Initiation of transcription by RNA polymerases I and II (Pol I and Pol II) requires the assembly of general transcription factors on promoters. The Pol II transcription factor TFIID, comprised of the TATA-binding protein (TBP) and 14 TBP-associated factors (TAF(II)s), recognizes promoters and directs preinitiation complex assembly. The RNA Pol I selectivety factor SL1 is analogous to TFIID, is composed of TBP and three TBP-associated factors (TAF(I)s) and is required to direct initiation from rRNA promoters. Recently, several novel TAFII-containing complexes have been identified, such as the TBP-free TAFII Complex (TFTC), the P/CAF complex and the Spt3-TAFII31-GCN5-Acetylase Complex (STAGA). These complexes contain several common TAFs and at least one component with histone acetyltransferase activity (GCN5 or PCAF). The existence of these complexes has raised the question of whether they have distinct or overlapping roles in gene control. We propose to investigate the specificity and dynamics of assembly of RNA Pol I and Pol II complexes their function in cellular regulatory processes in the nucleus of living cells. Specifically, I plan to: 1) probe protein-protein interactions amongst TAFs by FRET in living cells (Fluorescence Resonance Energy Transfer), 2) determine the involvement of specific pol I and pol II TAF-containing complexes in two nuclear processes (transcription and DNA repair) by studying the potential co-localization of these complexes with other components of the transcription apparatus and the DNA repair machinery, 3) measure the kinetics of TAF-complex movement in living cells by FRAP (Fluorescence Recovery After Photobleaching) to determine whether complexes exist prior to engagement on promoters, and 4) analyze assembly dynamics of the TAF-containing complexes in living cells of synthetic or endogenous model genes in relationship to the sequential recruitment of the individual components of the preinitiation complex and transcription initiation.

In mitosis the duplicated chromosomes are split in half and pulled by the spindle apparatus to opposite poles of the cells. This process is governed by a protease, separase, which cuts the proteinous glue holding chromosomes together. Compelling evidence argues that separase has additional important functions to ensure faithful transmission of the genetic material. These include positioning and stabilization of the mitotic spindle and regulation of mitotic exit. However, no additional separase substrates have been identified in higher eukaryotes to date. Not surprisingly, a multitude of regulations act on separase, one being the binding of a protein inhibitor of anaphase, securin. We will develop a novel method for in vitro evolution of a temperature-sensitive securin to streamline the generation of active human separase. It will then be used to identify as-yet-unknown targets. Small pools of in vitro expressed proteins and fractionated proteins from isolated mitotic spindles will be screened for proteins that are cleaved by separase. To find interaction partners of separase that are not cleaved a novel method of highly specific photocrosslinking will be applied. These biochemical approaches will be followed by the in-depth characterization of the identified proteins by a broad spectrum of cell-biological techniques. The successful accomplishment of the proposed work will result in a detailed analysis of separase function and open up new aspects of mitosis research. It will represent a further important contribution to my achievements in the field of chromosome biology and establish my group in the cell biology community as an attractive partner for future collaborations.

1) At early stages of development, the heart is a single tube. In lower vertebrates such as fish, it remains as a two-chambered linear tube, but in higher vertebrates such as mammals, the heart is divided into four chambers (left/right, ventricles/atria) separated by septa. The morphology of each chamber is specific to its individual function, and the separation of chambers into left and right components is required for separate pulmonary and systemic circulations. A few molecules are expressed in a polar fashion in the heart, but the molecular mechanisms that specify the identities of each chamber and the positions of septa have remained unclear. My project is to find the molecular mechanisms of chamber formation during cardiovascular development with as aim to explain the evolution of the vertebrate heart from a simple two-chambered structure to a four-chambered organ adapted to dual circulation, and how this organ forms aberrantly in human disease. 2) So far I have studied limb, eye and heart development using chick embryos. Chick is very useful to study morphogenesis, because methods of in ovo manipulation have been established. However in chick genetic methods have not been established, and it develops differently from mammals. I am interested in human disease, and I hope to apply my findings to therapy of heart disease. 3) My project allows me to learn advanced mice genetics and microarray techniques. I will be able to develop new methodologies to study mouse embryology by combining my manipulation techniques with mouse genetics. The study of the heart is still unexplored, and this will allow me to expand my research horizons towards therapy of human disease.

