Physical forces such as mechanical, electrical and Ca2+ impulses, have long been implicated as instructive cues in cell-cell communication, but the mode of action of these signals during early embryonic patterning and differentiation remain poorly understood. Here, I propose to address the question of how early embryonic function coordinates cell-cell interactions of distinct cell populations that result in the formation of a single organ, the cardiac outflow tract. This region represents the transition phase, morphologically and functionally, between the heart and the vasculature and has long been found to be uniquely susceptible to a broad range of genetic and environmental perturbations. Using multidisciplinary approaches, I plan to characterize the morphology of the cardiac outflow tract and combine it with quantitative functional analysis at the cellular resolution. I propose a sophisticated set of experiments to perturb mechanical and electrical signaling with high temporal and spatial control that will allow me to test their role in regulating outflow tract development. Ultimately, the goals of this project are to understand the fundamental impact of mechanical and electrical stimuli on cellular differentiation and tissue morphogenesis, and to begin deciphering the signals encoded by such forces and their integration with established developmental programs. I believe that proposed project will open a new area of investigation and will allow me to build a career as an independent research scientist.

What is the smallest set of genes sufficient and necessary to maintain a functional cell? Genome reduction projects seek to answer this question by simplifying the genomes of existing microbes with the aim of arriving at functional cells with favourable properties for biotechnological applications. Understanding how far a cell can be simplified is closely linked to fundamental problems such as how mutations can be tolerated and how genes interact with each other and the environment to produce the cellular phenotype. Despite recent methodological advances in genome engineering, genome reduction efforts still face several formidable challenges. In general, it remains difficult to identify genes that are not needed under defined conditions and only a better understanding of the impact of multiple mutations could resolve this challenge. I propose to combine genome engineering with computational systems biology models and bioinformatic analyses of functional genomic data to i) investigate how reliably the effects of multiple gene deletions can be predicted and ii) develop integrated methods to construct minimized cellular subsystem. We will use the metabolism of Saccharomyces cerevisiae as a model system due to the availability of large-scale mathematical models and an unparalleled amount of data on genetic interactions. I believe that the project will advance not only our ability to rationally design minimal cellular circuits, but also our understanding of the evolution of mutational robustness. The CDA award would provide the scientific independence and financial support to set up my research group as well as help me to maintain a multidisciplinary collaborative network.

Serotonin (5-hydroxytryptamine, 5-HT) projections from the brainstem raphe nuclei show a variable degree of degeneration and superimposed plastic changes in Parkinson´s Disease (PD). The main objective of this research project is to uncover plastic adaptations of the 5-HT system upon neurodegenerative lesion and pharmacological treatment in a rat model of PD. The project is based on preliminary data from the host laboratory, indicating that antiparkinsonian pharmacotherapy induces sprouting of 5-HT varicosities in the striatum, and that this effect is correlated to the development of abnormal movements (dyskinesia). PLAN: Rats with unilateral 6-hydroxydopamine (6-OHDA) lesions of the medial forebrain bundle will be used as a model. Part of the animals will be chronically treated with the antiparkinsonian medication, L-DOPA, and examined for the occurrence of dyskinesia. 5-HT neuroplasticity will be examined using a multidisciplinary approach including (i) ultrastructural analyses of the morphometric and junctional features of preexisting and newly formed (sprouted) 5-HT axon varicosities; (ii) confocal microscopy analysis of neuroplasticity markers in 5-HT-positive axon varicosities; (iii) in situ hybridization and radioligand binding assays to evaluate the expression and function of the 5-HT transporter; (iv) studies of depolarization-induced 5-HT release in brain slices, and its modulation by the glutamatergic system. SIGNIFICANCE: Results generated by this new international collaboration will bring insights into the relationship between morphological and functional alterations affecting the serotonergic innervation to the basal ganglia.

