturation of the follicle.

selected Publications Duit S, Mayer H, Blake SM, Schneider WJ, Nimpf J. Differential functions of ApoER2 and very low density lipoprotein receptor in Reelin signaling depend on differential sorting of the receptors. J Biol Chem. 2010 Feb 12;285(7):4896908. n 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):3069-80. n Andrade N, Komnenovic V, Blake SM, Jossin Y, Howell B, Goffinet A, Schneider WJ, Nimpf J. ApoER2/VLDL receptor and Dab1 in the rostral migratory stream function in postnatal neuronal migration independently of Reelin. Proc Natl Acad Sci U S A. 2007 May 15;104(20):8508-13.

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Johannes Nimpf team

Christine Ehresheim Alexander KaltenbĂśck Christian Leeb Harald Rumpler

R e s e arc h

G ro u ps

Egon O gris

Enzyme biogenesis and monoclonal antibodies

Egon Ogris team

Bhumika Bhatt Ingrid Frohner Wolfgang Hintringer Thomas Kupka Marie Lang Katharina Maderböck Ingrid Mudrak Stefan Schüchner

Cells employ posttranslational modifications such as phosphorylation/dephosphorylation to regulate almost all aspects of their life. These modifications are catalyzed by protein kinases and phosphatases, respectively.

A key phosphatase in the cell is protein phosphatase 2A (PP2A), a known tumor suppressor and target of cancer causing viruses. PP2A comprises a family of protein phosphoserine/-threonine phosphatases with prototypical multisubunit architecture, in which a catalytic subunit achieves substrate specificity through the interaction with regulatory subunits. The PP2A family consists of over 70 different holoenzymes which target probably many hundred substrates in the cell. How holoenzyme assembly is regulated and what the substrates of these different holoenzymes are is largely unknown.

A pathological decrease of PP2A activity has been linked to the development of human diseases such as cancer or Alzheimer. Thus, our major research goals are to understand the molecular mechanisms of PP2A regulation and to identify the substrates and processes regulated by PP2A.

PP2A activity is decreased in Alzheimer brain tissue suggesting a potential causal role for PP2A in Alzheimer pathogenesis. In collaboration with Estelle Sontag (University of Newcastle, Australia) we are investigating whether dysfunction of PP2A biogenesis might be involved in Alzheimer disease development.

PP2A substrate identification is difficult with the currently available methods due to the transient enzyme-substrate interaction during catalysis.

In collaboration with the Ammerer laboratory we developed a novel two-hybrid system termed M-Track, with which it is possible to detect, validate and identify transient enzyme-substrate interactions such as the ones between PP2A and its substrates.

Immunofluorescence of laminopathy nucleus with a general anti Lamin A/C antibody (red) and the point-mutant specific anti R453W antibody (green). Fluorescence intensities of the lamin A/C stainings are shown below the respective images.

Cartoon of an M-Track assay: Bait protein “Y”, prey protein “X” human 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).

Our study of PP2A biogenesis in yeast led to a model, in which the generation of the active enzyme is tightly coupled to the assembly of substrate-specific holoenzymes. This process is under surveillance of the PP2A methylesterase, PPE1, which seems to control the correct order of the PP2A activation process. We are currently investigating how PP2A biogenesis is regulated by different signaling pathways.

The second more business-oriented focus of the lab is the generation of monoclonal antibodies against human disease-linked point-mutant proteins.

We show that antibodies with such exquisite specificity represent a new type of research tools for the analysis of disease mechanisms. Our lab also provides a monoclonal antibody service for the generation of custom-tailored monoclonal antibodies to internal and external customers (please see report of Monoclonal Antibody Facility on page 82).

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. n Roblek M, Schüchner S, Huber V, Ollram K, Vlcek-Vesely S, Foisner R, Wehnert M, Ogris E. Monoclonal antibodies specific for diseaseassociated point-mutants: lamin A/C R453W and R482W. PLoS One. 2010 May 13;5(5):e10604. n Sontag JM, Nunbhakdi-Craig V, Mitterhuber M, Ogris E, Sontag E. Regulation of protein phosphatase 2A methylation by LCMT1 and PME-1 plays a critical role in differentiation of neuroblastomacells. J Neurochem. 2010 Dec;115(6):1455-65.

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R e s e arc h

G ro u ps

R ain e r P ro h aska

Stomatin, membrane domains and neuroacanthocytosis We are interested in and working on two areas. One is the structure and function of the membrane protein stomatin, and the other is the pathomechanism leading to the neurodegenerative disease neuroacanthocytosis.

European Multidisciplinary Neuroacanthocytosis (EMINA)

Initiative

on

An ERA-NET/E-RARE network coordinated by Prof.

Adrian Danek (LMU, Munich) comprises six partner organizations in Germany, Austria, The Netherlands, France and Turkey, and aims to take a significant step forward in both basic research and applied clinical research into the neuroacanthocytosis (NA) syndromes. These syndromes are a group of Stomatin-specific microdomains rare, currently incurable, neurological illnesses which affect mostly young adults. NA is associated Stomatin, originally found in erythrocyte membranes, with neurodegeneration in the brain, similar to is an oligomeric, cholesterol-binding, lipid raftHuntington’s Disease, but can be distinguished by associated, palmitoylated integral membrane the presence of spiky erythrocytes (acanthocytes) protein that is localized to the cytoplasmic side in the blood. Several candidate genes for various of the plasma membrane and late endosomes of NA forms have been identified: VPS13A (vacuolar many cell types. We are studying these features protein sorting 13A) for chorea-acanthocytosis and stomatin’s interaction with other membrane (ChAc), XK (Kell system) for McLeod Syndrome (MLS), proteins, such as the glucose transporter GLUT1 JPH3 (junctophilin 3) for Huntington’s Disease-Like and stomatin-like protein-1. Using in situ chemical 2 (HDL2), and PANK2 (pantothenate kinase 2) for cross-linking, isolation of stomatin complexes Pantothenate Kinase-Associated Neurodegeneration and mass spectrometry, we recently identified the (PKAN). We recently reviewed the scientific interacting partners GLUT1, anion exchanger-1, basis of the related syndromes, NA and NBIA aquaporin-1, ferroportin-1, urea transporter-1, (neurodegeneration with brain iron accumulation). nucleoside transporter-1, the calcium-pump (CaOur group participates in EMINA basic research ATPase-4), CD47, LW/ICAM-4, and flotillin-1 and -2 by focusing on the identification of acanthocyte in erythrocyte membrane domains. These findings membrane domains that differ in composition from are in line with the hypothesis that stomatin plays the normal, discocyte membrane. We hypothesize a role as a membrane-bound scaffolding protein that the VPS13A defect will affect vesicle sorting modulating transport proteins in cholesterol-rich and autophagy in late stage erythropoiesis and membrane microdomains. thus lead to changes during reticulocyte membrane reorganization.

Similar sorting d e f e c t s may affect autophagy in neurons leading to accumulation of protein aggregates and cell death.

Schematic model of stomatin in the red cell membrane. Stomatin (STOM) interacts with itself (at A follow-up C-terminus) to form dimers and oligomers and with several transporters (e.g. glucose transporter ERA-NET/EGLUT1), channels (e.g. aquaporin-1/AQP1), the anion exchanger (band 3), and pumps in the RARE project membrane and appears to modulate their activities in a cholesterol-dependent manner.

(EMINA-2) will be investigated by young scientists including Mario Mairhofer at AKH/MUV as Austrian partner.

selected Publications Rungaldier S, Oberwagner W, Salzer U, Csaszar E, Prohaska R. Stomatin interacts with GLUT1/SLC2A1, band 3/SLC4A1, and aquaporin-1 in human erythrocyte membrane domains. Biochim Biophys Acta. 2013 Mar;1828(3):956-66. n Prohaska R, Sibon OC, Rudnicki DD, Danek A, Hayflick SJ, Verhaag EM, Vonk JJ, Margolis RL, Walker RH. Brain, blood, and iron: perspectives on the roles of erythrocytes and iron in neurodegeneration. Neurobiol Dis. 2012 Jun;46(3):607-24. n Mairhofer M, Steiner M, Salzer U, Prohaska R. Stomatin-like protein-1 interacts with stomatin and is targeted to late endosomes. J Biol Chem. 2009 Oct 16;284(42):29218-29.

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Rainer Prohaska team

Stefanie Rungaldier Claudia Roos

R e s e arc a r c h

G ro r o u ps p s

F riE D R I C H P ropst

The neuronal cytoskeleton in axon guidance

Friedrich Propst team

Anton Kamnev Waltraud Kutschera Rajeshwari Meli Zsuzsanna Orban-Nemeth Thomas Völk Petronela Weisová

Axon extension, axon branching, and axon retraction are major morpho­ logical changes that neurons have to execute to accomplish correct wiring of the nervous system during development and regeneration after injury.

