Inhibitor of DNA binding (Id) family members are key regulatory proteins in several cellular processes including proliferation, differentiation, angiogenesis and metastasis, that act as dominant negative inhibitors of the basic helix-loop-helix (bHLH) transcription factors. Id1 and Id3 are important for angiogenesis during development, as Id1-/-Id3-/- mice are not viable due to hemorrhage in the forebrain. Id1/Id3 expression in endothelial cells is also required for tumor angiogenesis. Xenograft tumors in Id1+/-Id3-/- mice show angiogenic defects and fail to growth and/or metastasize. Reduction of Id dosage in tumor mouse models also leads to abnormal tumor vascularization. Other studies with cultured cells suggest that Id expression in the tumor itself could also contribute to the neoplastic phenotype of tumor cells by inhibiting differentiation and stimulating proliferation and invasiveness. The low postnatal expression of the Ids and their roles in tumorigenesis mark them as attractive targets for cancer therapy. We will use Cre/LoxP technology to conditionally inactivate Id1 in a temporal and spatial controlled manner. We plan to conditionally inactivate Id in a mouse model of breast cancer. Conditional disruption of Id in the breast epithelium or endothelial cells will establish the requirement for Id expression in either the vasculature or the tumor in an in vivo model system. We will also use the conditional Id knockout mice to determine if Id loss after tumor initiation results in tumor regression and to analyze the effects of complete loss of Id1/Id3 in adult animals, important considerations for developing cancer therapies using Ids as targets.

Glial cell function in Drosophila nervous system Wuping Ge (China) Department of Physiology, University of California, San Francisco Glial cells constitute 90% of the cells in our brain, but their functions are largely unknown. During the past decade, what we know about glial cell function is mainly derived from mammals or cell culture system. To understand the role of glial cells in the brain, it is important to analyze their contributions to neural circuit formation and activity modulation, but these studies are very difficult to perform in mammals. Drosophila offers the possibility to express a light-gated channel fused with green fluorescent protein-GFP (ChR2-GFP) in different subsets of glial cells and to manipulate glial cell activity easily in the intact insect or in the dissected whole brain. During my PhD studies I investigated properties and the function of a novel glial cell population, the NG2+ cell, and found long-term potentiation, a cellular explanation for learning and memory, also exist in the synapses of NG2+ cells, by carrying out electrophysiological studies of mammalian brains (rat). In this proposal I will use a new model system with genetic and molecular techniques to investigate the role of glial cells in neural circuit formation and function in Drosophila nervous system. Jan lab is a leading group in developmental neurobiology. It will expose me in many new approaches to address the question. The successful completion of this project in Drosophila will shed light on the research of the glial cells in mammals, for Drosophila glial morphology and functions are similar to those in mammals.

Intricate networks of weak interactions that stabilize native three-dimensional structures, make protein folding an inherently complex process. In the conventional paradigm, the problem is inaccessible to experiments because protein domains are assumed to fold in an all-or-none process. In addition to limitations in connecting energetics with structure, mutational methods have significant experimental uncertainties and are unable to map complex networks of interactions. In contrast, analytical theory predicts small barriers to folding and the possibility of downhill folding. These theoretical predictions have been confirmed experimentally in recent years, including the observation of global downhill folding. Using nuclear magnetic resonance (NMR) techniques: structural determination by triple-resonance NMR experiments, protein dynamics by NMR relaxation measurements and determination of residual dipolar couplings at various degrees of unfolding, we will investigate how small globular proteins with marginal barriers (less than 3RT), such as BBL and gpW proteins from Escherichia coli, unfolds atom by atom starting from a defined three-dimensional structure. This view confirms the statistical nature of folding, and exposes the interplay between hydrogen bonding, hydrophobic forces, backbone conformation and side-chain entropy. Those data allow also determine a map of the interaction network in this protein, which reveals the source of folding cooperativity.