A crucial step during cellular division is the accurate segregation of chromosomes to progeny. This is accomplished by an elaborate bipolar microtubule-based structure called the mitotic spindle. Centriole duplication and assembly play a crucial role in spindle formation by organising and defining the number of spindle poles. Defects in centriole biogenesis can therefore lead to aneuploidy, a condition associated with cancer formation. This application is rooted in our goal to better understand at the molecular level proteins required for centriole duplication and to define the complement of human proteins required for this process. Towards this goal, we propose a cross-disciplinary approach encompassing functional genomics, cutting-edge microscopy and automated 3D image analysis. We will begin by screening a small set of 111 proteins biochemically enriched on centrosomes for aberrations in centriole number using automated image acquisition and analysis of RNAi-treated cells. Our assays will be further refined using this set before progressively scaling-up, our longer-term goal to reach genome coverage. Second, we will use RNAi-resistant epitope-tagged bacterial artificial chromosomes of identified genes to perform genetic rescues, sub-cellular localization and dynamic analyses in live cells. Finally, we propose to carry out epistatic-like analyses to determine the relationship between the novel centriole duplication regulators identified and known regulators of centriole duplication. This will help us delineate where in the centriole duplication pathway these novel proteins are required as well as to increase our overall understanding of centriole duplication in mammals.

Amyotrophic Lateral Sclerosis (ALS) is an invariably fatal neurodegenerative disorder of motor neurons, often associated with mutations in the superoxide dismutase (SOD1) gene. Transgenic mice carrying mutant human SOD1 develop motor neuron disease resembling ALS, through the gain of a yet unidentified toxic property, potentially mediated by misfolded mutant SOD1 protein aggregates. Despite the apparent selectivity for motor neurons, it has been recently recognized that non-neuronal cell-types, such as microglia and astrocytes, contribute to pathogenesis and disease progression in SOD1-mediated neurodegeneration. Emerging evidence implicates the innate immune system in the pathogenesis of various neurodegenerative and protein misfolding diseases. Cells of the innate immune system express Toll-like receptors (TLRs), which are an evolutionary conserved family of receptors that recognize structural motifs on a wide array of pathogens, referred to as pathogen-associated molecular patterns, as well as some endogenous molecules. TLRs are widely expressed in the CNS indicating that these receptors may participate in CNS autoimmunity, tissue injury and neurodegeneration. I plan to investigate the role of TLRs in the pathogenesis and disease progression of SOD1-mediated disease. I hypothesize that certain TLRs recognize misfolded forms of mutant SOD1 protein aggregates or other components associated with deteriorating motor neurons and trigger a signaling cascade leading to activation of microglia and other inflammatory responses contributing to disease onset and/or progression.

Bacterial species that live in symbiosis and disease relationships with host organisms must sense and respond to each other and to the environment. Such regulation is achieved by quorum sensing through hormone-like molecules known as autoinducers. As these signaling events are known to influence the regulation of a number of virulence genes, disruption of quorum sensing would lead to an unorganized bacterial infection, and thus decreased virulence. Here we aim to study the various components of a novel quorum sensing pathway recently discovered in Salmonella. The details of this pathway are most likely to be revealed through structural analysis coupled to in vivo analysis of whole animal infection. The resolution of such a pathway will shed light on bacterial signaling and provide a species distinct template for antimicrobial design as not to promote antibiotic cross-resistance. The ability of bacteria to sense the environment is also demonstrated by the evolution of antibiotic resistance. A dangerous example of environmental sensing is Methicillin Resistant Staphylococcus Aureus, or MRSA. MRSA resists antibiotics by the expression of beta-lactamase and PBP2a. The expression of these proteins is regulated by BlaR1/MecR1 which conveys a signal to upregulate the transcription of beta-lactamase and PBP2a in the presence of antibiotics. To combat MRSA, studies will be undertaken to reveal the mechanisms of signal transduction by solving the crystal structure of BlaR1/MecR1 complemented by biochemical analysis. This will elucidate the mechanism by which BlaR1/MecR1 regulates antibiotic resistance, which will in turn be exploited for the creation of new antibiotics.

