During the transition from S phase to mitosis, epithelial cells displace their nuclei which subsequently migrate from the basal membrane to the apical surface of the neuroepithelium, a phenomenon termed interkinetic nuclear migration (INM). The nucleus moves from the basal zone to the apical surface in G1 before it replicates its DNA in S-phase. It then returns to the basal zone in G2 and undergoes mitosis. The cellular mechanisms that underlie INM are elusive. So far nuclear positioning has been mainly studied in unicellular organisms. However, applying the knowledge coming from these studies to investigate nuclear positioning during INM in a multicellular organism will establish new nuclear positioning concepts in higher eukaryotes. It will also help to gain insight into related processes like cell migration. I want to study mechanics that trigger INM on a molecular level in the retinal neuroepithelium of the zebrafish embryo. 4D-microscopy will be used to follow nuclei, microtubules and centrosomes as well as actin filaments in the developing embryo. Molecular details of the microtubule-nucleus interactions will be investigated in detail as nuclear positioning depends on microtubules in many organisms. Anchor and motor proteins will be studied using genetic methods mainly morpholino approaches. Knocking down motor proteins as well as molecules that could link microtubules to the actin cytoskeleton and determining their influence on INM will give us insights into the molecular composition of this process. The outcome of these studies will lead to a general understanding of INM mechanics and will open doors to a better understanding of how it is linked to cell cycle events.

Splicing is a fundamental gene regulation process that removes the non-coding regions and joins the coding regions of pre-mRNA to produce proper mature mRNA for protein translation. Most of the eukaryotic genes display alternative splicing patterns that allow the production of multiple proteins from the same pre-mRNA, significantly enriching the complexity of the proteome. Even though the chemistry of the splicing reaction is known, comparatively little is known about the splicing code, namely how splice sites are chosen and splicing decisions are made by the cellular splicing machinery. There is evidence that in mammals constitutive and alternative splicing events are regulated by trans-acting proteins that are recruited by cis-acting elements along the RNA sequence such as exonic and intronic splicing enhancers as well as exonic and intronic splicing silencers. Although substantial effort has been recently put into the identification and characterization of cis-acting elements that regulate splicing, a detailed and comprehensive study of trans-acting proteins recruited by the intronic/exonic splicing enhancers/silencers in mammalian cells remains to be explored. Therefore, this project aims to establish functional relationships between the known cis-acting elements and trans-acting factors, study the biological function of the splicing isoforms of the trans-acting factors and discover novel trans-acting factors by an RNA interference medium-throughput screen. Since many genetic diseases arise from mutations in cis-acting elements, a comprehensive library of the associated trans-acting factors might point to novel therapeutic targets.

Pre-mRNA splicing, the removal of introns from mRNA precursors, is indispensable for the expression of most genes in eukaryotes. Splicing involves two transesterification steps within the spliceosomal complex, an elaborate molecular ensemble formed by the ordered interaction of four small nuclear ribonucleoproteins (snRNPs), U1, U2, U5, and U4/U6, and numerous splicing factors with the pre-mRNA. To date, six kinds of proteins are identified that are specific to U5 snRNPs Snu40p, Dib1p, Prp28p, Snu114p, Brr2p, and Prp8p in yeast. Notably, Prp8p (280 kDa) is highly conserved among species and essential for nuclear pre-mRNA splicing in vitro and in vivo. Prp8p can be crosslinked not only to loop1 of U5 snRNA which tethers the 5’ and 3’ exon during splicing reaction, but also to 5’ splice site (5’SS), branchpoint, and 3’ splice site (3’SS) of the pre-mRNA, perhaps acting as a cofactor in RNA catalysis. In addition, Prp8p binds strongly with the ATP-dependent RNA unwindase, Brr2p, and, EF-2 homologous GTPase / putative translocation inducer, Snu114p in yeast. Moreover, it is interesting that specific mutations near the C-terminus of Prp8p cause the retina disorder, Retinitis Pigmentosa. With this fellowship, I will do a dramatic shift from my previous studies first, I will purify this ternary complex by S.cerevisiae expression system and tri-(U4/U6)•U5 snRNP by tandem affinity purification (TAP) method, then I will determine the structure of Prp8p/Brr2p/Snu114p ternary complex by X-ray crystallography and the envelope of the tri-(U4/U6)•U5 complex by cryo-electron microscopy, which will provide important insights into the catalytic core of the spliceosome.