1) The integrin family of transmembrane adhesion receptors are the main components of an adhesion complex that is important for the attachment of cells to the extracellular matrix. It has been previously shown that integrin function is dependent on its ability to physically interact with the cytoplasmic protein talin. However, it is still not clear how talin contributes to integrin function. I propose to use Drosophila to address this problem, I will generate constructs that contain tagged version of different domains of talin and generate transgenic lines carrying these constructs, these lines will be used to analyze the function of talin. In addition I propose to use a protein tag to biochemically isolate the native adhesion complex associated with Talin. 2) My previous work concentrated on a unique type of cell behaviour, epithelial polarization, by studying how cell polarity is established in flies. In my future work I will shift my emphasis to another type of cell behaviour, namely, the study of how cells interact with the extracellular matrix. The study of Cell-ECM interactions mediated through integrins is a large and highly specialized field that is distinct from my previous interests. The work I propose will afford me the opportunity of working on non-epithelial systems such as the muscle, and will allow me to train in biochemistry which I have not done before. 3) This project will allow me to study a novel biological problem using a combination of tools, at least some of which will be learned and acquired during the work proposed. In addition, this proposal would allow me to develop an indepedent resrearch program which I could pursue in the future.

Electrophysiological analyses and optical imaging have been used to investigate excitable cell firing and calcium transient in acute brain slice, cultured brain slice or cultured neurons, but both techniques are incapable of recording from multiple neurons with single-cell resolution in vivo. To overcome this limitation, we propose to introduce genetically-encoded neural activity probes such as Yellow Cameleons, Clomeleon or Flash to a population of cortical neurons by in vivo electroporation technique and measure ensemble of neural activity with two-photon microscopy. Two-photon microscopy has enabled fluorescence imaging in the intact brain of freely-moving animals. This approach will establish the method to image neural activity of alive animals during a behavioral tasks. I will utilize the rodent somatosensory barrel cortex as a model system, which provides us with a good opportunity to analyze how the sensory stimuli are processed by neural networks. There are many accumulated information from previous works of electrophysiological analyses and optical imaging, and I can compare my data with preivious results. My graduate and postdoctoral training were concerning mammalian developmental biology. My background of developmental biology, mouse genetics and molecular biology allowed me to pursue this goal. Especially, in vivo electroporation technique is indispensable for this project, which is an efficient method of gene delivery specifically to bulk of specific subtypes of mature neurons and not to glial cells. A HUMAN FRONTIER SCIENCE PROGRAM would allow me to develop and expand my research interests in the information processing of mammalian cortex.

Ascidians are invertebrates with a basic chordate body plan. The extent to which the ascidian larval central nervous system (CNS) diverged from the complex vertebrate CNS provides information about key innovations during chordate CNS evolution. Gene expression patterns of members of the Hedgehog (Hh) signaling pathway in ascidians suggest conservation of signaling pathways involved in chordate CNS development. The application of technologies now available in ascidian research permits functional studies and the characterization of regulatory networks underlying formation of the CNS. Here we would like to focus on Shaggy/GSK3ß, which acts at the interface of the Hh and wingless/Wnt signaling pathway in both Drosophila and vertebrates. Analysis of Shaggy/GSK3ß function in the ascidian Ciona intestinalis will provide insight in the evolution of signaling pathways important for CNS development. Research on these signaling pathways in ascidians also offers a significant advantage over mouse research transgenic animals can be produced much faster, and the genome of ascidians is far less complex. In addition, the host laboratory is the only institute in Europe that performs genetic screens with ascidians. This presents the unique opportunity to screen for ascidian mutant phenotypes on a single cell level, which is just not possible in any vertebrate system. The host laboratory also has a long-standing interest in ascidian research and will provide an excellent environment to study ascidian biology. In combination with the research opportunities and recent advances in this field, this fellowship creates the possibility to become an independent researcher in a new promising field.

Although cell membranes are important sites of biological regulation, function of membrane proteins is poorly understood. Intramembrane-cleaving proteases (I-CliPs) are a recently-discovered class of enzymes involved in regulating membrane protein activity. I-CliPs are novel polytopic membrane proteins that catalyse proteolysis in the hydrophobic membrane bilayer, unlike all other hydrolytic enzymes. Despite their central roles in regulating fundamental biological processes in all branches of life, little is known about how I-CliPs function mechanistically, and about their full roles in different species. I propose to investigate how these novel proteases function in the membrane bilayer using chemical inhibitors that I will develop for this purpose. In a complementary approach, I will probe their function using biochemical and biophysical methods. Finally, I will use these inhibitors to analyze the biological function of one large family of I-CliPs whose members are conserved in all branches of life. My doctoral research was focussed on investigating the roles of cell signalling in embryonic development using the Drosophila model system. Through a combination of genetics and cell biology, I discovered that a key developmental regulator encodes an I-CliP. Unlike my previous experience where I used a genetic and cell biological model to understand cell signalling and development, I now propose to study the biochemical function of this unusual class of enzymes using chemical and physical methods. I have only limited experience in these approaches, and acquiring these skills will be essential to my long-term goals of investigating membrane proteins involved in cell signalling.

