The release of neurotransmitter at synapses requires the fusion of synaptic vesicles with the plasma membrane. Synaptic vesicle exocytosis, like the fusion of membranes at all stages of vesicular transport, is mediated by SNARE proteins. These SNARE proteins have been shown to be sufficient for fusion in vitro, whereas genetic experiments indicate that other components are also essential in vivo. Traditionally, G protein pathways are thought to be only modulatory in synaptic vesicle release however, genetic evidence from the nematode C. elegans indicates that it could be an essential process. Indeed, mutations in Gq-alpha lead to a severe paralysis of the worms, for which one possible cause is a lack of synaptic vesicle release. Alternatively, this paralysis could be caused by defects in other molecular pathways or by defects in the muscles. If Gq-alpha is actually essential to synaptic exocytosis, vesicle release should be eliminated in Gq-alpha null mutants, whereas if it is only a modulatory component, vesicle fusions should persist. We propose to address this issue by determining (i) whether mutants missing presynaptic Gq-alpha lack synaptic exocytosis, (ii) whether constitutive activation of Gq-alpha increases the number of synaptic fusions, and (iii) the identity of the signaling components downstream of Gq-alpha. For this purpose, I plan on using an interdisciplinary approach, mixing molecular, genetic and electrophysiological approaches. My previous experience in electrophysiology will be useful, whereas I will learn genetic and molecular techniques in the Jorgensen laboratory. I will then be prepared to run my own laboratory using this experimental system.

The central nervous system can be modified by experience and environment. The distinct environment-dependent modification of the mature nervous system is best exemplified by the recent finding that neurogenesis in the adult hippocampus can be regulated by environment. This adult neurogenesis adds new neurons specifically in the dentate gyrus of the hippocampus, which projects to the hippocampal CA3 region via mossy fibers (MFs). In the proposed study, I will examine the role of MFs in regulation of the brain functions by environment. I will examine effects of changes in qualitatively different environmental factors on the MF synaptic transmission and behaviors in mice, to reveal the correlation between synaptic and behavioral changes. I will use behavioral tasks considered to reflect anxiety and despair, because the neurogenesis has been implicated in the impairment of mood regulation. Furthermore, I will examine the contribution of peptide transmitters in MFs, whose expression is also regulated by environment, as well as the adult neurogenesis to synaptic and behavioral changes. I expect that the proposed study will reveal novel aspects of hippocampal functions. During my graduate and postdoctoral studies, I have been examining hippocampal functions using electrophysiological techniques. In the proposed study, I will carry out behavioral experiments in addition to electrophysiological recordings. Thus, the CDA will help me to learn new methodology and provide an opportunity to comprehensively investigate the functional significance of the MF synaptic transmission, which is my long-term research subject.

Defining the DNA damage response at chromosome ends This project is aimed at understanding how cells detect and process dysfunctional telomeres. When telomeres are shortened in primary human cells, the DNA damage response is activated leading to apoptosis or senescence. Although this pathway is thought to be important for aging of human cells and contributes to certain aspects of human cancer, little information is available on how dysfunctional telomeres are detected. In this project, the molecular details of this response will be defined. The approach will use a combination of genetics, cell biology, biochemistry, and proteomics to define the proteins associated with dysfunctional telomeres and the signaling pathways involved. The research will provide me with training in cell biology and genetics and educate me in the areas of telomere biology, aging, and cancer.

Mechanosensitive (MS) ion channels are the simplest mechanosensors. MscS is a bacterial MS channel, which is also voltage dependent and therefore is an extremely interesting molecule for studying structure–function relationship of both voltage- dependent and mechano-dependent gating. The main goal of the project is to study the conformation changes of the transmebrane segments of MscS channel, which probably constitute channel’s primary stretch and voltage sensors. Various techniques will be employed. Classical cysteine accessibility method will give information about relative position in the membrane of a given residue. State-of-the art fluorescence methods, including patch-clamp fluorometry (PCF), will allow real-time monitoring of conformation changes of specific channel domains. And a new optical nano-tweezers technology will enable us to induce conformational changes of known magnitude in specific domains of the channel. The project is an innovative combination of PCF and optical nano-tweezers to detect and induce local conformational changes in the MS channels. The establishment of this methodology will be of great importance for future research on MS channels as well as on other channels and transporters in the native bacterial membranes. It will also expand my personal knowledge from classical electrophysiology into a multidisciplinary approach for studying structural dynamics using optical techniques.

