Biosynthesis of polypeptide chains at the ribosome is coupled with their folding into the native tertiary structure. The folding process often requires the assistance of molecular chaperones to proceed efficiently. In bacteria, the ribosome-associated chaperone Trigger Factor and the soluble heat shock protein DnaK directly bind to nascent polypeptide chains and are thought to assist co-translational folding. How the interaction of the chaperones with the nascent chain affects the folding events is unclear. Do individual domains of a polypeptide immediately fold when the nascent chain exits the ribosomal tunnel, or is folding postponed through the action of chaperones until the complete amino acid sequence has been produced and released from the ribosome? We propose to address this question in experiments involving in vitro transcription-translation and proton-deuteron exchange measurements. In particular, we will focus on i) determining the effects of Trigger Factor and DnaK on the folding of substrates in solution, independently of translation, ii) investigating the mechanism of chaperone-assisted co-translational folding of nascent chains, and iii) characterizing the functional cooperation of Trigger Factor and DnaK in assisting folding of newly synthesized proteins at the ribosome. This research program will expand my experience with chaperone systems, and provide training in techniques of molecular biology, biochemistry and biophysics, to build on my NMR expertise. Moreover, the broad range of expertise available in the laboratory will not only provide support for the experimental work, but also enable my training in designing creative interdisciplinary experiments.

I will study the role of PP2A, ser/thr protein phosphatases, in the establishment of cell polarity in C.elegans embryo. In a genome-wide RNAi screen, inhibition of three different subunits of PP2A caused defects in mitotic spindle localisation. Moreover, RNAi of one PP2A subunit results in abnormal localisation of PAR-2 indicating that PP2A might act to establish and transduce polarity information. For the first step, I plan to characterise the effects of PP2A knock-down on the organisation of the mitotic spindle and on the localisation of different PAR proteins, both of which are key events in establishing embryonic cell polarity. Secondly, by generating specific antibodies, I will investigate whether the three different PP2A subunits are associated in the same complex. If so, I aim to study the localisation and the dynamics of this complex during early cell divisions. Finally, I will carry out a new RNAi screen and a two-hybrid screen to identify new proteins involved in the cell polarity pathway that depend on PP2A activity. This project is a logical extension of my interest and training in the mechanisms that control polarity establishment in culture cells, with a change from molecular and cell biological approaches to genetic and developmental biology in a whole organism. From my post-doctoral experience, I will acquire expertise in genome-wide RNAi develop in Ahringer's laboratory to identify gene function in C. elegans and in the excellent nematode embryo model for dissection of signalling pathways involved in cell polarity. These new competences will allow me to develop ambitious projects in my future academic career as an independent scientist.

Recent research has demonstrated the plasticity of the auditory system. This work has focused on changes in auditory processing mechanisms, and has not considered how these may relate to cognitive factors such as attention. In the proposed project I will characterise the neural bases of auditory perceptual improvements at a number of different levels, from those that relate to ‘low-level’ sensory processing, to those that relate to ‘higher-level’ cognitive mechanisms, such as attention. To achieve this I will acquire expertise in the neurophysiological techniques of EEG and MEG, which record the electric and magnetic fields at the human scalp during sound presentation. Using complex analysis tools developed at the host laboratory, I will be able to isolate components from these recordings that relate to different levels of sound processing, and will also be able to assess the effects of attention on sound processing. This work will provide me new skills in the field of human neurophysiology, which will complement my previous experience in auditory psychophysics. This will enable me to conduct comprehensive studies assessing auditory processing using multi-disciplinary research techniques, combining both neurophysiological and psychophysical approaches. In addition, it will form the basis of my research endeavour to study auditory perceptual processes in relation to cognitive functioning, a topic that is currently much neglected.