These transformations are guided by extracellular signals which ultimately need to be translated into the rearrangement of the neuronal cytoskeleton. We study signaling mechanisms and posttranslational modifications of microtubuleassociated proteins and other components of the cytoskeleton that regulate the orchestrated reorganization of microtubules and actin in response to extracellular signals. Our approach combines gene ablation in the mouse with cell biological and molecular analyses in cultured neurons and other primary cells.

One focus of our research is the role of microtubuleassociated proteins of the MAP1 family. In a recent study we found that one of these proteins, MAP1B, is a component of a pathway that links calcium influx and activation of neuronal nitric oxide synthase to the reconfiguration of axonal microtubules. Thus, MAP1B might mediate some of the physiological and pathological effects of nitric oxide in the brain. Since calcium and nitric oxide contribute to growth cone steering through repulsive axon guidance cues, this study also suggested that MAP1B is an essential component of axon guidance signaling to the cytoskeleton.

We have since obtained evidence that MAP1B is necessary for signal transduction of several unrelated repulsive guidance cues. To elucidate molecular details we investigate posttranslational modifications of MAP1B, identify its binding partners and perform structural analyses on the MAP1B light chain.

We have also analyzed the functional properties of other MAP1 proteins and found that the light chains of these proteins determine to some extent their functional characteristics. Moreover, we characterized a novel member of the MAP1 family, which we termed MAP1S. MAP1S is expressed not only in the brain, but also in a wide range of other tissues and represents the nonneuronal counterpart of MAP1A and MAP1B. We have generated MAP1S deficient mice and are exploring the role of this protein in cell division, cell migration, and tumorigenesis.

MAP1B is essential for nitric oxide-induced axon retraction. a) Cultured neurons from adult wild-type mice (dorsal root ganglion) were treated with the nitric oxide donor SNAP, fixed, stained for tubulin and analyzed by confocal fluorescence microscopy. Cellular morphology was scored as unchanged (left) or retracted (right). b) Cultured neurons from adult MAP1B+/+ or MAP1B/- mice were treated with SNAP for 1 h and processed as above. Microtubule configuration was classified as unchanged (compared to untreated cells) or displaying retraction hallmarks (sinusoidal bends along the axon, a trailing remnant, and a retraction bulb). MAP1B-/neurons displayed a severely reduced capacity to respond to SNAP by axon retraction.

selected Publications Orbán-Németh Z, Henen MA, Geist L, Zerko S, Saxena S, Stanek J, Koźmiński W, Propst F, Konrat R. Backbone and partial side chain assignment of the microtubule binding domain of the MAP1B light chain. Biomol NMR Assign. 2013 Jan 22. n Fuhrmann-Stroissnigg H, Noiges R, Descovich L, Fischer I, Albrecht DE, Nothias F, Froehner SC, Propst F. The light chains of microtubule-associated proteins MAP1A and MAP1B interact with α1-syntrophin in the central and peripheral nervous system. PLoS One. 2012;7(11):e49722. n Stroissnigg H*, Tranc`´íková 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 Sep;9(9):1035-45. *Equal contribution 44

R e s e arc a r c h

G ro r o u ppss

F lorian R aibl e

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

make use of the remarkable transparency of Platynereis to observe cells and molecules in action.

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

The hormonal control of reproduction and regeneration What could be the function of ancestral-type hormones in Platynereis? One of the systems that we aim to dissect is the hormonal machinery orchestrating reproduction and regeneration.

Platynereis is an excellent object for this analysis, as it has been a central model for the link between chronobiology and reproduction. Our molecular analyses have identified a spectrum of conserved hormones in Platynereis. Thanks to the establishment of new molecular tools, we are now able to systematically assess the impact of these candidates on the development and maturation of the animals.

These experiments are supported by an ERC starting grant (HOR.MOON) as well as the interdisciplinary research platform “Marine Rhythms of Life“ that has recently been established at the University of Vienna, and provide us with first molecular insight into the enigmatic hormone machinery underlying circalunar reproductive periodicity.

Exploring a new marine model system Over the past years, Platynereis has emerged as a very promising “next-generation” model system.

We have pioneered transgenic technology in Platynereis that allows us to mark and interrogate cell types with unprecedented precision. Moreover, we explore transcriptomic technology and functional assays to dissect the logic of hormone regulation and action in the animal. Finally, we

Florian Raible team

Benjamin Backfisch Stephanie Bannister Mingliu Du Anna Maria Kurzreiter Alessandra Polo Roger Revilla-i-Domingo Sven Schenk Elisabeth Steinkellner Agnė Valinčiūtė Ancestral-type hormones in a simple invertebrate.

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

Visiting Scientist

Vitaly Kozin

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 regulation of stem cells in worms and sponges. Our vision is to firmly establish Platynereis as a reference species for marine biology.

Hormonal orchestration of regeneration and reproduction by the medial Platynereis brain. (A) Classical transplantation studies revealed that immature Platynereis heads are the source for an endocrine brain hormone inhibiting maturation and supporting regeneration. (B) Implantations of small brain fragments (blue) map the source of the brain hormone to the medial Platynereis brain. The new molecular tools established in the lab allow us for the first time to identify and study the cellular circuits and hormonal cues responsible for these functions.

selected Publications 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 USA. 2013 Jan 2;110(1):193-8 n Tessmar-Raible K, Raible F, Arboleda E. Another place, another timer: Marine species and the rhythms of life. Bioessays. 2011 Mar;33(3):165-72. n Christodoulou F, Raible F, Tomer R, Simakov O, Trachana K, Klaus S, Snyman H, Hannon GJ, Bork P, Arendt D. Ancient animal microRNAs and the evolution of tissue identity. Nature.

2010 Feb 25;463(7284):1084-8.

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a r c h R e s e arc

r o u ps p s G ro

Hans R ot h e n e d e r

Cell cycle regulation and DNA damage response

Hans Rotheneder team

Karim Abiri Susanne Schweiger

My laboratory is focused on the mechanisms controlling the growth and cell cycle of the mammalian cell. They respond to perturbations such as replication errors or DNA damage by inducing cell cycle arrest, senescence, or apoptosis.

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

We have recently identified and characterized a protein that we called EAPP (E2F Associated PhosphoProtein). EAPP interacts with E2F1-3, comprising the activator group of E2F proteins, and modulates E2F-dependent transcription.

Tumour cells often overexpress EAPP, indicating that it confers a selective advantage to these cells. EAPP levels increase upon DNA damage and higher EAPP levels seem to protect cells from apoptosis. This protection can also be achieved by the 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 knock down of p21 reduces the anti-apoptotic activities of overexpressed EAPP. This suggests that p21 at least in part mediates this activity of EAPP. EAPP stimulates p21 expression by binding to its promoter and seems to be required for the assembly of the transcription initiation. The knock down of EAPP facilitates apoptosis and goes along with reduced p21.

Our findings suggest that EAPP is indispensable for the survival of a cell. The required amount of EAPP seems to depend on the environmental conditions.

Preliminary evidence suggests that the role of EAPP in transcription is not limited to the p21 promoter.

Active promoters are occupied by multiple types of complexes and EAPP seems to be an important component of at least some of them. Lowering EAPP levels influences the expression of some of the genes examined including important cell-cycle regulators. To examine which genes are influenced

by EAPP and to scrutinize the biochemical details of its activity will be our future focus.

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

selected Publications Andorfer P, Schwarzmayr L, Rotheneder H. EAPP modulates the activity of p21 and Chk2. Cell Cycle. 2011 Jul 1;10(13):2077-82. n 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. n 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.

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a r c h R e s e arc

r o u ppss G ro

P e t e r S c h l ö g e l h of e r

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

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.

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 specialized, two-step cell division that ensures the reduction of the genome prior to the formation of generative cells. During meiosis, homologous centromeres are segregated during the first, and sister centromers during the second division. As there is no intervening DNA replication between the two meiotic divisions, each of the final division products contains only half of the initial DNA content. For a given diploid organism the developing generative cells are then haploid. It is important to note that during meiosis, genetic information between maternal and paternal chromosomes is mutually exchanged, leading to novel combinations of genetic traits in the following generation.

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 (BioOptics, DeepSequencing, Mass Spectrometry, Bioinformatics).

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 makeup.

Novel combinations between parts of paternal and maternal chromosomes are generated through the process of homologous recombination (HR). A pre-

Our latest achievement is the development of a technology that allows us to map meiotic DSBs with nucleotide resolution. We have already successfully used this novel method to map DSBs genomewide in the yeasts Saccharomyces cerevisiae and Schizosaccharomyces pombe. Currently, we are optimizing the protocols for mapping DSBs genome wide in Arabidopsis thaliana. Furthermore, we have recently established that two central proteins in the meiotic DNA repair process, the recombinases RAD51 and DMC1, are differentially loaded at meiotic DSBs. This differential loading is under the control of two important DNA-damagesignaling kinases, ATM and ATR. Currently, we are concentrating on identifying the downstream targets of these kinases in higher plants.

