abstracts | talks EMBO Fellows' Meeting, 13 - 16 June 2013, Heidelberg

A genomic binding atlas for MBD proteins reveals methylation- dependent and -independent targeting principles

Tuncay Baubeca,*, Robert Ivanekb, Florian Lienertc, Dirk Schübelera a Friedrich Miescher Institute b Department of Biomedicine, University of Basel, Mattenstrasse 28, 4058 Basel, Switzerland c Department of Systems Biology, Harvard Medical School, 200 Longwood Ave., Boston, MA 02115, USA

Recent advances in sequencing have identified the genomic distribution of DNA methylation in unprecedented resolution while our knowledge about the binding of trans- acting factors still lacks behind. We have established a genome-wide comparative mapping approach that enables quantification of protein structure contribution to genomic localization of DNA binding factors. This we utilized to generate a comprehensive binding atlas for the entire methyl-CpG binding domain (MBD) family of proteins, including disease-relevant mutants, domain deletions and tissue-specific isoforms, revealing modular binding preferences determined by structural composition. By comparing wild type with mutant MBD proteins we demonstrate that the recognition of methyl-CpGs by functional MBD domains is necessary and sufficient for targeting MBD proteins to methylated regions in vivo. Quantitative assessment and modeling of DNA methylation-dependent targeting reveals that binding occurs as a linear function of local methylation density regardless of cell type, sequence composition or histone modifications. By systematic analysis of domain contribution to genomic binding, we further identified specific preferences that are determined by domains required for interactions with DNA or protein complexes. Notably, these recruit a subset of MBD proteins to tissue-specific regulatory regions in a DNA methylation-independent manner. In summary, this quantitative framework exposes a complex binding behaviour determined by structural composition and revises current models of DNA methylation readout through MBD proteins. EMBO Fellows' Meeting, 13 - 16 June 2013, Heidelberg

Spore Photoproduct Lyase, a radically different DNA repair enzyme for bacterial spore extreme UV-resistance

Alhosna Benjdiaa,*, Korbinian Heilb, Thomas Barendsa, Thomas Carellb, Ilme Schlichtinga a Department of Biomolecular Mechanisms, Max-Planck Institute for Medical Research, Jahnstrasse 29, 69120 Heidelberg, Germany b Department of Chemistry, Center for Integrated Protein Science (CiPSM), Ludwig-Maximilians University, Butenandtstrasse 5-13, 81377 Munich, Germany

Bacterial spores possess an enormous resistance to ultraviolet (UV) radiation. This is largely due to a unique DNA repair enzyme, Spore Photoproduct Lyase (SP lyase). Unlike DNA photolyases which use light energy and a fully reduced flavin cofactor to repair UV-induced DNA damages, SP lyase employs an alternative and light-independent direct reversal mechanism to repair photolesions. SP lyase belongs to the emerging superfamily of radical S-adenosyl-L-methionine (SAM) enzymes and involves a reduced [4Fe-4S] cluster and SAM to initiate the repair reaction. We solved the first crystal structure of this enigmatic enzyme in complex with its [4Fe-4S] cluster and its SAM cofactor, in the absence and presence of the DNA lesion, the spore photoproduct (1). The high resolution structures provide fundamental insights into the DNA binding mode and the enzyme active site. The DNA lesion recognition involves a β-hairpin structure and its binding is mostly driven by interactions with the bases. In the active site, we show that SAM and a conserved cysteine residue are perfectly positioned for hydrogen atom abstraction from the 5'-ring of the lesion and donation to the 3'-ring of the spore photoproduct, respectively. Based on our structural and biochemical characterizations, we substantiate the role of this cysteine as a hydrogen atom donor. In addition, we identify another critical residue, tyrosine 98, which is likely involved in the regeneration of the enzyme cofactor for the next repair cycle. Our structure reveals how SP lyase, in a unique fashion, combines specific features of radical SAM and DNA repair enzymes to enable a complex radical-based repair reaction to take place (2).

(1) Benjdia* et al., Nucleic Acids Res. 2012 ;40(18):9308-18. (2) Benjdia*, Curr Opin Struct Biol. 2012 ;22(6):711-20. [email protected] EMBO Fellows' Meeting, 13 - 16 June 2013, Heidelberg

Chemical approaches to Biology and Medicine

Gonçalo Bernardesa,*, Dario Nerib a Department of Chemistry, University of Cambridge, Lensfield Road, Cambridge CB2 1EW, United Kingdom Institute of Molecular Medicine, Faculty of Medicine, University of Lisbon, Av. Prof. Egas Moniz, 1649-028 Lisbon, Portugal. b Department of Chemistry, University of Cambridge, Lensfield Road, Cambridge CB2 1EW, United Kingdom

This abstract features reaction engineering for chemical site-selective protein modification, the construction of homogeneous, vascular targeting antibody-drug conjugates (ADCs) for cancer therapy and the development of novel carbon monoxide (CO) releasing molecules (CORMs) for the delivery of therapeutic CO in vivo.

Reaction engineering at cysteine: From disulfides to thioethers, a range of novel reactions and methods that enable controlled, specific modification of proteins at cysteine were developed.[1,2]

Traceless, homogeneous ADCs for cancer therapy: Using a vascular targeting approach, it was demonstrated that a chemically defined, non-internalizing antibody-drug conjugates (ADCs) may be used to mediate strong anti-tumor activity in vivo.[3,4]

Delivery of therapeutic CO in vivo: CO-releasing molecules (CO-RMs) have the potential to constitute safe treatments if CO release in vivo can be controlled in a spatial and temporal manner.[5] First, we studied the interaction of known CORM-3 with proteins. This results in the loss of a chloride ion, glycinate, and one CO ligand.[6] These findings drove the development of a novel CORM with improved solubility and stability that distributes preferentially to the liver and fully protects mice against severe forms of malaria.[7]

[1] G. J. L. Bernardes et al., J. Am. Chem. Soc. 2008, 130, 5052-5053. [2] J. M. Chalker, G. J. L. Bernardes, B. G. Davis, Acc. Chem. Res. 2011, 44, 730-741. [3] G. J. L. Bernardes et al. Angew. Chem. Int. Ed. 2012, 51, 941-944. [4] M. Steiner, G. J. L. Bernardes* et al., Chem. Sci. 2013, 4, 297-302. [5] C. C. Romão, W. A. Blättler, J. D. Seixas, G. J. L. Bernardes*, Chem. Soc. Rev. 2012, 41, 3571-3583. [6] T. Santos-Silva, A., G. J. L. Bernardes* et al., J. Am. Chem. Soc. 2011, 133, 1192-1195. [7] A. C. Pena, G. J. L. Bernardes* et al., Antimicrob. Agents Chemother. 2012, 56, 1281-1290. EMBO Fellows' Meeting, 13 - 16 June 2013, Heidelberg

Non-invasive high speed imaging of fast physiological processes in awake and freely moving mice

Oliver Brunsa,*, Thomas Bischofa, Daniel Harrisa, Moungi Bawendia

MIT - Department of Chemistry

Non-invasive imaging of physiological processes like heart rate, breathing motion, blood perfusion and dynamic behavior of the gastro intestinal tract is of great interest forpreclinical research related to cardiovascular disease, hypertension or behavioral sciences.

So far the imaging techniques available to study this processes in high resolution all require animals to be fixed in one position. Therefore anesthesia is used if live animals are studied. This has great limitations in terms of experiment time. More importantly, the anesthesia massively disturbs the physiological processes desired to study.

SWIR or NIR-II imaging (between 900 nm and 1700 nm) had been shown to be the ideal modality for non-invasive imaging. Due to the suboptimal emission properties of the materials used previously (e.g. single walled carbon nanotubes (SWCNTs)) fast imaging rates of 25 to 50 frames per second could not be archived.

Here we use InAs/CdZnSe core-shell quantum dots, which emit in the SWIR region with a quantum yield of 30% after functionalization in physiologic solution. This emission intensity is two orders of magnitude higher than that of SWCNTs used previously for non-invasive SWIR imaging. With this quantum dots we have enough emission signal to move away from anaesthetized mice to study physiological processes.

Now we can image physiological parameters like heart rate, breathing rate and perfusion as well as energy metabolism in conscious and freely behaving mice. EMBO Fellows' Meeting, 13 - 16 June 2013, Heidelberg

The Geminin and Idas coiled coils form a preferentially heterodimeric structure which affects Geminin function in DNA replication licensing

Christophe Caillata,*, Eleftheria-Dafni Pefanib, Peter J. Gillespiec, Zoi Lygeroub, Anastassis Perrakisd, et al. a Unit for Virus Host-Cell Interactions (UVHCI), 38042 Grenoble, France b Laboratory of Biology, School of Medicine, University of Patras, 26505 Rio, Patras, Greece c Centre for Gene Regulation and Expression, College of Life Sciences, University of Dundee, Dundee DD1 5EH, United Kingdom d Division of Biochemistry, The Netherlands Cancer Institute, 1066 CX, Amsterdam, The Netherlands

In metazoans Geminin is an important regulator of proliferation and differentiation. Geminin prevents genome over-replication by inhibiting the DNA replication licensing factor Cdt1, but the mechanisms coordinating cell proliferation to differentiation processes remain unclear. Two proteins containing a coiled-coil domain similar to Geminin : Idas (or Multicillin) and GEMC1 were recently identified. Idas and GEMC1 being also involved in cell proliferation and/or cell differentiation we investigated their possible cooperation with Geminin. We show that Geminin preferentially forms stable coiled-coil mediated heterodimers with its homologue, Idas. In contrast to Idas:Geminin heterodimers, the Idas homodimers are thermodynamically unstable and Idas is unlikely to exist as a stable species on its own under physiological conditions. The crystal structure of a complex of the homology regions of Idas and Geminin forms a tight head-to-head heterodimeric coiled-coil that explains well the properties of the complex. This Idas:Geminin heterodimer binds Cdt1 less strongly than Geminin:Geminin, still with high affinity, and with notably different thermodynamic properties. Consistently, in Xenopus egg extracts, Idas:Geminin is less active in licensing inhibition compared to a Geminin:Geminin homodimer. In human cultured cells, ectopic expression of Idas leads to limited over-replication, which is counter-acted by Geminin co-expression. The properties of the Idas:Geminin interaction suggest it as a possible mechanism to modulate Geminin activity in cells. EMBO Fellows' Meeting, 13 - 16 June 2013, Heidelberg

Rapid reactivation of X-linked genes upon reprogramming of human somatic cells by cell fusion

Irene Cantonea,*, Hakan Bagcia, Matthias Merkenschlagera, Neil Brockdorffb, Amanda G Fishera a MRC Clinical Sciences Centre, Imperial College of London, London (UK) b Department of Biochemistry, University of Oxford, Oxford (UK)

X chromosome inactivation is a dosage compensation mechanism by which female somatic cells ensure gene expression from only one of the two X chromosomes in mammals. Repression of genes on the inactive X chromosome (Xi) is a major example of stable silencing that is maintained for lifetime. Reactivation of the Xi is associated with pluripotency and has been widely used as a model to study the erasure of epigenetic memory in mouse cells. In human, the status of the two X chromosomes in embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs) show considerable epigenetic variation depending on culture conditions, thus preventing the analysis of the molecular mechanisms that lead to X reactivation in pluripotency and reprogramming. Here, we used an inter-species cell fusion system whereby diploid female human fibroblasts (hF) are reprogrammed via fusion with mouse ESCs and showed that chromatin structural changes and biallelic expression of X-linked genes on the Xi are detected in the human nucleus of heterokaryons early during reprogramming. Delocalization of XIST and loss of H3K27me3 enrichment from the Xi are the first events that occur two days after fusion, concomitantly with de novo expression of pluripotency genes. Sequential RNA/DNA-FISH and SNP-expression analysis show that these changes on the Xi are followed by promoter DNA demethylation and reactivation of at least some X-linked genes. Biallelic expression of X-linked genes occurs in a subset of cells in which XIST has been delocalized, thus suggesting that the reorganization of XIST/H3K27me3 chromatin domain is not sufficient, although necessary, for gene reactivation. Our findings suggest a step-wise reactivation process that may entail only specific heterochromatic regions of the Xi. Future studies will therefore investigate the reactivation of genes along the Xi by analyzing the expression of polymorphisms, during reprogramming of clonal fibroblast populations, via allele-specific RNA sequencing approaches. EMBO Fellows' Meeting, 13 - 16 June 2013, Heidelberg

Real time dynamics of RNA Polymerase II clustering in live human cells

Ibrahim Cissea,*, Ignacio Izeddinb, Sebastien Causseb, Lydia Boudareneb, Adrien Senecalb, et al. a Howard Hughes Medical Institute, Chevy Chase, MD, US Ecole Normale Superieure, 46 Rue d'Ulm, 75005 Paris, France b Ecole Normale Superieure, 46 Rue d'Ulm, 75005 Paris, France

Transcription has been reported to be spatially compartmentalized in transcription factories hypothesized as clusters of RNA Polymerase II (Pol II). However, little is known of when these foci assemble, and how stable they are. We report a single-cell quantitative method to characterize protein clustering both spatially and temporally, with single-molecule sensitivity in live eukaryotic cells. We obtained super-resolution images consistent with clustered Pol II in the nucleus. Yet, Pol II clusters formed very transiently, with an average lifetime of 5.1 (± 0.4) s, refuting their notion as statically assembled nuclear substructures. Stimuli affecting the cells transcriptional state yielded orders of magnitude changes in the distribution and dynamics of Pol II clusters, implying that clustering is regulated and plays a role in the cells ability to effect rapid response to external signals. Our observations suggest transient crowding of enzymes may aid in rate-limiting steps of multi- component macromolecular assembly in vivo. EMBO Fellows' Meeting, 13 - 16 June 2013, Heidelberg

Cell type-specific Gene Regulatory Network of the Polycomb Repressive Complex 2 (PRC2) in Arabidopsis

Miguel De Lucasa,*, li pua, François Roudierb, Siobhan bradya a Department of Plant Biology and Genome Center, University of California - Davis, Davis, USA. b Institut de Biologie de l'Ecole Normale Supérieure, Centre National de la Recherche Scientifique (CNRS), Paris, France

Chromatin is a dynamic structure that responds to numerous stimuli to regulate DNA accessibility. The covalent modification of histones is the principal process used by the cell to modulate nucleosome mobility and turnover. The Polycomb Repressive Complex 2 (PRC2) catalyzes the trimethylation of Histone 3 at lysine 27 (H3K27me3), which can act as a repressive chromatin mark. The plethora of key developmental processes regulated by PRC2 proteins has raised interest from a wide range of fields.

PRC2 composition is conserved from humans to plants, but their spatiotemporal regulation and function has not been comprehensively characterized. Arabidopsis thaliana has 12 homologs of Drosophila PRC2 subunits. The three PRC2 complexes described in Arabidopsis control the expression of key genes involved in developmental transitions. It is also known that PRC2 complexes are required to maintain organ identity and to keep cells appropriately differentiated. However, their role in these processes remains unknown.

Our goal is to describe the role of PRC2 in controlling cell proliferation and differentiation in the Arabidopsis root.