Asymmetric distribution of mRNA is an intrinsic mechanism targeting proteins to their site of function. Axis formation in Drosophila requires the localisation of a transcript encoding a TGF alpha homologue, gurken (grk), first to the posterior of the oocyte and later to the dorsal anterior corner. The mechanism of grk mRNA localisation is poorly understood because of the lack of in vivo assays for localisation and the complexity of the process. My project is based on a new in vivo assay, which consists of injecting fluorescently labelled grk RNA in the oocyte and visualising its movement in particles in real time. My goal is to characterise the minimal cis-acting sequences necessary for grk mRNA particles assembly, movement and anchoring. To achieve this, I will inject truncated grk RNAs and study their movements in the oocyte. In parallel, I will develop a new technique, which we call in vivo-SELEX, to define short RNA sequences localising in the oocyte. Another objective is to determine trans-acting factors specific for the different steps of grk movement. I will first investigate the role of known proteins whose mutations induce grk mislocalisation. Moreover, the grk RNA localisation signals will be used to isolate specific binding proteins from oocyte extracts. The candidates will be analysed by mass spectrometry and their role investigated in vivo using mutants or antibodies. This work will allow me to expand my knowledge in cellular biology and biochemistry. The use of powerful imaging systems will complete my knowledge of fluorescent microscopy and of the different acquisition systems, which is essential for my future research carrier.

Cyclin E is one of the activators of Cdk2, expressed near the G1-S phase transition and critical for initiation of DNA replication and other S-phase functions. Protein levels of cyclin E peak at the G1-S boundary, achieved by periodic transcription followed by auto-phosphorylation dependent proteolysis. Deregulation and elevated levels of cyclin E are associated with malignancy possibly through genomic instability. Recently is has been shown that degradation of phosphorylated cyclin E depends on a SCF-complex containing the newly identified human F-box protein hCdc4. hCdc4 is homologous to the yeast Cdc4p and contains WD40 repeats involved in substrate recognition. Both in humans and in mice, three splice variants of Cdc4 are expressed. Biochemical evidence from S. cereviciae and S .pombe indicates that the hCdc4 homologues need to form homo- or heterodimers respectively in order to function properly. Here it is proposed to test dimerization between the different splice variants of hCdc4. Experiments have been designed to reveal the role of the hCdc4 dimerization in relation to substrate specificity, activity of the SCFhCdc4 complex and the possible autoregulation of hCdc4 levels. The proposed project will focus on the role of Cdc4 dimerization, both in lower eukaryotes and in higher eukaryotic systems. Therefore, this project will extend my training to gain expertise with different model systems and further improve my knowledge of standard and novel biological techniques with a particular focus on the biochemistry and cell biology of mammalian tissue culture systems.

Heterochromatin is a specialized chromatin structure present in all eucaryotes with essential functions in chromosome segregation, maintenance of genome stability and transcriptional gene silencing. Recently, a surprising link between heterochromatin formation and the RNA interference (RNAi) pathway was discovered in the fission yeast Schizosaccharomyces pombe. RNAi is a widespread silencing mechanism mostly known for its post-transcriptional activity that degrades target mRNAs or blocks their translation, but studies in plants, Drosophila, Tetrhymena and S.pombe clearly demonstrated that RNAi also acts at the chromatin level. The aim of this proposal is to identify and dissect mechanisms through which RNAi acts at the level of chromatin in S.pombe. Using a protein purification strategy, we have recently purified a complex, that we named RITS (RNA-induced Initiation of Transcriptional gene Silencing)(1). The identification of RITS provides a direct physical link between RNAi and heterochromatin formation machineries and our first studies suggest that RITS complex targets specific genomic regions through an RNA-based pairing mechanism. We are now focusing on understanding how this targeting mechanism works and how it is regulated, using in vitro and in vivo approaches. 1. A. Verdel et al., Science 303, 672-6 (Jan 30, 2004).

