1) Timing of mitosis relies on the E3 ubiquitin-ligase anaphase-promoting complex (APC). APC is activated by association with CDC20-like substrate recruitment factors. Whereas CDC20 functions in early mitosis, it is later replaced by its homolog CDH1 ensuring proper APC activity until the G1/S boundary. The few known APC(CDH1) substrates all play a role in cell cycle control. However, since APC(CDH1) has been shown to be active during differentiation and in postmitotic cells, it is likely that APC controls processes in cells that are not actively dividing. The intention of the proposed project is to characterize APC(CDH1) in both differentiating and in differentiated cells as follows: (a) elucidation of novel APC(CDH1) substrates, (b) identification of processes during differentiation that are governed by APC(CDH1), and (c) analysis of the regulation of APC(CDH1) activity in response to differentiating stimuli. These studies should assist in elucidating the contribution of APC(CDH1) to neuronal differentiation and to preservation of a differentiated state. 2,3) This work will allow me to extend my research experience from mechanistic studies in yeast to investigations in developmental biology of higher eukaryotes. Using novel approaches and employing new model organisms, it will focus on the processes that are controlled by ubiquitination and proteolysis during development and in differentiated cells. Dr. Kirschner’s lab is one of the very few laboratories with an outstanding reputation in both biochemistry and developmental biology. I will thus be able to complement my biochemical skills with a profound education in developmental biology of higher eukaryotes.

The hippocampus is widely believed to be involved in the storage or consolidation of long-term memories. Synaptic plasticity in the hippocampus is the leading experimental model that may underlie learning and memory. Indeed synaptic plasticity has been extensively studied in the hippocampus, mainly at the glutamatergic synapses on CA1 and CA3 pyramidal cells. The past few years have seen an increasing number of studies on hippocampal interneurons. Hippocampal interneurons are suggested to play an essential role in timing the occurrence of pyramidal cell activity by imposing “context” for the “content” carried by networks of principal cells. These observation raise an important question which is the goal of this project : How does the pyramidal cell population communicate with interneurons and is this synaptic communication modifiable? To address this issue parallel-recorded hippocampal pyramidal cells in the CA1 area and interneurons will be monitored in the behaving animal combined with microstimulation of single pyramidal cell. Three groups of experiments will be performed: experiment 1. (in vivo) We will test the hypothesis whether synaptic strength between pyramidal cells and interneurons is modifiable by behavioral experience. experiment 2. (in vivo) This experiment will examine whether pairing the discharge of a postsynaptic interneuron with that of a presynaptic input can persistently modify the strength of its afferent synapses. experiment 3. (in vitro) Hypothesis: The temporal relationship between the firing pattern of a presynaptic pyramidal cell and a postsynaptic interneuron is critical for the induction of principal cell-interneuron synaptic plasticity.

The molecular mechanisms that drive cellular life and death decisions have been the subject of intense research because of their primary roles in tissue development and maintenance. Excessive or insufficient survival signals can lead to various disorders, including cancer. Activation of the MAPK/ERK signaling pathway is induced by several stimuli and can result in cell proliferation, differentiation and survival. RSK has been shown by our group to be an important effector of this pathway, however, relatively little is known about the regulation of RSK activity. Using biochemical approaches, I intend to elucidate the molecular mechanisms involved in RSK activation. I propose, a) to identify novel upstream kinases that regulate RSK, b) to study the regulation of RSK binding partners, and c) to identify novel RSK downstream substrates. Ultimately, understanding the molecular mechanisms involved in the regulation of RSK will be essential for the generation of therapeutic agents capable of modulating cell survival and death. The laboratory of Dr. John Blenis at Harvard Medical School focuses on cellular growth, survival and differentiation, and has made outstanding contributions in the area of signal transduction mechanisms. Working on RSK signaling within this group will provide me with sufficient experience to pursue an academic career in signal transduction, preferably in molecular neurosciences, which has been the focus of my Ph.D. studies. My postdoctoral work, described in the accompanying proposal, builds on my areas of interest - signal transduction, cell survival and proliferation - and will undoubtedly further enhance both my research and teaching potential.

