Microtubules play essential roles in various cellular functions such as cell division, organelle transport, cell polarization, and cell motility. Although centrosomes are the major sites of microtubule nucleation in animal cells, microtubules are also generated independent of centrosomes but dependent on existing microtubules. Microtubule-dependent microtubule generation is important for cytoplasmic microtubule network construction in yeast and plant cells. However, little is known about the molecular mechanism of this process or the roles of these microtubules in cellular functions in animals. Through a genome-wide RNAi screen in Drosophila cells, I recently identified 5 novel genes (named Dgt genes) required for microtubule-dependent microtubule generation in the mitotic spindle by localizing gamma-tubulin—a microtubule nucleator—to existing microtubules. The identification of these genes will enable the study of the roles and molecular mechanisms of this microtubule generation pathway in animals. Together with my laboratory members and collaborators, I propose to elucidate (1) the precise role of non-centrosomal microtubules in mitosis and cell polarization through RNAi and mutant analyses of Dgt genes in cultured cells and whole animals and (2) the molecular mechanism underlying the involvement of the Dgt proteins in the localization and activation of gamma-tubulin by developing an in vitro microtubule nucleation assay. This project will provide new insights into numerous dynamic cellular events utilizing microtubules. The support of the CDA is critical for establishing my independent research program and will enable me to assemble a team to conduct experiments.

Proteins contain one or several structural and functional regions called domains. In prokaryotic organisms, it is most common for a protein to consist of one domain, whereas in eukaryotic cells it is more common to have multidomain proteins. The domains are responsible for the molecular interactions, both internally, within the protein, and externally, to other proteins. The interaction domains are often represented in cellular interaction networks and are, for instance, involved in protein subcellular localization, signal transduction, and control of the activity, conformation and substrate specificity of enzymes. Generally, the scientific question we want to address is how the topology of domain interaction networks correlates with other types of biological networks, and how it defines function and ultimately determines phenotype. More specifically, we want to focus on the interactions involving membrane proteins and evolutionary events.

The emerging Hippo signal transduction pathway restricts cell proliferation and promotes apoptosis. Two serine/threonine kinases, Hippo (Hpo) and Warts / Large Tumour Suppressor (Wts/Lats) constitute the core of the Hippo pathway, which is conserved throughout the animal kingdom. Wts, the most downstream kinase, phosphorylates and thereby inactivates the transcriptional coactivator Yorki (Yki). Yki promotes the expression of genes that serve proliferative and anti-apoptotic functions. Loss of activating Hippo pathway components results in tumour phenotypes both in Drosophila and mammals, indicating a conserved tumour-suppressive function of the Hippo pathway. The small, non-catalytic protein Mats is an essential component of the Hippo pathway, as it is required for Hpo-mediated activation of Wts kinase. The mechanism of Wts kinase inhibition and its activation by Mats are poorly understood. I propose to elucidate the crystal structures of human and Drosophila Wts and the Wts-Mats complex. This shall give insights into the autoinhibited state of Wts and the allosteric effects that occur upon Mats- and Hpo-mediated Wts activation. Structural insights will be tested biochemically as well as cell-biologically. In collaboration with developmental biologists, we will study in vivo implications of Mats-mediated Wts kinase activation in Drosophila melanogaster.

The training will be focused on study of approaches, methods, mathematical models and software that are used for complex genetic data analysis. The main goal, which is planed to achieve is to learn how to design a project aiming to study genetics of complex trait(s) in humans. As a part of the training, it is suggested to participate in on-going consortia projects to identify genetic risk factors for complex disease (CHARGE, ENGAGE) as a part of Genetic Epidemiology Unit (Erasmus Medical Center). To achieve the home institutions needs, it is planned to perform a comprehensive statistical analysis of data from fine-mapping of three chromosomal regions that was found to be involved in regulation of pathophysiological traits during allergic diseases to map genes of these complex traits. The planed training would allow Dr. Elena Gusareva to get acquainted with outstanding scientists who are very experienced in bioinformatics and who have been leading projects aiming to map complex diseases genes. The training will most likely bring to establishment of a new collaboration between the Department of Molecular Immunology, Institute of Molecular Genetics, v.v.i., Academy of Science of the Czech Republic, Prague, Czech Republic and Genetic Epidemiology Unit, Erasmus Medical Center, Rotterdam, The Netherlands.