Sperm competition occurs when ejaculates from two or more males compete to fertilise a given set of ova, and typically selects for males to produce and inseminate large numbers of sperm. An obvious way to increase sperm production rate is to increase testis size, and most empirical work has focussed on this parameter. However, recent experimental evidence suggests that, within species, at least some of the phenotypic plasticity in sperm production rate displayed in response to environmental cues of sperm competition level occurs independently of testis size. This could be due to variation in the speed with which each spermatozoon is produced, but whether animals are capable of modulating the kinetics of spermatogenesis in this way has not been experimentally investigated. The proposed project will (1) develop a spermatid-specific antibody/BrdU double-labelling technique to track the process of spermatogenesis in the marine flatworm Macrostomum lignano, and (2) apply this technique to test whether the kinetics of spermatogenesis in M. lignano display phenotypic plasticity in response to changes in mating group size that reflect sperm competition level in this species. Such a finding would overturn the prevailing view that the kinetics of spermatogenesis are largely fixed within species, with widespread implications for our understanding of variation in male fertility and sperm competitive ability in natural populations. In addition to this potential scientific impact, the project will aid the professional development of the Short-Term Fellow through exposure to new experimental techniques (immunocytochemistry, confocal microscopy) and research in a new model system.

The innate immune system detects ”molecular patterns” on microbes or viruses and initiates fast responses to contain the infection. In addition, innate recognition stimulates adaptive immune responses. Therefore, the efficiency of innate detection largely determines immunity to infection. Pattern recognition receptors are integral components of the innate immune system and recognize molecules specific to pathogens. RIG-I is a DExD/H-box RNA helicase sensing viral RNA in the cytosol of mammalian cells. Activation of RIG-I induces the expression of antiviral cytokines. Previous work from the host laboratory and others shows that RIG-I agonists are RNA molecules phosphorylated at their 5’-end. During my postdoctoral training, I would like to further characterize the molecular determinants that allow RIG-I to distinguish self from viral RNA. Two lines of investigation will answer this question: (i) I would like to start with in vitro experiments to identify optimal RIG-I ligands, and I want to confirm these results by testing their agonistic activity in vivo. (ii) Next, I am planning to identify physiological RIG-I agonists by purifying, and subsequently cloning, RIG-I-associated RNAs from cells infected with different viruses. Notably, the influenza A virus NS1 protein interacts with RIG-I and inhibits RIG-I-mediated responses. Therefore, I will also analyze the mechanism of inhibition by testing (i) the role of RNA ligands in the association of NS1 with RIG-I and (ii) possible effects on downstream signaling components. Collectively, these steps will significantly extend our knowledge of RIG-I function and innate recognition, and may suggest novel anti-viral strategies.

Recent findings have shown that aging, the major determinant of our lifespan, is a highly plastic process and subject to extensive regulation. This regulation, also known as longevity control (LC), resides in the hands of a complicated network whose elucidation is the aim of this proposal. Due to the network’s complexity I have to employ systematic approaches that can not be conducted in mammalian systems. I propose the use of the much more accessible nematode C. elegans, where even a comprehensive set of ~340 candidate genes involved in LC has already been identified. However, mechanistic context and molecular functions of these genes and their protein products are largely unknown. I would like to take this analysis to a next level by organizing all these candidates into aging-regulatory pathways and by hopefully also extending their pool, eventually working towards a first comprehensive map of the LC network. This map will be comprised of multiple kinds of relationships amongst the candidates - relationships derived from a reiterative approach employing novel proteomics and molecular epistasis analysis. I aim to identify a) overlaps and dependencies in expression and subcellular localization amongst candidates, b) physical binding amongst candidate proteins, as well as c) physical binding of candidate proteins to currently unknown players in LC. The resulting map will be of tremendous value, as it should give us a first mechanistic overview over the regulation of aging. Even more, the potential translatability of the map into mammalian systems might provide the grounds for new directed approaches on the prevention of aging and its dependent pathologies in humans.