We demonstrate that PRC2 subunits are differentially expressed among the different root cell types, being highly present in the dividing/pluripotent cells of the meristem and the cambium. To better understand PRC2 spatiotemporal regulation and function, we have identified a transcriptional regulatory network that controls PRC2 expression in the root vasculature. We have also generated vascular-specific profiling of the H3K27me3 and H3K4me3 marks and correlated it with vascular- specific gene expression. This multi-layered transcriptional regulatory network (TF-PRC2genes- H3k27me3) will allow us to understand how epigenetic modifications are dynamically regulated in space and time to control cell type and tissue specification, differentiation and function. EMBO Fellows' Meeting, 13 - 16 June 2013, Heidelberg

Identification of biological variation between single-cells using RNA-Seq

Idan Efronia,*, Ip Pui-Lenga, Nawy Tala, Mello Alisona, Birnbaum Kennetha

New York University, Center for Genomics and Systems Biology, 12 Waverly Place, 10003, USA

Global profiling of single cells has the potential to reveal coordinated transcriptional regulation at the cellular level that is otherwise obscured by sampling of heterogeneous cell populations. However, single-cell experiments are characterized by high levels of technical noise, and traditional methods for identification of differentially expressed genes are inapplicable to single-cell assays, as they rely on biological replicates. We developed an approach to identify biological variation that exceeds technical noise in single-cell experiments. Technical noise distribution is determined empirically from pooled-and-split cell samples, and the resulting probability function is used to derive confidence measures for biological cell-to-cell variation. We applied this method to cells from two tissues of the Arabidopsis root: the highly homogenous Quiescent Center (QC), which supports the root stem cell niche, and from the meristematic stele, which includes vascular cells at early stages of differentiation. Cell-type specific markers, including transcription factors with known functions, were among the most highly expressed genes, and the identity of single cells could be robustly determined. Using our statistical approach, we have detected hundreds of genes that vary significantly among cells of the same cell type. Notably, we detected multiple coherent gene modules that varied together, such as ribosomal proteins and auxin biosynthesis genes in the QC, and validated this coregulation with single root data. Thus, our method allows the detection of biological variation among single-cell profiles and shows promise in the characterization of novel cellular states. EMBO Fellows' Meeting, 13 - 16 June 2013, Heidelberg

Thymic epithelial cell expansion through matricellular protein CYR61 boosts progenitor homing and T-cell output

Yalin Emrea,*, Magali Irlaa, Isabelle Dunand-Sauthiera, Romain Balleta, Christian Vesinb, et al. a University of Geneva, Pathology and Immunology, Switzerland b University of Geneva, Physiology and Metabolism, Switzerland

Thymic epithelial cells (TEC) are phenotypically and functionally heterogeneous stromal cells that generate microenvironments required for the formation of T-cells within the thymus. TEC control the migration, proliferation and selection of differentiating thymocytes, and defects in TEC impair the normal development of T-cells leading to immunodeficiency or autoimmunity. Here we show that treatment with CYR61 (cysteine-rich protein 61), a matricellular protein implicated in cell proliferation and migration, improves thymus function and T-cell development. We identify TEC as the major source of CYR61 and show that binding of CYR61 to LFA1, ICAM1 and integrin α6 supports the adhesion of TEC and thymocytes as well as their interaction. Treatment of thymic lobes with recombinant CYR61 expands the stromal compartment by inducing thymocyte-independent proliferation of TEC via activation of the Akt signaling pathway. In addition, engraftment of CYR61 overexpressing thymic lobes into athymic nude mice drastically boosts the yield of thymic output via expansion of TEC. This increases the space for the recruitment of circulating hematopoietic progenitors and the development of T-cells. Our discovery paves the way for therapeutic interventions designed to restore thymus stroma and T-cell generation. EMBO Fellows' Meeting, 13 - 16 June 2013, Heidelberg

Role of par-1 serine/threonin kinase in synaptic transmission

Clara Essmanna,*, Emma Hileya, Stephen Nurrisha

MRC LMCB University College London

During my EMBO long-term fellow ship I worked with Stephen Nurrish at the MRC Laboratory for Molecular and Cellular Biology in London to study regulation of synaptic function using C. elegans. I have been able to demonstrate a novel function of the polarity gene PAR-1 in control of activity at the neuromuscular junction. Splicing produces many different PAR-1 isoforms. I identified a mutation (nz90) as being defective in the expression of a new spliceform, PAR-1L. PAR-1 belongs to the family of par-proteins (“partitioning defective” par-1-6) that are highly conserved key players to establish cell polarity during the first cell division. Moreover, par-proteins and their orthologues have been shown to mediate and maintain cell polarity in polarized cells such as neurons, where they are thought to be involved in the development of Alzheimers disease. However, until now, a role for PAR-1 in C. elegans neuronal function has not been demonstrated. Contraction of C. elegans muscles is caused by release of acetylcholine (ACh) by cholinergic motorneurons, relaxation is caused by release of GABA from GABA-ergic motorneurons. par-1(nz90) has a defect in Ach-release as demonstrated by both resistance to acetylcholinesterase inhibitors, and by electrophysiological recordings of the muscle. However, rescue experiments demonstrate that PAR-1 acts in the GABA-neurons. Rescue does not require PAR-1 kinase activity suggesting PAR-1 acts as a scaffold in neuronal signalling, again a novel property for the PAR-1 protein. Experiments with synaptic markers suggests a small but significant increase in pre-synaptic release sites in the GABA-neurons of par-1(nz90) mutants. Given the effect on Ach-release by par-1(nz90) we speculate that the GABA-neurons are forming extra synapses onto the cholinergic motorneurons and cause their inhibition. In support of our model par-1(nz90) inhibition of Ach-release requires GABA-synthesis. Currently we are identifying the GABA-receptors by which the par-1(nz90) mutation acts to inhibit ACh release. EMBO Fellows' Meeting, 13 - 16 June 2013, Heidelberg

A two-step mechanism for epigenetic specification of centromere identity and function

Daniele Fachinettia,*, Diego Folcoa, Arshad Desaia, Lars Jansenb, Don Clevelanda a Ludwig Institute for Cancer Research and Department of Cellular and Molecular Medicine, University of California at San Diego, La Jolla, CA 92093, USA b Instituto Gulbenkian de Ciência, Rua da Quinta Grande 6, P-2780-156, Oeiras, Portugal

The basic determinant of chromosome inheritance, the centromere, is specified in many eukaryotes by an epigenetic mark. Using gene targeting in human cells and fission yeast, chromatin containing the centromere-specific histone H3 variant CENP-A is demonstrated to be the epigenetic mark that acts through a two-step mechanism to identify, maintain and propagate centromere function indefinitely. Initially, centromere position is replicated and maintained by chromatin assembled with the centromere-targeting domain (CATD) of CENP-A substituted into H3. Subsequently, nucleation of kinetochore assembly onto CATD-containing chromatin is shown to require either CENP-A’s amino- or carboxy-terminal tails for recruitment of inner kinetochore proteins, including stabilizing CENP-B binding to human centromeres or direct recruitment of CENP-C EMBO Fellows' Meeting, 13 - 16 June 2013, Heidelberg

Identification of new pathways inducing axonal degeneration

Konstantin Feinberga,*, Alessandro Dattib, David Kaplana, Freda Millera a The Hospital for Sick Children, Molecular Biology Department, Toronto, Canada b Samuel Lunenfeld Research Institute - Mount Sinai Hospital, Toronto, Canada

The cognitive and motor dysfunction that occurs in neurodegenerative disorders and neurotrauma is due to the loss of appropriate functional neural connections as a consequence of the neuronal death and/or degeneration of axons. My objectives are to identify the molecular mechanisms that underlie axonal degeneration and to discover an effective way to prevent it. To address this, I asked whether kinase inhibitors could be identified that prevent axon degeneration in cultured primary sympathetic or sensory neurons withdrawn from survival factors. Using a library of 480 kinase inhibitors, including over 110 in clinical trials or use, I identified a number of drugs that dramatically prevented axon degeneration, including inhibitors of the activities of growth factor receptors such as EGFR, VEGFR and c-MET, intracellular kinases including GSK3β and c-Abl, and kinases that regulate the cell cycle and DNA replication, such as Chk and Cdk. Three of the most potent drugs used for cancer indications and that are known to cross the blood brain barrier, Ponatinib and Bosutinib (Abl/Src inhibitors) and Foretinib (VEGFR/c-MET inhibitor) were chosen for further analysis. These drugs delayed or prevented axonal death for up to seven days when applied only to axons in compartmented chambers. Foretinib also delayed neuronal degeneration caused by chemotherapeutic treatment, and delayed Wallerian degeneration, where axons are physically detached from their cell bodies. We are now identifying using phosphoproteomics the targets of these drugs in axons, and determining whether they prevent axon degeneration in rodent models of Parkinson’s Disease and diabetic neuropathy, and the degeneration of septal cholinergic neurons that are preferentially vulnerable in Alzheimer’s disease (fimbria-fornix lesions). We believe that this work will result in the discovery of novel treatments for neurodegeneration caused by disease or trauma. EMBO Fellows' Meeting, 13 - 16 June 2013, Heidelberg

Tollip modulates EGFR signaling

Malgorzata Garstkaa,*, Ilana Berlina, Hans Janssena, Magdalena Szczygiela, Jacques Neefjesa, et al.

NKI-AVL, Amsterdam, The Netherlands

Tight control of Epidermal Growth Factor Receptor (EGFR) signaling is required for proper cell growth and development. To terminate EGFR signaling, receptor is ubiquitylated and delivered to endosomes where upon action of ESCRT proteins EGFR is incorporated into intralumenal vesicles of the Multivesicular Body.

Tollip depletion results in increased EGFR surface levels and delays EGFR degradation. Tollip depletion also causes significant morphological changes of the endocytic pathway - abnormally enlarged MVB compartment characterized by a decreased amount of intralumenal vesicles compared to the control.

Tollip localizes to cytosol and throughout the endocytic pathway – from the plasma membrane to lysosomes. Tollip binds to Tom1L1, and two of ESCRT components - Eps15 and Hrs, and participates in the delivery of ubiquitylated EGFR from the plasma membrane to the endosomes. Membrane binding, ubiquitin binding and dimerization of Tollip are required for its function.

Tollip controls the vesiculation at the Multivesicular Body and is involved in ESCRT-dependent EGFR delivery to lysosomes. EMBO Fellows' Meeting, 13 - 16 June 2013, Heidelberg

SwissSidechain: a molecular and structural database of non-natural sidechains

David Gfellera,*, Olivier Michielina, Vincent Zoetea

Molecular Modeling, Swiss Institute of Bioinformatics, Lausanne, Switzerland

Amino acids form the building blocks of all proteins. Naturally occurring amino acids are restricted to a few tens of sidechains. Yet, the potential chemical diversity of amino acid sidechains is nearly infinite. Exploiting this diversity using non-natural sidechains has recently found widespread applications. With the SwissSidechain database (http://www.swisssidechain.ch), we offer a central and curated platform for non-natural sidechains. SwissSidechain provides biophysical, structural and molecular data for hundreds of commercially available non-natural amino acid sidechains. We further provide plugins to seamlessly insert non-natural sidechains into peptides and proteins using molecular visualization software, as well as topologies and parameters compatible with molecular mechanics software. Recent experimental results on a phage display optimized ligand of urokinase plasminogen activator show how expanding the building blocks of peptides and proteins with non-natural sidechains is powerful to develop potent inhibitors of protein interactions. EMBO Fellows' Meeting, 13 - 16 June 2013, Heidelberg p27Kip1 is a microtubule-associated protein that promotes microtubule polymerisation during neuron migration

Juliette Godina,*, Sophie Laguessea, Noémie Thomasa, Lina Malinouskayaa, Pierre Closeb, et al. a GIGA-Neurosciences Interdisciplinary Cluster for Applied Genoproteomics (GIGA-R), University of Liège, C.H.U. Sart Tilman, Liège 4000, Belgium b GIGA-Signal Transduction Interdisciplinary Cluster for Applied Genoproteomics (GIGA-R), University of Liège, C.H.U. Sart Tilman, Liège 4000, Belgium

The migration of cortical interneurons is characterized by extensive morphological changes that result from successive cycles of nucleokinesis and neurite branching. Their molecular bases remain elusive and the present work describes how p27Kip1 controls cell cycle unrelated signaling pathways to regulate these morphological remodelings. Live imaging reveals that interneurons lacking p27Kip1 show delayed tangential migration resulting from defects in both nucleokinesis and dynamic branching of the leading process. At the molecular level, we described p27Kip1 as a novel microtubule-associated protein that promotes polymerisation of microtubules in extending neurites, thereby contributing to tangential migration. Furthermore, we show that p27Kip1 controls actomyosin contractions that drive both forward translocation of the nucleus as well as growth cone splitting. Thus, p27Kip1 cell-autonomously controls nucleokinesis and neurite branching by regulating both actin and microtubule cytoskeletons. EMBO Fellows' Meeting, 13 - 16 June 2013, Heidelberg

Mitochondrial protein import caught in the act: a study by electron cryo-tomography

Vicki Golda,*, Raffaele Ievab, Martin van der Laanb, Nikolaus Pfannerb, Werner Kühlbrandta a Max-Planck-Institute of Biophysics, Frankfurt, Germany b Institute for Biochemistry and Molecular Biology, Freiburg, Germany

Mitochondria import a plethora of proteins into four distinct compartments, via complex and versatile networks of dynamic targeting pathways. A major gateway into mitochondria is via the TOM and TIM23 translocases, which form macromolecular complexes spanning the outer and inner membranes respectively. Due to an intensive biochemical effort, the fundamentals of protein transport by TOM/TIM23 are understood, yet there is inadequate structural information. Our goal is to directly visualise the protein import machinery in action within whole mitochondria, by the technique of electron cryo-tomography. This is achieved by stalling a pre-protein substrate through both channels concurrently, creating the bona fide import supercomplex.

A novel substrate has been designed, which by the virtue of a tightly folded domain (DHFR), has the ability to arrest whilst crossing both complexes and membranes simultaneously. It contains a specific C-terminal electron dense tag to assist localisation on the mitochondrial surface when viewed in the microscope. Overexpression and purification of the substrate has been successfully optimised in E. coli. Biochemically, this preprotein has been shown to be functional by in vitro import into energised S. cerevisae mitochondria and subsequent detection of the TOM/TIM23 supercomplex by native-PAGE.

Tomograms have been collected which show the number and distribution of pre-protein import sites on the mitochondrial surface. Early data pinpoints specific sites of protein translocation, which we now aim to investigate in more detail. The protein complexes found at these sites will be analysed with the ultimate goal of carrying out subtomogram averaging. This information will be used to determine the three dimensional organisation of translocation sites in situ. EMBO Fellows' Meeting, 13 - 16 June 2013, Heidelberg

Structure determination of membrane proteins in a native environment

Franz Hagna,*, Manuel Etzkorna, Thomas Raschlea, Gerhard Wagnera

Harvard Medical School, Department of Biological Chemistry and Molecular Pharmacology, Boston, MA 02115, USA

Structural studies of membrane proteins are still hampered by difficulties in finding appropriate membrane mimicking media that maintain protein structure and function. Here, I will describe a novel membrane system, called phospholipid nanodiscs, that consists of a patch of lipids and two copies of a lipid-binding protein that wrap around these lipids and form disc-shaped particles. Using this system, membrane proteins can be investigated in a real detergent-free and native-like lipid environment. I will show our recent success in performing structure determination of membrane proteins in this system with NMR spectroscopy. Furthermore, I will show unpublished data on structural studies of mitochondrial membrane proteins that play a key role in diseases like heat failure and cancer. Finally, I will present data on our studies of G-proteins that bind to the pharmaceutically important class of G-protein-coupled receptors and will eventually outline our plans for future work. EMBO Fellows' Meeting, 13 - 16 June 2013, Heidelberg

Structural basis of the translational regulation of msl2 mRNA by SXL and UNR during dosage compensation in Drosophila

Janosch Henniga,*, Cristina Milittib, Gregorz Popowiczc, Iren Wanga, Miriam Sonntaga, et al. a Institute of Structural Biology, Helmholtz Zentrum München, 85764 Neuherberg, Germany & Center for Integrated Protein Science Munich (CiPSM) at Biomolecular NMR, Department Chemie, Technische Universität München, 85747 Garching, Germany b Centre for Genomic Regulation (CRG), Gene Regulation, Stem Cells and Cancer Programme, 08003 Barcelona, Spain c Institute of Structural Biology, Helmholtz Zentrum München, 85764 Neuherberg, Germany

The protein Upstream of N-Ras (UNR) is a key regulator of gene expression at the translational level in both humans and Drosophila. In Drosophila, the role of UNR in dosage compensation is well characterized. UNR and the female-specific protein Sex-lethal (SXL) bind cooperatively to the 3’ UTR of msl2 mRNA, which encodes the rate-limiting subunit of the dosage compensation complex. This interaction represses the translation of msl2 mRNA thereby blocking dosage compensation in female flies. To unravel the molecular mechanisms of these interactions we studied the structural basis for the assembly of the SXL-UNR-msl2 ribonucleoprotein complex.

UNR consists of five cold-shock domains, where only the first of these domains (CSD1) is required for the interaction with msl2 mRNA and SXL. On the other hand, only the RNA binding domains of SXL, contained in a fragment called dRBD4, are required for the interaction.

Here, we present the crystal structure of the complex of dRBD4-CSD1 bound to its interaction region in msl2 mRNA, validated by NMR spectroscopy and small angle neutron/X-ray scattering. The structure reveals new insights into how the cooperative and novel interaction between an RNA recognition motif (Sxl), a cold shock domain, and mRNA triggers translational repression in the development of fruit flies. Based on this structure, a detailed mutational analysis provided further insights which will be presented. EMBO Fellows' Meeting, 13 - 16 June 2013, Heidelberg

Uncoupling of the endocannabinoid signalling complex in a mouse model of fragile X syndrome

KM Junga, M Sepersb, Chris Henstridgec,*, O Lassalled, D Neuhoferd, et al. a Department of Anatomy and Neurobiology, University of California, Irvine, California 92697, USA. b INSERM U862, Circuit and Dendritic Mechanisms Underlying Cortical Plasticity Group, Neurocentre Magendie, 146 Rue Léo-Saignat, F 33077 Bordeaux Cedex, France. c Institute of Experimental Medicine, Hungarian Academy of Sciences, H-1083 Budapest, Hungary. d INSERM U901, Marseille 13009, France.