The molecular mechanisms regulating cell fate and pattern formation during plant development belong to one of the most fundamental questions in developmental biology. Stomata, specialized epidermal structures regulating gas exchange between plant tissues and the atmosphere, constitute an excellent model to study these universal questions. To understand the complete circuit for stomatal development and patterning, we will need to identify genes that positively and negatively regulate multiple divisions and signaling events. Our present knowledge about positive regulators is poor. In this project I will clone a newly identified positive regulator of stomatal development. By performing detailed molecular and functional characterization of the gene I will identify at which stages it is expressed and required and will elucidate genetic interactions between it and known regulators of stomata development. As a complementary approach, I will explore cellular targets of the stomatal development network. Particularly, I want to investigate the dynamic organization of the cytoskeleton, organelle behavior and cell polarity organization during stomata development. By taking advantage of new stomatal-lineage-specific enhancer elements and the phenotypes created by overexpression of key stomatal regulators, I will be able to resolve cellular behavior in a never before seen way. This project is an integrated approach to study genetic and cellular mechanisms regulating stomata development in the model system Arabidopsis thaliana. The output of this work will provide valuable information about molecular and cellular mechanisms regulating cell differentiation and specification in plants.

The synapse is a specialized structure involved in the transfer of information in the brain. Thus, any regulation of the synapse will modulate the quality of the information that is communicated within the brain. Recently, it has been shown that astrocytes take an active role in regulating synaptic function. It is now widely accepted that the synapse comprises three components, not only pre- and postsynaptic neurons, but also processes sent by astrocytes. Astrocytes extend numerous processes contacting many types of synapses where synapse-glia interactions occur and where the information processing is performed. Although some of the general principles of the contribution of astrocytes to synaptic transmission are now becoming established, it is still unknown how a single astrocyte handles the functioning of the different synapses enwrapped by its processes. Thus, it is crucial to understand how individual astrocytic processes talk to their associated synapses and how the activation of individual processes occurs in relation to synaptic signaling. Using state-of-the-art cellular imaging techniques and synaptic recordings, activity of individual astrocytic processes will be monitored to study synaptic cross-talk between astrocytes and synapses. I will test whether activity in individual astrocytic processes is related to synaptic activity, if the glial processes are independently activated and whether they are specialized for one type of synapse. Finally, the ability of the same astrocyte to release different neuromodulators will be tested. This project should further our understanding of the role of glial cells in the regulation of the transfer of information in the brain.

X-chromosome inactivation (XCI) in female mammals is a classic example for epigenetic gene regulation. In mice, early preimplantation embryos and the placenta display imprinted XCI where the paternally inherited X is inactive. At the blastocyst stage, embryos reactivate their paternal X to undergo later random XCI where either the maternal or paternal X gets inactivated. In the proposed experiments I want to study the X-reactivation process, which is currently poorly understood. The noncoding RNA Xist is specifically expressed on the inactive X and crucial in the silencing process. On the active X, Xist is repressed by expression of its antisense partner Tsix. My first aim will be to test, if induced Tsix-expression is sufficient to cause Xist downregulation and X-reactivation at different stages of development. This will show, if X-reactivation is limited to the blastocyst-stage or if it is exclusively under control of Tsix-expression. In a complementary approach, I will use single-cell microarrays to screen for genes specifically linked to the X-reactivation process. This unbiased genome-wide screen will allow me to identify new factors involved in X-reactivation which can be further analyzed by functional assays routinely used in the Lee-lab. My scientific background lies in the fields of germ cell determination, maternal effect genes and neurobiology. In the proposed projects I will extensively widen my scientific horizon by gaining new experiences in chromatin biology, non-coding RNAs and dosage compensation. The new approaches I will learn will aid me significantly in my future scientific career.

Vesicle budding into the endosomal structure gives rise to multi-vesicular bodies (MVB). The protein machinery involved carries out a variety of cellular/pathological processes e.g. receptor down regulation and enveloped virus release. A number of studies led to the discovery that CHMP (charged MVB proteins) are implicated in the final step of membrane fission. However, our understanding of lipid and protein rearrangements involved in the fission process is extremely limited. The CHMP3 structure determined by the host laboratory revealed a flat helical arrangement with a basic surface required for membrane interaction and two homo-oligomerisation interfaces. I now aim to determine the structure of: (i) monomeric, cytosolic, full length CHMP(s) in their proposed closed conformation for understanding the molecular basis for activation and membrane targeting and (ii) heterodimeric CHMP complexes for identification of CHMP-CHMP and CHMP-membrane interaction interfaces. Membrane binding and deformation properties will be addressed on solid supported bilayers of defined composition: (i) I will quantify CHMP-bilayer and lateral CHMP-CHMP interactions on bilayer using surface sensitive label free and fluorescence techniques (ii) mechanistic basis of curvature generation in bilayer e.g. induced phase-separation and/or intercalation of side chains into the bilayer will be addressed by neutron reflectometry. My training in chemical biology and biophysics will be useful for understanding the protein and lipid rearrangements involved in the fission process. At the same time this proposal offers an excellent opportunity for learning structural biology.