Controlling action is fundamental to behavior. In response to environmental requirements, the brain can mediate appropriate behaviors by selecting appropriate types of action. Goal-directed actions result from choosing of many possible actions the one, which gets closest to the goal. These actions are flexible but require a judgment on the expected outcome of the action. Following a habit on the contrary, demands little effort, but only permits limited behavioral flexibility. The neural circuits for these two disparate behaviors are not entirely separated in the brain. In contrast, the striatum, a substructure of the basal ganglia, is critically involved in both goal-directed and habitual actions. Dopaminergic input to the striatum has been related to rewarding stimuli and is supposed to play a modulatory role in the choice of action. However, the exact function of this modulation is not clear. To address this question, I will use the newly developed technique for optical control of neural activity and selectively stimulate and block activity of dopaminergic axon terminals in the striatum of rats performing a two-alternative choice odor discrimination task. I will modify task conditions to assess the contribution of dopamine-related neural activity during the performance of habitual and goal-directed actions, and follow how goal-directed actions transform into habits. Given the involvement of the striatum in a number of human neuropsychiatric disorders such as Parkinson or schizophrenia, I envision this work will also contribute to our understanding of cognitive dysfunctions of the human brain.

Cellular neurobiologists have been investigating neuronal long term potentiation (LTP) fpr twenty years and still, no concensus on the underlying molecular mechanisms has emerged. However, the main hypothesis is that a massive influx of calcium ions entering through glutamate receptors leads to an increased and sustained number of glutamate receptors at the synapse. In this project, we will build a computational model integrating the various cellular processes linked to LTP: receptors electrical activity, kinases activation and phosphorylation effects, local protein synthesis and regulation of receptor trafficking. The computational model will be incrementally constrained by data from the literature and from undergoing experiments at our laboratory. In the process, the model will also generate new hypotheses to be experimentally tested. The model will first be implemented as a unidimensional compartment represented by ordinary equations. The model will then be transformed into partial differential equations to incorporate geometrical effects due to the spine and dendrite shapes. A major innovation of this model is that it will combine the neuron electrical and biochemical activities. This comprehensive and realistic model should provide the first mechanistic explanation of long term potentiation. We will also make predictions that will be experimentally validated.
Prof Upinder S. Bhalla has developed in the late nineties the first models of neurobiological systems, pioneering the field of computational neurobiology. His expertise is instrumental to this project. He will help me interpret my experimental data.

I developed an approach to quantifying the orientation of DNA polymerases by observing the mutational patterns of strand replication in sequenced genomes. The replication terminus is clearly identified by these methods but the replication origin is not. Contrary to 50 years of biochemical characterization, a range of overlapping replication pattern indicates that initiation of replication at the origin can occur at multiple locations. I therefore hypothesize that subpopulations of bacteria exist that initiate DNA replication outside of the standard origin initiation region. Sub-population dynamics in bacteria are an important subject our ability to effectively design antibiotics to prevent bacterial growth depends not only on the bulk behavior of the population but also on individual susceptibility. The origin has been used as a target for drug design. Incomplete population control, a possible result of failing to recognize variability, can lead to the spread of antibiotic resistance and this variability should be investigated. I will first molecularly characterize the size of the sub-population of cells that initiate replication outside of the terminus region in Escherichia coli in liquid growth experiments and through fluorescent microscopy. Second, I will molecularly determine the specificity and range of potential replication start locations using ectopic insertion of a replication termination system. In addition to this molecular work I will undertake a careful bioinformatic analysis to estimate the proportion of replication starts at different locations and compare this with my molecular findings, thereby fusing molecular and bioinformatic approaches.