Non-small cell lung cancer (NSCLC) is a devastating disease carrying a 5 year overall survival of less than 5%. K-RAS is often mutated in human cancers, activating cell proliferation and survival signals. In vivo, oncogenic K-RAS triggers a potent oncogene induced replicative senescence response (OIS), a form of growth arrest that prevent cancer progression. In this setting, inactivation of the promyelocytic leukemia gene (PML), a tumor suppressor that is frequently lost in human NSCLC, leads to the development of a more aggressive phenotype due to OIS defect and up-regulation of the phosphorylated ribosomal S6 protein. It is of great interest that oncogenic K-RAS induced NSCLC completely regresses due to apoptosis when K-RAS expression is experimentally shut off. This observation implies that oncogenic K-RAS maintains an aberrant pro-survival signaling network upon which cancer cells are critically dependent, defined as “oncogene addiction”. I hypothesize that oncogene addiction plays a pivotal role in NSCLC pathogenesis and that OIS is a key tumor suppressor mechanism in K-RAS induced NSCLC tumorigenesis. To confirm these hypothesis I will: 1. test in vivo the anti-tumor effects of specific inhibitors of the RAS signaling cascade and whether Pml null tumors are more sensitive to this strategy 2. identify the apoptosis effector molecules responsible for tumor regression upon K-RAS withdrawal 3. characterize the mechanism of action of PML in OIS. The scope is to identify the cellular mechanisms required for tumor maintenance and shed light on the molecular networks governing OIS response providing the framework for the development of novel targeted anti-cancer drugs.

The overall goal of this proposal is to visualize in vivo behavior and characterize functional mechanisms of MOG specific T regulatory cells during EAE. We have generated several novel tools: FoxP3KI mice and MOG 35-55/IAb tetramers, which together will allow us to determine whether antigen specific FoxP3+, tetramer + T cells are induced in vivo and understand the mechanism by which T-regs inhibit functions of encephalitogenic T cells and regulate autoimmune tissue injury in the CNS. To directly test hypotheses that arise from studies using of the FoxP3KI mice and MOG tetramers, we propose to take advantage of mice in which a DTR/GFP (Diphteria toxin fused with the Green Fluorescent Protein) reporter has been introduced into the endogenous FoxP3 locus, allowing conditional depletion in vivo of FoxP3 expressing cells through administration of diphteria toxin. Using these mice we will be able to study the generation and the trafficking behaviour of MOG specific T-regs, as well as their in vivo function, by deleting them at different stages of EAE and evaluating their involvement in regulating the course of the disease. Finally we have generated a novel reporter mouse IL-17 DTR RFP in which the IL-17 expressing cells express concomitantly the DTR-RFP fusion protein, therefore we can at the same time track in vivo the Th17 cells by measuring the RFP expression and delete them at will via administration of DT. Based on GFP and RFP expression and using flow cytometry analysis, we can follow T-regs and Th17 effector cells in vivo at single level and we will be able to determine how these are generated and how they counter regulate each other during EAE.

Understanding how cells establish and maintain cell polarity, the asymmetric distribution of molecules in a cell, is a prime challenge in biology. Although many conserved players in the polarity network are identified and assigned to distinct complexes, our current picture is mostly static. However, polarity requires the continuous enrichment of proteins and their activities in defined boundaries in order to orchestrate the local cytoskeleton and vesicular transport machinery. Cdc42 plays a central role in the initiation of polarity. It engages the Par6/aPKC complex, a major polarity regulator, and thereby triggers localized signaling when GTP-bound. This project aims to understand the mechanisms that maintain high Cdc42-GTP levels in restricted cellular areas during polarization. This key problem in the field is not trivial given the dynamic nature of Cdc42 membrane interaction and the finite local residence time of its regulators and effectors. I will perform an integrative study combining live cell imaging with proteomics and high throughput approaches. The work involves 1) the characterization of the diffusion properties of Cdc42, its regulators and effectors in cells in order to estimate local complex formation dynamics and stability 2) the establishment of a biosensor to monitor spatio-temporal activities of Cdc42 3) the generation of a comprehensive RhoGEF/GAP library and the functional characterization of regulators relevant in polarity, involving imaging, mass spectrometry and siRNA 4) studies on the dynamic interplay of immediate downstream proteins between separated polarity devices in order to understand additional determinants of transient local recruitment.