The initiation of DNA replication is a complex and highly regulated process, which requires the ordered assembly of proteins at origins of DNA replication. The experiments proposed in this research project aim to dissect and order the processes, which start after assembly of the prereplicative complex (pre-RC) and lead to activation of the origin and thus the establishment of a replication fork. I will employ an in vitro-assay in which the pre-RC is assembled on origin DNA using extracts of Saccharomyces cerevisiae cells arrested in G1, which are additionally supplemented with pre-RC proteins. Using this assay I will study the recruitment of factors acting downstream of the pre-RC starting with the Mcm10 protein. Requirements for this association will be determined both within the pre-RC as well as in Mcm10. Analyzing the recruitment of proteins at different stages (e.g. binding to the Mcm10/ pre-RC assembly) will allow me to order the events that ultimately lead to the formation of a replication fork. In a second approach I will investigate a proposed role of polymerase epsilon in the initiation of DNA replication. To this end I will purify polymerase epsilon associated proteins from the initiation stage. Furthermore I will use the temperature-sensitive degron technique to downregulate polymerase epsilon in cells arrested in G1 and to assay for defects in replication initiation following the release from the arrest. These approaches should significantly increase our knowledge of how eukaryotic replication origins are activated.

This proposal addresses the important issue of how the DNA damage response takes place in the context of chromatin, the physiological substrate for DNA repair and checkpoint signalling machineries in eukaryotic cells. In response to DNA double-strand breaks, the phosphorylation of the histone variant H2AX has been widely documented. The phosphorylated species, referred to as gammaH2AX, specifically binds chromatin modifiers and DNA damage response proteins. However, how these factors work together in a coordinated manner to promote an efficient damage response is still unclear. I propose to pursue two complementary approaches to address this issue in mammalian cells. First, I will investigate the chromatin-associated function of damaged chromatin interactors with a particular focus on the checkpoint mediator MDC1 (Mediator of DNA Damage Checkpoint 1), a well characterised gammaH2AX binding factor. I will search for chromatin modifying activities associating with MDC1 in vivo and of relevance to the DNA damage response. Second, I will analyse the dynamic binding of chromatin modifiers to damaged chromatin, focusing on Poly(ADP-Ribose) Polymerase 1 (PARP1) binding and FACT (Facilitates Chromatin Transcription) release upon H2AX phosphorylation. Through this work, I expect to gain mechanistic insights into how molecular partners of gammaH2AX operate, both in the DNA damage response and on chromatin structure. Not only does this project build on my previous experience in the chromatin field, but it will also allow me to develop novel and complementary skills in the field of the DNA damage response, which will greatly benefit me in my future career as an independent scientist.

The influence of parasites on the evolution of living organisms is a central topic in all disciplines in evolutionary biology since all organisms are at risk from parasitism. Even if some studies provide evidence that parasites are able to alter the host’s phenotype to enhance their fitness, modification of host nutrient intake by parasites has never been evoked as an adaptive strategy to enhance parasites’ growth and survival at least once inside the host. Recent advances in nutritional theory offer an opportunity, for the first time, to quantify the relationship between parasitism, nutritional state and immune responses, and to explore the extent to which hosts can self-medicate through altering their diet choice to fight infection, or instead are manipulated to enhance parasite’s development. To tackle such questions I will investigate the prediction that the host’s nutritional behaviour is expected to change with the nutritional burden provided by the parasite. In so doing, I will monitor and compare the feeding behaviour and the immunity responses of a generalist leaf-feeding caterpillar, Helicoverpa armigera infected by either a macro- and/or a microparasite with regard to its ability to self-select diets containing a protective nutrient balance or a protective toxin. Diets will include differentially radio-labelled compounds to establish the extent to which nutrients ingested by the host are diverted to parasite’s growth. This novel and interdisciplinary approach will have considerable impact on our understanding of the evolution of hosts’ life history traits and important implications in the emerging field of Darwinian medicine.