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 Kurzbauer MT, Uanschou C, Chen D, Schlögelhofer P. The recombinases DMC1 and RAD51 are functionally and spatially separated during meiosis in Arabidopsis. Plant Cell. 2012 May;24(5):2058-70. n Edlinger B, Schlögelhofer P. Have a break: determinants of meiotic DNA double strand break (DSB) formation and processing in plants. J Exp Bot. 2011 Mar;62(5):1545-63. n Kurzbauer MT, Schlögelhofer P. Retinoblastoma protein goes green: the role of RBR in Arabidopsis meiosis. EMBO J. 2011 Feb 16;30(4):631-3.

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Peter Schlögelhofer team

Ines Dittrich Mona von Harder Manuel Hofer David Horner Michael Janisiw Maximilian Kramer-Drauberg Marie-Therese Kurzbauer Katja Schneider Clemens Uanschou

a r c h R e s e arc

r o u ps p s G ro

W olfgang S c h n e id e r

Molecular mechanisms of dyslipidemias and atherogenesis The work of my group focuses on receptor-mediated endocytotic processes.

Wolfgang Schneider team

Raimund Bauer Sabine Fürlinger Philipp Tondl Fan Zhang

In detail, we investigate molecular genetic, cell biological, and biochemical details of (i) the low density lipoprotein (LDL) receptor gene family in oocyte growth and embryo development, and of the receptor LR11 in smooth muscle cell biology, (ii) avian lipases and transfer proteins (e.g. the lipolytic proteome) of the granulosa cells surrounding the chicken oocyte as well as of the extra­embryonic yolk sac, which mediate yolk lipid deposition and subsequent utilization by the embryo, respectively, (iii) the molecular genetic basis for human atherosclerosis caused by single-gene mutations that reduce or abolish receptor-mediated transport of lipoproteins and/ or cholesterol, and (iv) the role of the recently discovered apolipoprotein, apo-AV, in the etiology of human pathological hypertriglyceridemia.

In regards to (i), we have elucidated the role of the LDLR family member LR11 in Ang-II stimulated vascular smooth muscle cell migration. A circulating soluble form of LR11, sLR11, is a novel marker of carotid IMT (intima-media thickness), and targeted disruption of the LR11 gene greatly reduces intimal thickening of arteries through attenuation of Ang-II-induced migration of SMCs. In the projects related to (ii) we have shown, for the first time in any system, that hepatic arylacetamide deacetylase (AADA), like the key lipase ATGL, is upregulated by fasting, and that its affinity for insoluble carboxylester substrates is compatible with an in vivo function similar to that of ATGL. Unknown heretofore, hepatic expression of chicken AADA is estrogen-

responsive, and is induced to the same degree as the stimulation of hepatic VLDL-production by estrogen. These observations support the hypothesis that chicken ATGL, PNPLA3, and AADA play roles in acylglycerol metabolism related to the high rates of VLDL synthesis essential for reproduction. We have characterized patatin-like phospholipases, and revealed their unique tissue distribution patterns in the laying hen.

In project (iv), we have achieved the first molecular and functional characterization of a nonmammalian ApoA-V, and have described a novel mechanism for modulation of triglyceride levels by ApoA-V proposed based on discovery that the apo binds to LRs. Finally, we have established that a new chondroitin sulfate-modified collagen forms a follicular membrane which to date has been assumed to be a bona-fide basement membrane.

VLDL particles in coated structures of oocytes. The electron micropraph shows serum-derived lipoprotein particles (VLDL) in clathrin-coated pits (c.p.) being internalized via invagination and pinching-off of coated vesicles (c.v.) in a chicken oocyte. The receptor gene family involved has been extensively characterized in my group. (Adapted from M.M. Perry and A.B. Gilbert, J. Cell Sci. 39:257, 1979.)

selected Publications Bauer R, Plieschnig JA, Finkes T, Riegler B, Hermann M, Schneider WJ. The developing chicken yolk sac acquires nutrient transport competence by an orchestrated differentiation process of itsendodermal epithelial cells. J Biol Chem.

2013 Jan 11;288(2):1088-98. n Dichlberger A, Schlager S, Lappalainen J, Käkelä R, Hattula K, Butcher SJ, Schneider WJ, Kovanen PT. Lipid body formation during maturation of human mast cells. J Lipid Res. 2011 Dec;52(12):2198-208. n Mikl C, Peters J, Trapp M, Kornmueller K, Schneider WJ, Prassl R. Softness of atherogenic lipoproteins: a comparison of very low density lipoprotein (VLDL) and low density lipoprotein (LDL) using elastic incoherent neutron scattering (EINS). J Am Chem Soc. 2011 Aug 31;133(34):13213-5.

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R e n é e S c h ro e d e r

RNA aptamers and RNA chaperones RNA is at the center of all steps of gene expression. Cells can be defined by their transcriptomes, not by their genomes. We are interested in discovering many regulatory elements that are part of the RNA regulon and in identifying their interacting partners and their targets.

To achieve this goal we adapted the classical SELEX procedure to be used in combination with genome sequences and deep sequencing. Genomic systematic evolution of ligands by exponential enrichment (SELEX) allows the isolation of p r o t e i n -b in din g RNAs independently of computational predictions and expression conditions.

and human genomes. These experiments delivered thousands of genomic RNA aptamers that regulate gene expression. We are currently analyzing the mode of action of these aptamers. Another focus in our laboratory deals with proteins that promote RNA folding: RNA chaperones.

As model examples we are analyzing the mode of action of the E. coli protein StpA and the HIV-1 Tat peptide. While StpA promotes RNA annealing and strand exchange, HIV-1 Tat only promotes

Renée Schroeder team

Lucia Aronica Ivana Bilusic Anna Grushkevich David Fima Ruchman Indacochea Meghan Lybecker Katarzyna Matylla Kulinska Maximilian Radtke Johanna Stranner Nadia Tukhtubaeva Adam Weiss Robert Zimmermann The HIV-1 Tar RNA hairpin undergoes refolding during transcription of the HIV genome. The refolding is promoted by the tat peptide.

We used genomic SELEX with an E. coli library to isolate RNA aptamers against RNA polymerase and the regulator protein Hfq. We further selected RNA polymerase-II-binding aptamers from the yeast

RNA annealing. Using biochemical and biophysical methods (NMR) we are studying the structural dynamics of both RNA and protein.

Overview of SELEX procedure. A genomic RNA library derived from DNA is submitted to several rounds of selection (b-e) and amplification (f-g,a) until a pool enriched in desired sequences is obtained. Deep sequencing of enriched pools allows the annotation of genomic aptamers to analysed genomes. selected Publications Stampfl S, Doetsch M, Beich-Frandsen M, Schroeder R. Characterization of the kinetics of RNA annealing and strand displacement activities of the E. coli DEAD-box helicase CsdA. RNA Biol. 2013 Jan 1;10(1). n Doetsch M, Stampfl S, Fürtig B, Beich-Frandsen M, Saxena K, Lybecker M, Schroeder R. Study of E. coli Hfq‘s RNA annealing acceleration and duplex destabilization activities using substrates with different GC-contents. Nucleic Acids Res. 2013 Jan 7;41(1):487-97. n Matylla-Kulinska K, Boots JL, Zimmermann B, Schroeder R. Finding aptamers and small ribozymes in unexpected places. Wiley Interdiscip Rev RNA. 2012 Jan-Feb;3(1):73-91.

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G ro r o u ps p s

J oac h im S e ip e lt

Virus host cell interactions We are interested in the molecular interplay between viruses and host cells.

Joachim Seipelt Joachim Seipelt has been on university leave since July 2010 and works at the biotech company AVIR Green Hills Biotechnology AG. AVIR is developing a novel intranasal influenza vaccine (deltaFLU).

Our virological focus is centered on human rhinoviruses that are the main causative agent of common cold. Its closest relatives are important pathogens found in humans, such as poliovirus or coxsackievirus, and in animals, e.g. foot and mouth disease virus. These small and simple viruses can very quickly subvert eukaryotic cells into virusproducing machines, killing infected cells. Cells try to mount defensive reactions against these viral intruders, but viruses have evolved complex strategies to adapt to host cells and their defensive reactions. Analysis of this interplay between host and virus can provide interesting insights into both viral and cellular functions.

