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.

In most eukaryotes, cytokinesis is mediated by a contractile actomyosin ring (CAR), which assembles at the onset of mitosis. I wish to address the mechanisms coordinating the temporal and spatial reorganization of the actin cytoskeleton during the cell cycle, using the fission yeast Schizosaccharomyces pombe as a model organism. Fission yeast cells are rod-shaped and grow at their ends before dividing symmetrically. Both polarized growth and cytokinesis rely on the actin cytoskeleton, which undergoes major rearrangements during the cell cycle. In S. pombe, the onset of septation is controlled in part by the septation initiation network (SIN), a signal transduction pathway regulated by the Spg1 GTPase. The site of cytokinesis is defined by the release of the protein Mid1p/Dmf1p from the nucleus. I propose to investigate the relationships between SIN signaling, CAR assembly and the growth machinery, following two lines of research based on the use of new genetic screens and advanced method in live imaging. First, I will make use of the overexpression of Spg1p, which induces ectopic septation, to investigate the regulations of CAR assembly and contraction. Second, I will exploit the overexpression of Mid1p, the spatial cue for CAR assembly, to address the mechanisms underlying the spatial definition of cell growth and cytokinesis. Both the subject of study and the model system will differ from my previous research, thus allowing me to acquire a complementary expertise essential for my career development.

The refinement of synaptic connections is crucial for the development and function of the nervous system. It is coordinated by a combination of synapse formation and disassembly of previously formed connections, and abundant evidence supports that both of these processes require neural activity. Our preliminary experiments have shown that myocyte enhancer factor 2 (MEF2), an activity-regulated transcription factor, acts as a negative regulator of synapse number in developing cultured hippocampal neurons. This finding suggests two possible effects of MEF2 on synapse development: 1) MEF2 decreases synaptogenesis or 2) MEF2 promotes active synapse disassembly. To distinguish between these possibilities, we plan to utilize a regulated gene activation system that will allow temporal control of MEF2 activation at the time of synapse development. In addition, to characterize the molecular mechanisms by which MEF2 mediates synapse development, we will employ a genome-wide screen to identify neuronal targets of MEF2. Finally, we propose to generate transgenic mice to explore the physiological role of MEF2 in synapse development and subsequent effects on animal behavior. My previous research has focused on identifying the molecular mechanisms by which immune cells recognize changes in their environment. In the proposed research, I plan to analyze the signal transduction pathways that operate in cells of the nervous system and mediate their responses to environmental stimuli. This research experience will provide me with a great opportunity, not only to acquire interdisciplinary knowledge and techniques, but also to investigate the fundamental principles that govern cell function.

My post-doctoral research focus is the elucidation of the molecular mechanisms involved in regulation and signaling of Epac, a guanine nucleotide exchange factor for the Rap1 G protein. My first goal is to understand the mechanism by which cAMP regulates Epac, which will have consequences for our comprehension of cAMP regulation in many cellular responses. Using NMR spectroscopy, both structural and dynamic effects of cAMP on the cAMP-binding domain will be examined and then studied in the context of the catalytic GEF domain to describe how signaling information is transmitted. Second, I want to characterize the role of the second cAMP-binding domain in the Epac2 protein, whose function is unknown. Using mutagenesis and ligand screening, insights into the biological function can be achieved. My third goal is to identify new effector proteins of the Epacs. This will be done through a combination of immunoprecipitation and mass spectrometry, in an effort to discover new signaling pathways of this protein. Characterization of components involved in Epac-mediated signaling pathways will delineate the biological roles of this novel cAMP-binding protein as well as provide general insight into cAMP-mediated regulatory mechanisms. As Epac is the only other ubiquitous protein known to bind cAMP apart from PKA, it is important to discriminate Epac-mediated effects of cAMP signaling. This research will differ significantly from my previous chemistry and biophysics endeavors by allowing me to extend into the fields of biochemistry and cell biology. This will develop my biological experience, allowing my future research in signaling to be placed in a wider context.