Fragile X syndrome (FRAX), the most common inherited form of mental disability, is caused by loss of fragile X mental retardation protein (FMRP), an RNA-binding protein that controls glutamate mGlu5 receptor-dependent signaling. Enhanced long-term depression (LTD) is widely considered as the major synaptic pathophysiological consequence of exacerbated mGlu5 signaling. However, unexpectedly, we found that another mGlu5- and also endocannabinoid-dependent form of LTD is completely absent in a mouse model of FRAX. We found that spatial and functional uncoupling of the endocannabinoid signalosome, a perisynaptic macromolecular complex present in dendritic spines that links mGlu5 receptors to the 2-arachidonoylglycerol (2-AG)-producing enzyme diacylglycerol lipase-α, accounts for this impaired synaptic plasticity at excitatory synapses in several forebrain areas. To assess anatomical uncoupling, we performed a detailed electron microscopy study, which revealed that the perisynaptic accumulation of mGlu5 remained intact in both groups. In contrast, DGL-α distribution was concentrated perisynaptically at glutamatergic synapses in wild type, but not in FMRP-KO mice. Instead, the levels of DGL-α within the spine head cytoplasm were significantly higher in FMRP-KO animals and intriguingly, increased DGL-α labeling was found in the spine neck in FMRP-KO mice. This spatial alteration in the endocannabinoid signalosome may explain reduced versus enhanced endocannabinoid signaling at glutamatergic or GABAergic synapses reported earlier, respectively, and may lead to a shift in the balance of excitatory/inhibitory synaptic plasticity in FMRP-knockout animals. To test whether pharmacological enhancement of 2-AG signaling normalizes this perturbed plasticity in FMRP-KO mice, we blocked either MGL or ABHD6, two 2-AG degrading enzymes, which restored LTD at glutamatergic synapses and reversed behavioural abnormalities in the FMRP-KO. Thus, the regulation of 2-AG metabolism may provide a new approach to alleviate some phenotypic consequences of FRAX. EMBO Fellows' Meeting, 13 - 16 June 2013, Heidelberg

Toward a structural understanding of kinetochore architecture

Jenny Kellera,*, Arsen Petrovica, Anika Altenfelda, Andrea Musacchioa

Max Planck Institute of Molecular Physiology, Otto-Hahn-str.11, 44227 Dortmund, Germany

High fidelity of chromosome segregation during mitosis is essential for maintaining the integrity of the genome. If the process is perturbed, cells can gain or lose chromosomes and become aneuploid. In some cases, surviving cells may proliferate without regulation leading to chromosome instability (CIN) and tumor formation (Cleveland et al., 2003; Diaz-Rodriguez et al., 2008). Deciphering the molecular mechanisms of accurate chromosome segregation is therefore important to understand tumorigenesis. Kinetochores are multi-protein complexes that play a central role in the correct separation of chromosomes during mitosis (reviewed in Santaguida and Musacchio, 2009). A conserved complex known as the KMN (for Knl1 complex, Mis12 complex, Ndc80 complex) network is essential for kinetochore/microtubule attachment and for the feedback mechanisms (Spindle Assembly Checkpoint, SAC) that help to maintain ploidy in mitosis. At present, high-resolution structural information on the KMN network is only available for the NDC80 complex (whose structure was determined in the Musacchio laboratory), whereas the remaining 6 subunits have not been characterized. In the work presented, we show the structural organization of the KMN network and its role in the recruitment of the Rod-Zw10-Zwilch (RZZ) complex, which is essential for the SAC. EMBO Fellows' Meeting, 13 - 16 June 2013, Heidelberg

Host cell entry of Respiratory Syncytial Virus involves macropinocytosis followed by proteolytic activation of the F protein

Magdalena Krzyzaniaka,*, Ari Heleniusa

ETH Zurich, Institute of Biochemistry, Schafmattsrasse 18, Zurich 8093, Switzerland

Respiratory Syncytial Virus (RSV) is a highly pathogenic member of the Paramyxoviridae that causes severe respiratory tract infections. Reports in the literature have indicated that to infect cells the incoming viruses either fuse their envelope directly with the plasma membrane or exploit clathrin-mediated endocytosis. To study the entry process in human tissue culture cells (HeLa, A549), we used fluorescence microscopy and developed quantitative, FACS-based assays to follow virus binding to cells, endocytosis, intracellular trafficking, membrane fusion, and infection. A variety of perturbants were employed to characterize the cellular processes involved. We found that immediately after binding to cells RSV activated a signaling cascade involving the EGF receptor, Cdc42, PAK1, and downstream effectors. This led to a series of dramatic actin rearrangements; the cells rounded up, plasma membrane blebs were formed, and there was a significant increase in fluid uptake. If these effects were inhibited using compounds targeting Na+/H+ exchangers, myosin II, PAK1, and other factors, no infection was observed. The RSV was rapidly and efficiently internalized by an actin-dependent process that had all hallmarks of macropinocytosis. Rather than fusing with the plasma membrane, the viruses thus entered Rab5-positive, fluid-filled macropinosomes, and fused with the membranes of these on the average 50 min after internalization. Rab5 was required for infection. To find an explanation for the endocytosis requirement, which is unusual among paramyxoviruses, we analyzed the fusion protein, F, and could show that, although already cleaved by a furin family protease once, it underwent a second, critical proteolytic cleavage after internalization. This cleavage by a furin-like protease removed a small peptide from the F1 subunits, and made the virus infectious. EMBO Fellows' Meeting, 13 - 16 June 2013, Heidelberg

Strong heterogeneity in mutation rate causes misleading hallmarks of natural selection on indel mutations

Erika Kvikstada,*, Laurent Duretb a The Wellcome Trust Centre for Human Genetics, University of Oxford, United Kingdom b LBBE, UCBL- Universite Lyon 1, Lyon, France

Elucidating the mechanisms of mutation accumulation and fixation is critical to understand the nature of genetic variation and its contribution to genome evolution. Of particular interest is the relative impact of selection vs. non-adaptive forces operating on insertions and deletions (indels). Here, we take advantage of recent population-scaled sequencing efforts, together with whole-genome sequence alignments, that provide unprecedented datasets of indel polymorphism and divergence for the analysis of indel fixation dynamics. Briefly, we investigated the derived allele frequency (DAF) spectra for short (1-50 base pairs) indel variation identified in the 1000 Genomes Pilot 1 data. In conjunction with comparisons of indel diversity vs. divergence in a modified McDonald-Kreitman approach, our analyses revealed an apparently higher fixation probability for insertions than deletions. This fixation bias scales with the local mutation rate and is particularly pronounced at indel hotspot loci. In non-repetitive (NR) regions, the DAF and McDonald-Kreitman tests are incongruent with the action of widespread selection on indels. Furthermore, we identified an unprecedented number of loci in NR contexts with evidence for multiple indel events in the primate phylogeny. According to a model of uniform indel rate evolution, such loci are 60-fold more frequent than expected, thus providing evidence of as yet unidentified “cryptic” indel mutation hotspots. We propose that indel homoplasy, at known and cryptic hotspots, produces systematic errors in determination of derived vs. ancestral alleles that in turn lead to misidentification of insertions vs. deletions via parsimony. Given the strong heterogeneity in indel mutation rate across the genome, classic selection tests should be interpreted with caution due to the sensitivity of parsimony methods to indel homoplasy. These results are predicted to have great impact on additional studies seeking to infer evolutionary forces operating on indels observed in closely related species, since such mutations are traditionally presumed to be homoplasy-free. EMBO Fellows' Meeting, 13 - 16 June 2013, Heidelberg

Angiogenesis controls neurogenesis in the developing cerebral cortex

Christian Langea,*, Ruben Boona, Christine Wub, Calvin Kuob, Peter Carmelieta a Laboratory for Neurogenesis and the Neurovascular Link, Vesalius Research Center, VIB and KU Leuven, Leuven, Belgium b Division of Hematology, School of Medicine, Stanford University, San Francisco, USA

Neural stem cells (NSCs) regulate their expansion and differentiation according to instructive signals from their cellular microenvironment, the stem cell niche. Blood vessels are thought to be an integral part of the stem cell niches in the of the developing and the adult brain. However, the functional significance of blood vessels for NSC maintenance and the underlying molecular mechanisms are poorly defined. Here, we show that angiogenesis is coupled with the switch from NSC expansion to neurogenesis during development of the mouse cerebral cortex. Moreover, specific inhibition of brain angiogenesis reduces the switch to neurogenesis in the cortex, demonstrating for the first time a causal role of blood vessels in this process. Contrary to previous models, we find that reduction of blood vessels in the NSC niches augments proliferation and expansion specifically of NSCs, while reducing their differentiation. Finally, our results suggest oxygen delivery as a major mechanism of blood vessel influence on the switch to neurogenesis since mimicking hypoxia signaling in the NSCs, phenocopies the effect of angiogenic inhibition. Taken together, these findings provide novel, unprecedented insights how blood vessels regulate NSC proliferation and differentiation in their in vivo niche. EMBO Fellows' Meeting, 13 - 16 June 2013, Heidelberg

Bone-derived Osteocalcin regulates muscle mass and function

Kathrin Julia Lauea,*, Olga Sumaraa, Marc McKeeb, Gerard Karsentya a Columbia University, New York, US b McGill University, Montreal, Quebec, Canada

Sarcopenia, the decline of muscle mass and function, is a main characteristic of organismal aging and a major public health problem. Remarkably, the onset of muscle decline during aging is concurrent with a loss of bone mass suggesting that both tissues could share one or several regulatory mechanisms. However, the signals that coordinate the functions of the musculo-skeletal system are poorly understood. Osteocalcin, a peptide hormone secreted by osteoblasts, regulates physiological processes known to decline with age such as glucose homeostasis and male fertility. During exercise, the circulating levels of active osteocalcin increase. We therefore tested whether osteocalcin and its putative receptor, Gpcr6a, could regulate muscle mass and function. Indeed, upon physical challenge on a treadmill, the distance and time Osteocalcin-/- mice can run are significantly lower compared to their wild type littermates. Accordingly, these mice exhibit decreased oxidative capacity and defects in the uptake of nutrients in early adulthood. At later stages, osteocalcin-deficient mice show a decrease in muscle mass and myofiber diameter as well as a reduction in structural protein levels. In addition, osteocalcin is required for muscle regeneration after chemical-induced injury. Our findings identify osteocalcin as one of the long sought after molecules linking bone and muscle tissue suggesting that a decrease in osteocalcin level during aging could contribute to the development of sarcopenia. EMBO Fellows' Meeting, 13 - 16 June 2013, Heidelberg

Structure of Biological Supramolecular Assemblies Solved by Solid-State NMR

Antoine Loqueta,*, Nikolas Sgourakisb, Karin Gillera, Jean Philippe Demersa, Rashmi Guptac, et al. a Max-Planck-Institute for Biophysical Chemistry, 37077 Göttingen, Germany b University of Washington, Seattle, WA 98195, USA c Max-Planck-Institute for Infection Biology, 10117 Berlin, Germany

The assembly of multiple protein subunits into large supramolecular complexes plays a key role in many cellular processes, notably in bacterial virulence, neurodegenerative disease-related mechanisms, and as a part of nanomachines. 3D structures of these supramolecular assemblies are still rare as they remain often recalcitrant for atomic-resolution techniques. First, their elongated shape, complexity, and lack of long-range order are severe obstacles to grow single crystals amenable to X-ray crystallography. Second, their inherent insolubility restricts the use of solution NMR.

Here, I will show that state-of-the-art Solid-State NMR methodology (1-3) is able to reveal the supramolecular interfaces and ultimately the complete atomic structure of a protein assembly. Our approach is demonstrated on the Salmonella typhimurium Type III Secretion System Needle, a bacterial filament that mediates the injection of pathogen effector proteins to the cytosol of host cells during bacterial infection. Using an integrated approach combining Solid-State NMR, STEM, and computational modeling, we present an atomic resolution model of the Needle (4) in its native filamentous state.

(1) Loquet et al., J. Am. Chem. Soc. 2010 (2) Loquet et al., J. Am. Chem. Soc. 2011 (3) Loquet et al., Acc. Chem. Res. 2013 (4) Loquet et al., Nature 2012 EMBO Fellows' Meeting, 13 - 16 June 2013, Heidelberg

The yeast Mph1 helicase alters telomere structure by disrupting the protective fold-back structure

Sarah Luke-Glasera,*, Brian Lukea

Zentrum für Molekulare Biologie der Universität Heidelberg, DKFZ-ZMBH Allianz, Im Neuenheimer Feld 282, 69112 Heidelberg, Germany

The yeast helicase, Mph1 is similar in sequence to human FANCM and displays many overlapping in vitro biochemical properties with human RTEL1. Recently, RTEL1 has been implicated in the resolution of t-loop structures at telomeres. Through chromatin immunoprecipitation experiments we found that Mph1 localizes to yeast telomeres. Furthermore, over-expression of Mph1 results in the loss of a fold-back structure at natural telomere 6R. Consistent with the fold-back structure providing a protective role, we observed toxicity in CST mutants and rapid senescence in telomerase mutants upon Mph1 overexpression due to exacerbated nuclease-mediated resection within the telomeric repeats. Importantly, when we overexpressed an allele of Mph1 (Mph1 delta 60) that is unable to interact with Smc5, we did not detect telomere dysfunction phenotypes. The SMC5/6 protein complex forms a cohesion-like ring structure that has also been shown to localize to telomeres. smc5/6 temperature sensitive mutants display rapid cellular senescence in the absence of telomerase and this is rescued upon the further deletion Mph1. Furthermore, smc5/6 mutants, like Mph1 over-expression, show a defect in telomeric fold-back structure. Together these data suggest that the SMC5/6 complex may be responsible for the tight regulation of the Mph1 helicase at telomeres in order to resolve the telomere fold-back structure at the appropriate time and place. EMBO Fellows' Meeting, 13 - 16 June 2013, Heidelberg

Investigation of phosphorylation mediated signaling in mitosis by targeted mass spectrometry approaches

Alessio Maiolicaa,*, Fabrizio Villab, Erwin Schoofc, De Metondo Mariaa, Ruedi Aebersolda, et al. a ETH, Zurich, Switzerland b IEO, Milano, Italy c Technical University of Denmark, Lyngby, Denmark

The mitotic cell cycle is a strictly ordered sequence of events that determine dramatic changes in the cells. A central regulatory system accurately coordinates, mainly by means of phosphorylation and protein degradation the ordering of all the steps by initiating each event at the appropriate time. Advances in phosphoproteomics made possible the identification of thousands regulated phospho sites in mitosis, offering a view of the mitotic phosphoproteome regulation at a “systems level”. However very little is known about the contribution of every kinase to the complex regulatory landscape of all mitotic events. Phosphorylation mediated signaling events are, virtually always, organized as cascades of reactions where each kinase or phosphatase can modulate the activation of other signaling molecules by phosphorylation or de-phosphorylation events. Reproducible quantification of every phosphorylation site is therefore required for a complete understanding of biological process such as mitosis. Here we report a novel strategy, base on kinase reaction screening, for the identification of kinases’ substrates by targeted mass spectrometry methods.

The approach consists of four phases:

1)Kinase purification Functional enzymes are purified from stable cell lines inducibly expressing affinity-tagged kinases and/or recombinantly expressed in ectopic system.

2)Peptide library construction Substrate peptides are opportunely selected form the literature and chemical synthetized in their unphosphorylated form on a large scale.

3)Multiplex in vitro kinase assay on peptide libraries The phosphopeptides, formed by the in vitro reaction with specific kinase, are identified by shotgun mass spectrometry.

4)Validation of candidate substrates The peptides library is used in its phosphorylated and unphosphorylated forms to develop assays for selected reaction monitoring (SRM) and SWATH. The assays are then used for detection and validation of the phosphorylation sites in cellular systems.