Genome integrity is maintained by different mechanisms that prevent excess of deoxyribonucleotides (dNTPs), the building blocks required for DNA synthesis. Indeed, excess of dNTPs lead to increased mutation rate that can be deleterious for the cells. However, mutations are essential to promote the genetic variability that is required for the evolution of species. Indeed, studies showed that increased dNTP pools help yeast cells to survive DNA damage. The idea that mutagenesis is increased under stress conditions is novel and particularly interesting in term of evolution, because it suggests that gene mutations do not occur at random but are induced by the environment, allowing a better survival of the population in these conditions. My long-term plans are to understand i) how are dNTPs levels regulated to maintain genome integrity as well as to induce mutagenesis ii) what is the molecular mechanism that triggers induced mutagenesis. We propose to combine in vivo and in vitro experiments using genetic approaches in conjunction with biochemical and structural studies to address these questions in Escherichia coli, our model organism. This work shall contribute to our knowledge of the conserved process of DNA replication and may shed light on the molecular basis that promote cancer and neurodegenerative diseases. The HFSP fellowship award supported the emergence of this project and a CDA award would provide the financial support and scientific independence to develop it. It will help me to set up my laboratory in an appropriate and competitive environment as well as help me to maintain and organize a collaboration network trough laboratories with complementing expertise.

Cbl-interacting protein of 85 kDa (CIN85) and CD2-associated protein (CD2AP) are adapter molecules controlling a variety of cellular processes including receptor endocytosis, neuronal cell survival, immunological synapses in T cells and kidney glomerular filtration. I have recently shown that CIN85 is required for the fly development. In order to understand the physiological and pathological roles of CIN85 in mice, I propose to ablate CIN85 and its multiple isoforms by a gene targeting approach followed by the molecular characterization of observed phenotypes. Mice lacking wild-type and extra large CIN85 isoforms in brain are viable and fertile displaying a hyperactivity syndrome. By targeting exon 12 of CIN85 I will remove the full size and all major SH3 domain-containing CIN85 isoforms. I'm interested to constitutively and conditionally use these mice to molecularly characterize cell-specific functions of different CIN85 isoforms and to understand the biology underlying the phenotype in classical CIN85 knockouts, as well as to investigate CIN85 function in T cells. In addition, CIN85/CD2AP double knock-out mice will be created to investigate overlapping functions during development and adult life. Experimentally, mice and embryos will be subjected to histopathological analysis and primary isolates of various cell types will be used to study dynamics and posttranslational modifications of CIN85-containing protein complexes (predominantly in dendritic spines and T-cells), using biochemical methods, mass spectroscopy and bioinformatics. Taken together this proposal aims to determine the unique and common functions of different CIN85 isoforms and CD2AP in vivo.

A challenging frontier in neuroscience is to understand the molecular pathways that regulate the organization, function and plasticity of circuitry mediating specific behavioural responses to environmental stimuli. Using pheromone-initiated behaviours as a model, the present study proposes a methodology to investigate the molecular pathways regulating the organization and function of the female versus male pheromone-mediating circuitry that leads to sexual versus aggressive behaviours, respectively, in adult males. Transgenic mice will be generated in which neurons activated in response to female versus male pheromones can be recognized by their expression of a fluorescent reporter. Using established single-cell gene expression profiling techniques, I propose to isolate and analyze pheromone receptor-expressing neurons in the vomeronasal organ and within higher order central nervous system (CNS) structures to address two fundamental questions: (1) What is the combinatorial pheromone receptor code that mediates processing of female versus male pheromones? (2) What are the distinct molecular pathways that regulate the organization and function of higher order circuitry processing female versus male pheromone information? This study is anticipated to significantly advance our understanding of the molecular logic that underlies female versus male pheromone-initiated behaviours in adult males. Further, the project proposes a general methodology for the investigation of molecules regulating the organization, function and plasticity of circuitry in the adult CNS.