While the influence of histone lysine methylation on chromatin structure and function has been extensively studied, there is little known about the occurrence and function of this post-translational modification in non-histone proteins. In order to gain insight in the functional significance of non-histone lysine methylation I propose the following line of investigation: First, I will identify non-histone substrates by immunoprecipitations with a pan-methyl-antibody. Next, the impact of lysine methylation on described functions and properties of these candidate proteins will be analyzed by biochemical and cell-biological means. Finally, I will address the in vivo function of lysine methylation of selected candidates by replacing the wild-type gene with a non-methylatable mutant allele. This research plan will be carried out in two distinct eukaryotic model systems. The generation of knock-in mice for the most exciting mammalian non-histone substrates will reveal the roles of their methylation during mammalian development. In addition, I will also take advantage of the powerful genetics of a lower eukaryote (Tetrahymena thermophila) to rapidly examine the involvement of non-histone lysine methylation in basic cellular processes. The proposed research significantly and logically extends my previous work on histone methylation. This project will offer me valuable exposure to two genetic model organisms with which I have no experience so far. Furthermore, the focus on non-histone proteins will launch a novel and exciting area of research that will widen the importance of lysine methylation to numerous biological questions outside of histone biology.

Mice infected with low numbers of wild type (wt), but not of SecA2-deficient intracellular bacteria Listeria monocytogenes (LM), develop CD8+ T cell-mediated long-lasting immunity, and are resistant to secondary infection with high numbers of wt LM. SecA2 is an auxiliary secretion system of Gram-positive pathogenic bacteria which controls the secretion at least fifteen bacterial proteins. Although not protected, mice immunized with SecA2- LM mount robust secondary CD8+ T cell responses. Also, dendritic cells (DC) are required to prime T cells. Based on this, we hypothesized that one of the SecA2-secreted protein triggers an unknown intracytosolic pattern recognition receptor necessary for protective CD8+ T cells differentiation. Our first aim will assess whether immunization with DC coexpressing a SecA2-secreted protein and an LM antigenic peptide mount antibacterial protective memory responses. Our second aim will determine which intracytosolic receptor is targeted by this bacterial protein. Long-term approaches will involve knocking-in this receptor in vivo and study how it impacts protective memory induction. Our third aim will focus on real-time dynamics of bacteria, bacteria-infected cells and T cell activation using biphotonic microscopy. Specifically, we will test whether SecA2- or wt LM-primed CD8+ T cells undergo distinct dynamic phases during priming. During my HFSP-sponsored postdoctoral period, I improved my CV and was granted an Assistant Professorship in France. Since then, I built-up my research system and accumulated new data. A CDA would further secure my position and funding for the coming years as well as support my carrier long-term perspectives.

Cohesion between sister chromatids is essential for the faithful inheritance of chromosomes through cell division. Cohesin, the protein complex responsible for tethering sister chromatids was extensively characterized in yeast cells by genetic and biochemical methods. Together these data led to a comprehensive hypothesis of the cohesin cycle: the loading of the ring-shaped complex onto chromatin in G1, the establishment of cohesion in S and the release by cutting of the cohesin ring in M phase (Uhlmann, 2004, Exp Cell Res 296, 80-85). However, the biochemical assays utilized in the past lack the spatial and temporal resolution required to directly prove or refute the proposed mechanism and to generate new mechanistic models. I will therefore establish an experimental system combining biochemical and quantitative imaging methods to directly visualize the functional dynamics of cohesin on chromatin in Xenopus egg extracts as well as in cultured mammalian cells. I will develop fluorescence based reporters, to assay the local composition, conformation and dynamics of cohesin complexes. With these tools, I will focus on two major questions. First, how, when in the cell cycle and at which chromosomal locations does cohesin loading occur? And second, how do cohesin complexes establish sister chromatid tethering during replication? These experiments will advance the mechanistic understanding of the essential function of the cohesin complex. In addition, as the majority of previous work was carried out in yeasts, my studies in vertebrate systems will supply important information about the conservation of the mechanism of sister chromatid cohesion during evolution.

During the last decades biology has decomposed cellular systems into genetic, functional and molecular networks. It has become evident that these networks consists of components with specific functions (e.g. proteins and genes). This has generated a considerable amount of knowledge and new hypotheses. The idea presented here is to test part of this knowledge by building, through reverse engineering, new synthetic biological systems from the known components. Two projects are proposed: 1. A synthetic biology project, aimed at creating new modular signaling proteins: Proteins in signaling networks are modular in nature. I propose that the modularity of these proteins are now so well understood and comprehensively cataloged that we can begin to use the modules as building blocks in new synthetic modular systems and proteins. Designing and successfully introducing synthetic proteins into cellular signaling networks would provide us with a powerful research tool with many applications, such as development of biosensors, protein drugs and rewiring of biological pathways. 2. Modular decomposition of protein interaction networks, aimed at deriving network topology and dynamics: Deriving the topology and dynamics of protein-protein interaction networks in cellular signaling systems under different conditions (e.g. DNA damage response and disease states) will be useful for the understanding of robustness, complexity and checkpoint mechanisms of biological systems. I will perform integrative studies, based on synthetic biology, proteomic assays and bioinformatics techniques, of the topology and dynamics of both biological and engineered synthetic signaling protein interaction networks.