Mitochondria are the primary site of cellular energy generation, and as such are essential for viability. Their morphologically varied structures, which depend on a balance between opposing fission and fusion events to maintain their form, reflect differential cellular requirements and environmental conditions. The goal of the proposed project is to elucidate the molecular mechanisms involved in facilitating and regulating mammalian mitochondrial fusion. The proposal focuses on the mitofusin proteins, GTPase-like fusion components located in the mitochondrial outer membrane that have been implicated in cell development and disease. Under the proposed research plan, the functional roles of various mitofusin domains in mitochondrial fusion will be investigated by both in vivo confocal microscopy analyses of cells expressing defined mitofusin mutants, and complementary in vitro nucleotide hydrolysis assays and mitochondrial fusion assays to analyze regulatory mechanisms of fusion events. As well, the project will explore the hypothesis that mitofusins assemble into protein complexes and seeks to identify novel components of the fusion machinery. The analysis of mitochondrial morphology provides a significant opportunity to learn techniques in mammalian cell biology and confocal microscopy. In turn, the project will be complemented by my past research experience in the area of protein chemistry.

The number of species on the earth is estimated to be tens of millions, and the evolution of genes/genomes which resulted in this diversity of organisms is one of the most interesting topics in biology. I plan to work on cichlid fishes, instead of medaka, which have evolved astonishing variation within a very short period of time, in order to find out what types of genomic/genetic changes have been evolved to achieve this diversity. Because colours and colour patterns on the body surface are among the most divergent and readily recognizable features of animals, I chose pigment-cell-associated genes as the best targets of this research proposal. The aim of this study is to identify the number and chromosomal locations of genes responsible for body-colour determination as well as mating preference in cichlids for later positional cloning and functional studies. Several pairs of fishes which 1) have distinct body colour differences, 2) reach maturity quickly, 3) lay enough eggs for fine locus/QTL mapping, and 4) are closely related to each other and whose F1s are known to be fertile (e.g., The Normal and Gold morphs of A. citrinellus) are crossed/hybridised in the laboratory, and the phenotypes (i.e., body colour and mating preference) of their backcross siblings are analysed to be grouped. I perform AFLP on their genomic DNAs and phenotype-specific bands are used for STS construction. Linkage and recombination distances between the responsible locus/QTL and STSs are evaluated. Tens of backcross siblings are enough for this rough mapping, but I will construct maps as fine as possible for future gene identification by the forward genetic approaches.

All motile organisms possess means of sensing and responding to changes of the external temperature. Given a choice, an animal moves towards a cooler area when overheating, and towards a source of warmth when faced with a cold environment. This simple behaviour demonstrates the main task of the thermosensory system: to define the thermal quality of a stimulus (hot versus cold), and to trigger appropriate behavioral and physiological responses. In addition, extreme temperatures are sensed as noxious stimuli, and as such evoke unique, very rapid reactions. Despite the essential nature of thermosensation, little is known about the central mechanism of temperature discrimination and the circuits involved in behavioral choices and output. The study of temperature coding is further complicated in mammals by the existence of multiple layers of autonomic, endocrine and skeletomotor responses that are interconnected to the sensory pathways to regulate the production of endogenous heat. I propose to employ an integrated genetic, molecular and behavioural approach to elucidate the logic of temperature perception in Drosophila, a system ideally suited for a comprehensive dissection of complex circuits and behaviors.

The mammalian cerebral cortex is characterized by a modular functional organization: distinct cortical areas with specific patterns of input and output projections are devoted to different functions. In particular, sensory information of distinct modalities (vision, audition, somatosensory perception) is relayed from the periphery via specific dorsal thalamus nuclei, which project to distinct cortical areas. Recent studies have shown that the initial targeting of thalamic projections to specific cortical areas is first regulated at the level of an intermediate target, the basal ganglia and subsequently shaped within the cerebral cortex. Whereas sensory ascending pathways, including thalamic projections, form in a stereotyped manner during embryogenesis, they can be remarkably modified in response to activity. In particular, when a modality is depleted, corresponding areas can be recruited for the processing of the intact modalities (cross-modal plasticity). Understanding how thalamocortical connectivity is established and shaped during normal and pathological development is an essential question that remains to be fully investigated at the cellular and molecular levels. To this aim, we propose to use a combination of in vivo and in vitro approaches in mice to achieve three specific objectives: i) to determine the mechanisms controlling the initial targeting of thalamocortical axons in the basal ganglia, before they enter the cortex ii) to elucidate the role of regionalized cortical cues in the guidance, refinement and maturation of thalamocortical projections iii) to establish a model of cross-modal plasticity with physiological relevance.