The approach has been used for the identification of novel substrates of different mitotic kinases namely: AuroraB, Plk1, Cdk1 and Haspin. EMBO Fellows' Meeting, 13 - 16 June 2013, Heidelberg

Control of colonic epithelium damage by NAIP

Kendle Maslowskia,*, Jurg Tschoppa

University of Lausanne, Department of Biochemistry, Switzerland

Neuronal apoptosis inhibitory protein (NAIP, or BIRC1) is a member of the IAP (inhibitor of apoptosis) and NLR (Nod-like receptor) families. Humans contain one functional copy of the NAIP gene, whereas, in mice, multiple homologues of Naip exist which varies depending on the background (the C57B/6 mouse strain posses five: Naip 1, 2, 5, 6 and 7). We have developed complete Naip knockout mice (Naip1-7–/–), which have enabled us to study the in vivo function of the Naip family. In humans and mice, NAIP is highly expressed in the colonic epithelium, which suggests a role of Naip within the colon. The AOM/DSS model of colitis-associated CRC was used to induce colonic tumors in Naipfl/fl and Naip1-7–/– mice. Naip1-7–/– mice showed an increase in tumor burden, despite having reduced colitis. In tumors from Naipfl/fl mice, expression of Naip was decreased, compared to the surrounding colonic tissue. This suggests that a reduction of Naip is a feature of tumor development. Whilst inflammation is most often linked directly to tumor progression, certain protective factors during colitis have tumor-promoting properties, such as IL22, which we found was increased in Naip1-7–/– mice. AOM induces O6-methylguanine adducts, resulting in G to A nucleotide transitions; early after administration a wave of apoptosis induced by accumulation of damaged DNA occurs. Failure to repair DNA or clear AOM-initiated colonic epithelium can lead to increased tumorigenesis. Therefore, we analyzed colonic extracts 8 hrs after AOM injection and found that Naip1-7–/– had defects in DNA-damage response, which could underpin the increased tumorigenesis observed in these mice. In summary, we have generated novel Naip1-7–/– mice, which has enabled us to determine a critical role for Naip in regulating responses to epithelial damage and induction of colitis and colorectal cancer. EMBO Fellows' Meeting, 13 - 16 June 2013, Heidelberg

Coordinating epithelial cell division with tissue organization and polarity

Eurico Morais-de-Sáa,*, Claudio Sunkela

IBMC, Porto, Portugal

Epithelial cells form a monolayered tissue that controls exchanges between the different compartments of multicellular organisms. The architecture and function of this tissue relies on the asymmetric localization of cellular junctions and the formation of cortical domains polarized along the apico-basal axis. During proliferation, epithelial cells have to coordinate the re-organization of their polarity with the cell shape changes associated with mitosis. Here, we use clonal analysis and live imaging of the Drosophila follicular epithelium to explore how the polarity machinery is remodelled during cell division. Furthermore, we show that cytokinesis is asymmetric along the division plane of follicle epithelial cells, positioning the midbody near the apical domain. In this context, asymmetric cytokinesis is independent of the intrinsic asymmetry of actomyosin ring components. Instead, it relies on apical adherens junctions formed between the mitotic and neighboring cells, which maintain the contact with the contractile ring during cytokinesis and determine midbody position in partially depolarized daughter cells. We further show that ectopic midbody localization alters epithelial shape due to the mispositioning of the new apical interface formed between daughter cells. Thus, we propose that the AJ acts as a positional cue maintained within a proliferating tissue, which transfers cell architecture to the new daughter cells via the positioning of the cytokinetic machinery. EMBO Fellows' Meeting, 13 - 16 June 2013, Heidelberg

How SR proteins connect splicing to mRNA export in vivo

Michaela Müller-McNicolla,*, Michaela Steinera, Valentina Bottia, Holger Brandla, Karla Neugebauera

Max Planck Institute of Molecular Cell Biology and Genetics, Pfotenhauerstr. 108, 01307 Dresden, Germany

The SR protein family comprises seven regulatory RNA-binding proteins that play essential roles in pre-mRNA splicing. Some SR protein family members connect splicing to subsequent steps in gene expression, such as mRNA export and translation, but their endogenous mRNA targets are largely unknown. To systematically investigate the role of individual SR proteins in mRNA export in vivo, we established murine P19 stem cell lines, expressing seven GFP-tagged SR proteins from bacterial artificial chromosomes (BACs) without overexpression. As a prerequisite for a function in mRNA export, we first quantified the capacity of individual SR proteins to shuttle between the nucleus and the cytoplasm. We observed that in contrast to HeLa cells, where SRSF2 and SRSF5 do not shuttle, in P19 cells all SR proteins shuttle, however with different shuttling capacities. Investigating this further, we found that the shuttling capacities correlate with the phosphorylation status of individual SR proteins, their interaction with the mRNA export factor NXF1 and their presence in translating polyribosomes. To identify mRNAs that depend on individual SR proteins for their export to the cytoplasm, we performed knockdown and cell fractionation followed by microarrays or RNA-Seq. We identified export targets for all SR proteins and observed that the shuttling capacities correlate well with the number and expression levels of exported mRNAs. Furthermore, the export targets are functionally related and point to an important function of SR proteins in the regulation of different cell fates. To confirm that export targets bind directly to their cognate SR protein we performed UV crosslinking and immunoprecipitation (iCLIP) followed by deep sequencing. Our study reveals the basis for the differential contribution of individual SR proteins to mRNA export in P19 cells and presents for the first time endogenous export targets for all SR proteins. EMBO Fellows' Meeting, 13 - 16 June 2013, Heidelberg

Identifcation of novel molecular targets in colon cancer

Paloma Ordóñez-Morána,*, Joerg Huelskena

Swiss Institute for Cancer Research, École Polytechnique Fédérale de Lausanne, Switzerland

The Wnt pathway appears to be one of the most central mechanisms in colon cancer initiation based on its frequent constitutive activation (~90%). We and others have reported that the main effector of this pathway β-catenin binds to different transcription factors enhancing the expression of alternative sets of target genes. Forkhead box O3 (FOXO3a), downstream effector of PI3K/AKT pathway, is one of those for which β-catenin acts as a transcriptional coactivator. In cancer, FOXO transcription factors have been considered to be tumour suppressors because they can induce cell-cycle arrest and apoptosis.

Recently, we have described the function of these transcriptional effectors in colon cancer progression. Unexpectedly, their simultaneous nuclear accumulation promotes cell scattering and metastasis by regulating a defined set of target genes. Furthermore, β-catenin confers resistance to FOXO3a-mediated apoptosis induced by PI3K/AKT inhibitors. So, FOXO3a does not behave as a tumor suppressor but rather as a metastasis inducer activated by PI3K/AKT inhibitors when acting in concert with β-catenin. In this context, we have evaluated SPRY2 regulation by both proteins, and our results reveal SPRY2 as a new direct β-catenin/FOXO3a target gene that is expressed in Wnt-activated cells in normal and tumoral tissues and correlates with shorter overall survival of colon cancer patients. Thus, we propose that it would be important to evaluate β-catenin status before deciding on treatment and that SPRY2 should therefore be considered as a prognosis marker.

Furthermore, intestinal stem cells have been described as the cells-of-origin of cancer. At present, we investigate undescribed β-catenin targets (possible tumour suppressors or oncogenes) that modulate the intestinal stem cell compartment. For these targets we define their effects on stem cell self-renewal versus differentiation, long-term maintenance and lineage fate. We therefore are identifying regulatory functions controlling cancer stem cell maintenance which could be relevant for tumourigenesis. EMBO Fellows' Meeting, 13 - 16 June 2013, Heidelberg

Asymmetric inheritance of primary ciliary membrane in dividing neural progenitors

Judith Paridaena,*, Michaela Wilsch-Brauningera, Wieland Huttnera

Max Planck Institute of Molecular Cell Biology and Genetics, Dresden, Germany

The primary cilium is a microtubule-based protrusion of the cell that is involved in mediating extracellular signals, such as Sonic Hedgehog (Shh). The primary cilium is nucleated by the centrosome containing the eldest centriole (mother centriole). Prior to mitosis, the primary cilium is disassembled in order for the centrosomes to serve as the poles of the mitotic spindle. Recent findings show that asymmetric inheritance of the centrosome containing the mother centriole is related to cell fate decisions during mammalian neurogenesis. However, it is unknown whether centrosome-associated structures such as the primary cilium are involved in this asymmetrical regulation of cell fate. Therefore, we investigated the fate of the primary cilium upon cell division of developing neural progenitors and the possible involvement of the primary cilium in subsequent cell fate decisions in the developing mouse neocortex. We found that in mitotic neural progenitors and cultured cell lines, a membrane structure containing the ciliary small GTPase Arl13b is associated with the centrosome that contains the mother centriole. By surface biotinylation of the apical membrane in explanted mouse telencephalic hemispheres, we show that this structure contains ciliary membrane and is derived from the primary cilium that was present at the cell surface prior to mitosis. Using live imaging, we find that upon completion of mitosis, this centrosome-associated ciliary membrane is asymmetrically inherited by one of the daughter cells. Furthermore, we show that inheritance of the ciliary membrane causes earlier reestablishment of the primary cilium after division. Interestingly, the association of the ciliary membrane with one centrosome in mitotic neural progenitors decreases as neurogenesis proceeds. We hypothesize that the observed asymmetry in primary cilium reassembly after mitosis might differentially expose daughter cells to extracellular signals, such as Shh. Therefore, we speculate that inheritance of the ciliary membrane affects cell fate decisions during mammalian neurogenesis. EMBO Fellows' Meeting, 13 - 16 June 2013, Heidelberg

Gabaergic projection neurons improve naive olfactory discrimination of close odors in drosophila

Moshe Parnasa,*, Wolf Huetterotha, Gero Miesenböcka

University of Oxford, UK

Most neurons involved in perceptual judgments are at least two synapses removed from sensory receptors. Psychophysical models that link perception to the physical qualities of external stimuli are thus black boxes. Opening these black boxes is challenging and requires comprehensive estimates of activity in many neurons carrying perceptually relevant signals. Because sensory representations are distributed over large numbers of neurons, such estimates have generally remained elusive. Here, we take advantage of the well-characterized olfactory system of fruit flies to relate knowledge of the neuronal population representations of odors to behavioral measures of odor discrimination. Flies detect odors using ~50 types of olfactory receptor neurons (ORNs). ORN axons segregate anatomically by receptor type and transmit signals via separate synaptic relays, to discrete classes of excitatory projection neurons (ePNs). Previously, ORN responses to odors and a transformation estimating PN spike rates from measured ORN spike rates were presented. ePNs project to the mushroom body, and the lateral horn (LH). The LH, thought to be responsible of naïve behavior, also receives input from a functionally uncharacterized group of GABAergic inhibitory PNs (iPNs). The fact that iPNs target exclusively the LH hints at a possible function of these inhibitory neurons in naïve behavior. We formulate and test a simple model of innate odor discrimination that takes as its input the estimated PN signals projected onto the LH and generates as its output a prediction of whether two odors can be distinguished. We show that the main determinant of discriminability is the distance between the PN activity patterns evoked by two odors. Experimental manipulations of this distance have graded predictable perceptual consequences. We further show that, inhibition by iPNs makes closely related odors easier to distinguish, in all likelihood by imposing a high-pass filter on ePN output that stretches the distances between partially overlapping odor representations. EMBO Fellows' Meeting, 13 - 16 June 2013, Heidelberg

Translational coordination of protein folding at the ribosome

Sebastian Pechmanna,*, Judith Frydmana

Department of Biology and BioX, Stanford University, USA

Newly synthesized proteins must be folded properly to become functional and avoid aggregation. Complex cellular mechanisms, including regulation of local translation rates and recruitment of protein folding and translocation components, influence co-translational folding events at the ribosome. The choice of codons in the mRNA sequences can influence local translation kinetics during protein synthesis. However, whether codon preference is generally linked to co-translational regulation of polypeptide folding remains unclear. Here, we derive a revised translational efficiency scale, which allows us to uncover the evolutionary conservation of codon optimality across ten eukaryotic organisms. This analysis reveals distinct and universal patterns of conserved optimal and non-optimal codons, often in clusters, which associate with the secondary structure of the translated polypeptides independent of the levels of expression. Quantifying the substrates of molecular chaperones at the ribosome further suggests a link between the translational efficiency and the coordinated binding and specificity of co-translationally acting chaperones. Our analysis thus suggests an evolved function for codon optimality in regulating the rhythm of elongation to facilitate co-translational nascent chain folding, beyond its previously proposed role of adapting to the cost of expression. These findings establish how mRNA sequences are generally under selection to optimize the co-translational folding of corresponding polypeptides. EMBO Fellows' Meeting, 13 - 16 June 2013, Heidelberg

Extensive transcriptional heterogeneity revealed by isoform profiling

Vicent Pelechanoa,*, Wu Weib, Lars Steinmetzc a Genome Biology Unit, European Molecular Biology Laboratory (EMBL), Meyerhofstrasse 1, 69117 Heidelberg, Germany. b Stanford Genome Technology Center, Stanford University, Palo Alto, CA 94304, USA. c Genome Biology Unit, European Molecular Biology Laboratory (EMBL), Meyerhofstrasse 1, 69117 Heidelberg, Germany. Stanford Genome Technology Center, Stanford University, Palo Alto, CA 94304, USA.

Transcript function is determined by sequence elements arranged on an individual RNA molecule. Variation in transcripts can affect mRNA stability, localization, and translation, or give rise to truncated proteins with differing subcellular localizations and functions. Given the existence of overlapping, variable transcript isoforms, determining the functional impact of the transcriptome requires identification of full-length transcripts, rather than just the genomic regions that are transcribed. Here, by jointly determining both transcript ends for millions of RNA molecules (TIF-Seq), we reveal an extensive layer of isoform diversity previously hidden among overlapping RNA molecules. Variation in transcript boundaries appears to be the rule rather than the exception, even within a single population of yeast cells. Hundreds of short coding RNAs and truncated versions of proteins are concomitantly encoded by alternative transcript isoforms, increasing protein diversity. In addition, most genes express alternative isoforms that vary in posttranscriptional regulatory elements, and tandem genes frequently produce overlapping or even bicistronic transcripts. This extensive transcript diversity is generated by a relatively simple eukaryotic genome with limited splicing, and within a genetically homogeneous population of cells. Our findings have implications for genome compaction, evolution, and phenotypic diversity between single cells. They also suggest that isoform diversity as well as RNA abundance should be considered when assessing the functional repertoire of genomes. EMBO Fellows' Meeting, 13 - 16 June 2013, Heidelberg

Root system development and plasticity

Benjamin Pereta,*, Laurent Nussaumea, Malcolm Bennettb a Unité Mixte de Recherche 7265, Commissariat à l’Energie Atomique et aux Energies Alternatives, Centre National de la Recherche Scientifique, Aix-Marseille Université, Laboratoire de Biologie du Développement des Plantes, Université d'Aix- Marseille, France b Division of Plant and Crop Sciences, University of Nottingham, Sutton Bonington Campus, Loughborough, Leicestershire LE12 5RD, UK

Plants have evolved extraordinary adaptive mechanisms to cope with a heterogeneous environment and to compete for water and nutrient acquisition. One key aspect is the continuous production of new organs throughout the plants life to compensate for their inability to move. As a consequence, plant development is characteristically highly plastic both at the shoot and root level. In the past years, I have focussed my research on root development and more recently on its plasticity in the context of the EMBO fellowship. In my presentation, I will give an overview of the molecular and hormonal mechanisms controlling the formation of roots. More precisely, I will explain how highly specific expression patterns can be achieved through positive feedback loops. Auxin is a plant hormone that triggers lateral root formation, acting as a signal to induce and synchronize its development. At the tissue level, the hormone auxin is transported by influx and efflux transporters. The study of the influx carrier LAX3 led to the identification of the mechanism that controls cell separation during this critical developmental process. Another key aspect of plant development is the hydraulic properties of tissue that largely control cellular elongation and growth. Aquaporins are membrane channels that allow water transport in both animals and plants. I will describe how aquaporins are regulated by auxin during lateral root development and how the most abundant root aquaporin PIP2.1 controls lateral root growth. Finally, I will describe how the environment controls the lateral root formation process. Phosphorus is a major element that is present in most organic molecules (ATP, DNA, RNA, Phospholipids, Phosphorylated proteins…). It is absorbed by plants in the form of inorganic phosphate (Pi) and its availability strongly affects root architecture. I am now studying how Pi deficiency is perceived by the plant to induce lateral root formation. EMBO Fellows' Meeting, 13 - 16 June 2013, Heidelberg

MicroRNA regulation of naïve and primed pluripotent states

Barbara Pernautea,*, Juan Miguel Sanchez-Nietoa, Thomas Spruceb, Kimberley Smithc, Tristan Rodrigueza a National Heart and Lung Institute, Imperial College London, UK b MRC National Institute for Medical Research, London, UK c Comparative and Biomedical Sciences, Royal Veterinary College London, UK

Understanding how cells exit the pluripotent state and initiate the path to differentiation is a key question in developmental and stem cell biology. To date the first identified step of differentiation in the mammalian embryo is the transit from a of naïve state of pluripotency, found in the pre-implantation epiblast and in embryonic stem cells (ESCs), to a pluripotent state that is primed for differentiation, and that can be found in the post-implantation epiblast and in epiblast stem cells (EpiSCs). These two different states of pluripoteny are thought to be regulated by different mechanisms as different combinations of growth factors are required for their maintenance both in vivo and in vitro. We have found that during the transition from the naïve to the primed state of pluripotency there is a critical change in the regulation of the apoptotic machinery. MicroRNAs (miRNAs) are small non-coding RNAs that repress gene expression post-transcriptionally. We show that with the onset of differentiation the apoptotic pathway becomes primed for cell death and is only kept in balance by an increase in the expression levels of miRNAs. Furthermore, we find that key to the priming of the apoptotic pathway is the pro-apoptotic protein Bim, that becomes competent to induce cell death as cells initiate differentiation. Our work provides insight into how miRNAs regulate the different pluripotent states and contributes to the understanding of the regulatory networks involved in stem cell homeostasis, pluripotency and differentiation during early embryo development. EMBO Fellows' Meeting, 13 - 16 June 2013, Heidelberg

Telomeric repeat-containing RNA (TERRA) promotes telomere shortening through Exonuclease 1-mediated resection of chromosome ends

Verena Pfeiffera,*, Joachim Lingnera

EPFL-Ecole Polytechnique Fédérale de Lausanne, School of Life Sciences, ISREC-Swiss Institute for Experimental Cancer Research, 1015 Lausanne, Switzerland

Telomeres protect chromosome ends from unwanted DNA repair activities and they prevent chromosome end shortening due to the end replication problem by engaging telomerase [1, 2]. Telomeres from animals and fungi are transcribed into the long noncoding telomeric repeat containing RNA (TERRA) [3]. To elucidate TERRA function in Saccharomyces cerevisiae, a doxycycline regulatable promoter was inserted upstream of the transcriptional start site of TERRA at telomere 1L. Induction of TERRA led to shortening of telomere 1L but not other chromosome ends. TERRA interacts with the Ku70/80 complex which represses the 5’-3’ Exonuclease 1 (Exo1) at telomeres [4]. Deletion of EXO1 fully suppressed the TERRA-mediated telomere shortening. Telomeric DNA in S. cerevisiae has a loose consensus sequence (TG1-3). Sequence alignment of sister telomeres allows distinction of a centromere proximal zone that is faithfully replicated by semiconservative DNA replication and a telomere distal zone at which sequences diverge due to new telomeric sequences that are added by telomerase or DNA recombination. In addition to Exo1-mediated telomere shortening, TERRA transcription increases the frequency of telomere healing events involving telomerase and Rad52-dependent recombination. Therefore, this data indicates that telomere transcription regulates cellular lifespan through modulation of chromosome end processing activities.