Within this theme, we are working on several topics: We have in the past analyzed interactions of viruses with the cytoskeleton. We have found that a viral proteinase 2A cleaves cytokeratin 8 during virus multiplication. Another area of interest is cell-death (apoptosis) during virus infection. We have found significant differences in the induction of apoptosis when analyzing different serotypes of HRVs. These data provide further evidence on the substantial biological diversity of these viruses. Furthermore, we have identified a set of antiviral compounds that show striking properties. In collaboration with Karl Kuchler, MFPL, we have used yeast as a model system to identify underlying mechanisms.

HeLa cells (left) afer infection with human rhinovirus (right). Actin is shown in red, cytokeratin 8 in green.

selected Publications Krenn BM, Gaudernak E, Holzer B, Lanke K, Van Kuppeveld FJ, Seipelt J. Antiviral activity of the zinc ionophores pyrithione and hinokitiol against picornavirus infections. J Virol. 2009 Jan;83(1):58-64. n Wacheck V, Egorov A, Groiss F, Pfeiffer A, Fuereder T, Hoeflmayer D, Kundi M, Popow-Kraupp T, Redlberger-Fritz M, Mueller CA, Cinatl J, Michaelis M, Geiler J, Bergmann M, Romanova J, Roethl E, Morokutti A, Wolschek M, Ferko B, Seipelt J, Dick-Gudenus R, Muster T. A novel type of influenza vaccine: safety and immunogenicity of replication-deficient influenza virus created by deletion of the interferon antagonist NS1. J Infect Dis. 2010 Feb 1;201(3):354-62. n Landstetter N, Glaser W, Gregori C, Seipelt J, Kuchler K. Functional genomics of drug-induced ion homeostasis identifies a novel regulatory crosstalk of iron and zinc regulons in yeast. OMICS. 2010 Dec;14(6):651-63.

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G ro r o u ppss

C h ristian S e is e r

Chromatin modifiers in development and disease Histone modifications affect the chromatin structure and thereby the accessibility of particular genomic regions and control fundamental biological processes such as transcription, replication and DNA repair.

Our group is specifically interested in the impact of reversible histone acetylation and phosphorylation on gene expression, differentiation and development.

One project deals with the role of the class I histone deacetylases HDAC1 and HDAC2 in development and disease. HDAC inhibitors induce cell-cycle arrest, differentiation or apoptosis in tumor cells and therefore HDACs are potential targets for anti-tumor drugs. We have originally identified mouse HDAC1 as a growth factor inducible factor in cytolytic T-cells. By loss-offunction studies in the mouse we have shown that HDAC1 is crucial for embryonic development, repression of tumor suppressors and unrestricted proliferation of embryonic stem cells. Therefore, HDAC1 is a promising target for cancer therapy.

To examine the function of HDAC1 and its closest homologue HDAC2 as regulators of proliferation, differentiation and tumorigenesis we have generated conditional knockout mice to ablate HDAC1/HDAC2 in epidermis, brain and T-cells (in collaboration with Patrick Matthias, FMI Basel and Wilfried Ellmeier, Medical University of Vienna).

Deletion of HDAC1 and HDAC2 in the mouse brain leads to severe developmental problems and embryonic lethality. Hematoxylin/eosin staining of representative paraffin sections from wildtype (A) and HDAC1/HDAC2deficient (B) mice at embryonic day 18.5. Simultaneous loss of HDAC1 and HDAC2 results in reduced proliferation, increased apoptosis, DNA damage, impaired differentiation and severe cerebral hemorrhage.

In the second project, we examine the role of histone phosphorylation during transcriptional activation of mammalian genes by stress and growth factors. The presence of histone H3 phosphorylation marks at promoters correlates with the induced expression of dozens of target genes in mammalian cells. We have recently shown that 14-3-3 zeta can act as a reader protein for S10- and S28-phosphorylated histone and have identified the phosphatase PP2A as chromatinassociated transcriptional repressor which removes the active H3S10ph mark from specific target genes. We are currently analyzing in a genomewide approach RNA polymerase II recruitment and changes in specific histone modifications at the promoters of stress-induced genes.

Transient transcriptional derepression by histone H3 phosphorylation. In the absence of signals the gene is silent due to recruitment of HP1γ by local H3K9-dimethylation. Signal-dependent phosphorylation of H3S10 leads to dissociation of the HP1γ repressor, acetylation of H3K14 and binding of 14-3-3ζ. The resulting recruitment of RNA polymerase II and transcriptional activation occurs in the presence of the repressive H3K9me2 mark. Deacetylation, dephosphorylation and subsequent dissociation of 14-3-3ζ and binding of HP1γ reconstitute the repressed state of the promoter. selected Publications Sawicka A, Seiser C. Histone H3 phosphorylation - a versatile chromatin modification for different occasions. Biochimie. 2012 Nov;94(11):2193-201. n Grausenburger R*, Bilic I*, Boucheron N, Zupkovitz G, El-Housseiny L, Tschismarov R, Zhang Y, Rembold M, Gaisberger M, Hartl A, Epstein MM, Matthias P, Seiser C, Ellmeier W. Conditional deletion of histone deacetylase 1 in T cells leads to enhanced airway inflammation and increased Th2 cytokine production.

J Immunol. 2010 Sep 15;185(6):3489-97. (* equal contribution, Wilfried Ellmeier and Christian Seiser are co-senior authors) n Lagger S, Meunier D, Mikula M, Brunmeir R, Schlederer M, Artaker M, Pusch O, Egger G, Hagelkruys A, Mikulits W, Weitzer G, Muellner EW, Susani M, Kenner L, Seiser C. Crucial function of histone deacetylase 1 for differentiation of teratomas in mice and humans. EMBO J. 2010 Dec 1;29(23):3992-4007.

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Christian Seiser team

Alexandra Bedeir Astrid Hagelkruys Dominik Hartl Julia Krahmer Katharina Mattes Mirjam Moser Anna Sawicka Tina Meischel Simon Weissmann Mircea Winter

R e s e arc a r c h

G ro r o u ps p s

T obias S i e b e r e r

Signaling networks in shoot organ formation In contrast to animals, plants show an indeterminate mode of development, where organ formation mainly occurs post-embryonically and is highly adaptive to the environment.

Tobias Sieberer team

Wenwen Huang Lisa Kappel Olena Poretska Martina Rupp Nikola Winter Karin Zwerger

The organs of higher plants above ground are generated through the activity of stem cell centres, the so-called shoot meristems. To ensure correct growth, the plant must tightly balance the ratio between pluripotent stem cells and differentiating cells, which are consumed by organ formation.

This requires constant intercellular communication achieved by complex interactions of numerous signaling molecules. In addition to this short range communication in the meristem, plants have also developed sophisticated long-range signals to communicate between meristems to concert overall growth. We are interested in the molecular nature and function of these signals, which in some respects represent a communication system analogous to the neuronal network of animals.

Apart from gaining essential new insights into the molecular control of plant growth, the knowledge we acquire should be applicable to improve the production of food and renewable energy resources.

Shoot system architecture affects light harvesting potential, the synchrony of flowering and seed set, and the number of flowers and seeds per plant.

Thus, changes in architectural characteristics have been, and continue to be central breeding targets in agriculture, horticulture and forestry.

to define novel pathways and mechanisms, which control the growth rate and architecture of shoots.

Besides using modern functional genomic tools to identify and characterize genes of interest, we perform phenotypic and reporter-based screens to find small molecules affecting these processes.

Those compounds are then used to determine their molecular targets in the plant by biochemical and

Development of an interdisciplinary toolset to identify novel regulators of shoot organ formation.

genetic means.

Currently we are focusing on the following projects: 1) the AMP1 pathway, which controls stem cell pool size and leaf initiation rate. 2) the strigolactone pathway, which regulates the outgrowth of lateral branches.

We pursue an highly interdisciplinary approach Plants mutated in the AMP1 gene (B) form leaves quicker than wildtype plants (A). We identified a small molecule that can enhance the leaf initiation rate in wild-type plants (C: control plant, D: treated plant) to a similar extend as the amp1 mutation.

(E) Plants lacking the hormone strigolactone generate more shoot branches (right) than wild-type plants (left).

selected Publications Poppenberger B, Rozhon W, Khan M, Husar S, Adam G, Luschnig C, Fujioka S, Sieberer T. CESTA, a positive regulator of brassinosteroid biosynthesis. EMBO J. 2011 Mar 16;30(6):1149-61. n Crawford S, Shinohara N, Sieberer T, Williamson L, George G, Hepworth J, M端ller D, Domagalska MA, Leyser O. Strigolactones enhance competition between shoot branches by dampening auxin transport. Development. 2010 Sep 1;137(17):2905-13. n Anzola JM, Sieberer T, Ortbauer M, Butt H, Korbei B, Weinhofer I, M端llner AE, Luschnig C. Putative Arabidopsis transcriptional adaptor protein (PROPORZ1) is required to modulate histone acetylation in response to auxin. Proc Natl Acad Sci U S A. 2010 Jun 1;107(22):10308-13.