Constitutional dynamic chemistry (CDC) takes advantage of both supramolecular and covalent reversible bonds and can be employed to create dynamic, adaptive structures. Dynamic combinatorial chemistry emerged from the transposition of the dynamics of supramolecular systems to molecular chemistry by introducing reversible bonds into molecules. Applying CDC to polymer science gives rise to dynamers, which are constitutionally dynamic polymers capable of modifying their structure in response to external stimuli. I propose to investigate the use of monomers featuring naturally occurring moieties such as saccharides to obtain dynamic analogues of biologically relevant polysaccharides. These materials have the added advantage of featuring both the biorecognition properties of the naturally existing polymers and the adaptive nature inherent to dynamers. As oligo- and polysaccharides are key players in a number of biological processes such as cell adhesion, the dynamers obtained will be used for the study and control of these complex events. The design of building blocks will be guided both by computer modelling and biological as well as structural intuition. Upon synthesis of the building blocks using standard synthetic methods, the assembly of the biopolymers will be achieved using CDC, followed by careful characterisation using a range of biophysical and chemical techniques. Reorganisation following the introduction of a biologically relevant template, will lead to selection and amplification of the polymer with the most interesting biological properties. The resulting polymers can be considered “smart” materials and will be applied e.g. in the controlled release of a drug.

Abscisic acid (ABA) is a vital plant hormone that controls gas exchange during photosynthesis and orchestrates a variety of developmental and stress response mechanisms. Recently, a chloroplast-localized receptor (ChlH) for ABA has been identified. ChlH is a dual function protein as it also generates Mg-protoporphyrin, an important intermediate in chlorophyll biosynthesis. Mg-protoporphyrin triggers a key signal transduction pathway between the plastid and the nucleus and alerts the nucleus when the plastid is damaged. I propose to study the connection between ABA perception and Mg-protoporphyrin signaling using a multi-disciplinary approach that combines structural biology, quantitative biochemistry and plant genetics. Specifically, this project aims to identify the ABA and Mg-protoporphyrin binding domains in ChlH, to characterize their structure, and to find and analyze mutations that impair both ChlH functions in vitro. Then I will use plant genetics, to study the impact of these mutations on typical ABA functions, on chlorophyll biosynthesis and on plastid-to-nucleus communication. This project sets the stage to uncover the molecular mechanisms of intracellular ABA perception in plants and its connection to downstream signaling events at both the atomic and cellular level.

A class of water-soluble chlorophyll-binding proteins (WSCPs) present in the water-soluble fraction of chloroplasts is protected against photogeneration of singlet oxygen by an as yet unknown mechanism that does not involve other pigments as excited-state quenchers. The physiological role of WSCPs also remains undetermined. They have been hypothesized as chlorophyll transport proteins in biosynthesis/biodegradation pathways. The project will study the photoprotection mechanism and chlorophyll binding behaviour in WSCPs by a combination of biochemistry/molecular biology, advanced ultrafast spectroscopy and project-specific instrument development. The Fellow will undergo substantial biochemical training, as well as expanding his spectroscopic expertise to include the ultrafast time-domain methods of the Host. Site-directed mutagenesis will be used to target specific conserved amino acid residues. In particular, two conserved tryptophan amino acids are potentially involved in the photoprotection mechanism. Ultrafast transient absorption and development of an ultrafast fluorescence apparatus will be used to study the potential role of these residues in the photoprotection mechanism. The peptide backbone carbonyl of a conserved proline amino acid acts as the chlorophyll ligand and this residue may influence the unique chlorophyll binding behaviour of WSCPs. Monomeric apo-protein WSCPs form tetrameric protein complexes upon binding chlorophyll. To investigate this process the Fellow will develop an instrument combining stopped-flow/continuous-flow methods with ultrafast spectroscopy. Our novel approach will be used to study quaternary protein structure formation.