[1] Jain D., Cooper J.P. (2010) Annual Review of Genetics 44: 243-269 [2] de Lange T. (2009) Science 326: 948-952 [3] Feuerhahn S. et al. (2010) FEBS letters 17: 3812-3818 [4] Pfeiffer V., Lingner J. (2012) PloS Genet. 6: e1002747 EMBO Fellows' Meeting, 13 - 16 June 2013, Heidelberg

Genetic Handicap Principle: Low Mutation Burden of Down Syndrome Population

Konstantin Popadina,*, Reza Sailania, Audrey Letourneaua, Federico Santonia, Stylianos E. Antonarakisa

Department of Genetic Medicine and Development, University of Geneva Medical School, 1 rue Michel-Servet, 1211 Geneva, Switzerland

A handicap is a circumstance that makes progress or success difficult (Oxford Dictionary). As a genetic handicap we refer to trisomy of chromosome 21, which is highly deleterious due to 1.5 fold increased level of expression of 300 genes from this chromosome. Since selection eliminates organisms with increased mutation burden, Down Syndrome (DS) fetuses should be preferentially filtered out due to severe effect of the trisomy. Indeed, the rate of spontaneous abortions is significantly increased for DS fetuses (80% versus 8% in euploid fetuses), corroborating this selection. Thus live-birth DS individuals have to possess initially low mutation burden, which helped them to tolerate the harmful effect of trisomy and survive. According to the handicap principle, we expect that live-birth DS cohort should be initially higher fitted as compared to Control cohorts, where there was no so strong selection. Previously, we have observed a low expression variation in 8 live-birth DS versus 8 Control individuals, that can be interpreted as an evidence of increased fitness (ASHG 2012: http://abstracts.ashg.org/cgi-bin/2012/ashg12s?author=Popadin&sort=ptimes& sbutton=Detail&absno=120122658&sid=564830). Now we support this hypothesis by genetic data. Genome-wide variation in frequencies of SNPs between 380 unrelated European DS individuals and 215 controls demonstrated that DS population has a deficit of rare derived alleles as compared to our control population. This leads to deficit of both heterozygous and homozygous DS genotypes with rare derived alleles. It is noteworthy that trisomic SNPs, reconstructed for chromosome 21 demonstrate even more pronounced deficit of rare derived alleles in DS. Overall, our results (more optimal expression pattern in live-birth DS and deficit of rare, potentially more deleterious, alleles in DS) support the hypothesis of highly-fitted genomes of DS and suggest that during embryogenesis there is a preferential elimination of DS fetuses, highly contaminated by slightly-deleterious mutations. EMBO Fellows' Meeting, 13 - 16 June 2013, Heidelberg

Analysis of the yeast plasma membrane using soluble fluorescent reporters

Dušan Popov-Čeleketića,*, Frans Bianchia, Gemma Moiset Colla, Febrina Meutiawatia, Bert Poolmana

Biochemistry Department, University of Groningen, The Netherlands

In budding yeast S. cerevisiae proteins and lipids of the plasma membrane (PM) are heterogeneously distributed. Yeast PM exhibits high complexity and prominent patterns of membrane compartmentalization that have been extensively studied. Most studies on the PM organization in vivo largely rely on the localization of integral membrane proteins. These proteins, however, diffuse very slowly, are prone to aggregation, low expression and non-specific localization. The aim of this study is to develop new tools for the analysis of architecture and dynamics of the yeast PM. As model proteins we used amino acid permeases (AAP) residing in the yeast PM. Using analysis in silico followed by a series of mutational and truncational analyses we have found that the C-terminal sequences derived from nine amino acid transporters (Gap1, Bap2, Hip1, Tat1, Tat2, Mmp1, Sam3, Agp1, and Gnp1) were associated with the PM. Two reporters derived from Gap1 and Hip1 were selected for further studies based on their contrasting behaviour; the former being homogenously distributed along the membrane and very mobile and the latter present in patches in the membrane and static. Using super-resolution microscopy (photoactivated light microscopy, PALM) our reporters have enabled us to analyze the PM at the resolution of 50 nm. In parallel, we have been observing changes in the dynamics of our reporters under various conditions using Fluorescence Recovery After Photobleaching (FRAP). These short and soluble sequences (48-62 aa residues) are less prone to aggregation and exhibit higher expression than the full-length proteins they are derived from. Importantly, the reporters exhibit different patterns of localization and move along the membrane with various speeds making them excellent tools to study the factors that contribute to the organization of the yeast plasma membrane. EMBO Fellows' Meeting, 13 - 16 June 2013, Heidelberg

Magnetic resonance imaging of tumor glycolysis using hyperpolarized 13C-labeled glucose

Tiago Rodriguesa,*, Eva Serraoa, Brett Kennedya, De-en Hua, Mikko Kettunena, et al.

CRUK Cambridge Institute and Department of Biochemistry, University of Cambridge, Li Ka Shing Centre, Robinson Way, Cambridge CB2 0RE, UK

High rate of aerobic glycolysis in tumors provides opportunities for tumor detection and treatment monitoring using metabolic imaging such as FDG-PET and hyperpolarized [1-13C]pyruvate. In the current study we investigated the possibility to use hyperpolarized [U-2H,U-13C]glucose to detect glycolysis in vivo.

EL4 tumor-bearing mice were imaged before and 24h after treatment with etoposide (67mg/kg). Hyperpolarized [U-2H,U-13C]glucose sample (3.55M) was dissolved with 3ml of 2H2O-saline. MR data were collected at 7 T using a surface coil placed over the tissue of interest (tumor, heart, kidney) 15s after the start of the glucose injection. A series of 13C spectra were collected: 4 spectra collected from the lactate region followed by 1 collected from the glucose region every second for 40s. In some animals, a glucose and lactate 13C chemical shift selective images were collected. The ratios of the signal integrals from lactate (183-187ppm) and glucose (60-100ppm) were calculated.

Signal from labeled [U-13C]lactate was observed in EL4 or LL2 tumor spectra but was not observed in other tissues. Consistent with this, lactate was predominantly located tumor in 13C chemical-shift image. Low levels of dihydroxyacetone phosphate, 6-phosphogluconate and bicarbonate , likely produced by 6-phosphogluconate dehydrogenase, were also observed in the tumor spectra. This experiment could therefore give simultaneous information on both glycolysis and Pentose Phosphate Pathway activity. The flux of 13C label from glucose to lactate was decreased at 24 h after treatment with the labelled lactate/glucose ratio decreasing from 1.82±0.42 to 0.69±0.11 after treatment (n=6, p=0.026). The current results suggest that despite relatively short T1 (~9s), lifetime of hyperpolarized glucose is long enough for real-time in vivo imaging of glycolysis. The capacity to non-invasively measure the glycolytic pathway may provide more profound understanding of the altered metabolism of cancer and empower the identification of targets for diagnostic imaging, prognosis or therapy. EMBO Fellows' Meeting, 13 - 16 June 2013, Heidelberg

Suppression of protophloem differentiation and root meristem growth in Arabidopsis by CLE45 requires the receptor-like kinase BAM3

Antia Rodriguez-Villalona,*, Stephen Depuydta, Luca Santuaria, Christian Hardtkea

University of Lausanne, Switzerland

Peptide signaling occupies central role in plant development, yet only few concrete examples of receptor-ligand pairs that act in the context of specific differentiation processes have been described. In this work, we report that second-site null mutations in the Arabidopsis leucine-rich repeat receptor-like kinase gene barely any meristem 3 (BAM3) perfectly suppress the postembryonic root meristem growth defect and the associated perturbed protophloem development of the brevis radix (brx) mutant. The roots of bam3 mutants specifically resist growth inhibition by the CLAVATA 3/endosperm surrounding region 45 (CLE45) peptide ligand. Wild-type plants transformed with a construct for ectopic overexpression of CLE45 could not be recovered, with the exception of a single severely dwarfed and sterile plant that eventually died. By contrast, we obtained numerous transgenic bam3 mutants transformed with the same construct. These transgenic plants displayed a wild type phenotype however, supporting the notion that CLE45 is the likely BAM3 ligand. The results correlate with the observation that external CLE45 application represses protophloem differentiation in wild type, but not in bam3 mutants. BAM3, BRX and CLE45 are expressed in a similar spatio-temporal trend along the developing protophloem, up to the end of the transition zone. Induction of BAM3 expression upon CLE45 application, ectopic overexpression of BAM3 in brx root meristems and laser ablation experiments suggest that signaling homeostasis through intertwined regulatory activity of BRX, BAM3 and CLE45 guides the proper transition of protophloem cells from proliferation to differentiation, which in turn determines post-embryonic growth capacity of the root meristem. EMBO Fellows' Meeting, 13 - 16 June 2013, Heidelberg

Deep sequencing the RNA encapsidated by viruses: mapping of RNA recombination; discovery of functional motifs; and packaging of host RNAs including retrotransposons

Andrew Routha,*, Johnson Johna

The Scripps Research Institute, La Jolla, USA

RNA recombination within viral genomes is a powerful driving force behind the evolution and adaption of RNA viruses and has been attributed to be the source of outbreaks of new virus strains including Echovirus and Poliovirus as well as the emergence of entirely new viruses such as SARS-CoV. Using Deep Sequencing (RNAseq) we have set out to analyse the genomic polymorphism and quasi-species present within an array of eukaryotic +ssRNA viruses, including Flock House Virus, Cricket Paralysis Virus, Sindbis Virus and Human Rhinovirus.

We present a method to map viral RNA recombination events with single-nucleotide precision and have applied this to reveal prolific viral RNA recombination providing a detailed and quantitative description of the complex mutational landscape of the transmissible viral genome. Moreover, as recombination events that remove functional motifs are negatively selected, the distribution of recombination events reveals the genomic motifs required for viral replication and encapsidation. Consequently, functional viral motifs can be discovered de novo without the requirement for prior characterisation of the viral lifecycle.

In addition to their viral genomes, we found that some viruses also package host mRNAs, rRNAs, non-coding RNAs and retrotransposons. The packaging of these host RNAs elicits the possibility of horizontal gene transfer between hosts that share a viral pathogen. EMBO Fellows' Meeting, 13 - 16 June 2013, Heidelberg

Generation and modulation of B-cell memory in human volunteers subjected to experimental malaria infection

Anja Scholzena,*, Anne C. Teirlincka, Wiebke Nahrendorfa, Else M. Bijkera, Robert W. Sauerweina, et al.

Department of Medical Microbiology, Radboud University Nijmegen Medical Centre, Nijmegen, The Netherlands

Naturally-acquired immunity to the malaria parasite Plasmodium falciparum (Pf) is thought to be inefficient and short-lived. Impaired immunological memory is partly due to immune evasion strategies of Pf, but there is also evidence that the parasite actively modulates the immune system. A striking feature in individuals from malaria-endemic areas is the expansion of a rare, novel B-cell subset, termed atypical memory B-cells (MBCs). The mechanisms underlying their expansion and their function in malaria-infection remain unknown. We analyzed the acquisition of antigen-specific MBCs and the modulation of B-cells in the unique setting of controlled malaria-infection in human volunteers.

Project 1: We analyzed the human B-cell compartment in blood samples collected prior to, during and after a primary and low-grade malaria- infection. Atypical MBCs were expanded and proliferating immediately after parasite exposure. Their proliferation associated with elevated levels of B-cell activating factor, a key cytokine driving B-cell differentiation. After resolved infection, however, atypical MBC proliferation and proportions were comparable to pre-infection levels.

Project 2: The acquisition of antigen-specific MBCs was analyzed in volunteers subjected to multiple Pf exposures under curative drug cover, which efficiently induces protection from challenge infection. Pf-specific MBCs were efficiently and step-wise generated by this immunization regimen, and stably maintained. In this setting, we also observed repeated expansion of atypical MBCs, but they did not accumulate over time.

Project 3: To get further insights into the role and function of atypical MBCs, we analyzed their phenotype in human blood and spleen. They were phenotypically clearly distinct from other B-cell subsets, expressing a unique pattern of receptors mediating B-cell survival, activation, migration and T-cell interaction. The strong similarity of blood and spleen atypical MBCs and the much greater absolute number of this rare subset in spleen now enables us to perform more in-depths functional analysis of these cells, which is currently underway. EMBO Fellows' Meeting, 13 - 16 June 2013, Heidelberg

A steroid receptor-microRNA switch regulates longevity in response to germline ablation

Yidong Shena,*, Adam Antebia, et al.

Max Planck Institute for Biology of Ageing, Cologne, Germany

While the gonad primarily functions in procreation, it also affects animal longevity. Ablation of germline stem cells in C. elegans or D. melanogaster significantly extends lifespan, suggesting that gonadal longevity could be evolutionarily conserved. This longevity depends on the presence of somatic gonad and is therefore not a simple tradeoff between fertility and survival but under the control of careful signaling. Our results show that germline ablation actively triggers the production of dafachronic acids (DAs) production, which switches on a molecular module of DAF-12/NHR steroid receptor and two microRNAs of let-7 family. The microRNA targets, AKT-1 and LIN-14, are subsequently inhibited to extend lifespan through enhanced DAF-16/FOXO activity. Notably, the core components of this steroid receptor-microRNA switch are evolutionarily conserved and also regulate developmental progression in worm larvae. Therefore, these studies suggest that metazoan lifespan is coupled to the gonad through elements of an early-life developmental timing switch. EMBO Fellows' Meeting, 13 - 16 June 2013, Heidelberg

Polymerase epsilon nonessential subunits prevent toxic recombination during DNA replication

Jennifer Svendsena,*, Jillian Youdsa, Simon Boultona

DNA Damage Response Laboratory, Clare Hall, London Research Institute, South Mimms, EN6 3LD, UK

Of paramount importance to organism survival is the faithful duplication of a cells genetic material through the concerted action of DNA replication and repair factors. Using a genome-wide RNAi screen we identified a genetic interaction between the DNA repair helicase RTEL-1 and the nonessential subunit of the leading strand replicative polymerase epsilon, POLE-4. RTEL-1 is an anti-recombinase that prevents inappropriate recombination by unwinding D-loop intermediates during DNA repair, meiosis, and at telomeres. POLE-4 is a nonessential subunit of polymerase epsilon that promotes polymerase processivity. Our studies reveal that C.elegans pole-4 mutants are 100% embryonic lethal when combined with the rtel-1 mutation. One possibility is that the absence of POLE-4 decreases the processivity of polymerase epsilon leading to an increased number of aberrant fork structures that must be counteracted by RTEL-1. In this case, loss of both POLE-4 and RTEL-1 would lead to persistent aberrant replication fork structures that may be processed by structure-specific nucleases. Consistent with this hypothesis pole-4; rtel-1 lethality is associated with a significant decrease in DNA replication within the mitotic zone of the worm germline. Coincident with the decrease in DNA replication is activation of the replication checkpoint and the accumulation of spontaneous double strand breaks, DSBs, marked by RAD-51 and RPA. Furthermore the accumulation of DSBs in pole-4; rtel-1 worms is dependent on the structure-specific nuclease MUS-81 and the RAD-51 paralog RFS-1 suggesting that DSBs occur at stalled replication forks following processing by MUS-81. These data suggest that POLE-4 and RTEL-1 function to prevent the accumulation of toxic recombination intermediates during DNA replication. Recent generation of a pole-4-/- mouse model has revealed insight into the function of POLE-4 in development and ageing. Future plans will be aimed at further elucidating the relationship between POLE-4 and disease and the genetic interaction between pole-4 and rtel-1 in mice. EMBO Fellows' Meeting, 13 - 16 June 2013, Heidelberg

Chromatin proteins and sequence evolution in eukaryotes and prokaryotes

Tobias Warneckea,*, Fran Supeka, Ben Lehnera, Marc Facciottib, Corey Nislowc, et al. a Centre for Genomic Regulation, Barcelona, Spain b UC Davis Genom Center, CA, USA c University of British Columbia, Vancouver, Canada

What is the relationship between genetic and epigenetic change? Do epigenetic changes simply reflect changes that occurred at the genetic level? Or may epigenetic features prompt such changes in the first place (e.g. by affecting mutation dynamics and DNA repair)?

The presence of nucleosomes, for example, can promote or impede the formation of DNA lesions or interfere with efficient DNA repair, so that some mutations may occur more, others less frequently in a nucleosomal context. Conversely, evolution at the sequence level can affect nucleosome architecture. Notably, as nucleosomes form preferentially on more bendable DNA, mutations that render the DNA template less bendable can prompt the nucleosome to shift to a position that is more conducive to nucleosome formation.

In short, nucleosomes (and other DNA-binding proteins) can both affect and reflect sequence evolution. But what is the dominant direction of causality in genetic versus epigenetic change? And what are the implications of chromatin architecture for mutability and longer-term patterns sequence evolution?