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G ro r o u ppss

T im S k e rn

How do viruses hijack cells?

Most viruses interfere with or modulate host systems to ensure successful replication. My group has been looking at proteins from footand-mouth disease virus (FMDV), human rhinovirus (HRV), tick-borne encephalitis virus, West Nile virus and vaccinia virus to learn more about the mechanisms of these interactions.

Using oligopeptides prepared by our collaborators in Brazil, we have recently examined the specificity of the leader proteinase of FMDV and could show that the enzyme can recognize a basic residue before or after the cleavage site. However, a peptide containing basic residues before and after the cleavage inhibited the enzyme. Using this information, we were able to develop a nanomolar inhibitor of the enzyme. We are using sitedirected mutagenesis to examine the basis for the unusual specificity of the enzyme. We have also used NMR to demonstrate that a self-processing reaction performed by the leader proteinase is an intramolecular reaction, facilitated by the flexibility of the C-terminus of the protein. We have also examined, both in vivo and in vitro, the substrate specificity of the HRV2 2A proteinase

Tim Skern team

N NMR signals of the wild-type leader proteinase and the C-terminal P187A mutant

15

and could show that the enzyme is not recognizing a particular amino acid at a particular position in the cleavage site but rather the overall shape of the substrate.

The vaccinia virus protein A46 is an immunomodulator, capable of interfering with MyD88-mediated signaling by interacting with specific protein components of the signaling cascade. To investigate these interactions, we have been successful in generating a modified form of the protein that can be crystalised. Diffraction patterns of the crystals resolve at about 2.6Ă… and should allow solution of the structure in 2013.

The structure will allow an understanding of the mechanism of interaction with its partners and reveal whether the protein truly has a novel fold, as indicated by protein prediction servers.

The foot-and-mouth disease leader proteinase.

selected Publications Kurz M, Stefan N, Zhu J, Skern T. NS2B/3 proteolysis at the C-prM junction of the tick-borne encephalitis virus polyprotein is highly membrane dependent. Virus Res. 2012 Sep;168(1-2):48-55. n Schrauf S, Kurz M, Taucher C, Mandl CW, Skern T. Generation and genetic stability of tick-borne encephalitis virus mutants dependent on processing by the foot-and-mouth disease virus 3C protease. J Gen Virol. 2012 Mar;93(Pt 3):504-15. n Nogueira Santos JA, Assis DM, Gouvea IE, JĂşdice WA, Izidoro MA, Juliano MA, Skern T, Juliano L. Foot and mouth disease leader protease (Lbpro): Investigation of prime side specificity allows the synthesis of a potent inhibitor. Biochimie. 2012 Mar;94(3):711-8.

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Martina Aumayr Sofiya Fedosyuk Flavia Leite David Neubauer Tomas Sara Ulrike Seifert Jutta Steinberger

a r c h R e s e arc

r o u ps p s G ro

D e a S lad e

DNA damage response

Dea Slade team

Tanja Kaufmann

DNA damage response (DDR) is a complex regulatory network that involves DNA damage sensing, signaling 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.

The dynamics of the DDR protein network is

PARPs and sirtuins regulate stress response after DNA damage.

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 and inflammatory stress, which alter NAD levels.

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 DNAdamaging 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 PARPmediated 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 NADconsuming 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.

PARPs and sirtuins modify proteins: PARPs synthesize poly(ADP-ribose) , while sirtuins remove acetyl groups.

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. n 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):1326171.

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K ristin T e ssmar - R aibl e

Lunar periodicity and inner brain photoreceptors Circadian clocks do not function in isolation, as evidenced by their plasticity to environmental factors and the interplay between clocks in central and peripheral tissues.

Ever since the dawn of life, eco-systems have been governed by periodic changes in light conditions that act as reliable cues to synchronise biotic processes. However, it still remains unclear how organisms internally orchestrate multiple clocks of different period lengths (e.g. circadian, circalunar, circannual) for the synchronization of the same process. Most, if not all organisms feed this light information into molecular clockworks that allow them to anticipate rhythmic changes in their environment and to synchronize specific biological events.

For decades, efforts to study the underlying molecular mechanisms have focused almost exclusively on land model species and their most prominent light-dark cycle: the circadian rhythm.

A key finding of this work was that in eukaryotes the underlying common core mechanism consists of negative transcriptional/translational feedback loops formed by a set of regulatory genes (‚clock‘).

the lunar clock in Platynereis? And thirdly, what are the moon light sensors on the cellular and molecular level?

Platynereis possesses multiple Opsin-based photoreceptor cells, some of which are part of the eyes, while others are located in the medial region of its forebrain. We have started to especially focus on the latter as candidate moon light receptors.

Interestingly, such inner brain photo­receptor cells also exist in the forebrain of vertebrates. In fact, we were able show that these cells belong to the most ancient cell types present in the vertebrate brain.

Yet we know nothing about their function. We have also started to solve this riddle, using the zebrafish and medakafish. We approach the function of these Opsins using biochemical analyses, zincfinger mediated mutagenesis, transgenesis, whole mount in situ hybridization, immunocytochemistry, as well as electron microscopy.

Kristin Tessmar-Raible team

Olga Antonova Enrique Arboleda Vinoth Babu Veedin Rajan Marcus Dekens Ruth Fischer Bruno Fontinha Natalia Hallay Tobias Kaiser Julia Steger Juliane Zankte

However, life evolved in the sea, and a rich body of literature describes rhythmic phenomena in marine organisms that are unparalleled on land. These exceed circadian rhythms and include tidal, lunar and semilunar rhythms. Yet, albeit important and widespread, not a single molecule has clearly been implicated in a moon-entrained clock, due to the lack of a suitable molecular model species.

We aim to fill this gap by analyzing the molecules that govern the lunar reproductive periodicity of the marine bristle worm Platynereis dumerilii. Its large sequence resources and molecular tools, along with its ancestral-type nervous system make it an ideal starting point not only to unravel the molecular principles of its circalunar and circadian clocks, but also to place both clocks in an evolutionary context. Using these worms, we have started to address the following three main questions: firstly, how are lunar and circadian clock interconnected? Secondly, what is the nature of

Lunar-controlled rhythms are widespread and of fundamental importance for marine organisms. Simplified phylogeny of metazoan groups with representatives exhibiting moon-controlled rhythms. In most mentioned cases evidence for a free-running lunar clock mechanism exist. All mentioned groups represent marine species. Lunar-controlled rhythms have also been described outside metazoans in green and brown algae (bottom).

selected Publications Tessmar-Raible K, Raible F, Arboleda E. Another place, another timer: Marine species and the rhythms of life. Bioessays. 2011 Mar;33(3):165-72. n Dray N*, Tessmar-Raible K*, Le Gouar M*, Vibert L, Christodoulou F, Schipany K, Guillou A, Zantke J, Snyman H, Béhague J, Vervoort M, Arendt D, Balavoine G. Hedgehog signaling regulates segment formation in the annelid Platynereis. Science. 2010 Jul 16;329(5989):339-42. * equal contribution. n Tessmar-Raible K, Raible F, Christodoulou F, Guy K, Rembold M, Hausen H, Arendt D. Conserved sensory-neurosecretory cell types in annelid and fish forebrain: insights into hypothalamus evolution. Cell. 2007 Jun 29;129(7):1389-400.

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alipas h a v aziri

Multi-scale understanding of biological function

Alipasha Vaziri

We aim to understand biological function on a multi-level scale: the fundamental principles of protein and biomolecular dynamics on the nanoscale, and the dynamic interactions in cellular networks giving rise to macroscopic function and behavior on the organism level.

team

Christoph Götz Robert Prevedel Ugur Sezer

The success of this endeavor will critically depend on the development of new techniques and approaches, which represents a main focus of our research.

Biological function at the quantum limit Has nature evolved to be able to take advantage of any quantum mechanical effects in biological function? We are interested in answering this question experimentally by pushing the real time mechanistic understanding of the function of ion channels and pumps as nano-scale “protein

High resolution optogenetic mapping of neuronal circuits The ability to stimulate neuronal activity in a noninvasive and cell specific fashion is necessary to study a wide range of fundamental neuroscience questions. Genetically expressible photoactivatable ion-channels such as channelrhodopsin have enabled the optical control of neural activity.

However, given the low channel conductance, the initiation of action potentials is only possible when a sufficiently large number of channels are activated at the same time, which has made single cell resolution of optogenetic activation a major challenge. Using the scanningless technique of temporal focusing for which the axial beam profile can be controlled independently of its lateral distribution, large number of channels on individual neurons can be excited simultaneously leading to strong (up to 15mV) and fast (≤1ms) depolarisations.