Mediator is a large multi-protein complex critical for eukaryotic gene regulation. It is critical for gene regulation in all promoters since it functions as an interface between a wide variety of transcription factors bound to upstream regulatory sequences and RNA polymerase II. The mechanism by which Mediator controls gene regulation, however, remains obscure. Structural and functional studies on Mediator are essential to elucidate the Mediator mechanisms. However, the size (over 1 Mega Daltons), and the complexity (21 subunits) have prevented from such studies over the years. I will take a full advantage of the robust multi-protein expression system established in Takagi’s lab to obtain a large quantity of Mediator subcomplex, termed “Head module” (7 subunits, 223 kDa), and solve its structure by X-ray crystallography and EM. Additionally, I will apply genome-wide yeast two-hybrid array, and proteomics, and yeast genetics to identify the factors interacting with Head module. As Head module controls the action of Mediator, combined results will shed a light on how Mediator controls transcription.&#12288 Previously, I revealed the nuclear transport mechanism by solving the structure of human Transportin 1 (single protein) complexed with its substrates by X-ray crystallography. My proposed project will not only extend my own expertise of X-ray crystallography from solving the structure of a single protein to multi-protein complex, but also to learn new techniques such as protein complex expression, biochemical assays, yeast genetics and EM through collaboration to broaden my expertise. Thus, these will be indispensable for my career development.

Consolidation is the process through which newly learned information becomes a long-lasting memory. Classical views proposed that, once consolidated, memories are stable and resilient to disruption. However, this view has been challenged by findings that established memories become labile when recalled and require another phase of protein synthesis to be maintained. Therefore, it has been proposed that each time a memory is reactivated it undergoes again another process of stabilization, named re-consolidation. The aim is to determine to what extent both consolidation and reconsolidation involve the same molecules and brain areas (circuits) with similar temporal dynamics. Preliminary studies have identified a number of molecules required for inhibitory avoidance (IA) memory consolidation. These include, among others, CCAAT enhancer binding protein beta (C/EBPb), insulin-like growth factor II (IGF-II), muscle-specific tyrosine kinase receptor (MuSK) and brain-derived neurotrophic factor (BDNF). In this Aim, we will use protein synthesis inhibitors and selective and temporally-restricted disruption of C/EBPb, IGF-II, MuSK and BDNF in the BLA, NAc or aCC to determine similarities and differences in anatomical and temporal molecular requirements between consolidation and reconsolidation of IA.

Understanding the physics of the biological systems is of great importance to understand their complexity in vivo. Many processes within the cell are driven by the mechanics of its components. The biomechanics of the cell nucleus is known to be closely related to its functioning. Cells in the body feel their environment, experiencing a variety of external forces and by using complex intracellular pathways they transmit feedback signals to their nucleus and alter their gene expression. This mechanism is particularly important for stem cell differentiation. Stem cells are non-specialised cells, which can be directed to become specific if exposed to the appropriate conditions. A key step in the utilization of stem cells is to trigger them towards the desired lineage in a controlled way. To date, cells and tissues subjected to rather global physical forces have been studied and the effect of the external stimulation on differentiation has been shown. Nevertheless, the mechanism behind this process still remains elusive. The goal of this research is to elucidate the process of force transduction within the cell from the external stimulus to the nucleus and use this knowledge to mimic natural mechanical stimuli to influence stem cell differentiation. I will use well-controlled mechanical stimulation exerted by an AFM tip on individual differentiating cells to manipulate their biomechanics. A combination of AFM and fluorescent imaging will be used to gain information about mechanical properties and protein dynamics of both the cell and its nucleus upon stimulation. The outcome of this research will be of great importance for our fundamental understanding of stem cell biology.

Behavioral studies have shown that in addition to three-dimensional (3D) shape, two dimensional (2D) surface reflectance information in faces is important for face recognition. The neural representation of these two types of information, however, is not well understood. It is unclear weather shape and surface reflectance retain unique representations, or integrate into a single representation in the human brain, and if so along which time course. The goal of this project is to investigate the neural correlates of 3D shape versus 2D reflectance independently using neuroimaging (fMRI) and electrophysiological (EEG) methods. I will first determine the general spatial and temporal characteristics of neural responses to faces that vary only in 3D shape or in 2D reflectance. I will then “decode” neural response patterns to shape versus to reflectance using a pattern-based classification approach. The results of this project should provide insight into the respective role of 3D shape versus 2D reflectance, both at behavioral and neural level. My previous research experience is in human psychophysics and computational modeling. This project will allow me to master all aspects of neuroimaging and electrophysiological methods, and implement them in conjunction with behavioral measures and computational models. If successful, these converging techniques can be applied as a powerful combination in my future work on other high-level cognitive functions. The host laboratory will provide me with an excellent opportunity to learn neuroimaging and electrophysiological techniques and will allow me to shift my research focus to a neural domain.