The Arabidopsis root has a simple radial structure, organised in concentric single layers. This radial pattern is elaborated during embryogenesis and maintained postembryonically through asymmetric cell divisions of initials in the root apical meristem. The SHORT-ROOT (SHR) gene, encoding a putative transcription factor, is required for the asymmetric cell division of the cortex/endodermis initials, and for endodermis specification. Recent results have revealed that the functions of SHR in root radial patterning depend on intercellular movement of the SHR protein. The aim of this proposal is to find new components of the SHR radial patterning pathway. A genetic screen will be performed to identify mutations that perturb the spatial distribution of the SHR protein. These might affect genes encoding regulators of either SHR expression or SHR protein trafficking. The most interesting mutants will be genetically and phenotypically characterised to determine the role of the mutated genes. One or two mutants will be chosen for map-based molecular cloning. In parallel, conditional expression of SHR coupled to microarray technology will be used to find direct targets of SHR. The function of the putative targets will be analysed by characterising the effect of their disruption or misexpression. During my previous research experience, I become interested in the mechanisms controlling plant development. So far my work was essentially focused on genetic and cytological approaches. This proposal would allow me to look at plant development from a more molecular and genomics point of view. I would acquire tools to identify and to study new genes involved in a developmental pathway.

Brassinosteroid (BR) is multifunctional growth regulator in plants.The aim of this project is to identify new components in the BR signaling cascade, using two different approaches, genetic and genomic, that are based on specific tools available in the host laboratory. Classical genetic based on loss-of-function mutation is not suitable in this particular case due to genetic redundancy, or alternatively to the fact that signaling components may be encoded by essential genes. Genetic analyses will therefore be carried out to isolate gain-of-function mutation, by activation tagging, conferring resistant to BR biosynthesis inhibitor brassinazole. This overexpression of genes at random in the genome is a convenient way to identify components involved in the signaling cascade of physiological events. In parallel, a genomic approach using the Affymetrix technology will be developed to identify genes involved in the signaling pathway and in the early response to BR treatment. The skills required for this project are totally new for me as my PhD and my former experience were mainly characterization of genes involved in a cellular response. This project would allow me to acquire expertise in the field of signal transduction and genomic. Moreover, this project will require bioinformatics for the analyses of genomic data. The skills required can serve to any topic in the field of molecular biology. I have the opportunity, after this postdoctoral position, to come back in the team where I did my PhD as a researcher. I therefore would like to design genetic screen to unravel the signaling cascade leading to the activation of iron transporters in roots described in the achievements.

This project aims to investigate the interactions between the cytoskeleton and trafficking of MHC class I/II molecules in DCs during maturation and contact with T cells in vivo by real-time confocal imaging of MCMV-infected cells. This will be achieved by combining the expertise of the host lab in the area of cell biology and the newly established MCMV-BAC-based mutagenesis methods invented by the applicant. DCs capture antigens in the periphery, migrate to lymphoid organs and present processed antigens via MHC complexes to T cells. Recent in vitro data indicates that during all these processes major changes in the cytoskeletal network of DCs are important(1,2,3). Since in vitro culture systems do not adequately reflect the situation during these processes as they occure in live cells, the project will investigate DCs both in vitro and in the context of the skin and lymphoid organs of the live mouse. We aim to achieve this by using recombinant MCMVs as expression vectors for fusions of GFP (or yFP, cFP) with MHC class I/II, actin, fibronectin, fascin and tubulin. Engineering of MCMV can be easily performed using BAC technology. Since several markers will be co-expressed from one MCMV vector and since MCMV infects various cell types including DCs in mice, this project will for the first time allow us to monitor intracellular interactions in vivo, without laborious generation of transgenic mice. My current research work concentrates on genetics of herpesviruses and on investigation of virus-host cell interactions. In contrast, the proposed project intends to exploit recombinant MCMV as an expression vector for real-time investigation of DCs both in vitro and in vivo.

