My general scientific interest is to understand how adhesion between early embryonic cells affects embryonic morphogenesis. A basic hypothesis suggests that differences between cells, regarding the identity and/or quantity of their cell adhesion molecules (CAMs) may regulate embryonic morphogenesis. Although studies aimed to understand the role of selective adhesion have been carried out in several developmental systems, none of these can be tested at the cellular and the genetic level at the same time. Here I propose to use C. elegans as a model for studying embryonic cell adhesion, by combining cell manipulation strategies with the advantages of an excellent genetic system. In this proposal I aim to test how strongly different embryonic isolated cells adhere to each other and set these experiments as a framework to study the role of known molecules involved in cell adhesion at the cellular level. As very little is known about the identity of CAMs in C. elegans, I will use a reverse genetic approach to identify new molecules, which will be further studied for their functions during embryogenesis. I plan to use this postdoc training in order to gain the necessary practice and take advantage of the vast knowledge obtained in the Goldstein lab and to continue afterwards, establishing my own lab in Israel. Not only is the Goldstein lab a leading lab in the fields of embryology and cell manipulation, but also working there will expose me to state-of-the-art equipment, techniques and a scientific environment that are not currently available to me. In addition, such experience in another country is a strict requirement for being an independent investigator in my country.

Metastasis accounts for a large part of cancer malignancy. Invasion is the initial step of metastasis, and acquiring aggressive invasiveness is important for the subsequent dissemination of tumor cells into circulation. Changes in the invasiveness of tumor would require harmonized changes in the action of several factors either inside, surface or outside of the tumor cell, indicating that exhaustive analyses like gene expression profiling are effective in studying this process. Recently, it has been shown that a transcription factor Twist, which plays major roles in embryonic morphogenesis, also plays an essential role in the regulation of tumor invasion. Twist enhances tumor invasiveness possibly by inducing an epithelial-mesenchymal transition (EMT) of tumor cells. In the proposed study, I will set up a system to express Twist in an inducible manner in cells of the transplanted tumor. It is expected that Twist triggers an EMT of the tumor cells and promotes invasion of the transplanted tumor. It will therefore provide an experimental system to study how the invasiveness of tumor is enhanced. Subsequently, to address the molecular basis supporting the enhancement of invasiveness, I will analyze the time-scale changes in the gene expression profile of the tumor cells after Twist expression. As a PhD student, I have studied p53-mediated apoptosis, an intracellular mechanism concerned with tumorigenesis. In contrast, through this fellowship experience, I will mainly study the behavior of tumors in the tissue context. Therefore, this experience will particularly help me in obtaining a comprehensive view on the process of malignant tumor progression.

By dynamically controlling the assembly and disassembly of their cell-cell junctions, epithelial sheets can stratify, branch, form tubular structures or move as a unit. Such morphogenetic events are critical to normal development, and abnormalities in intercellular adhesion are associated with a variety of human disorders, including cancers. Cadherin-mediated adherens junctions (AJs) are essential for organizing epithelial sheets. An emerging prerequisite to AJ formation is the assembly of the transmembrane protein nectin, which through afadin, connects to small GTPases and the actin cytoskeleton. The mechanisms by which the nectin-afadin network governs AJ assembly are poorly understood. By conditionally targeting the single-copy afadin gene in skin epithelial stem cells, I will explore the physiological significance of this network to intercellular adhesion and epithelial morphogenesis. Primary keratinocytes cultured from these mice will allow me to elucidate mechanistically how afadin promotes AJ formation. Finally, by generating epidermally-expressing GFP-afadin and RFP-actin mice and culturing their keratinocytes, I will explore the dynamics of afadin-associated cytoskeletal rearrangements and cell-cell adhesion. The project applies my experience in mammalian GTPases to address the mechanisms governing intercellular adhesion in tissue morphogenesis and cancers. The combination of my postdoctoral and PhD training will equip me for a future career in scientific research, and enable me to tackle a problem beginning with whole animal studies, moving to primary cell culture studies and finishing at the molecular level.