The olfactory system is a great example of neuronal diversity and specificity of both neuron identity and connectivity. Sensory neurons expressing the same olfactory receptor (OR) make connections with the same target glomerulus in the antennal lobe in the brain. This creates the challenging task of correlating OR expression with targeting specificity of the neuron. I am interested in the genetic program that governs neuronal diversification and links it to a neuron’s connectivity, and ultimately allows the construction of functional neuronal networks. I will use the relatively simple and genetically tractable olfactory system of Drosophila melanogaster to identify and characterize the molecular components of this genetic program. First, I will continue to analyze a few of the most interesting mutants I have previously discovered in a genetic screen that have defects in either neuron specification or neuronal connectivity. Second, I will analyze candidate groups of molecules using RNAi gene targeting to identify the proneural and neuron-specific transcription factors involved in neuronal diversification, and the targeting receptors and ligands required for connection specificity. Finally, I will characterize the role of FGF receptor Breathless in the specification and/or connectivity of one specific ORN class. This research is likely to gain new insights in aspects of neuronal diversification and connectivity in all kinds of complex nervous sytems. The CDA will help me to establish myself as an independent researcher, and to build a research group of substantial size and impact and competitive financial support.

The soil bacterium Burkholderia pseudomallei (B.ps) is a major health risk in tropical Australia and SE-Asia as it causes the serious and emerging disease melioidosis in humans and animals. Its natural habitat is unknown which is a major limitation for public health education programs.
We have developed a sensitive and specific molecular assay to detect B.ps in soil and performed a pilot study collecting 800 soil samples in rural Darwin, Australia to study the habitat of B.ps. An association between B.ps and grass-rich areas was found and an increase of B.ps load at these sites in the wet season when the root mass largely increases. B.ps was also detected in soil closely associated with roots of Tully grass (Brachiaria humidicola cv Tully). This suggests the rhizosphere of some grasses to be a potential reservoir of B.ps.
By a newly set up collaboration with Dr. Michael Schmid (Helmholtz Zentrum Muenchen, German Research Center for Environmental Health, Department Microbe-Plant Interactions), we aim to study the colonization behaviour of B.ps in the rhizosphere of selected grasses from rural Darwin using optical techniques such as fluorescence in situ hybridization (FISH) and confocal laser scanning microscopy (CLSM). During my visit of Dr. Michael Schmid’s laboratory in June 2009, I will learn and apply FISH as well as CLSM.
This interdisciplinary project combines our previous findings based on real-time PCR based methods with the latest imaging technology and will largely contribute to the elucidation of the reservoir and microhabitat of B.ps. This will be important to inform effective public health measures to contain the spread of this potentially deadly bacterium.

The vascular system is essential for animal survival as it transports nutrients and oxygen throughout the body. Although several recent breakthroughs have enhanced our understanding of the genetic control of vasculogenesis and angiogenesis, the molecular mechanisms of vascular development still remain largely unknown. In general, forward genetic screen is one of the most powerful tools to reveal new clues of vascular development, and zebrafish is the best suited animal for such genetics screen among the animals with a closed circulatory system. Moreover, the transparency of zebrafish embryos and larvae enables one to observe the entire image of three-dimensionally expanded vascular system and to perform in vivo live imaging of blood vessel development. The accessibility of zebrafish allows various manipulations such as generation of chimeric fishes, pharmacological analysis. Therefore, we use zebrafish to reveal mechanisms of vascular development. Previous forward genetic screening identified the mutant with vascular defect, mts (Jin et al., 2007.) Through the analysis of this mutants, we propose to investigate the mechanisms of vascular development. Moreover, we will observe the individual steps of blood vessel development in detail, paying particular attention to individual cell behavior, cytoskeletal dynamics, and the process of endothelial cells polarization by in vivo time-lapse imaging. With the establishment of conditional knock down technology, we will attempt to reveal the mechanism of these steps. The analysis described above should give us further insights into blood vessel development.