Drawing on data from multiple eukaryotes but also bacteria and archaea, which encode their own unique complements of structural DNA-binding proteins, and summarizing results obtained over the last three years, I will dissect the complex relationship between sequence evolution and chromatin architecture and argue that the way genomes are packaged fundamentally biases trajectories of sequence evolution. EMBO Fellows' Meeting, 13 - 16 June 2013, Heidelberg

Dispersed PSM oscillator assay reveals the cell autonomous nature of the segmentation clock

Alexis Webba,*, Annelie Oswalda, Laurel Rohdeb, Daniele Soroldonib, Andrew Oatesb a MRC National Institute for Medical Research, London, UK b MRC National Institute for Medical Research, London, UK University College London, UK

Most current models of the segmentation clock postulate that the molecular oscillations driving somite formation are autonomous at the level of single cells, without the input of their neighbors or other tissues. But this assumption has not been explicitly tested and theories exist in the literature that can reproduce the observed tissue-level patterning without autonomous oscillators. We have developed a series of assays to systematically test whether the zebrafish segmentation clock contains an autonomous oscillator at the level of a single PSM cell. Using our cyclic gene reporter line, her1-YFP, we are able to monitor oscillations in gene expression in transplanted cells within the embryo, PSM explants, tailbud pieces, and dispersed and fully isolated cells. We have quantified properties that characterize oscillators, such as period, amplitude, number of cycles, and signal quality factor, from individual PSM cells in culture. We are also using single cells to directly address questions of molecular clock function that were previously inaccessible in the intact embryo. Our studies reveal the ability of single zebrafish PSM cells to oscillate autonomously in culture, independent of neighboring cells or tissues. They also demonstrate the tractability of the single cell model system for investigating the segmentation clock. EMBO Fellows' Meeting, 13 - 16 June 2013, Heidelberg

The Cotranslational Function of Ribosome-Associated Hsp70 in Eukaryotic Protein Homeostasis

Felix Willmunda,*, Marta DelAlamob, Sebastian Pechmannb, Taotao Chenb, Judith Frydmanb, et al. a Stanford University, USA/University of Kaiserslautern, Germany b Stanford University, USA

During protein biosynthesis, the processing and maturation of newly made polypeptides is performed by a highly complex machinery. As nascent chains emerge from the ribosome, they require a lineup of specific factors for further maturation and distribution throughout the cell. In contrast to the understanding of de novo folding in prokaryotes, little is know about the principles and selectivity of nascent chain binding factors in the cytosol of eukaryotes. We applied a system-level approach to analyze the role of the Hsp70 molecular chaperones in co-translational nascent chain binding. This enabled us to identify and to define the broad substrate set of the ubiquitous yeast Hsp70 homologue, Ssb1/2. We could determine for the first time, the cellular distribution and sequence characteristics of Hsp70-bound nascent chains. Our analysis revealed an important function of Hsp70 in protecting those emerging nascent polypeptides threatened for misfolding and aggregation by their slow co-translational folding kinetics. Thus, our system-level approach provides a powerful tool to interrogate protein homeostasis networks as they occur at the eukaryotic ribosome. EMBO Fellows' Meeting, 13 - 16 June 2013, Heidelberg

Impact of miRNAs on bone development

Nadine Wittkoppa,*, Suzanne W. Hoogstrateb, Lucas J. Kaaijc, Anneloes Dummera, Rene F. Kettinga, et al. a IMB, Mainz, Germany b Wageningen University, The Netherlands c Hubrecht Institute, Utrecht, The Netherlands

Bones give stability and at the same time flexibility to our bodies. To achieve those properties, a spatial and temporal developmental balancing is needed between mainly two cell types shaping the bone: the osteoblasts that secrete the extracellular matrix, which will later on mineralise and the osteoclasts, which resorb this matrix. The process of bone remodelling is active not only during development, but also during an organism’s adult lifespan. miRNAs are well-known players in development and fine-tune gene expression on a post-transcriptional level. Their role in bone formation has been investigated, but only on a piece- by-piece (bit by bit) basis and mainly in cell culture. We have aimed to unravel this gene regulatory network in a more systematic approach. Therefore, we have isolated early and terminal- differentiated/ mineralised osteoblasts during development. Employing a set of reporter lines we were able to distinguish and label the various stages and isolate those cells from whole zebrafish larvae (Danio rerio). We assessed the miRNA content of early osteoblasts as well as their mRNA expression profile. To complete the picture we generated Maternal Zygotic dicer mutants, which are deprived of almost all mature miRNAs and compared their expression profile to early and mineralised wildtype osteoblasts. This will help us to determine those mRNAs that are targeted by the identified miRNAs We found almost 100 annotated miRNAs enriched in early stage osteoblasts. From those we verified the expression of a couple in the bone forming tissue using In Situ hybridisation and knocked down their expression using morpholinos. So far we were able to observe a phenotype for two miRNA knockdowns; one of the phenotypes is indeed related to bone formation, the other one can be surprisingly observed in the lymphatic system. Currently, we are systematically testing candidate genes that are likely targeted by these miRNAs. EMBO Fellows' Meeting, 13 - 16 June 2013, Heidelberg

Ribosomal oxygenases (ROXs) - a new enzyme family of ribosomal regulators

Alexander Wolfa,*, Wei Gea, Tianshu Fengb, Peter J Ratcliffeb, Christopher J Schofielda a Chemistry Research Laboratory, University of Oxford, UK b Henry Wellcome Building for Molecular Physiology, University of Oxford, UK

Oxygen (O2) supply is crucial for most cells but hypoxia (low oxygen levels) occurs under physiological as well as various pathological conditions, like tumour growth, stroke and inflammation. A well-known cellular adaption to low O2-levels is the up-regulated expression of hypoxia response genes, mediated by the transcription factor HIF (hypoxia-inducible factor). In order to prevent HIF up-regulation under normoxic conditions cells have developed control mechanisms, including enzymes of the 2-oxoglutarate (2OG) dependent oxygenase superfamily. 2OG oxygenases catalyse hydroxylation reactions by transferring molecular O2 to a substrate.

We have identified a new set of 2OG oxygenases, the ROXs (ribosomal oxygenases), which target the ribosome. These ROXs modify ribosomal proteins posttranslationally by hydroxylating specific amino acids in the center of the ribosome. We have found ROX-catalysed modifications in human, yeast and E.coli ribosomes, indicating that this is a well-conserved process of protein synthesis regulation.

Ribosome action translates mRNA code into the amino acid sequence of a protein. In the decoding center (DC) of the ribosome mRNA and the appropriate aminoacyl-tRNA molecule bind via codon- anticodon recognition. Subsequently the amino acid is transferred to the growing peptide chain in the peptidyl transferase center (PTC) of the ribosome. ROX-catalysed modifications occur on Arg-, His- and Pro-residues facing either the PTC or the DC and can thereby affect translation. Altered translational accuracy in hypoxia for instance is due to a lack of prolyl hydroxylation in the DC.

The ribosomal oxygenases are a new subgroup of 2OG oxygenases having the potential for translational regulation. In E.coli the ycfD protein catalyses arginyl hydroxylation of the ribosomal protein Rpl16. Deletion of ycfD slows growth. Reduced expression of the human ycfD-homologs MinA53 and NO66 inhibits cell proliferation and both proteins are highly expressed in several cancers. abstracts | posters EMBO Fellows' Meeting, 13 - 16 June 2013, Heidelberg

Making sense of sensory stimuli on millisecond time scale

Nixon Abrahama,*, Alan Carletona

Laboratory of Sensory Perception and Plasticity, Geneva, Switzerland

The behavioral response to a sensory stimulus is preceded by the detection and discrimination processes. The temporal evolution of discrimination process of different odorants has been shown to be depending on the representation of stimuli in the olfactory bulb (OB) (Abraham et al., 2004) and the refinement of stimulus by the inhibitory circuits of OB (Abraham et al., 2010). These observations clearly show that discrimination is a complex process involving the evaluation of different properties of stimuli. The question if this detailed stimulus evaluation is essential for the detection process remains to be addressed. Here we investigate the evolution of temporal scales of detection and discrimination processes while the animal is in learning process of novel olfactory stimuli. Mice were trained, under head restrained conditions, on a go/no-go operant conditioning task to learn different odorants of varying complexity. Based on the performance levels of animals we classified the learning period as different stages, poor (≤ 60%), average (>60 and <80%) and good (≥80%) learning and analyzed the evolution of detection and discrimination times during these different epochs of learning. Even though animals learned all the odorants including enantiomers with very high accuracy, they took longer time periods to discriminate complex odorants compared to simple odorants, establishing the stimulus dependent discrimination times using a behavior read out towards positively rewarded odorants. We then asked if this reward value is analyzed by the animal before responding to a stimulus by measuring the detection times towards rewarded and non-rewarded trials. While monitoring the mice’ behavioral response on millisecond time scale we found out that the detection of both simple and complex stimuli precedes the real discrimination process even at highly learned stages. Therefore we conclude that mice need additional tens of milliseconds to evaluate the reward value of the stimuli after the detection process. EMBO Fellows' Meeting, 13 - 16 June 2013, Heidelberg

The cellular basis of visual motion detection in the fly optic lobe

Alexander Arenza,*, Alexander Borsta

Max-Planck-Institute for Neurobiology, Martinsried, Germany

Visual motion as a result of ego-motion (optic flow) is a crucial sensory stimulus for flight control in flies. While photoreceptors only respond to changes in brightness within their receptive fields irrespective of visual motion, large-field tangential cells in the lobula plate (LPTCs) of the fly optic lobe respond primarily to visual motion in a direction specific way. This feature extraction of visual motion direction appears to be achieved along processing lines comprising only a handful of steps. Based on anatomical evidence and genetic inactivation studies several neuronal cell types required have been identified, and it has emerged that two parallel processing streams exist, splitting visual information in an ON and an OFF pathway of visual motion detection.

However, how the computation and feature extraction is performed on a cellular basis remains largely unexplored, mainly due to the small size of the columnar elements. Here we address this question at the level of the presumed input elements of LPTCs, T4 and T5 cells. We performed whole-cell patch-clamp recordings from T4 and T5 cells in the optic lobes of Drosophila melanogaster and Calliphora vicina in order to determine the cellular and synaptic mechanisms involved in the processing of visual motion information. EMBO Fellows' Meeting, 13 - 16 June 2013, Heidelberg

Unlimited in vitro expansion of adult bi-potential pancreas progenitors

Paola Bonfantia,*, Meritxell Huchb, Sylvia F. Bojb, Harry Heimberga, Hans Cleversb, et al. a Diabetes Research Center, Vrije Universiteit Brussel, Belgium b Hubrecht Institute for Developmental Biology and Stem Cell Research, Utrecht, Netherlands

Novel cell therapy strategies in diabetes would greatly benefit from the availability of beta cell stem/progenitor cells. Lgr5 marks adult stem cells in multiple adult organs and is a receptor for the Wnt-agonistic R-spondins (RSPOs). Intestinal, stomach and liver Lgr5+ stem cells grow in 3D cultures to form ever-expanding organoids, which resemble the tissues of origin. Wnt signaling is inactive and Lgr5 is not expressed under physiological conditions in the adult pancreas. We report here that the Wnt pathway is robustly activated upon injury by Partial Duct Ligation (PDL), concomitant with the appearance of Lgr5 expression in regenerating pancreatic ducts. In vitro, duct fragments from mouse pancreas initiate Lgr5 expression in RSPO1-based cultures, and develop into budding cyst-like structures (organoids) which expand extensively for >40 weeks. Prospectively isolated duct cells can also be cultured into pancreatic organoids, containing Lgr5 stem/progenitor cells that can be clonally expanded. Pancreas organoids can be induced to differentiate into duct as well as endocrine cells, thus proving their bi-potentiality. Adult pancreas organoid cultures may represent a novel approach in regenerative medicine. EMBO Fellows' Meeting, 13 - 16 June 2013, Heidelberg

High resolution genome-wide analysis of small RNA mediated translational regulation in Chlamydomonas reinhardtii

Betty Chunga,*, Baulcombe Davida

Department of Plant Sciences, University of Cambridge, UK

The diverse natural roles for small RNAs in eukaryotes range from defense against viruses to the regulation of gene expression and chromosome structure via mechanisms affecting transcription, RNA stability and translational control. These processes are primarily guided by the evolutionarily conserved protein Argonaute. In plants, evidence for small-RNA-mediated translational control has emerged recently but little is known about the full complement of mRNAs that are affected or whether the mechanisms are the same as those that operate in animal systems. To address these issues, I am implementing ribosome profiling and HITS-CLIP on Argonaute-associated mRNAs in the green alga Chlamydomonas reinhardtii. It is not only the most established unicellular model organism for studying small RNA mediated pathways, but it can also be cultured synchronously and has simple genetics in the haploid vegetative phase, thus aiding follow-up genetic studies. EMBO Fellows' Meeting, 13 - 16 June 2013, Heidelberg

Co-transcriptional Drosha cleavage in terminal exon of liver specific lncRNA couples transcriptional termination with high mir-122 expression

Ashish Dhira,*, Nick Proudfootb, Catherine Joplingc a Sir William Dunn School of Pathology, University of Oxford, South Parks Road, Oxford, OX1 3RE, UK b Sir William Dunn School of Pathology, University of Oxford, South Parks Road, Oxford, OX1 3RE, UK c RNA Biology Group, Centre for Biomolecular Sciences, University of Nottingham, University Park, Nottingham NG7 2RD, UK

Long non coding RNA (lncRNA) that contain pre-miRNA are of obvious functional importance. Although only a minority of miRNA derive from exons of lncRNA host genes, highly expressed miRNA often derive from lncRNA terminal exons. Here, we present data for the liver specific miRNA, mir-122 that represents ~50% of the total miRNA population in liver. This highly expressed miRNA is located in the terminal exon of a ~5kb lncRNA. Nucleo-cytoplasmic fractionation followed by RT-PCR demonstrated the retention of this lncRNA in the nucleus. Furthermore, northern blot analysis revealed the presence of both spliced and unspliced transcripts that have non-canonical 3’ends defined by Drosha cleavage at the pre-miRNA stem loop region, as validated by RNase protection analysis. Consequently, these lncRNAs are non-polyadenylated and rapidly turned over as demonstrated by transcription block experiments with actinomycin D treatment. Interestingly, upon Drosha and DGCR8 knock down, mir-122 primary transcripts were extended at their 3’ ends and 3’ end processed at a canonical poly (A) signal (PAS). Deletion of the mir-122 stem region in cis confirmed the microprocessor knock down effect. Finally, ChIP analysis demonstrated low RNA polymerase II CTD Ser2P levels across the mir-122 lncRNA gene as compared to Ser5P levels. This suggests a non-productive environment for PAS recognition and illustrates that processing of terminal exonic pre-miRNAs can facilitate transcription termination. Altogether, our studies show that this lncRNA skews the RNA processing machinery towards the dedicated production of mir-122 resulting in very high levels of expression. EMBO Fellows' Meeting, 13 - 16 June 2013, Heidelberg

Illuminating the role of inhibitory microcircuits in higher-order olfactory processing in zebrafish

Thomas Franka,*, Yan-Ping Zhang Schaerera, Rainer W Friedricha

Friedrich Miescher Institute for Biomedical Research, Basel, Switzerland

The brain creates dynamic representations of the sensory environment by extracting stimulus features at early processing stages and synthesizing more abstract object representations in higher brain areas. However, the nature of the underlying neuronal circuits and their strategies to perform this task are still poorly understood. This research project aims at identifying neuronal computations representative for basic cortical circuits and uncovering the underlying cellular mechanisms. To this end, we visualize and manipulate different types of interneurons (INs) in the posterior zone of the dorsal telencephalon (Dp) of zebrafish, which is homologous to olfactory cortex in mammals and assumed to be involved in olfactory object representations and odor memory. Using transgenic marker lines, we measure odor responses of different IN types by 2-photon Ca2+ imaging and targeted whole-cell recordings. We analyze the synaptic architecture of Dp using electrophysiology and optogenetics, and derive hypotheses for the function of different IN types in the context of the neural circuit. These hypotheses are subsequently tested by reversibly silencing INs in transgenic lines, and the resulting cell-type specific effects are examined at the level of individual neurons and large-scale activity patterns. The results provide insights into canonical computations performed by basic cortical circuits. EMBO Fellows' Meeting, 13 - 16 June 2013, Heidelberg

RNA binding and RNA remodeling activities of the half-a tetratricopeptide (HAT) protein HCF107 underlie its effects on gene expression

Kamel Hammania,*, William B. Cookb, Alice Barkanc a CNRS, Institut de Biologie Moléculaire des Plantes, University of Strasbourg, France b Department of Biology, Midwestern State University, Wichita Falls, TX 76308 c Institute of Molecular Biology, University of Oregon, Eugene, OR 97403

The half-a-tetratricopeptide repeat (HAT) motif is a helical repeat motif found in proteins that influence various aspects of RNA metabolism, including rRNA biogenesis, RNA splicing, and polyadenylation. This functional association with RNA suggested that HAT repeat tracts might bind RNA. However, RNA binding activity has not been reported for any HAT repeat tract, and recent literature has emphasized a protein binding role. In this study, we show that a chloroplast-localized HAT protein, HCF107, is a sequence-specific RNA binding protein. HCF107 consists of 11 tandem HAT repeats and short flanking regions that are also predicted to form helical hairpins. The minimal HCF107 binding site spans ∼11 nt, consistent with the possibility that HAT repeats bind RNA through a modular one repeat–1 nt mechanism. Binding of HCF107 to its native RNA ligand in the psbH 5’ UTR remodels local RNA structure and protects the adjacent RNA from exonucleases in vitro. These activities can account for the RNA stabilizing, RNA processing, and translational activation functions attributed to HCF107 based on genetic data. We suggest that analogous activities contribute to the functions of HAT domains found in ribonucleoprotein complexes in the nuclear– cytosolic compartment. EMBO Fellows' Meeting, 13 - 16 June 2013, Heidelberg

Epithelial polarity factors are essential for the organization of muscle fibers

Aynur Kaya-Copura,*, Frank Schnorrera

Max Planck Institute of Biochemistry, Martinsried, Germany

Higher animals owe all their movements to their muscles. Muscle fibers achieve their contractile properties by subdividing their cytoplasm into a highly organised array of myofibrils, T-tubules and nuclei. Disorganisation of any of these components leads to many skeletal and cardiac myopathies. Similar to vertebrates adult Drosophila possess various muscle types such as heart, striated body muscles, and specialized indirect flight muscles enabling fast wing oscillations. Most structural components important for formation or function of myofibrils and sarcomeres are conserved from flies to vertebrates. Therefore we chose Drosophila for a systematic genetic analysis of muscle fiber morphogenesis in vivo, with a particular focus on myofibrillogenesis. To identify key regulators, we bioinformatically selected all genes with putative cytoskeletal or sarcomere related functions, and combined these with genome-wide RNAi data for muscle function [Schnorrer F et al 2010]. We analyzed muscle fiber phenotypes after muscle-specific gene knockdown of 195 candidate genes using fluorescently tagged sarcomeric markers. We focused on fibrillar indirect flight muscles and the morphologically distinct tubular leg and abdominal muscles. This screen identified many novel factors required for proper organisation of muscle fibers with some of them being exclusively important for formation of either tubular or fibrillar muscles.