Despite the extensive use of antibiotics and vaccination programs, infectious diseases continue to be a leading cause of morbidity and mortality worldwide. The intracellular bacterium Listeria monocytogenes is one of the most studied models for such infections. Its ability to spread from cell to cell allows it to escape antibody and complement activity. Resistance to this pathogen relies on both innate and adaptive immunity, but many aspects of host defenses against Listeria are still not completely understood. To gain deeper insight into the nature of the immune response, infectious agents need to be considered in the context of their complex mammalian host. For this reason, an in vivo approach will be undertaken in the present proposal. The main goal of my work will be to isolate mutations in genes of the mouse that specifically disrupt the resistance to Listeria using systematic ENU mutagenesis screening. The results gained by this approach are expected to substantially enhance our understanding of the immune response to infectious diseases in all organisms, including humans. This postdoctoral experience will greatly differ from my Ph.D. subject. Actually, it will be an opportunity to switch to a yet unknown model for me, the mouse, in which genetic techniques differ from those I used in flies. Moreover, my work will rely on a real host-pathogen interaction approach and will concern innate as well as adaptive immunity, which does not exist in Drosophila. Finally, this experience will provide me with a deeper knowledge in mammalian resistance reactions to intracellular pathogens, a field on which I wish to pursue a large part of my scientific career.

Regulated protein degradation plays an essential role in the development of all organisms. In particular, ubiquitin-mediated proteolysis has recently emerged as a central regulatory mechanism for many cellular processes. In this system, a poly-ubiquitin chain serves as a degradation tag, causing destruction of tagged substrate proteins via proteolysis in the 26S proteasome. This process is catalysed by ubiquitin-activating (E1), -conjugating (E2), and ligase (E3) enzymes. Generally, E3 ubiquitin ligases determine the specificity of substrate ubiquitylation. Recently, a new family of E3 ubiquitin ligases containing a RING finger-like domain called U-box has been identified. The Arabidopsis genome contains a large number (~63) of genes encoding U-box proteins (referred to as PUBs for Plant U-Box). However, to date, only a few U-box proteins have been functionally characterized. In this proposal, we test the hypothesis that specific Arabidopsis PUB proteins act as E3 ligases in signalling associated with the the phytohormone gibberellin (GA). The GA signaling pathway is emerging as an important paradigm for the functioning of the ubiquitin-proteasome pathway in plants. GAs are essential regulators of plant growth and development, and GA-deficient mutants exhibit a dwarfed, retarded growth phenotype. GA promotes plant growth by overcoming the growth-repressive effects of a family of nuclear repressor proteins known as the DELLA proteins, and GA-induced proteolysis of DELLA proteins is a key step in GA signaling. This proposal focuses on the relationship between Arabidopsis PUB proteins and the DELLA proteins. Considering the relatively high sequence conservation of the U-box domain among eukaryotes, this research will provide a good prototype for the understanding of U-box protein function in eukaryotes in general, and not just in plants. In addition, this work will provide important new information that is essential for a complete understanding of the molecular mechanisms of ubiquitin-mediated proteolysis in plants, particularly with respect to its role in plant hormone signaling pathways.

Establishment and remodeling of neuronal connections (synapse) require a spatially and temporally orchestrated control of neuronal morphology and fine-tuning of assemblies of signaling complexes. Recent evidence indicated that, even in mature brain tissues, neuronal activity may initiate and regulate the active modification of cell shape and the sorting of neuronal molecules. The targeting of protein cargo to functionally specialized areas of neurons is an important neurobiological and cell biological problem. Currently, many components of synapse have been isolated. The formation and remodeling of synapse are thought to be regulated by the targeting of functional unit (adhesion molecules, channels, and receptors) to functionally specialized areas of neurons. However, the molecular mechanisms that underlie the establishment of synapse remain unclear, in part owing to the inaccessibility of the vesicle transport machinery to direct experimentation. I am now trying to develop a neuronal, cell-free assay and an in vivo primary neuron system that accurately reconstitute known events in synapse formation. The importance of this assay system is that it provides a new previously unavailable tool to study synapse formation. This research may contribute significantly to elucidation of the mechanisms for synaptic plasticity, a principal mechanism of memory and learning, and basis for development of human intelligence. These investigations will provide me with many new ideas and cell biology approaches to elucidate the cellular mechanism of synapse formation to pursue future studies as an independent scientist.