LKB1 is a serine/threonine kinase with tumor suppressor activity and is mutated in the Peutz-Jeghers cancer syndrome. LKB1 is homologous to the Par4 polarity genes in C. elegans and D. melanogaster and has been shown to induce complete polarization in intestinal epithelial cells in which induced expression of its adaptor protein STRAD activates LKB1. The resulting polarization phenomena include formation of an apical brush border, redistribution of junctional proteins peripheral to the brush border and sorting of apical and basolateral markers to their respective membrane domains, all in the absence of cell-cell contacts. This implies that loss of epithelial polarity in Peutz-Jeghers tissues is the principal cause of cellular transformation. The objective of the proposed research is the elucidation of the molecular context through which LKB1 and STRAD act to control cellular polarity and its relationship to cancer. I plan to focus on constructing cell line models in which the different aspects of polarity can be analyzed independently by live-cell imaging. Using these tools I will try to unravel the hierarchy of the polarity events and to study downstream effectors of LKB1/STRAD through siRNA approaches, including a screen utilizing a genome-wide siRNA library. The proposed switch in research direction, from mechanistic studies of transcriptional regulation to more integrative studies of cellular differentiation, will, I hope, help me familiarize myself with new concepts and techniques, especially genome-wide approaches, and expand my experience and expertise, allowing me to pursue independent research in the future.

My proposal seeks to combine NMR spectroscopy and modelling methods to investigate the structure and dynamics of nascent chains and the ribosome stalk in intact ribosomes. In living cells, proteins are sometimes synthesized and folded concurrently on ribosomes – a process known as co-translational folding. It is fundamentally different from in vitro renaturation of unfolded proteins because of the interplay between the nascent chain and components in the ribosomal exit tunnel, reminiscent of molecular chaperones. Uncovering the mechanisms through which such processes take place may bridge the gap between in vitro and in vivo studies of protein folding and shed light on the misfolding-related human diseases. However, the inherent dynamics and structural heterogeneity of nascent chains hinder further elucidation by X-ray crystallography and cryo-EM. By contrast, the versatility of NMR spectroscopy relies on the underlying dynamics for characterization of such processes. Using the protocols recently established by Dobson and co-workers, I will use NMR to study intact E. coli ribosomes stalled following translation with nascent chains. NMR parameters such as chemical shifts will then be used as restraints for theoretical modelling, also developed in the Dobson lab, by which ensembles of structures, representing stages of co-translational folding events and interactions between ribosomal and auxiliary proteins, can be generated. Finally, I plan to complement the results by cryo-EM and other biophysical and biochemical methods, in collaborations if necessary, to yield a comprehensive description of the behaviour of a growing polypeptide chain attached to the ribosome.

Area LIP in the rhesus monkey has been implicated in the planning of eye movements, the allocation of spatial attention and decision formation. Many LIP neurons also respond selectively to two-dimensional (2D) shapes. Neurons in other areas in the lateral bank of the Intraparietal Sulcus (IPS) signal the 3D orientation of tilted surfaces (area CIP) or the presence of 3D objects (area AIP). This proposal focuses on the representation of 2D and 3D shape in posterior parietal cortex, and the functional interaction between posterior parietal and inferior temporal cortex. We will determine whether neurons in the lateral bank of the IPS respond selectively to 2D shapes irrespective of the visual cue defining the shape, and whether and how these posterior parietal neurons code for curved and tilted 3D surfaces defined by binocular disparity. This testing procedure will allow the first direct comparison between inferior temporal and posterior parietal cortex with respect to the representation of 2D and 3D shape. The main goal of this proposal is to study the functional interaction between dorsal and ventral stream areas during 3D object discrimination and grasping. We will implant chronic multielectrode arrays in different areas in the posterior parietal and in the inferotemporal cortex. These arrays will permit the simultaneous registration of different brain areas while the animals are either discriminating 3D shapes or grasping 3D objects. The incorporation of injection needles into the arrays will be necessary to inactivate a specific brain area while assessing both the behavioral consequences and the neurophysiological effects on different brain areas.

Chronic diseases such as type 2 diabetes, cardiovascular diseases and cancer are responsible for most deaths in industrialized societies. These diseases are often associated with the Western lifestyle which is characterized by a high-fat diet, lack of exercise and obesity. A group of nuclear receptors, the peroxisome-proliferator-activated receptors (PPARs) are linked to these diseases by regulating metabolism, fatty acid oxidation and lipogenesis at the transcriptional level. The exact mechanism, however, by which the PPARs mediate lipid metabolism and their relevance to human pathology and therapy is still unclear. Through the use of functional genomics this project aims to characterize the pathways through which PPARd and other nuclear receptors regulate lipid metabolism. During my PhD-research I have gained extensive experience in animal experimentation, pharmacological and physiological studies. With the current research proposal in the group of Dr. Ronald M. Evans at The Salk Institute I expect to improve my knowledge of the different aspects of molecular biology and functional genomics. I believe that experience with novel genetic tools such as the RNAi technology will be of great value for my development as an independent researcher. The combination of these fields with my current scientific background allows me to integrate different aspects and levels of biological research and will put me in an excellent position to develop novel and innovative lines of research. After the fellowship my aim is to start an independent research group studying the molecular biology of metabolic diseases.