Proteolysis of key regulators of the cell cycle is controlled by ubiquitin ligases called SCFs because each is formed by three major subunits: Skp1, Cul1 and one of many F-box proteins (Fbps). The substrate specificity of SCFs is dictated by different Fbp subunits that act as substrate recognition factors. The aim of the proposed project is to investigate the function of a particular Fpb, namely Fbl4, identified by Pagano’s group group (Cenciarelli et al., Current Biology. 1999). Since joining Pagano’s lab, I have shown that Fbl4 is localized on the centrosomes. I will use cell biology and biochemical methods to understand the reason for the centrosomal localization of Fbl4. In particular, I will explore the possibility that Fbl4 is required for centriole separation and/or subsequent steps of the centrosome cycle. The inhibition of the expression and function of Fbl4 in living cells by RNA interference or by neutralizing antibodies will allow me to determine whether a lack of function induces a defect in centrosome duplication. As future directions I intend to identify and characterize specific interactors of Fbl4.

Methylation of arginines is emerging as a novel type of protein modification important for the regulation of cell growth and gene expression at many levels. The product of the HMT1 and PRMT1 genes are the major arginine methyltransferases of the yeast Saccharomyces cerevisiae and of humans, respectively. Recent evidence suggests that Hmt1/PRMT1 affect several steps associated with pre-mRNA metabolism including transcription, capping, polyadenylation, and mRNA export. The proposed research is designed to test the hypothesis that Hmt1 associates with the RNA polymerase II transcription machinery where it methylates histone tails and specific RNA-binding proteins as they are recruited to nascent pre-mRNA transcripts. We also intend to identify novel targets and cellular pathways controlled by arginine methylation. Such findings will further our understanding of the function of this poorly characterized type of post-translational modification. My experience as an Honours and M.Sc student provided me with a strong background in virology, whereas my PhD in the field of telomeres and telomerase introduced me to the biology of cancer and aging. The development of the present proposal on a poorly characterized post-translational modification, namely arginine methylation, in Dr Silver's laboratory will complement my background in molecular biology and in mammalian cell systems via experience in proteomics as well as with genetic and cell biological techniques using yeast. At the end of the Fellowship, I will able to study different basic cell biological processes in both a genetically tractable system (yeast) and in mammalian cells.

During my Ph.D in Dr. Amon’s lab, I found that asymmetric segregation of signaling components regulates the cell cycle in S. cerevisiae. I developed a general interest in how asymmetries regulate cell division and other cellular processes. Based on these interests, I decided to conduct my post-doctoral studies in the lab of Dr. Schweisguth due to his expertise in asymmetric cell division in Drosophila. My post-doctoral work will provide training in the fly system and will generate reagents and projects that I will use to start my own lab. I will eventually address questions of how signal transduction and asymmetric segregation of cell fate determinants regulate developmental potential. The generation of daughter cells with differing developmental potentials often arises from the asymmetric segregation of cell fate determinants. Notch (N) signaling is a master regulator of developmental fates and is carefully controlled during the asymmetric cell divisions within the Drosophila sensory organ precursor (SOP) lineage. Neuralized (Neur) positively regulates Notch signaling by promoting the endocytosis of Delta. Recent work in the Schweisguth lab has demonstrated that Neur is asymmetrically segregated within the SOP lineage suggesting that, like other N signaling regulators, its localization is a critical step for appropriate N signaling and lineage specification (Le Borgne, manuscript in prep.). The goals of this proposal are to: 1.determine Neur domains important for asymmetric localization, 2. identify regulators of Neur through a candidate gene approach and yeast two-hybrid screen, and 3. test the function of Neur and regulators of Neur in an S2 cell line assay and in vivo.

1) It is widely believed that a long-lasting change in synaptic function is the cellular basis of learning and memory. The most thoroughly characterized example of synaptic plasticity in the mammalian nervous system is long-term potentiation (LTP). One of the major modifications responsible for LTP is the synaptic delivery of AMPA-type glutamate receptors (-Rs). The molecular mechanisms involved in regulated synaptic delivery of AMPA-Rs have not been well delineated. However, a region located at the C-terminal tail of AMPA-Rs GluR4 subunit that contains a retention signal relieved by PKA phosphorylation on Serine 842, has recently been identified. My objectives are: i) to identify proteins that interact with the retention motif of GluR4 ii) to demonstrate that the identified proteins interact with the GluR4 subunit in vivo and iii) to determine the role of the identified proteins in the synaptic delivery of GluR4 using a combination of electrophysiological recordings and two-photon imaging. The proposed experiments should identify AMPA-R-interacting proteins controlling the regulated synaptic delivery of AMPA-Rs during LTP. 2) My thesis focused on the characterization of protein networks interacting with serotonin receptors, which belong to the G-protein coupled receptor family. The present proposal aims to investigate the molecular events involved in the synaptic delivery of ionotropic receptors during plasticity and thus is all very new to me. 3) This proposal will offer me a unique opportunity to learn new technologies and acquire a solid background in the field of synapse physiology. This is certainly important for my future career in neuroscience.