Multilayer three-dimensional super-resolution microscopy of cellular protein distributions Photoactivated localization microscopy (PALM) is one of the techniques that has recently emerged for the optical imaging of protein distributions in biological samples at nanometer resolution. By combing PALM with temporal focusing, we have demonstrated that protein distributions deep (~10µm) within cells with a lateral localization precision better than 50nm can be imaged. Using genetically expressed fluorescent proteins, this technique permits the study of protein-protein and other biological interactions at ~30nm resolution.

machines” working at their structural and energetic limits. We aim to answer open, fundamental questions regarding the mechanisms that lead to the high level of specificity, directedness and efficiency of the ion selectivity and transport and discover to what extent quantum coherence and its interplay with environmental noise is responsible for these observed functional properties.

selected Publications Vaziri A, Plenio M. Quantum coherence in ion channels: resonances, transport and verification. New Journal of Physics. 2010;12(085001). n Andrasfalvy BK, Zemelman BV, Tang J, Vaziri A. Two-photon single-cell optogenetic control of neuronal activity by sculpted light. Proc Natl Acad Sci U S A. 2010 Jun 29;107(26):11981-6. n Vaziri A, Tang J, Shroff H, Shank CV. Multilayer three-dimensional super resolution imaging of thick biological samples. Proc Natl Acad Sci U S A. 2008 Dec 23;105(51):20221-6.

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G ijs V e rst e e g

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

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

The post-translational modifier ubiquitin is essential for both positive and negative immune regulation (see figure). 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 regulates innate immune cytokine expression. 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 signaling, suggesting that many of them act on different target molecules.

Over the next years my lab will focus 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.

Balanced immune regulation by ubiquitin is critical for preventing insufficient immune activation or autoimmunity.

selected Publications Rajsbaum R, García-Sastre A, Versteeg GA. TRIMmunity: The Roles of the TRIM E3-Ubiquitin Ligase Family in Innate Antiviral Immunity. J Mol Biol. 2013 Dec 12. pii: S0022-2836(13)00746-8. n Versteeg GA, Rajsbaum R, SánchezAparicio 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. n Oudshoorn D, van Boheemen S, Sánchez-Aparicio MT, Rajsbaum R, García-Sastre A, Versteeg GA. HERC6 is the main E3 ligase for global ISG15 conjugation in mouse cells. PLoS One. 2012;7(1):e29870.

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Gijs Versteeg team

Stefan Benke Tamara Engelmaier Thomas Hahn Sarjoun Sheety

a r c h R e s e arc

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r o u ppss G ro

C h ristina W aldsic h

Exploring RNA folding: from structure to function RNAs regulate biology: in the past years it has become increasingly evident that RNA is the driving force in most cellular processes.

Although these RNAs are highly diverse and fulfill very different tasks, they share their strict dependence on acquiring a specific 3D architecture to be functional. The process of folding describes how RNA undergoes the transition from a disordered, unfolded state to the native, functional conformation. Our research focuses on understanding this most essential aspect of RNA function by investigating RNA structure and folding pathways. Specifically, we aim to provide novel insights into protein-facilitated RNA folding and how RNAs fold in the living cell. Little is known about how RNAs fold in vivo and how they interact with their targets, despite RNA’s importance for cell viability. Therefore, it is of fundamental importance to gain insights into the forces driving RNA folding in vivo and to establish the contribution and impact of the cellular environment, in order to understand the basic mechanism of these RNA-dependent processes.

Catalytic RNAs, in particular group II introns, are the best-suited model system to study RNA folding in the living cell, as their structure and folding pathways are well characterized in vitro and formation of the native conformation can be measured as a function of catalysis.

Therefore, we investigate the intracellular folding pathway of the Sc. ai5γ group II intron.

Importantly, Sc. ai5γ and other yeast mitochondrial introns depend on trans-acting protein factors for efficient splicing in vivo. Consequently, we are interested in exploring how these proteins shape the folding of their target RNAs. This allows us to derive principles governing in vivo RNA folding facilitated by proteins and other cellular factors.

Aside from studying catalytic RNA, we are interested in telomerase, an RNP that has received considerable attention because of its significant up-regulation in the majority of cancer cells and its role in preventing chromosomal instability and senescence as well as in inherited human disorders.

In spite of the high level of interest in the biomedical importance of telomerase, telomerase RNA and protein components have largely eluded structural characterization.

In this regard, we are interested in exploring the structure of telomerase RNA and in studying the interplay of RNA folding and RNP assembly.

Ultimately, deciphering the rules governing RNA folding will advance our understanding of the basic mechanism of RNA-dependent processes, like selfsplicing and telomere addition, and the role of RNA in disease.

The folding pathway of the Sc. ai5γ group II ribozyme. In the unfolded state only the secondary structure is formed, while in case of the intermediate state Domain 1 (blue) compacts and forms a tertiary structure thereby providing the scaffold for docking of Domains 3 (green) and 5 (red), which completes folding to the native conformation (Pyle, Fedorova and Waldsich, TiBS 2007). selected Publications Cruz JA, Blanchet MF, Boniecki M, Bujnicki JM, Chen SJ, Cao S, Das R, Ding F, Dokholyan NV, Flores SC, Huang L, Lavender CA, Lisi V, Major F, Mikolajczak K, Patel DJ, Philips A, Puton T, Santalucia J, Sijenyi F, Hermann T, Rother K, Rother M, Serganov A, Skorupski M, Soltysinski T, Sripakdeevong P, Tuszynska I, Weeks KM, Waldsich C, Wildauer M, Leontis NB, Westhof E. RNA-Puzzles: a CASP-like evaluation of RNA three-dimensional structure prediction. RNA. 2012 Apr;18(4):610-25. n Tian N, Yang Y, Sachsenmaier N, Muggenhumer D, Bi J, Waldsich C, Jantsch MF, Jin Y. A structural determinant required for RNA editing. Nucleic Acids Res. 2011 Jul;39(13):5669-81. n Liebeg A, Mayer O and Waldsich C. DEAD-box protein facilitated RNA folding in vivo. RNA Biol. 2010 Nov-Dec;7(6):803-11.

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Christina Waldsich team

Franka Debeljak Stefan Handl Verena Heisig Andreas Liebeg Nora Sachsenmaier Michael Wildauer Georgeta Zemora

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G ra h am W arr e n

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

Graham Warren team

Lars Demmel Heather Esson Chris de Graffenried Katharina Havlicek Kyojiro Ikeda Ana Lozano Brooke Morriswood Katy Schmidt Marco Sealey Marek Suplata Sevil Yavuz

For some constituents, such as chromosomes, the underlying principles and the mechanistic details are relatively clear. For others, such as membranebound organelles, and particularly the Golgi, the principles and mechanisms have been controversial.

The primary aim of our work is to understand how the cell creates another copy of the Golgi and then partitions them equally between the two daughter cells, thereby ensuring that this organelle is propagated through successive generations.

As a model system, we have been using the protozoan parasite, Trypanosoma brucei, since it has a single Golgi whose biogenesis can easily be followed using imaging, biochemical and molecular biological techniques.

which helps determine the size and composition of the ER exit sites. Interestingly, increasing the size of the Golgi has no effect on transport or cell division times, suggesting that it is just the right size for maximal parasite growth.

We have also been studying the bilobe, a novel structure implicated in Golgi biogenesis and other functions likely related to the flagellum. We have carried out a detailed analysis of its structure, using both immuno-EM and biochemical techniques. The latter have recently allowed the characterization of seven new components. We have also been able to show that the single, polo-like kinase is involved in duplicating this bilobe, as well as other structures needed to support the growth of a new flagellum.

Lastly, we have found that phosphoinositides are involved in membrane traffic, perhaps through influencing endocytic activity at the flagellar pocket.

Our immediate future goals are to characterize the newly-discovered bilobe components and to set up a permeabilized cell system to study Golgi biogenesis, so that biochemical tools can be used to identify the needed components.

Golgi duplication involves transfer of material from the old to the new, a process that can be followed using photoactivatable GFP. We have also shown that the size of the Golgi can be manipulated by changing the levels of the sec16 scaffold protein,

A schematic view of the Golgi in relation to other organelles associated with the flagellum. The kinetoplast (containing the mitochondrial DNA), in the posterior part of the cell, is near the probasal body and basal body. The latter nucleates the flagellum in the pocket. The pocket collar closes the top of the flagellar pocket and partially overlaps with the bilobe. The flagellum attachment zone ensures adherence of the flagellum to the cell body. The Golgi localizes in the zone bounded by the nucleus, the bilobe and the flagellar pocket.