The cation channels TRPV1 and TRPA1 are implicated in pain perception. Both channels can be activated by pungent compounds and modulated by inflammatory factors. By analogy to TRP channels in the Drosophila eye, we hypothesize that TRPV1 and TRPA1 are components of supramolecular membrane-bound protein complexes that enable the channels to function specifically and effectively in a context dependent manner. We will use genetic, biochemical and functional screening paradigms to identify components of this putative modulatory complex. A modified yeast-two-hybrid system will be used to find direct interaction partners of the transmembrane TRP channels. A combined genetic-biochemical approach is intended to pull-down TRP channel complexes from native sensory tissue for further identification of constituents, and a functional screen will be established to shed light on TRP channel regulatory proteins, even those not directly associated with a physical signaling complex. All three different screening approaches will address different aspects of the hypothesized TRP channel complexes and they will therefore complement each other. The proposed research plan will help to elucidate TRP channel-based sensory perception in particular pain sensation. I previously worked on hearing and deafness genes and will switch now to the field of pain perception and ion channel research, which is new to me. Nevertheless, both fields of research are disciplines within sensory biology. By broadening my horizon in this way, I am confident that my postdoctoral training in Dr. Julius lab will allow me to mature as a scientist in the area of sensory neuroscience, a further step in my academic career.

The basal ganglia participate in motor initiation, selection and learning, as well as in cognition and attention mechanisms. Various innervation pathways involving the basal ganglia have been studied, yet the detailed properties of the microcircuits underlying these pathways are poorly understood. The striatum is the input layer of the basal ganglia, containing mostly medium spiny GABAergic neurons, which project to the output layer, the pallidum. The pallidum is tonically active, inhibiting the cortex and motor centers areas in the brainstem and thalamus. Input from the striatum inhibits pallidal neurons and releases these motor centers areas from inhibition. Projection from the striatum to the pallidum is mediated via two main pathways, the ‘direct’ and ‘indirect’ pathways. These pathways are antagonistic and there are various hypotheses regarding their function in movement scaling and selection of motor behavior. In order to understand the function of the basal ganglia, it is crucial to have a deeper understanding of the striatal microcircuit underlying these functional pathways. Striatal neurons of the direct pathway express D1 dopamine receptors and substance-P, whereas those of the indirect pathway express mainly D2 receptor types and enkephaline. These differences most likely reflect a multitude of other functional differences in their cellular and synaptic properties, which are currently unknown. It is therefore proposed to carry out a study aimed at revealing the detailed microcircuit organization of the striatum by using multiple whole cell recordings, single-cell RT-PCR, 3-D morphological reconstructions, anterograde and retrograde labeling.

The extracellular matrix (ECM) couples a cell with its environment and therefore directly regulates a cell’s fate via physical and biochemical cues. Rather than being a simple structure providing only cohesion and mechanical integrity to tissues, it is now believed that the conformation and signaling capacity of the ECM is changed by force. This force-dependent dynamic behavior of the ECM may underlie cellular sensation of mechanical stimuli, or mechanotransduction, via exposure of cell binding sites normally buried in the native fold of the protein. This study will determine how defined levels of externally-applied strain or alternatively cell-derived internally applied strain in cell cultures alters the conformation of the ECM protein fibronectin, investigate whether strain is detected by cells, and aid in our understanding of the pathways which regulate a cell’s growth, differentiation, and death. This proposal will utilize basic cell and molecular biological tools and fluorescence resonance energy transfer, an advanced imaging tool, to study how the conformation of proteins influences cell behavior at the nano-scale. These skills differ markedly, but will greatly enhance, my previous experience investigating fluid dynamics in vivo at the macro-scale using animal models such as intravital microscopy. These biological tools will bridge the gap between my work on how force is manifested within the body and a career investigating the impact of force on living cells.