The subject of the proposed research is the mechanism of mitochondrial fusion. Several factors have been implicated in this process, but their biochemical function and the mechanism of the reaction are not yet known. Therefore, I propose to characterise the biochemical activity of yeast fzo1p, a key player in this process and to purify interaction partners by affinity chromatography. Interacting proteins will be tested for a function in mitochondrial dynamics in vivo. To gain insight into the mechanism of mitochondrial fusion, I will establish a content mixing assay. With such a quantitative assay, the individual steps of the reaction and their different requirements will be determined. I will use yeast as a source material for the assay, because it allows combination of biochemistry with genetic methods. This approach has been extremely powerful in the dissection of the secretory pathway. The proposed research differs from my previous work in the aim of the project as well as the model organism used. However, it will draw on my experience with biochemical assays and similarly to my previous work, relies on the possibility to elucidate cell biological processes by reducing them to an in vitro reaction. This will be augmented by the possibility to validate findings in vivo using yeast genetics. I wish to later expand this research to the regulation of mitochondrial dynamics and its coordination with other organelles. This network can only be understood if one can know the quantities of all factors, which is at the moment only feasible in yeast. The proposed research would make this possible by introducing me to this model system and by expanding my biochemical knowledge.

1) c-Myc is a key regulator of proliferation that is frequently deregulated in tumours. Paradoxically, c-Myc also induces apoptosis. Mitochondria are the principal target for the apoptotic transcriptional action of c-Myc. To identify c-Myc effectors a proteomic approach will be employed to determine Myc-induced changes in mitochondrial polypeptides. These proteins will be identified and their roles in Myc-induced apoptosis assessed. 2) The project differs from my previous research in that I will study dividing cells and learn about the cell cycle, with which I am unfamiliar (my research so far has involved post-mitotic neurons). The focus on the c-Myc oncogene and cancer contrasts with my current work studying the excessive cell deaths that occur during neurodegeneration. I will also use unfamiliar techniques, such as 2D gel electrophoresis/mass spectrometry, the switchable ER(TAM) activation system (especially in transgenic animals) and confocal microscopy. 3) The project will aid my professional growth since I will learn about mechanisms of proliferation. Knowledge of the differences between proliferating cells and non-dividing neurons, and also neoplasias in contrast to excess apoptosis, should lead to new insights into the mechanisms of proliferation (and cancer) versus cell death (and neurodegenerative disorders). I will use the excellent ER(TAM) system in particular, switchable c-Myc activity in transgenic mice means that protein expression is not lacking throughout ontogeny, leading to a powerful in vivo system, which will be of use in future research. Access to unfamiliar techniques will enhance my skills and lead to new approaches to solving scientific problems.

1) While the architecture of different neural circuits has been well documented, the behaviour of complex networks is poorly understood. How the sensory nervous system builds a representation of the surroundings through colour vision is a challenging question. I will use a behaviour assay to address this issue in the exquisitely accessible visual system of Drosophila. Flies can be trained to associate a hue of colour and a stimulus. In this context, the powerful genetic tools of flies will allow me to manipulate the opsin content of the retina as well as the ability for the photoreceptor neurons to transmit the information to higher centres in the optic lobe. I will test the effect of such alteration on the behaviour of the fly. I will then use this assay to isolate new mutations affecting colour processing. This work should help us understand how a neural network is built to integrate colour information into a tri-dimensional representation of the world. 2) For my PhD, I used a unicellular organism to study a basic cellular function, how cells switch from proliferation to differentiation. I have now shifted my focus to the actual differentiation in a complex organism. I want to study how wiring of the nervous system is established during development and how the information from the sensory cells is integrated in the brain. 3) After my extensive training with mechanistic approaches of a relatively simple process, this research plan will help me switch my interest in genetic to the different mode of thinking of developmental biology and neurobiology. The techniques used will also be different and Drosophila is a powerful system for studying problems at the organismal level.

I propose to develop a new conditional tool to study the function of specific cell types based on their local environment in an intact organism. Transgenic expression of chimeric ligand and receptor complexes on cell surfaces will modify or ablate target cells based on their contact with other specified cells. This proposal outlines a cell-contact-mediated ablation method that will exploit the Drosophila Delta-Notch signaling mechanism. The bacterial endonuclease CdtB and CRE will be expressed as inactive moieties fused to the extracellular-transmembrane portions of Notch. A second gene encoding a Notch ligand will be expressed on cells interacting with the target cells. Contact will activate CdtB or CRE by intramembrane proteolysis followed by nuclear translocation. Preliminary data from in vitro cell killing assays suggest that this system tightly and specifically controls COS-7 and N2a cell ablation. I will test the usefulness of this system using microglia and their role in prion pathogenesis as a paradigm. Activation of microglia in the brain correlates to many neuropathological disorders, including Alzheimer`s and prion diseases. However, the specific function of microglia, be it physiological, pathological or both remains unclear. Compartmentalized ablation of microglia will help elucidate the significance of this cell type in these diseases.