Immuno-EM of an isolated flagellar preparation. The trypanosome bilobe (marked by gold particles) co-purified with the flagellum (F), basal body (black arrowhead) and probasal body (white arrowhead), and was labeled by antibodies against Tb-MORN1 (large gold particles) and TbCentrin4 (small gold particles). The two proteins exhibit distinct distributions on the structure (inset).

selected Publications Morriswood B, Havlicek K, Demmel L, Yavuz S, Sealey-Cardona M, Vidilaseris K, Anrather D, Kostan J, Djinović-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. n Ikeda KN, de Graffenried CL. Polo-like kinase is necessary for flagellum inheritance in Trypanosoma brucei. J Cell Sci. 2012 Jul 1;125(Pt 13):3173-84. n He CY, Pypaert M, Warren G. Golgi duplication in Trypanosoma brucei requires Centrin2. Science. 2005 Nov 18;310(5751):1196-8.

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G e org W e itz e r

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

Stem cells and progenitor cells are supposed to exist in niches, where they remain in an undifferentiated and quasi-dormant state until external signals stimulate commitment and differentiation to specific somatic cells, which may contribute to the repair or maintenance of homeostasis of an organ. Mimicking a stem cell niche of the heart in vitro, we succeeded in the isolation of somatic stem cells from postnatal murine hearts and were able to maintain these cells as monoclonal self-renewing cells lines expressing Oct4, Sox2 and Nanog for several years (see figure).

These cells are obviously committed to the

mesodermal lineage as they also express the early myocardial transcription factors Brachyury, Nkx2.5, GATA4, and Isl1 in the undifferentiated state. They differentiate spontaneously and exclusively 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 nearadult action potential. Stimulation of CVPCs with Activin A and Retinoic Acid did not yield any cell types of the endodermal and ectodermal lineage, respectively. The 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 the homeostasis of the heart.

Localisation of Oct4 protein during cell division of CVPCs. Immunofluorescence microscopy of CVPCs with Oct4 antibodies (green), and DAPI (blue). Bar: 15 Âľm.

Arrows, top, Metaphase; middle, Anaphase, and bottom, Telophase. Asterisks, Oct4 negative nucleus of a SNL76/7 feeder cell.

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.

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 Jan 22. n Fuchs C, Scheinast M, Pasteiner W, Lagger S, Hofner M, Hoellrigl A, Schultheis M, Weitzer G. Self-organization phenomena in embryonic stem cellderived embryoid bodies: axis formation and breaking of symmetry during cardiomyogenesis. Cells Tissues Organs.

2012;195(5):377-91. n Hofner M, HĂśllrigl A, Puz S, Stary M, Weitzer G. Desmin stimulates differentiation of cardiomyocytes and up-regulation of brachyury and nkx2.5. Differentiation. 2007 Sep;75(7):605-15.

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Georg Weitzer team

Christiane Fuchs Brigitte Gundacker Lucia Leitner Hannah Paar

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G e r h ard W ic h e

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

Gerhard Wiche team

Irmgard Fischer Rocio Garcia de la Cruz Valencia Karin Groß Eva Mihailovska Selma Osmanagic-Myers Marianne Raith Ilona Staszewska Gernot Walko Lilli Winter

In muscle cells, it forms the contractile apparatus, confers structural support and positions organelles; in neurons, it maintains the asymmetric cell shape and polarity; and in epithelial cells, it plays a pivotal role in maintaining cell and tissue integrity. We are interested in cytoskeletal linker proteins (cytolinkers), a family of multi-modular, highly versatile proteins of exceptional size, that by networking and anchoring cytoskeletal filaments regulate cytoskeleton dynamics and architecture. We are studying the role of cytolinkers in normal development, cellular stress response, and disease, combining mouse genetics with cell and structural biology.

Fig. 1: Electron microscopy of paracrystalline polymeric structures formed by lateral association of plectin’s alphahelical rod domain. Similar mechanisms are proposed to lead to compaction of hemidesmosomes.

Several years ago we discovered plectin, a ubiquitous cytolinker that became the prototype of what meanwhile is a whole family of similar proteins.

Plectin has key functions in shaping cell architecture, mechanical stabilization and polarization of cells, positioning of organelles, signal transduction and nerve conduction. Thus, loss or dysfunction of plectin leads to diseases affecting a variety of cell types and tissues.

Plectin’s versatility is based on an unusual diversity of isoforms differing in small N-terminal sequences that determine the localization of the protein. We have generated a panel of transgenic mouse lines, including full knockout (KO), single isoform and conditional/ tissue-restricted KO, and knock-in lines. Serving us

in analyzing isoform-specific functions and providing animal models for plectin-related human diseases, we use these systems focusing on: Myofibrillar myopathies.

We found myofiber integrity, including mitochondrial function, in skeletal muscle to be dependent on the proper targeting of desmin intermediate filament (IF) networks to strategic cellular sites via distinct plectin isoforms. Plectin-unanchored desmin networks collapse and form protein aggregates leading to dysfunctional myofibers. In addition, unbalanced plectin levels cause metabolic inadequacies. Other topics are plectin-related heart malfunction and failures in myofiber regeneration.

Skin disease.

Severe skin blistering (EBS) is the hallmark of most plectinopathies. The analysis of a knock-in mouse line mimicking the dominant plectin mutation of EBS-Ogna patients provides new insights into hemidesmosome(HD)-stabilizing mechanisms, revealing plectin-isoform-specific proteolysis and lateral self-association of plectin as novel mechanisms regulating HD-homeostasis (Fig. 1).

Role of plectin in neural cells.

Having identified the major neuronal plectin-isoform as a microtubule destabilizer, we are establishing its role in synaptic transmission, nerve conduction, axonal transport and metabolism.

Plectin‘s role in mechanotransduction and stress response.

We found plectin scaffolds to antagonize various stress-related perturbations of cell cytoarchitecture. Present studies are focused on plectin’s role in mechanotransduction in endothelial and fibroblast cells (Fig. 2).

Fig. 2: Cytoarchitecture of a fibroblast cell visualized by multiplecolor immunofluorescence microscopy.

selected Publications Winter L, Wiche G. The many faces of plectin and plectinopathies: pathology and mechanisms. Acta Neuropathol.

2013 Jan;125(1):77-93. n Teperino R et al. Hedgehog partial agonism drives Warburg-like metabolism in muscle and brown fat. Cell. 2012 Oct 12;151(2):414-26. n Walko G, Vukasinovic N, Gross K, Fischer I, Sibitz S, Fuchs P, Reipert S, Jungwirth U, Berger W, Salzer U, Carugo O, Castañón MJ, Wiche G. Targeted proteolysis of plectin isoform 1a accounts for hemidesmosome dysfunction in mice mimicking the dominant skin blistering disease EBS-Ogna. PLoS Genet.

2011 Dec;7(12):e1002396.

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A ng e la W itt e

fCh1, a model for gene regulation in haloalkaliphilic archaea The virus fCh1 was found by spontaneous lysis of a culture of the haloalkaliphilic, archaeon, Natrialba magadii, an isolate from the soda lake, Lake Magadii in Kenya.

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 two extremes: a high pH and high concentrations of salt. The sequence of fCh1, infectin­g the haloalkaliphilic archaeon Natrialba magadii, contains an open reading frame (int1) in the central part of its genome that belongs to the l integrase family of site-specific recombinases. Sequence similarities to known integrases include the highly conserved tetrad R-H-R-Y. The flanking sequences of int1 contain several direct repeats of 30 bp in length (IR-L and IR-R), which are orientated in an inverted direction. The invertible region encodes two structural proteins (gp34 and gp36, encoded

by ORF34 and ORF36) expected to represent the viral tail fibre proteins.

In vitro experiments using purified protein variants gp341 and gp3452 (containing the C-terminus of gp36) revealed exclusive binding of gp3452 but not of gp341 to cells of the cured strain Nab. magadii L13. This specific binding was inhibited by the addition of a-D-galactose. a-D-galac­tose also significantly reduced the infectivity of fCh1.

Binding experiments employing distinct domains of gp341 and gp3452 indicated the C-terminus to be responsible for binding to the receptor on the cell surface of Nab. magadii L13. This C-terminus contains a domain with similarities to the superfamily of galactose-like binding proteins. In summary, the experiments gave evidence that gp3452 represents the anti-receptor of fCh1 that binds to specific carbohydrate ligands located on the cell surface of Nab. magadii.

Angela Witte team

Bea Alte Judith Beraha Katharina Dimmel Daniel Kiesenhofer Michael Reiter Tatjana Svoboda Petra Till

Electron micrograph of fCh1 particle negatively stained with uranylacetate.