How acquired memories are stored and retrieved in functional neuronal networks in living animals is a fundamental question that is still largely unresolved. The current proposal offers an approach to gain insight into this question with two complementary strategies. The first is to develop and optimize a learning assay for larval zebrafish in which the animals learn to associate two distinct but intrinsically neutral visual stimulus patterns with danger and safety respectively. This is achieved by pairing the presence of the fish in a visually distinct area of the tank with the delivery of mild electroshocks. The second proposed strategy is to develop an in-vivo calcium imaging assay in those larvae to allow the monitoring of activity in large populations of neurons in response to these specific visual stimuli. The small size of the larval zebrafish brain in combination with the translucence of the preparation permits functional in-vivo imaging of any chosen part of the central nervous system. Initially, the well established methods of single cell electroporation of dextran dyes and the injection of calcium indicators in the form of AM-esters will be used. In addition, the established role of the zebrafish as a genetic model system will also allow targeting of specific populations of neurons for the expression of genetically encoded calcium indicators. The function and reliability of all these calcium indicators is analyzed by simultaneous in vivo patch clamp recordings of the labeled neurons. The general aim of these experiments is to seek and describe specific neuronal populations that respond differentially to correlates of acquired information in the vertebrate brain.

Formation of synapses is a central issue in neurobiological research. Since synapses in the brain are small and difficult to study, many players involved in synaptogenesis were originally identified in neuromuscular junctions (NMJ). These specialized synapses are about a hundred times bigger than those in the brain, with complex topology that can be easily evaluated by microscopy. Importantly, it was demonstrated recently that muscle cells are able to form autonomously complex postsynaptic specialization, without contacting a neuronal cell. During my post-doc, I will rely on my experience from yeast genome-wide visual screening, to set-up a high-through-put system to screen for novel regulators of the postsynaptic apparatus in muscle cells. In this project, I will use shRNA library that combines viral-based gene transfer with RNAi-mediated gene silencing and will allow me to affect expression of selected genes. I will start my investigation with 500 genes whose transcripts localize to the synapses. These genes are mostly expressed from the specialized synaptic nuclei, localized directly underneath the postsynaptic structures and are expected to be enriched in synaptic proteins. The ultimate goal of this project is to identify genes that regulate the formation of the NMJ so that I can extend the investigation to the less accessible synapses of the brain. I hope that this work will eventually be focus of my own laboratory.

Bird song is a stereotyped learned behavior that provides an experimentally tractable system for elucidating the neural mechanisms of motor learning. Models of motor learning require variability for exploration of different motor outputs and the selective reinforcement of those that match the learning target. Consistent with these models, song variability has been observed and appears to be important for song learning. In adult birds, song variability is modulated by social contexts, i.e. song variability is higher when the bird sings alone (undirected song) as compared to when the bird sings to another bird (directed song). Electrophysiological recordings and immediate early gene expression studies show social context dependent variability in the activity of neurons in a specialized pallial-basal ganglia circuit called the anterior forebrain pathway (AFP). Lesions of the output nucleus of the AFP eliminate social context dependent differences in song variability. Although these experiments suggest that the AFP is essential for generating song variation, they do not answer important questions concerning the source and control of neuronal variability. I propose to use a combination of single unit electrophysiological and pharmacological techniques to study the origin of and mechanisms underlying neuronal variability in the AFP. More generally, I hope to gain insights into brain mechanisms of motor learning. This grant will provide me with the opportunity to learn new techniques and explore bird song, a wonderful model for studying the neural basis of learning and memory.

Infectious viruses can enter the cell through several endocytic routes. These routes consist of a complex system of organelles and intracellular trafficking pathways. An integrated and comparative analysis of these routes is currently not available. The host laboratory is examining the role of the druggable human genome (7,000 genes) by RNA interference in five different infectious entry routes. At the moment, we are determining the role of these genes in particular steps in the routes by using quantitative light microscopy, fluorescent ligands, and marker proteins. The research goal is to gain a systems view on different endocytic pathways using infectious virus entries. We will develop data-analysis and graph theoretic methods for discovering which viruses hijack the same endocytic routes and groups of genes whose silencing leads to similar phenotypes. In addition, we will develop methods for integrating the measurement data with protein-protein and genetic interaction networks that are available in databases. We will assign the network nodes labels indicating in which sub-cellular structure they are active and study the features such networks with spatial information. Finally, we will examine in detail the genetic regulatory network of one endocytic pathway by silencing each gene of the pathway and measuring the altered expression profile of all other genes. The proposed research highly differs from my theoretical background in signal processing and complex systems. I will gain knowledge in cellular biology, laboratory work, and new measuring techniques. This combination of skills will be very important for my future scientific career in systems biology.