MicroRNAs (miRNAs) are a family of small non-coding RNAs that negatively regulate gene expression in a sequence-specific manner. They regulate aspects of development in multicellular organisms including Arabidopsis. However, our knowledge about roles of miRNAs in plant morphogenesis is still rudimentary. I will elucidate the biological role of the post-transcriptional regulation of the TIR1 gene and its homologs by an Arabidopsis miRNA, miR393 with respect to auxin signaling pathways. Auxin is an essential phytohormone that influences aspects of plant morphogenesis. The TIR1 gene is a pivotal positive regulator in auxin signaling pathway. Although the biological functions of the TIR1 homologs have not yet been characterized, similarities of their amino acid sequences suggest that the homologs are also required for auxin signaling. Therefore, miR393 is expected to negatively regulate a wide rage of auxin responses by eliminating its targeted transcripts. This research proposal will contribute to defining an important regulatory system in the auxin response pathway. As described above, I am interested in the mechanism of cell division control in plant development. Auxin is the most important phytohormone to control cell proliferation. An understanding the mechanism of auxin signaling is a prerequisite to unraveling the molecular mechanism of cell division control. In addition, the post-doctorial training will allow me to acquire technical skills and scientific knowledge analyzing multicellular system of plants, although my doctoral training has been primarily in plant cell biology.

The vomeronasal organ (VNO) functions as a second nose in higher vertebrates and is thought to be specialized in the detection of pheromones. Vomeronasal sensory neurons express genes of the V1r and V2r superfamilies. These genes encode seven-transmembrane proteins that are believed to be pheromone receptors. In addition, VSNs expressing V2rs also co-express proteins from another multigene family, termed H2-Mv. These are nonclassical class I genes of the major histocompatibility complex (Mhc). Interestingly, the nine H2-Mv genes are expressed differentially in subsets of neurons and in preferred combinations with specific V2rs. H2-Mvs may function as escort molecules in the transport of V2rs to the cell surface. However the functions of the V2r and H2-Mv complex on the cell surface, and the significance of the combinatorial co-expression of V2r and H2-Mv genes are not known. Here I propose a project that will uncover fundamental and much needed information on what Mhc genes might do in the vomeronasal sensory system. I plan to delete these genes with the chromosome engineering technique. The mutant mice will be subjected to electrophysiological and behavioral assays. I believe that this project will provide important information on how pheromonal communication and mate choice operate in mammals. This project will give me the opportunity to work on a very different model system (the mouse) compared to my present one (the fruitfly). In addition I will also be given the chance to learn advanced techniques in molecular neuroscience, skills that will be most valuable for my planned future career.

== Objective == I propose to examine the effects of the extremely high macro-molecular concentration (molecular crowding) and subcellular localizations of proteins on the pheromone response signaling pathway of S. cerevisiae, by combining in vivo measurements and computation. == Methodology == (1) In vivo mobilities of key proteins (including Ste5, Ste20, G-protein complexes and the pheromone receptor) in the crowded environment will be measured employing fluorescence correlation spectroscopy (FCS). FCS can deliver quantitative data about apparent diffusion constants, deviations from normal Brownian motion (such as anomalous diffusion and active transport), and local concentrations of proteins in vivo. (2) Quantities measured above or obtained from the literature will be compared with microscopic lattice gas automata simulations conducted under various macro-molecular concentrations. (3) This will aid in derivation of a meso-scopic reaction-diffusion simulation model that represents spatial organization and biochemical activity of the pathway at the whole cell scale. (4) The effects of space and crowding environments will be characterized by contrasting the spatial simulation with a classical, non-spatial model that the host institute has developed previously. == Training opportunity == The applicant's previous research did not involve biophysical experimentation or detailed biological modeling, both of which are essential for his professional development. The host institute has extensive and successful experience with experimental and modeling methods, thus substantial training and collaborative opportunities are expected.

Stereo vision is a model system for investigating the neural mechanism of information processing, because the computational task of the stereoscopic system is well-defined: a single neural representation of the 3D scene is derived from a pair of slightly disparate 2D representations for the left and right retinal images. The neural mechanism of the initial level of stereo processing is fairly well understood with a simple model, but it does not account for all aspects of stereoscopic depth perception. The neural representation must be further processed at higher extrastriate visual areas in order to fully account for the perceived stereoscopic depth. However, the neural mechanism of processing the initial representation of binocular disparity into a higher representation of stereoscopic depth is yet an open question. This project is aimed to bridge this gap. Computational modeling of a neuronal network of V1 and an extrastriate area will provide testable predictions of neural responses to grating patterns of various combinations of spatial frequencies and disparities, as well as their compounds. Then we will test these predictions by single-cell recordings of the responses of visual cortical neurons in awake, behaving monkeys. The research plan for this application is novel to the applicant in that it merges the theoretical and experimental approaches that he has so far experienced. Such comprehensive study of visual information processing is essential for a better understanding of the neural mechanism of complex cognitive functions of organisms such as perception and behavior. This is the direction of research that the applicant aspires to for a professional career.

