Membrane proteins of the Solute Carrier family 12 (SLC12), or Cation Chloride Co-transporter (CCC) family, mediate the transport of sodium and/or potassium ions together with chloride ions. In human, SLC12 transporters are omnipresent and fulfill important physiological roles in the central nervous system, kidney and other organs. Yet, characterization of SLC12 transporters is only poor. In order to obtain insights into the transport mechanism, we propose to determine the three-dimensional structure of a SLC12 transporter. X-ray crystallography is the method of choice to pursue this goal, but presents specific challenges for membrane proteins due to their problematic overexpression and large hydrophobic surfaces. Thus, to obtain large amounts of active detergent-solubilized membrane protein and regular crystal lattices containing both the protein and its surrounding detergent-micelle, alternative and novel approaches will be employed. Establishing an overexpression system for these transporters is not trivial as eukaryotic membrane proteins are often not expressed or produced in an aggregated state. Rather than screening only one expression host or one homolog, the expression screening will involve a multitude of homologs and four different expression systems, that are Escherichia coli, Lactococcus lactis, Pichia pastoris and baculovirus-Sf9 cells. A variety of detergents and stabilizing compounds such as inhibitors will be employed to obtain the protein in a stable detergent-solubilized state. Finally, during the extensive crystallization-screening, antibody fragments will be used to enlarge the hydrophilic surface of the protein to promote molecular interactions.

Much of our adult behavior reflects the neural circuits sculpted by experience during infancy and early childhood. At no other time in life does the surrounding environment so potently shape brain function as during these so called "critical periods". Research on autism spectrum disorders (ASD) has recently revealed striking similarities between critical period regulation and disordered neuronal circuit function. I will test the hypothesis that abnormalities of neuronal circuitry in ASD arise from a disruption of normal, postnatal experience-dependent synaptic plasticity. I will characterize neuronal circuit function in a defined brain region, the visual cortex, via in vivo electrophysiology and concomitant behavioral tasks across postnatal development in normal and particular mouse models of ASD (MeCP2, neuroligin mutants). This approach promises important insights into how neuronal circuits give rise to appropriate behaviors, and what might be the particular behavioral outcome of a given physiological abnormality. I will then attempt to rescue functional and behavioral impairments in these animal models of ASD through the manipulation of critical period plasticity and specifically by restoring a proper excitatory/inhibitory circuit balance. Rigorous consideration of a neurodevelopmental disorder as a neuronal circuit malfunction will yield a deeper understanding of the factors contributing to the basic establishment of normal brain function, as well as potentially novel therapeutic strategies for ASDs of diverse and complex etiologies.

This project aims at an integrative understanding of evolutionary novelty from the genetic mechanisms producing it to the ecological mechanisms selecting it. We rely on the co-evolution of a male-specific wing pigmentation spot present in some Drosophila species. Along with the spot, these males have evolved a wing-display behavior performed in front of the female they court. In recent work, we showed how the spot was gained by changing pigmentation genes expression, in particular by modifying their cis-regulatory elements (CREs). The proposal will expand from this work following 3 lines of research: 1) characterizing the gene regulatory network involved in the formation and evolution of wing pigment patterns and dissecting the CREs of this network to understand the logic of enhancer function and evolution. 2) testing whether sexual selection in the form of female preference is driving the evolution of this spot. 3) dissecting the neuronal network controlling male courtship behavior, and then addressing how these neurons have changed between non-displaying and displaying species. This research program address risky, yet fundamental and hardly explored evolutionary questions. I hope to unravel fundamental mechanisms of transcriptional regulation and behavior specification that will be of interest to biologists and biomedical scientists. I believe that HFSP funding would boost my endeavor at a stage of my carrier when I would rather concentrate on data production than endless funding applications. It would also increase my international visibility and help me to attract talented trainees from different fields, which will help me to implement the integrative approach I envision.

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.