The subject of the proposed research is the mechanism of circadian rhythms in mice. Several factors have been implicated in the central circadian clockwork, which consists of interwoven positive and negative feedback loops. At the core of this oscillator are the proteins period (PER) 1 and 2 and cryptochrome (CRY) 1 and 2, which rhythmically inhibit the heterodimeric transcription factor CLOCK:BMAL1. Insight into the precise molecular mechanisms of this process is still limited and lagging far behind the genetic progress made in the field. Therefore, I propose to characterise the PER:CRY complex with the aim of understanding how it acts as a transcriptional repressor. I will purify native PER- and CRY-containing complexes from synchronised tissue culture cells and determine their composition and the circadian changes they undergo. I expect to identify new complex components, which will be tested for a function in the circadian clock in a novel cellular system and by genetic loss-of-function studies in the mouse. To investigate how PER:CRY-mediated regulation of transcription is achieved I will analyse in detail the events taking place at regulatory sequences of genes that are under circadian control. In particular, I will address the question on how PER and CRY proteins can generate widely different phases of cyclic gene expression. In summary, the proposed combination of biochemical, molecular biological and genetic studies will make important contributions to the fields of molecular chronobiology as well as transcription regulation in general, since the PER:CRY complex appears to represent a novel type of transcriptional repressor.

DNA microarrays allow determination of global mRNA expression profiles of cells grown under various conditions. However, it is often not known how changes in mRNA expression are correlated with protein synthesis and the extent to which additional regulation occurs at the translational level. For instance, it is commonly assumed that developmental and oncogenic signaling achieve their phenotypic effects primarily by direct regulation of the transcriptional profile of cells. However, recent evidence indicates that direct effects on transcription may be preceded by effects on the translation efficiency of specific existing mRNA species. I want to establish a method for translational profiling, which involves the rapid purification of endogenously formed ribosomes and analysis of bound mRNAs with DNA microarrays. I will analyze how changes in steady state mRNA levels are related to changes in ribosome occupancy upon changing growth conditions in the yeast Saccharomyces cerevisiae. The method will also be established in mammalian cells with focus on HeLa and endothelial cells. Cells will be treated with pharmacological compounds or mixed with other cell types to explore the impact of intra-, and inter-cellular communication on translation. These studies will help to decipher the extent and nature of translational regulation and uncover new aspects of the gene expression program.

Tumor cells form metastases by invading and migrating into other tissues, via either elongated or rounded motility, which differ in that elongated motility needs degradation of the extra-cellular matrix (ECM) by protease activity, whereas rounded motility does not. Recently, the laboratory of Dr. Marshall has shown that the kinase ROCK, downstream of Rho, is specifically involved in rounded motility and that the constitutive activation of Rho is enough to make cell switch from elongated to rounded motility. Moreover, cells moving via elongated motility, once treated with ECM-protease inhibitors, switch to rounded motility and their migration was blocked only by simultaneous inhibition of both ECM proteases and ROCK. This is likely the reason why therapies targeting the ECM proteases turned out to be only limitedly effective. It is, therefore, of great interest to understand the pathways responsible for the establishment of rounded motility. As preliminary results suggest that ROCK is involved in rounded motility by keeping high levels of MLC2 phosphorylation and likely stimulating actomyosin contractility, the aim of this study is, indeed, to understand by which mechanism ROCK controls MLC2 phosphorylation, and therefore motility. Besides of its pure scientific importance, this research would help in designing novel strategies for anti-cancer therapy. This is an additional reason of my interest in this project, which differs from those I have been involved so far, as they were mostly concerned to normal cell behaviour. I believe that the excellent environment of the Marshall laboratory will strongly contribute to my scientific training and expand my career perspectives.

Our visual system regulates its sensitivity according to the prevalent characteristics of the visual scene. This adaptation allows efficient transmission of sensory information despite ever changing environmental conditions. A simple example is the accommodation to the average luminance level, for example when entering a dimly lit room. But adaptive phenomena reach far beyond the mean intensity. Recent experiments have shown that already neurons in the retina can selectively adapt to a surprising variety of stimulus correlations in space and time, such as orientation and spatial frequency of a stimulus. This suggests generic mechanisms that detect these correlations and regulate the network's sensitivity. The goal of my proposal is to investigate the biophysical machinery of this adaptive behavior. In particular, I will aim at distinguishing whether the observed changes in sensitivity result from neural fatigue of specific cells in the retinal network or from activity-dependent plasticity at the synapses. Electrophysiological recordings of the neural activity in an isolated retina will be used to characterize the adaptive phenomena and to identify synaptic and cell-intrinsic contributions. Mathematical modeling serves to test the consequences of the observed cellular characteristics for network function. Having worked before on single-cell analysis of sensory reception, the proposed project will extend my work to include dynamics within a neural network. The state-of-the-art experimental techniques that I will acquire as well as the new perspectives on sensory processing will be highly valuable experiences for my development as a scientific researcher.