The localization and organisation of the organs in the body depends on the patterning of the Left/Right (L/R) axis during embryogenesis. Around embryonic day E7.5 in mouse, an asymmetric signal generated in the node is transferred to the left lateral plate mesoderm (this mesoderm, located in the lateral region of the early embryo, contributes to the mesenchyme of various organs). This symmetry breaking event results in the establishment of L/R asymmetric gene expression patterns responsible for asymmetric organogenesis. If an increasing number of studies focus on the regulation of these early events, less is known about how asymmetric information is translated into L/R specific morphogenetic events driving later organogenesis. The aim of this proposal is to address this question using the developing gut as a model, in both chick and mouse embryos. I propose to identify the specific molecular mechanisms at work within the cells ultimately responsible for asymmetric morphogenetic events. To this end I will first characterize, in detail, the sequence of cellular events driving gut asymmetry. I will then identify genes implicated in the control of these events using a microarray approach comparing left and right part of the gut. Finally I plan to test the implication of the best candidate genes, coming out of the microarray screening i) using genetically modified mice lines, ii) by interfering with their function using retroviral misexpression of cDNAs and RNAi constructs in chick embryos. This study will bring new insights into the molecular regulation of asymmetric morphogenesis and enlighten the regulation of morphogenetic processes commonly utilized during embryogenesis.

The aim of the research described in this proposal is to develop a physical assay for chromosome packaging and compaction in vivo using Bacillus subtilis as model system. This assay would allow us to determine the requirements for chromosome condensation in vivo and test specific models for the organization of the bacterial chromosome by proteins like Smc and Spo0J. For that purpose we will first determine the dynamic distributions of Smc and Spo0J complexes along the chromosome and their dependence on DNA replication and transcription using high-resolution ChIP on chip technology combined with cell population synchronization. This analysis will reveal the temporal and spatial regulation of Smc’s and Spo0J’s association with DNA. We will then aim at generating a conditional allele of the Smc gene that can be switched on and off within seconds using drug (FK1012 or rapamycin derivatives) dependent dimerization of FKBP12 inserted into the Smc hinge domain. Artificial hinge dimerization is supposed to block de novo loading of Smc complexes onto DNA and might thus allow us to interfere with Smc function at specific points of the cell cycle. Finally, we want to adapt and optimize the chromosome conformation capture technique (3C) for use in bacteria and detect DNA loops (juxtapositioning of certain chromosomal regions) that depend on chromosomal proteins like Spo0J and/or Smc. This assay would allow us to record the formation of chromosomal loops at different points of the cell cycle and at specific regions of the chromosome in real time. Having detected loops we could then further dissect the molecular mechanisms of Smc and Spo0J dependent chromosome organization in vivo.

Cells are composed of many thousands of different molecules that interact, create and modify each other. Many of these molecules are proteins which are in turn subject to incessant fluctuations governed by a complex interplay between their thousands of atoms. While the intricate dynamics of both layers is readily acknowledged, little is known about the interface between molecular and network-level complexity. I plan to recombine modular protein domains into artificial network motifs with defined dynamic behavior. Special emphasis will be put on the design of swapable interfaces that allow for the exchange and 'rewiring' of the different components. Modular input devices that form or break specific interactions depending on external or internal signals will be interfaced with output devices whose activity is triggered by spatial proximity between two domains. The careful control and labeling of interacting players may, in combination with cellular and molecular modeling, reveal how the dynamics and modular architecture of proteins affects the functioning of natural networks. I have previously studied the interplay of molecular dynamics and protein-protein interaction with atomic detail simulations. Through this work, I hope to complement my meanwhile purely computational background with experimental techniques, systems biology know-how and strong collaborations that allow for independent research at the cross roads of molecular and cellular complexity.