Reactions mediated by the concerted action of protein kinases and phosphatases play a key role in cellular signal transmission and cell cycle regulation. The proteins involved are often inhomogeneously distributed in eukaryotic cells and this spatial dimension can be a source of rich and unexpected dynamics. My project focuses on in vitro reconstruction of the reaction cycle of the mitosis promoting factor (MPF). MPF is activated by an indirect autocatalytic dephosphorylation. The time evolution of autocatalytic reactions is known to be sensitive to inhomogeneity or compartmentalisation in the reaction medium. In addition, travelling waves can be generated. Therefore, we plan to engineer reactions within various spatial constraints and we will measure the reaction-diffusion process using fluorescence imaging methods. The results would provide important insight into the dynamics of the cell cycle and we might initiate complementary in vivo studies. The above project clearly differs from my previous research as I will perform in vitro experiments using biophysical methods like fluorescence correlation spectroscopy, fluorescence resonance energy transfer and creation of inhomogeneous media. Moreover, the analysis of stochastic reactions arising in inhomogeneous media will require an expertise in advanced stochastic mathematical methods. Prof Oudenaarden's lab is comprised of both experimental and theoretical physicists and therefore provides a stimulating environment for learning both biophysical and theoretical methods which would ideally complement my previous experience in cellular biochemistry, genetics and basic dynamical systems theory.

The assembly of neural connections is a prerequisite for the proper function of the nervous system. Connections of the first olfactory map in Drosophila are created in the antennal lobe (AL) during pupal development. Olfactory receptor neurons (ORNs) project convergent axons to specific glomeruli where they synapse with precisely targeted projection neuron (PN) dendrites. This targeting precision is a dramatic example of connection specificity in neurobiology. However, the molecular mechanisms regulating the targeting process are not understood. I propose to study the cellular mechanisms that underlie PN dendritic targeting in the Drosophila olfactory system. In particular, I plan to examine the mode of PN innervation to determine whether targeting either occurs directly to a prespecified region or to an initially larger area that is later refined. I will use both confocal and two-photon laser-scanning microscopy (TPLSM) to visualise PN dendrites in living pupae during AL development. Observing PN differentiation on a single cell basis will provide hints about underling molecular mechanisms. Finally, examining mutants of potential candidate genes such as cell surface receptors will shed light onto the nature of targeting precision. I will switch from cell biology into developmental neurobiology and use TPLSM as a novel technique. The HFSP fellowship would allow me to enter the field of neurobiology and image for the first time the development of a synapse from its formation on until adulthood. Brain wiring differs from my previous research topic on cell division. From studies of available candidate mutants I hope to develop independent future projects.

I intend to delineate the role of elements of the neuronal transcriptional machinery in the process of cognitive control. Specifically, the project will be focused on the role of the cAMP responsive element (CRE) and the serum response element (SRE), in working memory (WM) and memory extinction (ME) of conditioned fear in the rat prefrontal cortex (PFC). WM can be defined as the representation of items in consciousness during experiences or just after retrieval of previously acquired memories that guide goal-oriented behavior. ME is the process by which new learning representations are acquired on the basis of preexisting ones. Blocking CRE and SRE functions in the PFC will be attained by a novel molecular technique involving microinfusion of consensus binding-site oligos fused to cell-permeable peptide carriers into the target brain area. This new system allows a precise manipulation of the location and temporal effect of the genetic inhibition. The study of the molecular substrates underlying WM and ME in the PFC can be very enlightening, since affective changes related to posttraumatic stress disorders are accompanied by cognitive dysfunction and decreased neuronal plasticity in the PFC. The main characteristic of this project is its interdisciplinary nature. My previous research was mainly focused on the pharmacological aspects of learning, and not on the regulation of the genetic information. Understanding the neural basis of behavior definitely requires a multilevel system approach. This fellowship experience will greatly enrich my genetics and molecular biology knowledge, as well as enable me to merge cognitive neuroscience with advanced molecular biology.