Soft lithography represents a set of techniques that exploit elastomeric polymers such as poly(dimethylsiloxane) (PDMS) for the fabrication of various structures in the micro-scale. Microcontact printing, one of soft lithography methods, enables the patterning of mammalian cells using PDMS stamps. In this process the stamp is first "inked" with the cells and then pressed onto the surface to be patterned. The soft stamp enables a good contact with the surface and the cells are rapidly transferred from the stamp to the surface. This method allows the control over cell micron-scale spatial organization and can be used as a tool in various fields such as tissue engineering and cell biology research. An alternative material for stamp fabrication that was recently suggested is agarose hydrogel. It was demonstrated that by using agarose stamp, proteins and bacteria can be transferred to a surface while the stamp is providing an ideal "wet" environment. Moreover, it was shown that osteoblasts cells could be transferred onto porous scaffold for tissue engineering applications. The aim of this study is to establish the use of agarose stamps as a generic method for patterning various types of cells. In addition, other biocompatible hydrogels will be examined for this application. This will be followed by using hydrogel stamps to pattern cells inside micro-channels in order to explore the cells interaction with their environment.

The Mediator is a giant multi-subunit complex that is responsible for conveying regulatory signals from control elements to gene promoters in all eukaryotes from yeast to human. The Mediator is required for basal and regulated expression of nearly all Pol II- regulated genes and can therefore be regarded as a general transcription factor. Currently, the lack of detailed structural information on the Mediator prevents a deep understanding of its regulatory functions. The main purpose of this proposal is to determine the 3D structure of Mediator at high resolution. I will build upon an existing protocol in the Kornberg lab for the purification of milligram quantities of Mediator from S. cerevisiae. Suitable preparations of Mediator will be used for high-resolution structural analysis using both 2D and 3D crystallography techniques. Furthermore, I propose to investigate the structural and biochemical nature of the interaction between Mediator and transcriptional activators. A detailed understanding of Mediator structure should provide insights into the mechanisms underlying the transmission of regulatory information to the RNA Pol II and its associated factors. Previously, I characterised a number of fundamental structural properties of the 30nm chromatin fibre using EM. The proposed project will broaden my knowledge of the transcription field and provide training in a number of new techniques such as protein expression, the isolation and purification of large multi-subunit complexes and both 2D and 3D protein crystallography. In combination these techniques will provide powerful tools for future structural and biochemical studies of multi-subunit transcriptional complexes.

The Ski complex is essential in all the functions that are carried out by the cytoplasmic exosome, one of the major 3’-5’ degradation machinery in the cell. The principal aim of my work will be to determine the structure of the Ski complex. It is composed of 3 proteins, one of which containing an RNA helicase domain and two containing predicted protein-protein interaction domains. I also want to study structurally Ski7, an additional component that is likely to bridge the interaction between the Ski complex and the exosome. The structural information will be used to design specific mutations and test hypothesis on how it works in vivo in yeast. Finally, in the long term, it would be interesting to study the association of the Ski complex with Ski7 and the exosome in order to determine how all these different components are positioned and how the Ski complex can help the exosome in degrading mRNA.

Hace1 is a tumor suppressor, E3-ligase that is involved in protein folding and targeting misfolded proteins for degradation. The unfolded protein response (UPR) is proposed to be the link between the involvement of Hace1 in protein folding and cancer as tumor cells suffer from high unfolded protein load. Upon UPR activation, chaperons are up-regulated and misfolded proteins are either refolded or marked for degradation by the ubiquitin-proteosome system. UPR leads to apoptosis of untransformed cells and is growth promoting in transformed cells, hence a paradox. I propose to determine whether the tumor suppressor activity of Hace1 is related to reduction of misfolded protein load and reduced activation of the UPR. Two approaches will be taken to address this question: 1. Unfolded protein load will be induced in transformed and untransformed cells and the role of Hace1 in misfolded protein handling and cell survival will be determined. 2. Unfolded protein loads in live transformed an 'normal' cells will be determined by the diffusion parameters of RFP and the role of Hace1 in handling unfolded proteins in these cells will be studied. In my PhD thesis I studied the micro- and macro-localization of Ras in the cell using biophysical methods. My studies provided me with a strong basis in using a biophysical approach to study protein dynamics in live cells, which will contribute to the development of the novel method needed to determine unfolded protein load in live cells. By studding Hace1 I will have the opportunity to apply my skills to study how cells cope with physiological challenges and get acquainted with new approaches necessary to precede with my academic career.