Ribosome synthesis, one of the predominant biosynthetic activities in eukaryotic cells, consumes an important part of the cellular resources and must therefore be tightly coordinated with other fundamental cellular processes. Although ribosome biogenesis has been extensively studied, the nature of this coordination and the underlying mechanisms are poorly understood. Recent results indicate that several factors required for post-transcriptional steps of ribosome synthesis are also directly involved in cell cycle progression, suggesting that these factors mediate a connection between these two fundamental processes. The aim of this proposal is to systematically identify ribosome biogenesis factors also required for cell cycle progression both in yeast and mammalian cells. Characterization of their sub-cellular localization during the cell cycle and of the protein complexes they are associated with will provide clues on their mode of action and shed light on the molecular bases underlying the potential connection between ribosome synthesis and cell cycle progression. This project is part of a new line of research I wish to develop and the HFSP CDA would provide me with an independent source of funding to start developing this project and animating my own group. The CDA would indeed allow me to finance students or postdocs and to cover the heavy cost of some of the proposed experiments.

The bandoneon mutants exhibited simultaneous contraction of bilateral muscle due to a mutation in GlyRb. Interestingly, application of strychnine, an antagonist of GlyR, to early wild-type embryos (30 h treatment at 18 hpf) not only caused bandoneon-like behavior but also chronically blocked postsynaptic aggregation of GlyRs following treatment. In contrast, strychnine treatment at larval stages (30 h treatment at 96 hpf) acutely blocked GlyRs but did not alter clustering of GlyRs. These investigations indicate that the synaptic aggregation of GlyRs is dramatically regulated by activity during embryogenesis but not at later stages. I propose to study the regulation of glycinergic synapse formation by activity and its molecular basis. The effects of activity on development of glycinergic synapses will be quantified by immunolabeling of GlyRs, physiological recording of glycinergic currents and behavior. I have found that glycinergic synapses are most sensitive to activity during the period between 18 hpf and 36 hpf. I am generating transgenic fish to label GlyRs, glycinergic pre- and postsynaptic neurons by CFP, GFP and RFP, respectively. These zebrafish will be useful to visualize the GlyRs aggregation in vivo and to isolate postsynaptic neurons by FACS. I will collect mRNA from the postsynaptic neurons and perform microarray analysis to screen candidate genes that are up- or downregulated by the treatment. I would like to focus on cytosolic protein, which can be regulated by Ca. I will examine overexpression and knockdown of the candidates to analyze their function in synapse formation. This is a new, simple model to study development and plasticity of synapse in vivo.

Mice lacking an intrinsic immune system can be transplanted with human stem cells, allowing the development of human blood cells in a manipulable system. Such a system may provide a powerful tool for developing human therapeutics. In addition humanised mice could allow a greater analysis of human lymphocyte development in vivo. Natural Killer (NK) cells comprise part of the innate immune system and are strongly implicated in the recognition and clearance of tumour cells and pathogen-infected cells. I will attempt to generate humanised mice with a fully functional and stable NK cell compartment. Firstly, I will study the reconstitution efficiency of human NK cells in newborn alymphoid mice by intrahepatic injection of human stem cells of varying phenotype (CD34+CD10+/-CD7+/-CD38+/-) and origin (foetal liver, thymus or cord blood). Transgenic-expression of human class I MHC on a murine class I MHC-deficient background and enhanced in vivo concentrations of cytokines will also be tested for improved NK lymphopoiesis. Using this system I will investigate the phylogeny of mature human NK cell subsets and characterise cell surface phenotype, tissue distribution, turnover and function. Immature NK cell development in the bone marrow and liver and commitment of human progenitor cells to the NK linage will also be studied using transfer and cell tracking techniques, as will the role of the GATA family of transcription factors in human NK cell development in humanised mice. Techniques that will be used in this project include viral infection, small interfering RNAs, adoptive transfer, cytometry, cytokine and cytotoxicity assays and numerous human graft translation procedures.