MicroRNAs (miRNAs) represent a new class of pivotal regulators of development and differentiation in animals and plants, by acting as negative controllers of specific target genes. Embryonic stem (ES) cells are derived from early stages of embryonic development and have unique characteristics that make them different from all other cell types. They are able both to indefinitely proliferate by self-renewal and to give rise to all cell types and tissues. The aim of my project is the characterization of miRNAs with important roles in human embryonic stem cells and early vertebrate development. Initially, I will obtain a profile of miRNAs specifically expressed or enriched in human ES cells by microarray analysis of miRNAs expressed in undifferentiated and differentiated ES cells. Then, bioinformatic predictions of the targets of ES specific microRNAs will be validated utilizing a reporter assay. Moreover, miRNAs promoters will be characterized in order to find transcriptional factors and pathways regulating their specific expression. The third part of the project is based on the ectopic expression and antisense-mediated knock-down of specific ES microRNAs. These experiments will assess the role of miRNAs in maintenance of the stemness or differentiation of the ES cells. The Xenopus laevis embryo represents a classic model system of vertebrate development. The role of miRNAs in early development will be evaluated by experiments of overexpression and inhibition of specific miRNAs in Xenopus embryos followed by analysis of the phenotype. This work will deepen our knowledge about molecular pathways regulating stem cell biology and early vertebrate development.

The control of cell-specific gene expression is fundamental to multi-cellular organisms. Transcription initiation requires the assembly of multiple transcription factors and associated coactivators. Our ultimate goal is to use structural techniques, protein crystallography and small angle scattering, to define the protein-protein and protein-DNA interactions leading to expression of the insulin gene, which is expressed uniquely in pancreatic beta cells. While many of the important beta cell factors are known, how they assemble on the insulin promoter is not known. Structural biology is ideally suited for addressing these questions. To date we have determined structures of DNA/protein complexes with DNA binding domains alone. In order to expand our efforts to full length proteins and to isolate individual interactions for structural studies, I propose to develop an in vitro transcription assay.
An in vitro assay recapitulates transcription initiation using purified factors and HeLa cell extracts. Our assay will be modeled after an in vitro assay of a muscle-specific promoter developed by Dr. F.J. Dilworth at the Ottawa Health Research Institute. A central muscle transcription factor, E47/MyoD, belongs to the same family as a key insulin promoter factor, E47/NeuroD. The two systems appear to function similarly, including interactions with the coactivators p300 and pCAF, and the comparison will be informative. Once the assay is established, it will be expanded to include the other known insulin transcription factors. We believe the in vitro assay will be a resource for the diabetes community generally to investigate the regulation of insulin transcription.

Eukaryotic genomes comprise large heterochromatic regions associated with gene silencing. However, the molecular basis accounting for their formation and maintenance is poorly understood. Previous studies have suggested that the genome may intrinsically encode the nucleosomal organization with low and high affinity DNA sequences. Using the fission yeast S. pombe as model system, we propose to test the hypothesis that high affinity DNA sequences may function to precisely position nucleosomes to promote both heterochromatin formation and the inheritance of a silent state. To address this issue, the experimental strategy will be based on a combination of genomic and biochemical tools. First, I will establish a comprehensive map of nucleosome occupancy with tiling arrays and will identify DNA sequences involved in nucleosome positioning within heterochromatic regions. Second, I will investigate the role of silencing factors and DNA sequences in nucleosome positioning by using specific mutants. Finally, I will analyse the function of nucleosome positioning in the inheritance of a silent state through a cell-cyle dependent assay. Together, these complementary and independent strategies will give novel mechanistic insights on heterochromatin formation as well as the inheritance of a chromatin-based gene expression state, and will define a basis for future studies in higher eukaryotes.

The clathrin cage, composed of hexagons and pentagons engulfing a lipid bilayer, is the earliest membranous carrier described. The polymerization of clathrin into this cage was thought to generate the force needed to deform the membrane into a bud. After many years of research, this view has been challenged by two facts: first, in addition to the classical soccer ball structure, many other shapes have been observed ranging from flat lattices to tubules. Secondly, the binding of clathrin to the membrane is occurring through a wide family of adaptor proteins that have been shown to deform membranes and modulate clathrin polymerization. Our goal is to find the critical parameters (physical and biochemical) that control the shape and the polymerization rate of clathrin coats, and to measure the forces on the membrane applied by clathrin polymerization. - Little is known about the exact parameters that control the shape of clathrin coats. I will use a videomicroscopy assay to track the formation of clathrin coats on membranes from various mixtures of pure clathrin and adaptors. This. technique will also allow me to measure the dependence of clathrin polymerization rate on the shape of the coat. - To measure forces applied by clathrin coats on the membrane, I will counteract these forces by increasing membrane tension and/or rigidity and observe their influence on the final shape of the coat. Membrane tension can be modulated by the aspiration of Giant Liposomes into micropipettes, and membrane rigidity is easily tuned by changing the lipid composition. The CDA from the HFSP will help me start my independent career by providing the funds needed for establishing this project.