Establishment and maintenance of cell polarity belong to the most basic problems in cell and developmental biology. Polarity of cells is a necessary precondition for the asymmetric distribution of intracellular components. In plants, the polar localization of PIN proteins, which are recently reported by Dr Jiri Friml and colleagues, provides an excellent marker of cellular polarity. So far, the mechanism, which regulates cell polarity in plants is only poorly understood. In the presented project I propose a forward and reverse genetic approach in the model system Arabidopsis to specifically identify the factors regulating cell polarity in plants. The idea for the project on cell polarity emerged from my previous work, when two proteins (ACR4-GFP and ALE2-GFP), which I identified, represent themselves as novel markers for cellular polarity. For this project, an excellent marker for cell polarity as well as techniques in cell biology is essential. Dr. Friml’s group fulfils both of these requirements. Using well established genetic and cell biology methods in host laboratory together with an unique set of cell polarity markers (PIN-GFP, anti-PIN antibodies as well as ACR4-GFP and ALE2-GFP markers), components essential for generation and maintenance of cell polarity in plant will be identified. Through this project, I aim to acquire an expertise in cell biology. Beside, ZMBP Tübingen is a progressive institution with an unique organization structure. This will greatly add to my learning how to organize independent research. Thus training through this fellowship will give me an excellent opportunity for my professional growth and greatly help to achieve my scientific goal.

Ras proteins are small GTPases that play crucial roles in the regulation of several cellular processes such as growth, differentiation and transformation. Although Ras signaling was thought to occur uniformly on the inner leaflet of the plasma membrane, a growing body of evidence indicates that Ras activation happens dynamically within defined plasma membrane microdomains and at other specific intracellular compartments, thus ensuring the generation of distinct signal outputs. Yet the mechanisms that control the spatio-temporal segregation of Ras proteins remain poorly characterized. In this proposal, I aim to determine how the differential modification of Ras proteins by ubiquitination contributes to their location-specific signaling activities. The dynamics of Ras ubiquitination and the effect of this modification on signal propagation will first be assessed by fluorescence resonance energy transfer and biochemical approaches. The influence of ubiquitination on Ras subcellular localization and trafficking will next be monitored at the ultrastructural level by electron microscopy and by fluorescence recovery after photobleaching. Finally, the effect of ubiquitination on Ras transforming activity will be studied by conducting tumor xenograft experiments. A postdoc in Dr Bar-Sagi's laboratory will give me a unique opportunity to study the biology of small GTPases while learning new biochemical/biological approaches that have applied purposes. This fellowship experience will therefore greatly contribute to the strength of my understanding of intracellular signaling mechanisms and enhance my qualifications on this topic for the tenure of my own research group in France.

I wish to apply several new molecular tools, including genome wide location and expression analysis, to the nematode C. elegans to identify direct targets of master regulators of specific cell types. These experiments will allow me to investigate the extent of gene regulation in terminal differentiation. I plan to focus on the differentiation of the mechanosensory cells in C. elegans, a system that serves as a model for studying cell fate determination. The functional fate of touch-sensing cells is defined by the master regulator MEC-3, a homeodomain transcription factor. I will define the direct targets of this activator in two types of mechanosensory neurons. I will use expression analysis, which identifies differences in expression profiles between two or more conditions, and genome wide location analysis, a powerful method that identifies the genomic binding sites of transcriptional regulators. Analysis of the data collected by these methods should help identify the genes that are direct downstream targets of the master regulator MEC-3. Further analysis of such targets may lead to the characterization of new genes and to the unraveling of the way that these cells function towards mechanosensation. The particular research project offers me the opportunity to familiarize myself with modern molecular tools and develop new experimental skills while focusing on quite challenging questions of neural differentiation and cell fate determination. Both the experimental and theoretical background for this proposed research differs from my previous scientific experience, a fact that gives me the unique opportunity to expand my scientific and, hopefully, professional, horizons.