The physiologically important voltage-gated cation (Na, K, Ca) channels share a common architecture consisting of four subunits that generally contain 6 trans-membrane helices each. In each sub-unit, one of the helices is charged by lysine and arginine residues and acts as a voltage sensor. The goal of our project is to obtain structural insights on the channel opening to correlate its dynamical features with the trans-membrane voltage modulation and the displacement of the voltage sensor. Atomic force microscopy will provide a dynamic view of the solvent exposed portions of the channel, while the structure of the membrane embedded segment of the protein will be revealed by electron microscopy. In my previous research projects I used experimental data as an input for calculations Here, I wish to first produce some structural data and use them in further calculations. The present project, in which I will initially have to express proteins and prepare 2D crystals, will improve my knowledge of the molecular biology techniques that are essential to efficiently use the cell as a tool. Knowing more about these techniques will definitely raise my abilities to propose experiments that could verify hypothesis emerging from calculations or to even include some experimental work in projects that I will direct in the future. Through the current project I will also capitalize on my engineering studies during which I was introduced to different form of microscopy. Overall, this experimental study should provide me with new tools that will be useful in opportune time to overcome the main limitation of computational studies, i.e. the need of experimental structural data.

Specific mRNAs are targeted to distinct subcellular compartments of mammalian neurons, but the mechanisms and the biological significance of such a process are poorly understood. Given the strong conservation of mRNA targeting machineries between cells and species, I will use embryonic and larval Drosophila neuromuscular junctions as a genetic model to identify mRNA targeting factors. First, I will test the role of candidate proteins with conserved targeting function, using loss of function studies. Second, I plan to identify factors with polarized expression patterns, using a protein-trap genetic screen. Among such factors, products encoded by localized mRNAs and specific factors belonging to mRNA targeting machineries will be selected based on in situ hybridization pattern and sequence homology. Their biological role will be further studied using genetic loss of function. This project will allow me to use the experience I gained in Drosophila genetics and imaging to consider developmental biology at a subcellular level. While the questions I addressed during my Ph.D mainly concerned intercellular communication and specification of groups of cells on an organ scale (the ovary), the project I will develop aims at identifying factors involved in intracellular mRNA targeting mechanisms. Changing both the system and the scale of my studies will allow me to gain further insight into how a cell interprets and accomplishes its developmental program. In addition, both the experience of my host supervisor, and the technical and scientific environment of the EMBL will allow me to learn how to apply the most up-to-date molecular biological and cell biological methods.

1) PTP-PEST is an intracellular protein tyrosine phosphatase composed of one phosphatase catalytic domain and several protein binding domains. PTP-PEST has been involved as a regulator of focal adhesion (FA) and cleavage furrow turnover, as well as in the human inflammatory disease known as PAPA syndrome. However, the contribution of each PTP-PEST protein interaction domain in these different functions is still elusive. In Tremblay's lab, I will dissect the contribution of each protein binding domains to the FA and cleavage furrow phenotypes by generating PTP-PEST-/- cells re-expressing various mutants of PTP-PEST. Furthermore new fluorescent-reporter technology will be used to resolve the spatial and temporal function of PTP-PEST. To study the role of PTP-PEST in inflammatory disease, we will generate transgenic mice inducibly overexpressing dominant negative PTP-PEST specifically in the macrophage population. In parallel, conditional knock out mice are being generated. I will pursue this work by generating macrophage (and other relevant tissues) PTP-PEST conditional knock out. 2) The research opportunities within biochemistry, cellular biology, cell signaling and especially mouse genetics analysis offered in Tremblay's laboratory are outstanding complements of my own expertise. 3) I am convinced that combining my knowledge of live imaging, molecular biology with those aspects from Tremblay's laboratory and the outstanding research environment of the McGill Cancer Centre will lead to a productive and rewarding post-doctoral training. As a scientist, it is the best opportunity to complement my interest in cancer research with the ultimate ambition to develop my own lab.

Stem cells are natural units of tissue repair and homeostasis with enormous potential for clinical application. Multipotent skin stem cells reside in a niche, referred to as the “bulge,” located in each hair follicle. They give rise to epidermis, hair follicles and sebaceous glands during homeostasis and wound-healing. Elucidating their molecular properties is a prerequisite towards developing human skin stem cells for therapeutic purposes. Using mouse as a model, I provide a strategy to isolate skin stem cells. I will engineer transgenic mice expressing fluorescent proteins specifically in the bulge, so that pure populations of stem cells can be isolated by fluorescence-activated-cell-sorting. Purified cells will be assayed for whether they (1) retain stem cell character in-vitro, (2) retain their multipotent potential in-vivo, and (3) can be reprogrammed into other cell lineages. Microarray analyses will be performed on the stem cells to compare their expression profile with that of their progeny. This project differs dramatically from my prior research experience, where I studied the structure function relationship of CCR5, the principal HIV coreceptor. I used site-directed mutagenesis coupled to a panel of functional assays to understand HIV entry and how it might be blocked. My expertise in medicine, physiology, molecular and cell biology will be invaluable in tackling stem cell research. Training with a world leader in skin and stem cell biology at Rockefeller University will provide me with the necessary background to then return to my country of origin and launch my own lab, focusing on the biology and medical applications of epithelial stem cells.