C-di-GMP is a ubiquitous second messenger in bacteria that acts to regulate functions contributing to the virulence of bacterial pathogens of eukaryotes. A balance of synthesis catalysed by GGDEF protein domains, and degradation involving EAL and HD-GYP domains control the cellular levels of this nucleotide. The large numbers of different c-di-GMP signalling systems within individual species indicate the existence of considerable organisational complexity, which is poorly understood. In the plant pathogen Xanthomonas campestris pv. campestris (Xcc), the HD-GYP domain response regulator RpfG, positively controls the synthesis of virulence factors. Genetic and yeast two hybrid analyses indicate that RpfG has a functional interplay with other c-di-GMP signalling systems and that this may involve protein-protein interactions between the HD-GYP domain of RpfG and GGDEF domain-containing proteins. The work proposed will use advanced imaging technology to examine these interdomain interactions within Xcc. This will involve establishing cellular localisation of RpfG and (potential) target GGDEF domain proteins, the use of FRET and FRAP to monitor in vivo protein-protein interactions and examination of the role of conserved residues, including the GYP motif, in promoting such interactions. The experiments should test definitively whether interdomain interactions suggested by indirect methods occur in bacteria in vivo. The findings should contribute to a greater understanding of c-di-GMP signalling and its role in the complex of interactions between bacteria and their hosts during bacterial disease, which could have substantial implications for new approaches for disease control.

The vascular system is the first organ to form and function during embryonic development. Its correct development is essential for the proper formation of vertebrate embryos as well as for adulthood. Although much attention has been focused on the genetic requirements for early vascular development (vasculogenesis and angiogenesis), the mechanisms controlling later stages of blood vessel development (vascular maturation and blod vessel myogenesis) are largely unknown. In the past, I have successfully used the embryological and genetic attributes of the zebrafish model to study endothelial cell (EC) development: I identified regulators of blood vessel integrity and characterized vascular mural cells (MC). Here, I propose to identify new mechanisms involved in vascular maturation and blood vessel homeostasis focusing on the reciprocal relationship between EC and their surrounding microenvironment, including MC. Using a set of cellular, molecular, and genetic approaches as well as advanced microscopy techniques, I propose to elucidate how endothelial and mural cells cooperate to shape the vascular system and regulate vascular maturation and myogenesis. The long-term goal is to provide additional molecular entry points to further investigate EC maturation and MC development/differentiation in normal and pathological conditions. The HFSP long-term fellowship was essential in the establishment of my current research program. The CDA would offer me a unique opportunity as an independent young investigator to find the initial funding and scientific support to address new and important questions in vascular biology.

Mitosis is unquestionably one of the most spectacular events in cell biology, as it brings unparalleled visible changes in the cell. These changes are mostly dependent on the mitotic cyclinB:Cdk1 complex. Nevertheless, how this complex controls the sequential timing of these events remains a mystery. Quantitative studies in HeLa cells have shown that there is no correlation in the time intervals between consecutive events suggesting that the regulators of successive mitotic events act more like independent modules, than as a single mitotic engine or a cascade of dependent regulators. Here, we postulate that bistability in the activation of the cascades that govern these events underlies these observations. We will test this hypothesis by a) investigating whether a spatially-regulated bistable switch triggers the translocation of active cyclinB:Cdk1 from the cytoplasm to the nucleus b) examining two systems known to be bistable and determining to what extend they succeed in insulating downstream events from variability in the timing of upstream events and by c) modeling studies, which will first explore the circumstances under which a positive feedback promoting cyclinB:cdk1 abrupt nuclear import can function as a bistable switch and secondly explore how noise propagates in three different types of regulatory signaling cascades by comparing the modularity of their function. Such studies will provide deeper insights into the long-standing question on how activation of cyclinB:cdk1 controls the proper timing of mitotic events. Ultimately this project will shed light in the design principles at the heart of regulatory networks that control proper functioning of the cell-cycle.