Calcium is an important signaling molecule that modulates various cellular functions including gene expression and synaptic activity. We have identified and characterized a calcium/calmodulin-dependent protein kinase (CaM-K) cascade which is activated by calcium/calmodulin (CaM). CaM-kinase kinase (CaM-KK) phosphorylates and activates CaM-kinase I (CaM-KI) and CaM-kinase IV (CaM-KIV) as well as protein kinase B (PKB). In the present application, we will use a method for in vivo identification of CaM-KK signaling complexes using TAT-mediated protein transduction into the brain and the Tandem Affinity Purification (TAP) coupled with mass spectrometry. The TAT-mediated protein transduction method is a technique to introduce purified proteins into cells as well as into the brain of rodents. The TAP method is a protocol for purification of endogenous protein complexes under native conditions. Combining these two techniques, we propose to purify proteins complexed with CaM-KK from brain under physiological conditions. We will identify members of these CaM-KK protein complexes by mass spectroscopy and investigate the physiological regulation of these interactions as well as their effects on CaM-KK activity and subcellular localization. I have been extensively trained in the area of developmental biology. I want to research experience in different areas to expand my scientific background. The studies proposed in this application are not simply a change in research area, it will give me the diverse background and experience of modifying a new methodology. These experiences will greatly broaden my technical abilities and allow me to pursue my scientific interests.

Cell migration, pathfinding and target site selection are crucial processes in the development of a complex organism. Much progress has been made in elucidating the molecular machinery that underlies cell and growth cone migration, as well as the extracellular guidance cues that can steer this migration. However, the question of target site selection is still only poorly understood. I plan to study pathfinding and attachment site selection using the somatic muscles of the Drosophila embryo as a genetic model system. The embryonic hemisegments A2-A7 each contain 30 muscles, which are uniquely identifiable by their size, shape as well as where they attach themselves to specific sites in the epidermis. This is an ideal system to analyze myotube guidance and target selection. I will perform a large scale genetic loss of function screen using a subset of GFP marked muscles and score for muscle navigation defects in living embryos. The goal is to discover factors which selectively guide each particular muscle to its appropriate target. To complement this forward genetic approach, I will also use transgenic double-strand RNA-interference to systematically screen all putative transmembrane proteins in the Drosophila genome for a function in myotube guidance and target site selection. Compared to my previous studies, this project is approaching a very different field. Instead of focusing solely on intracellular processes, one of its main topics is cell-cell and cell-matrix communication. The integration of extracellular guidance cues with the intracellular machinery required for pathfinding and attachment site selection will significantly broaden my scientific horizons.

The goal of this proposal is to understand the mechanism of IL-2 mRNA stabilization in response to extracellular stimuli. In response to T cell activation signals, the half life of IL-2 mRNA is greatly extended. The 3'UTR of IL-2 mRNA contains AU rich elements (AREs) that mediate rapid IL-2 mRNA degradation. In order to study these cellular processes that modulate the IL-2 mRNA turnover during T cell activation, I propose to find transacting factors involved in the control of lL-2 mRNA stability in activated T cells. In conection with this, I have purified NF90 protein as one of the IL-2 ARE binding proteins in stimulated T cells, suggesting that NF90 modulates the activity of the degradation machinery that assembles on the 3'UTR stabilizes an mRNA containing the IL-2 ARE. To gain a better understanding how these trans-acting proteins including NF90 regulate gene expression at the post-transcriptional level, I will identify the nature of these cytoplasmic protein complex that control IL-2 mRNA stability using biochemical and genetic approaches. This project will contribute to obtain both unberstanding of general mechanism in the signal-regulated mRNA stabilization and methodology of defining the RNA binding protein related mRNA stability. Furthermore, understanding the regulation of IL-2 expression may provide us with new targets for screening for novel agents. Such drugs by inhibiting or promoting the expression of IL-2 should prove useful in the prevention of T-cell leukemia and may improve IL-2 mediated immunotheraphy for leukemia. Personally, this project provides me the progress in growing as an independent researcher in this important area.