Muscle-specific knockdown of an uncharacterized gene exclusively affects indirect flight muscles resulting in fewer flight muscles and flightless animals. The closest vertebrate homolog of this gene is required for establishment of polarity in epithelia by regulating vesicular trafficking. We find that our protein of interest also localizes to vesicular structures pointing to a similar function in muscle fiber organisation. Strikingly we discovered a role for various other genes implicated in epithelial polarity for correct flight muscle morphogenesis. Therefore, we propose a novel role for epithelial polarity proteins in the development and organization of muscle fibers. EMBO Fellows' Meeting, 13 - 16 June 2013, Heidelberg

Inter-species variation in transcription factor occupancy for essential developmental regulators

Pierre Khoueirya,*, Eileen Furlonga

European Molecular Biology Laboratory (EMBL), Heidelberg, Germany

Gene regulatory networks and environmental inputs determine cell fate choices during embryogenesis. Cis-Regulatory Modules (CRMs), the docking platforms for transcription factors, are at the heart of regulatory networks. They regulate gene expression at early developmental stages and later during differentiation processes. Genome-wide identification of in-vivo functional CRMs remains a challenge. Using Drosophila melanogaster as a model organism, our lab lately showed the ability to enhance spatio-temporal CRM activity prediction using in vivo transcription factor binding data for a set of 5 myogenic factors (Twist, Mef2, Tinman, Bapipe and Biniou) implicated in mesoderm specification (1). This approach unveiled a large number of CRMs linked to a wide range of putative mesodermal genes.

My project aims are 1) to filter the mesoderm network from likely non-functional CRMs and 2) to investigate the degree of evolutionary divergence in the targets and combinatorial binding of the five mesodermal factors in two Drosophila species.

For this, I am performing ChIP-seq analysis for the five myogenic factors on Drosophila virilis, a distantly related species to D. melanogaster (60 Million years). These experiments are carried out on five consecutive time points spanning mesoderm specification. Prior to ChIP, we generated D. virilis specific antibodies that showed the expected spatio-temporal activity in immunostaining assays. In parallel, I expanded and staged D. virilis embryos to match the corresponding stages in D. melanogaster. I then collected and fixed embryos, extracted chromatin and performed ChIP experiments. IP-purified DNA from different samples as well as input controls are then multiplexed for library preparation and subjected to sequencing with Illumina HiSeq. In parallel, I have begun to analyze the data obtained using a combination of in-house scripts and available tools for next generation sequencing analysis.

1: Zinzen, R.P., Girardot, C., Gagneur, J., Braun, M. & Furlong, E.E.M. Nature 462, 65-70 (2009). EMBO Fellows' Meeting, 13 - 16 June 2013, Heidelberg

Polarization of the Drosophila oocyte by a Cdc42-regulated actin cytoskeleton

Andrea Leibfrieda,*, Sandra Müllera, Anne Ephrussia

European Molecular Biology Laboratory, Heidelberg

The Drosophila oocyte is a polarized cell. Polarity establishment and maintenance is essential for correct development of the egg and future embryo. Microtubules are prominent regulators of polarity and were believed to mediate the establishment of polarity during oogenesis. The Par proteins Par-6, aPKC and Bazooka are needed to maintain oocyte polarity and localize to specific domains early in oocyte development. No upstream regulator or mechanism for localization of the Par proteins in the oocyte has been identified so far. We have analyzed the role of the small GTPase Cdc42 in oocyte polarity. We show that Cdc42 is required to maintain oocyte fate, which it achieves by mediating localization of Par proteins at distinct sites within the oocyte. We establish that Cdc42 localization itself is polarized to the antero-lateral cortex of the oocyte and that Cdc42 ensures the integrity of the oocyte actin network. Disrupting this network with Latrunculin A phenocopies loss of Cdc42 or Par protein function in early stages of oogenesis. Finally, we show that Cdc42 and aPKC interact genetically with regard to oocyte polarity and that loss of Par proteins reciprocally affects Cdc42 localization and the actin network. These results reveal a mutual dependence between Par proteins and Cdc42 for their localization, regulation of the actin cytoskeleton and, consequently, for the establishment of oocyte polarity. Our data allow ordering the complex sequence of microtubule and actin dependent events required for early oocyte polarity and its maintenance. Cdc42 and actin dominate in the establishment and maintenance of polarity in the young oocyte, whereas microtubules act in oocyte specification and late polarity events. EMBO Fellows' Meeting, 13 - 16 June 2013, Heidelberg

Regulation of pol III transcription by Id2 and E47 proteins

Dritan Likoa,*, Nikiforos Spandidosa, Robert Whitea, Owen Sansoma

Beatson Institute for Cancer Research, Glasgow, UK

RNA polymerase III (pol III) is responsible for transcribing short untranslated sequences, such as tRNAs and 5S rRNA. Transcription by pol III is vital for cellular maintenance and involves a number of intrinsic transcription factors, which recruit the polymerase and others such as c-myc, and HDACs that interact with members of the pol III machinery or modulate chromatin marks in order to influence pol III trancription. Furthermore a number of growth related kinases, oncoproteins and tumor suppressors directly influence pol III. Indeed a number of pol III transcripts are over-represented in cancer samples. Id2 is part of the Id family of proteins that are HLH motif-containing transcription factors. Ids interacting with factors such as MyoD and E47, inhibit differentiation and promote cell proliferation. They are highly expressed in rapidly proliferating cells, including tumour cells. A protein inhibited by Id dimerization is E47. It contains a bHLH domain and binds DNA to promote tissue specific differentiation. To date, all targets of Id2 and E47 regulation are genes encoding protein. Work presented below shows that attenuating levels of these proteins influences expression of short non-coding RNAs such as tRNA and 5S rRNA. Ids stimulate expression of tRNA and 5S rRNA, while E47 represses their expression. ChIP experiments position Id2 and E47 at tDNA and 5S rRNA genes arguing for a direct link between Id2/E47 and pol III transcription. Moreover, upon Id2 depletion promoter occupancy of pol III and factors at tDNAs genes is reduced. The opposite effect is seen upon E47 depletion. Taken together, these observations unearth a possible direct link between Id2, E47 proteins and pol III transcription machinery. Given that Id2, E47 and pol III are involved in tumorigenesis, it is conceivable that part of the effects Id2/E47 have on tumorigenesis are dependent on pol III and vice versa. EMBO Fellows' Meeting, 13 - 16 June 2013, Heidelberg

PknG mediated signalling pathways in Mycobacterium tuberculosis

Maria Natalia Lisaa,*, Nathalie Barilonea, Pedro Alzaria

Unité de Microbiologie Structurale, Institut Pasteur, Paris, France.

Mycobacterium tuberculosis (Mtb) is the causative agent of tuberculosis and is still a major world health problem. During its life cycle Mtb goes through distinct replicative stages and is also capable of lying in a dormant state for several years. Then, Mtb bacilli must possess efficient signalling systems to sense the environment and to adapt the bacterial physiology accordingly. Structural information about these mechanisms will thus contribute to the development of new drugs against Mtb.

Mtb possess eukaryotic-like serine/threonine protein kinases that regulate metabolic processes and the interaction with the host (Cole ST et al, 1998, Nature; Sassetti CM et al, 2003, Mol Microbiol). Among them, protein kinase G (PknG) is essential for mycobacterial survival inside macrophages (Walburger A et al, 2004, Science) and also controls the glutamate metabolism by regulating downstream partners (O'Hare HM et al, 2008, Mol Microbiol). Then, PknG is an attractive candidate for drug target against Mtb, and we are conducting structural studies to better understand the molecular determinants of the enzyme activity. PknG has a unique multidomain topology, with a central kinase domain flanked by N- and C-terminal rubredoxin and tetratrico-peptide repeat domains, respectively. Several high resolution X-ray crystal structures have been obtained of Mtb-PknG and also the homologous kinase from Corynebacterium glutamicum. All the obtained structures reveal most enzyme active-site signature elements adopting conformations that are characteristic of an active protein kinase (Huse M and Kuriyan J, 2002, Cell). However, a tilted and rotated orientation of helix alpha-C (a known hotspot for regulation) precludes the formation of a conserved and essential Lys-Glu ionic at the active site of PknG.

After analysis of the data we conclude that the different crystal structures obtained for the Ser/Thr protein kinase PknG represent an auto-inhibited latent conformation of the enzyme. A hypothesis about how a conformational switch may occur will also be provided as part of the discussion. EMBO Fellows' Meeting, 13 - 16 June 2013, Heidelberg tRNA wobble uridine hypomodification decreases the decoding efficiency of cognate codons in vivo

Danny Nedialkovaa,*, Sebastian Leidela

Max Planck Institute for Molecular Biomedicine, Max Planck Research Group for RNA Biology, Münster, 48149, Germany

Nucleotide modifications in tRNA are ubiquitous in all domains of life and those in the anticodon are important for accurate codon recognition during translation. Thiolation at the 2-carbon (s2) of the wobble uridine (U34) base is universally conserved in three tRNA species - tE(UUC), tK(UUU), and tQ(UUG). In the cytoplasm of eukaryotes, U34 also carries a 5-methoxycarbonylmethyl group (mcm5). Aberrant U34 modification is associated with increased stress sensitivity in many organisms. In yeast, the phenotypes can be suppressed by overexpressing hypomodified tK(UUU) and tQ(UUG), but the underlying molecular events are unknown. To delineate them, we analyzed the in vivo roles of U34 modification in translation by deep sequencing of ribosome-protected mRNA fragments (ribosome profiling) in Saccharomyces cerevisiae. This approach allowed us to quantitatively compare transcriptome-wide ribosome occupancy in wild type and U34 thiolation- deficient yeast (ncs2Δ) grown in rich medium or after diamide-induced oxidative stress. Diamide treatment led to widespread changes in gene expression of a similar magnitude in both strains. Lack of U34 thiolation thus does not seem to compromise the cellular response to diamide, despite the hypersensitivity of ncs2Δ yeast to this compound. The number of transcripts with altered ribosome loading in ncs2Δ cells, however, was larger in the expression response elicited by diamide. Strikingly, we detected significantly higher ribosome occupancy for codons read by the hypomodified tRNAs in the putative ribosomal A site within ribosome-protected fragments from ncs2Δ. This effect was discernible both in cells grown in rich medium or subjected to diamide- induced oxidative stress and was accompanied by smaller increases of cognate codon occupancy also upstream of the A site. Our data provides the first in vivo evidence that U34 hypomodification is associated with translational slowdown at cognate codons in endogenous transcripts. We are currently investigating whether this phenomenon is conserved in C. elegans and what its impact is on protein abundance and function. EMBO Fellows' Meeting, 13 - 16 June 2013, Heidelberg

Silver birch (Betula pendula): a novel model tree for molecular genetics

Kaisa Nieminena,*, Juha Immanenb, Risto Hagqvista, Katri Kärkkäinena, Ykä Helariuttab a Finnish Forest Research Institute, Vantaa Research Unit, Vantaa, Finland b University of Helsinki, Department of Biosciences, Helsinki, Finland

The aim of our research is to understand molecular mechanisms controlling tree development; we will explore natural variation in forest trees to identify novel genetic regulators of this process. Our model organism is an important forestry tree, silver birch, whose small diploid genome is currently being sequenced. This tree is monoecious, and already young seedlings can be induced to flower under greenhouse conditions. Birch brings the power of inbreeding and short generation times into tree genetics, enabling exploitation of advanced crossing schemes for genetic analyses. The main focus of our study is a collection of naturally occurring tree mutants: a diverse set of trees with atypical wood quality, cambial activity, branching pattern, or secondary metabolite content. We are currently most advanced in characterisation of a “swirly birch” mutant, which has a striking growth pattern. Its stem and branches grow initially normally, but when they get longer they are not anymore able to stay upright, and instead begin to bend downwards. Excitingly, this tree has an intriguing stem phenotype, where the tension wood formation is mutated into a rotating, “swirly” pattern. We aim, through a whole genome sequencing approach, map the causative gene behind this phenotype, and study its function in transgenic trees. Besides its fascinating basic science aspect (what makes a tree a tree?), understanding molecular mechanisms regulating tree development has immense applied value. Historically, due to their large size and long generation times, trees have not been easily accessible for traditional breeding. Detailed knowledge about regulatory mechanisms controlling tree traits will provide us with tools for their domestication. Boosting efficient production of woody biomass in commercial forests is essential for sustainable management of natural resources. With birch as our model, our project represents a novel approach of tree genetics with potential for ground-breaking insights into tree development and breeding. EMBO Fellows' Meeting, 13 - 16 June 2013, Heidelberg

Bacteriophage ΦCbK hijacks the Caulobacter crescentus cell cycle machinery

Gaël Panisa,*, Laurence Théraulaza, Patrick Violliera

Institute of Genetics & Genomics in Geneva (iGE3), Switzerland

Bacteriophages engage in a Trojan horse-style biological warfare with their host: destroying from the inside out by appropriating the host’s major biosynthetic machineries. Caulobacter divides asymmetrically at each cell cycle, giving two progeny cells with distinct morphologies and fates: the sessile replicating stalked (St) cell and the adventurous quiescent swarmer (Sw) cell that resides in G1-like non-replicative state and must differentiate into the St cell before proceeding to division. Consequently, differentiation and chromosomal replication are coordinated temporally and spatially by successive transcriptional waves of master regulators as CtrA and GcrA that are active in Sw and St cell stages, respectively, that might be targeted by phages. Phage ΦCbK is a virulent double- stranded DNA phage with a flexible noncontractile tail (Order Caudovirales, Family Siphoviridae) and a prolate cylindrical head encasing a 205 kb genome. As ΦCbK infection is restricted to the Sw phase the lytic cycle of ΦCbK might be directly coordinated with the Caulobacter cell cycle. Characterizing the lytic cycle revealed a burst size of 13 phage progeny per cell and that the integrity of the host chromosome is maintained. Using ChIP-Seq and immunoblotting approaches, we identified that ΦCbK phage infection reprograms the host transcriptional pattern and prevents expression of the host master regulator GcrA. Interestingly, the ΦCbK genome encodes a GcrA ortholog suggesting that host GcrA is replaced with phage variant. By contrast, CtrA is still present in Sw cells during ΦCbK infection and disappears concomitantly with the beginning of phage replication. ChIP-seq revealed that CtrA binds multiple promoters genes thought to encode phage DNA replication factors such as DNApolI, DNApolIII, TerSL …). This finding overturns the current dogma that in the lytic cycle that phage only manipulates the host. Instead, the phage also exploits intrinsic regulatory cues of the host cell cycle for its own replication. EMBO Fellows' Meeting, 13 - 16 June 2013, Heidelberg

Molecular mechanisms of chromosome segregation during meiosis, a genome-wide approach

Silvia Polakovaa,*, Zsigmond Benkoa, Juraj Gregana

Max F. Perutz Laboratories, Department of Chromosome Biology, University of Vienna, Austria