Currently our work concentrates on the identification and function of repressor and activator molecules encoded by the virus, gene regulation due to a recombination event, identification of the receptor for the virus on the cell surface of Nab. magadii and the transformation/shuttle vector system developed by the group. In addition the method is used to construct different mutants.

selected Publications Klein R, Rössler N, Iro M, Scholz H, Witte A. Haloarchaeal myovirus fCh1 harbours a phase variation system for the production of protein variants with distinct cell surface adhesion specificities. Mol Microbiol. 2012 Jan;83(1):137-50. n Iro M, Klein R, Gálos B, Baranyi U, Rössler N, Witte A. The lysogenic region of virus fCh1: identification of a repressor-operator system and determination of its activity in halophilic Archaea. Extremophiles. 2007 Mar;11(2):383-96. n Klein R, Baranyi U, Rössler N, Greineder B, Scholz H, Witte A. Natrialba magadii virus fCh1: first complete nucleotide sequence and functional organization of a virus infecting a haloalkaliphilic archaeon. Mol Microbiol. 2002 Aug;45(3):851-63.

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F ranz W o h lrab

Function of zona pellucida domain proteins

Franz Wohlrab

The first specific interaction between sperm and egg occurs at an extra足 cellular matrix (ECM) called the zona pellucida (zp). The zp does not only provide specific receptors for incoming sperm, but has other important functions.

It can induce the acrosome reaction on the sperm head, and by reacting to the release of cortical granules from the oocyte, provides the major block to polyspermy. It also serves as a protection for the fertilized egg and early mammalian embryo during its travel to the point of implantation. The zp then has to break open in a tightly controlled and timed program to allow the embryo to hatch and contact the maternal endometrium.

Studies in mice have shown that the zp consists of only 3 glycoproteins which all share a 260 amino acid domain called the zp domain. These proteins are coordinately expressed by the oocyte at the transition from the primordial to the primary stage. In other animals, however, they are often expressed by somatic tissues. In birds, we have shown that the two main components of the zp, ZP1 and ZPC, are made by the liver and granulosa cells, respectively. Similar extraoocytic expression of zp proteins is now established in many animals, including mammals. In these cases, the proteins have to travel to their final destination and then polymerize to form the growing zp. A major aim of this project is to investigate how these factors are targeted to the site of zp assembly. We have shown that purified native ZPC will selfassemble to larger structures if its concentration is sufficiently high, and focus our attention on optimal conditions for assembly of the zp in vitro. Special interest is being paid to the role of follicle-derived factors like GDF9, BMP15, activin, perlecan, etc. in this process.

In addition, the number of ovarian zp proteins besides the three canonical zp components is steadily increasing. Thus, in birds, we have up to now identified 8 follicular zp proteins. The function of these factors is entirely unknown. Another aim of this project is to delineate the roles these proteins play during the lifecycle of the ovary and in different organs. We have already shown that one of the liver-derived zp proteins, ZPAY, is not targeted to the ovary, but is associated in the brain and the kidney with cells lining tubular structures, such as cerebral smooth muscle cells, and the proximal tubulus, respectively.

A second project centers around the functions of the transcription factor YB-1.

Avian liver tubuli are lined with the zona pellucida protein ZPAY.

selected Publications Valiahdi SM, Jakupec MA, Daraei S, Wohlrab F, Keppler BK. Nuclear Translocation of YB-1 Protein Induced by Ultraviolet Light and the Investigational Gallium Drug GaQ3 in Melanoma Cells. Breakthroughs in Melanoma Research.

2011 Nov;481-494. n Stewart SG, Bausek N, Wohlrab F, Schneider WJ, Janet Horrocks A, Wishart GJ. Species specificity in avian sperm:perivitelline interaction. Comp Biochem Physiol A Mol Integr Physiol. 2004 Apr;137(4):657-63.

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I v an Y u d u s h kin

Functional imaging of signaling networks Adaptive character of responses to signals from the environment is the fundamental property of all living organisms. At the cellular level, it is brought about by the highly coordinated processes of signal transduction, orchestrated, in turn, by signaling enzymes.

How exactly the cells regulate and coordinate enzymatic activities to ensure specificity and the adaptive character of cellular signaling is not well understood. In our lab, we are using a combination of live cell microscopy and biochemistry to directly observe and manipulate the activity of several signaling enzymes in order to understand how they determine the kinetics and specificity of intracellular signaling.

Using FRET-based reporters for live cell imaging of the T cell receptor phosphorylation, we have previously demonstrated the existence of the dynamically regulated, spatially segregated pools of this important receptor in T cells. While the surface-bound receptor is needed to recognize antigen molecules on infected cells, the function of its intracellular pool was unclear. We have recently demonstrated that the intracellular pool

of tyrosine-phosphorylated T cell receptor recruited and activated the downstream kinase ZAP-70.

Furthermore, we were able to show that ZAP-70 trafficking through the endosomal recycling system was sufficient to induce expression of the early T cell activation marker CD69. Our data provide the first evidence that intracellular localization or trafficking of the T cell receptor complex may contribute to signaling specificity and suggest that internalized TcR/CD3 could serve as a long-term signaling compartment in activated T cells.

We are also investigating the mechanisms that regulate the activity and substrate specificity of protein kinases of the Akt and Src families. In collaboration with Thomas Leonard’s group, we are examining biochemical and cellular mechanisms that ensure the fidelity and efficiency of their activation and how channeling of substrates between these enzymes could help define signaling specificity. Our initial data indicate that we can specifically manipulate the timing and the extent of Akt activation in live cells. We are developing new methods to visualize the functional state of signaling enzymes in order to determine how their spatial distribution, regulation of specific activity and the corresponding changes in the flux of substrates via help determine their signaling function.

(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 FRETbased (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.

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. n 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.

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Ivan Yudushkin team

Michael Ebner Freia von RauĂ&#x;endorf Yue Xin

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B ojan Z agro v ic

Computational biophysics of macromolecules The function of biomolecules arises from the interplay between their structure, dynamics and interactions with the environment.

Bojan Zagrovic

We explore this principle through the use of computational and theoretical methods, such as molecular dynamics (MD) simulations, in close collaboration with experimentalists.

Second, all biomolecular processes occur in crowded, dynamic, constantly changing environments. We study how this affects protein-protein interactions and other basic processes such as folding or posttranslational modifications of proteins. In particular, we are interested in studying how binding partners find each other in the crowded cell and employ MD and Brownian dynamics simulations and structural bioinformatics methods to address this question.

team

Markus Fleck Matea Hajnic Mario Hlevnjak Antonija Kuzmanic Bianca Mladek Drazen Petrov Anton A. Polyansky Anita de Ruiter

Linear correlations decrease the overall quasi-harmonic entropy change (ΔSQH) in protein-protein interactions by a remarkably constant amount as revealed by MD simulations of complexes of ubiquitin (UBQ) with its binding partners such as UBM2 (PDB code: 2KTF) (Polyansky AA et al. Journal of Chemical Theory and Computation, 8, 2012).

Specifically, we are interested in the role of dynamics and conformational entropy in non-covalent protein interactions. Frequently, a change in conformational entropy of binding partners may be enough to alter their functional state without any associated rearrangement of their average structures. 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. Namely, biomolecular structures are typically static models derived from X-ray or NMR experiments performed on approximately 1020 dynamic copies of the same molecule. We use MD simulations to help interpret such time- and ensemble-averaged experiments and analyze the impact of conformational averaging on the derived structures.

Finally, we have recently discovered a remarkably robust correspondence between the pyrimidine content of mRNAs and the propensity of cognate protein sequences to bind pyrimidine mimetics.

We believe this provides strong support for the stereo-chemical hypothesis concerning the origin of the genetic code i.e. the idea that the genetic code evolved as a consequence of direct binding interactions between amino acids and the bases that code for them. What is more, our results give support to a novel hypothesis that cognate mRNAs and proteins may be physico-chemically complementary to each other and bind, especially if unstructured. We use different methods of computational biophysics including MD simulations, structural bioinformatics techniques and free energy methods to further explore this hypothesis.

NMR structure of protein S100-A1 (PDB code: 2L0P) colored according to the window-averaged pyrimidine content of its mRNA (left) or window-averaged affinity of its amino acids for a pyrimidine mimetic dimethylpyridine (protein polar requirement, right), together with superimposed sequence profiles of the two variables (Hlevnjak M et al. Nucleic Acids Research, 40, 2012). selected Publications Hlevnjak M, Polyansky AA, Zagrovic B. Sequence signatures of direct complementarity between mRNAs and cognate proteins on multiple levels. Nucleic Acids Res. 2012 Oct;40(18):8874-82. n Polyansky AA, Zubac R, Zagrovic B. Estimation of conformational entropy in protein-ligand interactions: a computational perspective. Methods Mol Biol.

2012;819:327-53. n Petrov D, Zagrovic B. Microscopic analysis of protein oxidative damage: effect of carbonylation on structure, dynamics and aggregability of villin headpiece. J Am Chem Soc. 2011 May 11;133(18):7016-24.

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www.mfpl.ac.at

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