The purpose of the proposed fellowship is to study the genetic basis of sexual dimorphism in the zebrafish, focusing on gonadogenesis. My graduate work has focused on aspects of early gonad development of the nematode, C. elegans, which are common to both sexes. I would like to expand these interests to investigate how dimorphic characters are established in a vertebrate system. Although much is known about the development of vertebrate organ systems, only a handful of genes are known to play a role in sexual development in mammals. By studying this problem in a genetically tractable organism, I will be able to add a great deal of knowledge to this field. During the course of this fellowship I plan to 1) determine if whether genes known to function in sex-specific development in mammals have conserved functions in the zebrafish gonad 2) isolate and characterize new genes that function to create dimorphic characters 3) create a genetic profile of sex in zebrafish. After completion of the fellowship, I plan to continue this line of research as principal investigator.

My aim is to understand the coordination of different brain modules in a complex task and I propose to investigate the problem of interference (the dual task problem) as a way to address it. Since this requires dynamic information of activity at the whole brain level, I accompany my proposal by a series of steps to develop a technique, based on phase analysis, to achieve high temporal resolution within fMRI. This new experience will differ from my previous research experience both in the methodology and in the concrete area of investigation. My research started in the area of dynamical systems, in applied math and from there I moved to computational neuroscience, more precisely in the workings of early vision. My work was mainly computational and behavioral. Since then the problem of representations and ultimately understanding the neuronal basis of thoughts has been my long-term goal. I am now in the process of a new jump to cognitive neuroscience. In this new stage of my career I want to work at the whole brain level, understanding the dynamics of streams of information. My work will be on fMRI, both in techniques development (one of the main aspects of this project) and in its application to the problem of dual task interaction. This project will require a series of steps, some of them more technical and others more conceptual. This fellowship should give me enough time to attempt this long term project and the opportunity to move to an environment where I may learn the technical aspects of fMRI and of cognitive sciences and provide the experience I have accumulated on dynamical systems and computational neuroscience over the first stages of my career.

The research objective is to study the dynamics of virus attachment to the plasma membrane. We will image interactions of individual virus particles with their biological receptors at the cell surface and on artificial surfaces in real time, and quantify the observed dynamics with computational methods. The acquired quantitative information will aid development of mathematical models. The results are expected to provide new evidence to support or refute current models of virus binding to its cellular receptor in the context the complex lipid-protein environment of the plasma membrane. This proposal differs from my previous research experience: 1) Virus biology, endocytosis, and glycolipid biology are new research interests, distinct from nucleocytoplasmic transport 2) Techniques including lipid reconstitution, virus propagation/ purification, image analysis, single particle tracking, in vitro flow cells and TIRF microscopy are new The mathematical modeling strategies proposed differ from those I used previously. My position in the Helenius lab is as a post-doctoral coordinator of an engineering-, biology- and mathematical modeling-based project. I have the responsibility to organize, and move along a large-scale program project involving ETH faculty and students with distinct research specialities. Thus, I extend my ability to work with technical and computational specialists. While I will likely expand my techniques repertoire, I also anticipate the intellectual freedom and responsibility to coordinate my research in the collaborative ETH environment will be a primary fellowship benefit and advance my long-term career goal of obtaining an academic research position.

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.