Sexual reproduction depends on meiosis, the process which produces eggs and sperm cells. During meiosis, the number of chromosomes is halved. Although we understand certain aspects of meiosis, some of the key regulators of meiotic chromosome segregation are still missing and only now we do have tools to identify these proteins and to determine their function. I will present our systematic screen of S. pombe knock-out library to identify genes involved in meiotic chromosome segregation. I believe that it is important to understand how the halving of the chromosome copy number during meiosis is achieved, because defects in this process cause miscarriages, infertility and genetic diseases such as Down syndrome. EMBO Fellows' Meeting, 13 - 16 June 2013, Heidelberg

Serotonin promotes neurogenesis following spinal cord injury in adult zebrafish

Angela Scotta,*, Thomas Beckerb, Catherina Beckerb a Department of Biology, McMaster University, Hamilton, Canada b Centre for Neuroregeneration, University of Edinburgh, Edinburgh, UK

Neurogenesis, the birth of neurons, occurs within central nervous system (CNS) of all adult vertebrates. Until recently, adult neurogenesis was believed to only take place in specific regions of the vertebrate brain. It is now known that, contrary to mammals, adult neurogenesis occurs within the spinal cord of adult zebrafish following a spinal lesion. Lessons from these natural ‘regenerators’ will significantly help to uncover signals important for successful endogenous neural regeneration that are not present in mammals, and identify potential courses of therapy for spinal cord injury. To date, the molecular factors responsible for spinal neurogenesis within the injured spinal cord are largely unknown. Previous work has shown that, following spinal cord lesion, serotonergic neurons are able to regenerate through the lesion site and re-innervate the tissue below the injury. Given this, we investigated the possible neurogenic role serotonin may play within the injured spinal cord. Our study demonstrated that serotonin treatment significantly enhanced neurogenesis of spinal motor neurons following complete spinal cord transection in adult zebrafish. A pharmacological screen highlighted the potential role of the serotonin receptor 5-HT1A in the generation and differentiation of spinal motor neurons in zebrafish embryos. We confirmed, in adult zebrafish, that the expression of 5-HT1A was upregulated at the level of the lesion site and the receptor was expressed on the neural progenitor cells within the spinal cord. In addition, the enhanced expression of this receptor was significantly correlated to the successful recovery of swimming performance at 2 weeks and 6 weeks following spinal cord injury in these fish. All together, these findings offer significant insight into the molecular mechanisms involved in the stimulation of adult neurogenesis and functional recovery in vertebrates following spinal cord injury. EMBO Fellows' Meeting, 13 - 16 June 2013, Heidelberg

Community genomics reveals novel members and complex time-series dynamics in the human microbiome

Itai Sharona,*, Michael J. Morowitzb, Brian C. Thomasa, Elizabeth K. Costelloc, David A. Relmanc, et al. a Department of Earth and Planetary Science, University of California, Berkeley, USA b School of Medicine, University of Pittsburgh, USA c Department of Microbiology & Immunology, School of Medicine, Stanford University, USA

The human microbiome has been associated with various medical conditions and physiological processes critically important to human health. However, the microbial communities that make up the human microbiome consist of both closely related and novel species of bacteria, neither of which can be resolved or studied using existing methods. Here, I will present the results of our two recent studies achieved using novel methods developed for the reconstruction of complete genomes from mixed microbial community DNA sequencing (metagenomics), an approach we call community genomics. These methods were successfully used to study gut colonization in a premature infant, where we tracked community dynamics over time and revealed strain-level differences between key members of the community. Furthermore, in the adult gut microbiome we were able to recover and characterize complete genomes from several members of an uncultivated clade of non-photosynthetic, phylogenetically diverse Cyanobacteria. These genomes shed light on the evolution of Cyanobacteria, an important microbial phylum responsible for the oxygenation of the Earth’s atmosphere. EMBO Fellows' Meeting, 13 - 16 June 2013, Heidelberg

Deletion of cavin genes reveals tissue-specific mechanisms for morphogenesis of endothelial caveolae

Carsten Gram Hansena, Elena Shvetsb,*, Gillian Howardb, Kirsi Rientob, Benjamin Nicholsb a Sanford Consortium for Regenerative Medicine and UCSD, 2880 Torrey Pines Scenic Drive, La Jolla, California 92037, USA b MRC Laboratory of Molecular Biology, Cambridge, UK

Caveolae, flask-shaped invaginations of the plasma membrane, are especially abundant in endothelial cells and among their other proposed functions, are thought to play important roles in endothelial permeability and transcytosis. The cavin proteins represent a family of novel key components of caveolae. Importantly, cavins have differential tissue distributions and ability to bind each other. Thus, we hypothesize that the ability to form complexes is crucial for their function in-vivo. We find that cavins form high molecular weight complexes in tissues and that apparent size of these complexes varies between tissues, when lung and fat contain smaller size complexes, while heart and kidney tissues contain larger size complexes. We next analyzed the formation of caveolin or cavins complexes in caveolin 1, cavin 1, cavin 2 and cavin 3 gene knockout mice. Caveolin 1 was not responsible for cavins complex formation, while cavin 1 was essential for normal oligomerisation of caveolin 1. Deletion of cavin 3 was found to slightly reduce the size of cavin1 complex. Deletion of cavin 2, however, resulted in a marked increase in the size of cavin 1-containing complexes. Importantly, deletion of cavin 2, but not cavin 3 caused loss of endothelial caveolae in lung and adipose tissue, but has no effect on the abundance of endothelial caveolae in the heart and other tissues. This correlated well with tissue-specific changes of endothelial function in cavin 2 null mice. To gain further insight into the role of cavin 2 specifically in lung endothelial cells, we established cultures of endothelial cells and further confirmed that cavin 2 controls the size of cavin complexes, and acts to shape caveolae. Our data reveal that endothelial caveolae are unexpectedly heterogeneous, and identify cavin 2 as a key molecular determinant of this heterogeneity. EMBO Fellows' Meeting, 13 - 16 June 2013, Heidelberg

The role of core RNA splicing factors in spindle assembly

Magdalena Strzeleckaa,*, Rebecca Healda

University of California, Berkeley, USA

The mitotic spindle is an intricate macromolecular structure required for accurate distribution of genetic material during cell division. Most research in the past couple of decades has focused on the protein components of the spindle. Interestingly, our lab has shown that an RNA component is also essential for spindle integrity. Furthermore, a number of genome-wide screens have implicated RNA processing factors in regulation of mitotic events. Interestingly, TPX2, a spindle assembly factor has been found to co-purify with active spliceosome, a multicomponent complex catalyzing RNA splicing reactions. In order to investigate whether spliceosome components play a direct role in mitosis I have been using transcriptionally silent and metaphase-arrested Xenopus egg extracts to reconstitute spindle assembly and study mitotic RNAs. Strikingly, molecular and biochemical perturbations of spliceosome assembly and function led to defects in spindle integrity. Moreover, next generation sequencing of RNAs from Xenopus tropicalis egg extract and of RNAs co-immunoprecipitated with spliceosomal small nuclear ribonucleoproteins (snRNPs) revealed that intron-containing pre-mRNAs are present at metaphase, suggesting that their processing might be required for mitotic progression. However, translation inhibition does not recapitulate spindle integrity phenotype caused by spliceosome perturbation, indicating that spliceosome plays a role in mitosis in the context of non-coding RNAs. Interestingly, we find that intron-containing non-coding RNAs are also associated with spliceosomal snRNPs in metaphase arrested extracts. Our data support the direct involvement of spliceosomal components in mitosis and suggest that it occurs through non-coding RNAs in splicing dependent or independent manner. EMBO Fellows' Meeting, 13 - 16 June 2013, Heidelberg

Host-parasite interactions in an anciently asexual invertebrate

Christopher Wilsona,*, Lara Meadea, Grace Mastersb a Imperial College London, Ascot, UK b Cornell University, Ithaca, NY, USA

Sex and recombination entail multiple evolutionary and genetic costs, yet they are nearly ubiquitous in plants and animals, whereas obligately asexual lineages rapidly become extinct. One explanation for this puzzle is that recombination enables populations to keep pace with relentlessly coevolving parasites and pathogens, by facilitating rapid adaptation. In contrast, asexual populations can only change their resistance phenotypes via sequential mutation. This 'Red Queen' hypothesis has considerable empirical support, but is challenged by the persistence of at least one lineage for over 30 million years without sex. Bdelloid rotifers are microscopic, filter-feeding invertebrates that have diversified into over 400 species and occupy almost every freshwater habitat worldwide. Like all animals, they suffer from pathogens, including deadly endoparasitic fungi. If the Red Queen hypothesis is correct, how have these hosts successfully resisted their natural enemies for so long without sex?

To investigate this question, I isolated and cultured bdelloid rotifers from the genera Habrotrocha and Adineta, differentiating clonal haplotypes by sequencing mitochondrial cytochrome oxidase I. Clonal populations were inoculated under controlled conditions with spores from fungal parasites in the genus Rotiferophthora, and mortality due to infection was quantified after 48 hours. Rotiferophthora isolates were found to have broad host ranges, but some rotifer clones in both genera were completely immune to certain parasite isolates. In quantitative comparisons, even closely related rotifer clones differed in their resistance to the same parasite isolate. These infectivity patterns suggest genotype by genotype interactions between hosts and parasites, which are required for reciprocal coevolution. I extracted total RNA from bdelloid rotifers at two time points after inoculation, in cases where hosts were either highly resistant or highly susceptible to the parasite. Transcriptomic data will help elucidate the genetic basis of resistance to parasites in hosts that have not undergone sex for millions of years. EMBO Fellows' Meeting, 13 - 16 June 2013, Heidelberg

KRAB/KAP1 epigenetic regulation in hematopoietic stem cells homeostasis and lineage commitment

Benyamin Yazdanpanaha,*, Isabelle Bardea, W. Benjamin Rauwela, Matthias Lutolfa, Didier Tronoa

École polytechnique fédérale de Lausanne EPFL, Switzerland

As multipotent progenitors, hematopoietic stem cells (HSC) undergo extensive epigenetic changes on the road towards terminal differentiation. Tightly regulated asymmetric cell divisions ensure the acquisition of lineage-commitment marks by one daughter cell and the maintenance of multipotency of the other. KAP1 is known as a transcriptional corepressor serving as universal cofactor for KRAB-containing zinc finger proteins (KRAB-ZFPs), a large family of sequence-specific DNA binding factors notably responsible for the early embryonic silencing of endogenous retroelements. We found that the hemato-restricted knockout of KAP1 in the mouse induced bone marrow failure, correlating with i) the immediate proliferation followed by the exhaustion of long-term repopulating HSC (LT-HSC) in vivo, with premature upregulation of genes specific of differentiated blood cells; ii) a proliferation defect of LT-HSC ex vivo; iii) an in vivo homing and ex vivo differentiation defect of KAP1 knockdown Lin- Sca1+ cKit+ cells; and iv) the binding of KAP1 to HSC- and other hemato-specific genes in human CD34+ cells. Furthermore, over 200 KRAB-ZFPs exhibited lineage- and stage-specific patterns of expression in murine and human hematopoietic cells, consistent with a role for this regulatory system at multiples steps of hematopoiesis. Further investigation of KRAB/KAP1 mediated epigenetic modifications is on the way to unravel the molecular mechanisms that link KAP1 genomic recruitment and spatio-temporal control of gene expression with blood differentiation. EMBO Fellows' Meeting, 13 - 16 June 2013, Heidelberg

Regulation and function of the TRPM6 Mg2+ channel – protein kinase

Jenny van der Wijsta,*, Joost GJ Hoenderopb, René JM Bindelsb, Dario R Alessia a The MRC Protein Phosphorylation and Ubiquitylation Unit, College of Life Sciences, University of Dundee, Scotland b Department of Physiology, Radboud University Nijmegen Medical Centre, The Netherlands

Introduction The transient receptor potential melastatin subtype 6 (TRPM6) has been identified as the Mg2+ entry pathway in the distal convoluted tubule (DCT) of the kidney. Mutations in the TRPM6 gene are linked to familial hypomagnesemia with secondary hypocalcemia, characterized by renal Mg2+ wasting. This finding highlights the critical role of TRPM6 in controlling Mg2+ homeostasis. Together with its closest homologue TRPM7, TRPM6 is unique as it consists of an ion channel merged with an alpha-kinase domain. It is classified as “atypical” as it has no sequence homology to any conventional protein kinase. To date, the function of this carboxyl-terminally fused alpha-kinase domain is poorly understood. My project, therefore, aims to study the regulation of the TRPM6 alpha-kinase. Methods To this end, in vitro kinase assays, peptide pull down studies and fluorescence polarization analyses were used. Results Wild type full length TRPM6 is able to phosphorylate MBP in an in vitro kinase assay, while truncating TRPM6 at M1719 blocked this kinase activity. This evidenced a so-called ‘dimerization/activation’ region that has previously been identified in TRPM7. Subsequent alanine scanning mutagenesis of residues L1700 to V1730 identified two conserved residues (L1718 and L1721) that are essential for kinase activity and binding within TRPM6 itself. A wild type biotin peptide, corresponding to residues 1700-1740, demonstrated binding to a region upstream of V1730, which is impossible using the equivalent mutant peptide (L1718/21AA). Reviewing the resolved TRPM7 alpha-kinase structure predicts the involvement of four residues within the alpha-kinase, and one downstream of the domain. Mutating these residues (L1743A, Q1832K, A1836N, L1840A, L1919Q) resulted in abolished kinase activity due to hampered binding. Conclusion The data generated in this project will lead to new fundamental understanding of the regulatory mechanisms of the TRPM6 alpha-kinase domain, contributing to development of novel therapies for hypomagnesemia and associated pathologies. EMBO Fellows' Meeting, 13 - 16 June 2013, Heidelberg

A TPR domain-containing N-terminal module of MPS1 is required for its kinetochore localization by Aurora B

WIlco Nijenhuisa, Eleonore von Castelmurb,*, Dene Littlerb, Anastassis Perrakisb, Geert JPL Kopsa, et al. a Molecular Cancer Research and Department of Medical Oncology, University Medical Centre Utrecht, 3584 CG, Utrecht, The Netherlands b Netherlands Cancer Institute, Department of Biochemistry, 1066 CX, Amsterdam, The Netherlands

The mitotic checkpoint ensures correct chromosome segregation by delaying cell cycle progression until all kinetochores have attached to the mitotic spindle. In this paper, we show that the mitotic checkpoint kinase MPS1 contains an N-terminal localization module, organized in an N-terminal extension (NTE) and a tetratricopeptide repeat (TPR) domain, for which we have determined the crystal structure. Although the module was necessary for kinetochore localization of MPS1 and essential for the mitotic checkpoint, the predominant kinetochore binding activity resided within the NTE. MPS1 localization further required HEC1 and Aurora B activity. We show that MPS1 localization to kinetochores depended on the calponin homology domain of HEC1 but not on Aurora B-dependent phosphorylation of the HEC1 tail. Rather, the TPR domain was the critical mediator of Aurora B control over MPS1 localization, as its deletion rendered MPS1 localization insensitive to Aurora B inhibition. These data are consistent with a model in which Aurora B activity relieves a TPR-dependent inhibitory constraint on MPS1 localization. EMBO Fellows' Meeting, 13 - 16 June 2013, Heidelberg

Structural and biochemical studies of the SLIP1-SLBP complex

Holger von Moellera,*, Rachel Lernerb, Adele Ricciardib, Claire Basquinc, William F. Marzluffb, et al. a Free University of Berlin, Germany b Program in Molecular Biology and Biotechnology, University of North Carolina, Chapel Hill, NC 27599, USA c Structural Cell Biology Department, Max Planck Institute of Biochemistry, Munich, 82152 Germany

Messenger RNAs (mRNAs) are produced as precursors that undergo multiple maturation steps (splicing, addition of a 5’-cap structure, 3’-cleavage and, in most cases, polyadenylation) before they can be exported from the nucleus to guide protein biosynthesis in the cytoplasm. These steps are interconnected and linked to upstream (transcription) and downstream (export, surveillance and translation) processes and are mediated by multi-protein and RNA-protein (RNP) molecular machines. Compared to the bulk of mRNAs the majority of metazoan replication-dependent histone mRNAs exhibit several distinct features: (1) Their genes are physically linked in clusters; (2) Their expression and translation is cell cycle regulated; (3) They are intronless and (4) They form a unique stem loop (SL) structure in their 3’-untranslated regions (3’-UTR). This specialized 3’-end is bound by SLBP, a multifunctional protein that participates in multiple steps of the histone gene expression pathway, including nuclear export and translation. The translational activity of SLBP is mediated by interaction with SLIP1, a MIF4G-like protein that connects to translation initiation. We determined the 2.5 Å resolution crystal structure of SLIP1 bound to the translation-activation domain of SLBP and identified the determinants of recognition. We discovered a SLIP1-binding motif (SBM) in two additional proteins: the translation initiation factor eIF3g and the mRNA-export factor DBP5. Pull-down assays confirmed the binding of SLIP1 to DBP5 and eIF3g and we determined the 3.25 Å resolution structure of SLIP1 bound to the SBM of DBP5. Our results suggest how the SLIP1 homodimer or a SLIP1-CTIF heterodimer can function as platforms to bridge SLBP with SBM-containing proteins involved in different steps of mRNA metabolism.