Nearly 100 years ago, a phenomenon termed heterosis was observed when hybrid plants outperformed their inbred parents for growth and vigor. Heterosis has been exploited in plant and animal food production, and it was a major contributor to the agricultural boom known as the ‘Green Revolution’. But heterosis has a more fundamental biological significance in the evolution of plant and animal species by providing a heterozygote advantage in naturally occurring hybrids. It has been shown that heterosis is a genome-wide phenomenon whose complex phenotypic effects involve the action of many genes. But the specific genes controlling heterosis remain unknown. Research in the laboratory of Dr. Dani Zamir at Hebrew University in Israel uses tomato as a model system to decipher the molecular basis of multigenic complex traits that exhibit a continuous range of phenotypes, as opposed to those that fall within discrete classes. My expertise lies in using molecular analysis to address the genome-wide regulation of gene expression through changes in chromatin (epigenetic) states. I intend to combine my knowledge with that of Dr. Zamir’s group, where I will develop new skills in quantitative genetics, statistics and bioinformatics. This combination of tools will enable me to trace heterosis to individual genes and study their molecular effects in a genomic context. This broad training will make me proficient in all aspects of analyzing complex traits, which I believe will be an exciting and leading area of research in the future.

The inter-cellular transfer of regulatory transcription factors and mRNAs has become a new paradigm in plant biology in recent years. The transcription factor KNOTTED1 (KN1) was the first such example of a developmental signal that traffics from cell to cell. Its conserved DNA binding domain, the homeodomain, is sufficient to mediate its own transfer from cell to cell in vivo, as well as promoting transfer of the KN1 mRNA. The molecular details and significance of this interaction as well as the significance of KN1 mRNA movement remain elusive. I therefore propose to investigate these questions by: (i) determining the sequences required for the interaction between KN1 and its mRNA, (ii) uncovering the molecular mechanisms of KN1/mRNA intra- and inter-cellular trafficking. (iii) Analyzing the physiological significance of KN1/mRNA trafficking in plant development. This interdisciplinary project aims to study the molecular mechanisms of a new paradigm in intercellular communication: the direct transfer of mRNA and transcription factors from cell to cell. My previous research experience has focused on evolutionary plant molecular biology and genetics. This project will train me in plant cell biology and biochemistry. Finally this project will provide the starting point for me to become an independent investigator.

During nervous system development, neurons have exquisite ability in selecting their synaptic partners among other adjacent cells and forming synapses only onto discrete and stereotyped subcellular regions of these targets. The molecular mechanism of such specificity is largely unknown. In C. elegans a genetic screen for mutants with defects in synaptic specificity has identified SYG-1 and SYG-2, two proteins of the Immunoglobulin superfamily that are both localized at synapses. In both syg-1 and syg-2 mutants, egg-laying motor neuron HSNL fails to recognize its synaptic partners and forms ectopic synapses onto abnormal targets, although its axon trajectory is normal. Interestingly the ectopic synapses in the syg mutants are still formed onto a selective group of targets, suggesting that synaptic specificity is altered but not eliminated in the absence of SYG-1/SYG-2. These data raise the possibility that synapses are formed as a result of synaptic competition between hierarchical potential targets. As a post-doctoral fellow in Dr Shen’s lab I propose to test this model using complementary cellular, molecular and genetic approaches in C. elegans. I will perform time-lapse experiments to ask if synaptic competition occurs during normal development. I will further test whether wild type synapses are required for suppression of ectopic vesicle clustering, by ablation and rescue experiments. Finally I will identify the factors downstream SYG-1/SYG-2 involved in synaptic competition using different genetic screens. In parallel I will develop trans-synaptic markers that will allow functional studies on synaptic specificity and neuronal networks development in C. elegans.

Thrombocytopenia, or low platelet count, is a serious problem in hematological and oncological clinical practice. The best mouse model for this disease is the Mpl-/- mouse, which lacks the receptor for thrombopoietin, and has only 10% of normal platelet levels. Recently, ENU mutagenesis-forward genetic screens have been used in the mouse to investigate gene-phenotype relationships. Thus far, these screens have almost exclusively used wild-type mice as a starting point. In contrast, we plan a large-scale forward genetic screen based on Mpl-/- mice. By screening the offspring of ENU-treated mice for elevated platelet counts we aim to find dominant suppressors of thrombocytopenia. These mutants will be identified and characterised biochemically. Like most small molecule therapeutics, the majority of ENU induced mutations lead to loss of function of proteins. Thus, in addition to the primary aim of significantly advancing our knowledge of platelet biology, these studies may identify new drug targets for the treatment of thrombocytopenia. During my PhD, I have used gene targeting technology or ‘reverse genetics’ to study signalling downstream of insulin/growth factor receptors. While this approach has been invaluable in investigating countless biological systems, it requires prior knowledge of the gene of interest. In contrast, a forward genetic screen selects for mutations in genes that have a function in the biological process of interest. A fellowship will allow me to acquire experience in murine forward genetic screening. I believe this, in combination with the techniques in reverse genetics learned during my PhD, will place me in a powerful position in the future.