All living organisms use ion channels to regulate the transport of ions across the cell membrane. Recently, the crystal structure of a voltage-dependent potassium channel from the hyperthermophile Aeropyrum pernix (KvAP) has been reported in complex with antibody Fab fragments. KvAP is a homotetramer of subunits that contains six hydrophobic segments, S1-S6, of which S5-S6 line the pore and determine ion selectivity, whereas S1-S4 form the voltage sensor (sKvAP). Based on the KvAP crystal structure it has been, somewhat controversially, suggested that the carriage of the gating charges, comprised mainly by the first four arginines in S4, through the membrane occurs through the movement of the S3-S4 helical segments against the membrane, leading to pore opening. The crystal conditions, tight packing and presence of Fab, have been described to lead to a non-native conformation at the intracellular surface of sKvAP, contributing to the gating controversy and demonstrating the need for new structures, preferably in solution. Here, I propose to determine the structure of the isolated, monomeric and functional 147-residue voltage-sensor domain in solution by NMR spectroscopy to obtain a deeper understanding of conformational changes accompanying changes in membrane voltage. Additionally, the characterization of protein dynamics and pertinent mutations of the sKvAP will complement this study. Besides the structure of sKvAP at zero membrane voltage, it will be attempted to define different voltage-dependent conformations and its folding pathway. In an important initial step of this project, I was able to obtain the sKvAP expression vector from Roderick MacKinnon.

Despite four decades of research characterizing the response properties of sensory neurons in primary cortical areas, we still do not have a good picture of how V1 really operates under realistic viewing conditions, i.e., how natural scenes are encoded by cortical activity patterns. We propose to compare the actual subthreshold and spiking activity of V1 neurons in response to natural scenery movies to predictions based on linear estimates of receptive field properties. The aim will be to design a computational model that can predict V1 neural responses to low complexity stimuli as well as natural stimuli. The specific steps of the project are the following: a) Investigate the receptive field (RF) structure of V1 neurons from their intracellular responses to natural image sequences. b) Characterize the influence of context in V1 sensory processing and information transfer. c) Determine, from the analysis of the acquired data, the dominant coding schema (time coding vs. rate coding) in V1 for natural scenery processing. d) Clarify the role of background ongoing cortical activity. e) Study the visual impact of oculomotor strategies in the active viewing of natural scenes. f) Develop and validate a computational model of V1 responses to natural stimuli. In this project, the applicant seeks to acquire advanced skills in experimental and computational neuroscience and further extend his initial training in Electrical Engineering and Computer Science.

Currently, there is very little understanding of the molecular mechanism that control the defined sizes of low copy large organelles such as spindle pole bodies (SPBs) or kinetochores. I will address this question using the budding yeast SPB as a model, because unlike other organelles it is of fairly simple design and its composition, structure and assembly properties are relatively well known. The available data suggest that the early assembling components like Spc42p play a role in setting the SPB size. I propose here to identify the factors that control SPB size genetically using GFP-tagged Spc42p strains and screening for ts-mutants with altered Spc42p-GFP signal intensities. In parallel, I will undertake affinity chromatography using Spc42p as well as other SPB core components as baits to identify novel factors involved in SPB size determination. Their impact on SPB size will be checked using in vivo methods and EM. Next, I will determine which of the identified factors bind to each other directly using in vitro methods like “pull-down” experiments. Then, I will try to reconstitute SPB size control in vitro. I will examine whether Spc42p is able to self-assemble using gel-filtrations, light scattering assays and EM studies and will extend this approach by including Spc42p interactors and other factors involved in SPB size control. To better understand the underlying molecular interactions I will try to determine the three-dimensional structure of the key components using X-ray crystallography. While being trained in in vitro methodology I will expand my expertise to performing a multi-disciplinary approach what will serve me greatly in my professional future.