The mechanisms underlying the evolution of the nervous system are poorly understood. In particular, it is not known, when the nervous system expands during evolution (for instance the frontal cortex of the human brain), how the connections of the new neurons are patterned in harmony with the pre-existing neuronal wiring. Yet, the organization of these new connections is critical to the function of the nervous system as a whole. The goal of this project is to understand how the projection to the central nervous system of a newly evolved neuron fits within the ancestral and highly organized wiring. I will use as a model system the simple and defined gustatory system of the flies Drosophila. In a few species, this otherwise conserved system has gained novel neurons and constitute a choice material to understand by which mechanisms neural connectivity evolves. How the connectivity evolves is a long-standing interest of mine, which I believe to be of general interest because changes in the nervous system reflects in most aspects of metazoan evolution. While the focus of my PhD was on neural development per se, my first postdoctoral experience, during which I have studied the evolution of pigment patterns in flies, gave me the opportunity to acquire the tools necessary to study how connectivity evolves. Therefore, I regard the current proposal as a first step of a larger investigation of how connectivity evolves at a cellular and genetic level that I am eager to pursue in the future.

We will identify and characterize novel proteins involved in synaptic vesicle (SV) fusion and recycling in C. elegans, to broaden our knowledge of these processes also in the mammalian nervous system. We will purify such proteins through their association with known SV proteins or members of the SNARE complex, using tandem affinity purification (TAP). Functional, TAP-tagged baits will be expressed in genetic backgrounds lacking the respective endogenous proteins, and subsequent large-scale purifications will allow mass spectrometric identification of co-isolated proteins. To test functional interactions of novel purified proteins with SV's or the SV fusion machinery, we will deplete them by RNAi, and subject those animals to a behavioural assay for defective neurotransmission. Specific phenotypes found will be verified after generation of the respective genomic deletion mutants. These mutants will be analyzed in detail by behavioural, pharmacological and, importantly, electrophysiological assays. Expression patterns of these proteins will be established using GFP-fusions and/or specific antibodies. Direct contacts between novel proteins and known SV components will be determined biochemically, or using in vivo assays. Deletion mutants will further facilitate structure/function analyses, i.e. characterization of individual domains of the new proteins. Finally, we will study potential genetic interactions with known proteins of SV's and the fusion machinery, utilizing available mutants. This work shall contribute to our knowledge of the highly conserved process of synaptic transmission, and may shed light on the molecular basis of certain genetic neurological diseases.

The survival of animals depends on their ability to sense their environment and respond by altering their behavior. In my post-doctoral studies, I will explore the mechanisms that enable animals to sense and respond to ambient oxygen levels and oxidizing and reducing (redox) environments. Life depends on electron flow between molecules. Reactions like photosynthesis and respiration require molecules that donate and accept electrons (reducing and oxidizing agents, respectively). However, these agents can also be dangerous. For example, although oxygen is vital for all animals, it generates oxygen radicals, which damage cells and may accelerate aging. I will use the nematode C. elegans as a model system. This organism offers a powerful range of molecular and genetic tools for analysis. It has a small, well-described nervous system so that I can analyze behavior at different levels of complexity: of genes and proteins, neurons and neural circuits, and the entire animal. Many genes expressed in the human brain have homologues in C. elegans - so I anticipate being able to generalize from information obtained in C. elegans to mammalian nervous systems. To identify molecules that sense and respond to redox stimuli I will primarily study aggregation of C. elegans into groups. This behavior is regulated by ambient oxygen levels. One focus will be on proteins of the soluble guanylate cyclase family that regulate both aggregation and oxygen sensing. Relatives of these proteins are expressed in almost every human cell and are important drug targets. They regulate many processes, including blood pressure, blood clotting, our immune response and learning and memory.