Cytokinesis, the physical separation of two cells at the end of mitosis, is a basic cellular process of immense importance: being indispensable for cell proliferation, cytokinesis is essential for the development of multi-cellular organisms. Although cytokinesis has drawn extensive research, its molecular mechanisms remain to be unraveled. The evolutionarily conserved centralspindlin complex, which consists of a mitotic kinesin-like protein and a Rho GTPase-activating protein, is a key regulator of cytokinesis. Centralspindlin localizes to the central spindle, a structure of bundled anti-parallel microtubules that forms between the separating chromosomes at anaphase. When either complex member is lost the central spindle is not formed and cytokinesis eventually fails. How centralspindlin contributes to central spindle formation and function is poorly understood. Furthermore, besides its involvement with the central spindle centralspindlin might act as a signaling platform orchestrating several molecular events during cytokinesis. To deepen our understanding of cytokinesis at the molecular level I therefore propose to identify centralspindlin-interacting proteins by drawing on both biochemical and genetic approaches. First, I will biochemically purify centralspindlin-associated proteins from mammalian cultured cells. Second, in a complementary genetic approach I will perform a genome-wide RNAi screen in C. elegans for suppressors and enhancers of the centralspindlin phenotype. Conserved interaction partners of centralspindlin will then be further characterized and analyzed for their role in central spindle formation and completion of cytokinesis.

The study of axonal growth is a central area of research in neurobiology. However, the cell-intrinsic mechanisms that regulate axonal morphogenesis remain largely to be discovered. The laboratory of Dr. Azad Bonni discovered that the ubiquitin ligase Cdh1-anaphase promoting complex (Cdh1-APC) controls axonal growth and patterning in the mammalian brain. In more recent studies, they identified the transcriptional corepressor SnoN and Id2 as key downstream targets of neuronal Cdh1-APC that promotes axonal growth. The focus of the proposal is to elucidate the mechanisms that regulate the Cdh1-APC-SnoN/Id2 cell-intrinsic pathway in postmitotic neurons and to determine how this pathway in turn controls axonal growth. To characterize the mechanisms that regulate Cdh1-APC in neurons, I will investigate the role of phosphorylation of Cdh1 and Cdh1-interacting proteins in axonal growth. To reveal the mechanisms by which SnoN and Id2 promotes axonal growth, I will determine transcriptional target genes regulated by SnoN and/or Id2, and reveal how these genes regulate the axonal growth. In addition, I would like to test the hypothesis that SnoN and/or Id2 might regulate the expression of microRNAs in neurons, because expression of several microRNAs has been recently shown to be regulated by transcriptional regulators and increasing evidence suggests that microRNAs regulate neuronal morphology. The postdoctoral research will be a great opportunity for me to learn knowledge and techniques of neuronal cell biology, signal transduction, and transcription regulation. HFSP fellowship will promote my professional growth through interdisciplinary research.

MicroRNAs (miRNAs) are short non-coding RNA molecules that post-transcriptionally regulate eukaryotic gene expression in a variety of cellular processes. Metazoan miRNAs recognise imperfectly complementary sequences in the 3' untranslated regions of target mRNAs and repress their translation by a mechanism that is poorly understood. The goal of my research project is to elucidate the mechanism of miRNA-driven translational repression. Using a combined biochemical and structural approach, I intend to dissect the molecular interactions that cause translational repression and ultimately obtain insights into their structural basis. To achieve this, I will (i) use in vitro reconstituted miRNA-induced silencing complexes (miRISCs) together with reporter mRNAs and a cell-free translation system to identify the stage of translation affected by miRNA-driven repression (ii) in parallel, biochemically test for direct interactions between miRISC and components of the translational machinery (iii) study the identified interactions by X-ray crystallography and electron microscopy. In addition, I plan to examine the link between miRNA-driven silencing and mRNA degradation in cytoplasmic processing (P) bodies, focusing on the interactions between P-body proteins and the miRISC protein Argonaute. Together, the outlined studies will bring significant molecular insights into the mechanism of RNA silencing pathways.