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.

Over the last few years there has been an enormous development in biological research and the birth of the fields of genomics and proteomics. One common requirement of these disciplines is the need for sensors that allow identification of specific DNA sequences or protein interactions. This proposal is focused on the development of new fluorescent tools and sensors for studying biological processes. First, using rational and combinatorial methods, new fluorescent GFP-like mini-proteins will be designed that can be incorporated at the DNA level into any recombinant protein. Second, fluorescent switchable sequence-specific DNA-binding peptides and protein complexes will be designed for the study of the basic mechanisms of DNA recognition. Finally, their application as probes for specific DNA sequences and gene expression control will be tested. Support from HFSP is fundamental for the development of these projects, since they close a gap between the purely chemical research carried out in our department and new biological-focused research in the frontier between chemistry and biology. The experience accumulated in the last few years in our department in the field of fundamental bioorganic chemistry gives us the opportunity to aim to more ambitious objectives, which can have practical implications in biological research.

DESIGNING A QUANTITATIVE MODEL OF THE CELL I propose to develop a quantitative model that captures main determinants of the molecular biology of the cell and its transitions between different states, for example during cell growth. This will lead to a more detailed understanding of how actions and interactions of proteins are controlled and how they determine the overall state of the cell. For these projects, I will join Prof. Edward Marcotte’s lab since it offers excellent facilities for both computational and wet-lab experiments in that field. First, I will explore a specific subset of yeast proteins in detail, e.g. the proteins of the mating pathway. This will reveal the possible dimensions of my analyses. Second, I will design a quantitative model of the cell that revolves around a series of transitions between discrete cell states, where these “states” are defined by gene expression profiles. I will identify the “basis set” of transcriptional patterns that best explain the majority of existing yeast expression data for each cellular state. The basis set will be used in a neural network that predicts expression profiles, e.g. for gene knockouts. It may even be possible to describe the cellular behaviour in a simple hidden Markov model which emits gene expression patterns for particular cellular states. Next, I will use the basis set of expression profiles to extract subnetworks from a probabilistic network of gene functional relationships and examine their properties. Finally, in order to better understand the robustness of these networks, I will analyse how gene duplicates are distributed across the network, e.g. how they conserve their position.

The selective expression of var genes forms the basis of immune escape and pathogenesis of the agent of severe malaria, Plasmodium falciparum. In order to study epigenetic mechanisms involved in the regulation of var gene expression, yeast orthologues involved in nucleosomal promoter repression will be disrupted in the parasite. Altered var gene expression will be assayed by measuring mRNA and protein levels and performing functional studies. Complementing this approach, two var gene promoters will be analysed. Wild type and mutated var gene promoters upstream from a resistance marker will be stably integrated into wild type and knockout parasites established in the first part. By enforcing expression through drug selection, elements involved in promoter activation or repression will be determined under different chromatin constraints. To identify cis-element binding factors, a pull-down assay of an episomally maintained plasmid, containing a var gene promoter positioned upstream of a resistance marker, will be performed. Bound proteins will be separated by denaturing gel-electrophoresis and subsequently analysed by mass-spectrometry. My previous research activities focussed on mosquito vector innate immune responses towards Plasmodium. The fellowship would enable me to extend my expertise towards the understanding of P. falciparum survival strategies in the human host. The profound training in parasite and vector biology would be optimal for my future scientific career in the field, enabling me to take novel and broad research paths to understand parasite development and transmission.

The brain detects noxious stimulation of the skin, viscera and other internal structures via different subsets of primary nociceptive sensory neurons. These subsets possibly engage distinct circuitry all the way up to the brain, explaining the ability to distinguish the bodily sites at which noxious stimuli are detected. The objective of this proposal is to get new molecular markers for such subsets and especially for viscera nociceptors that project to the gut and other internal organs and that form the basis of interoception, the brain’s perception of the body’s internal states. The ultimate goal is to use them, in conjunction with already identified markers for nociceptors innervating other targets, to compare their sensory maps, so as to illuminate the logic of brain decoding in the understudied visceral pain. In order to do that we will dissect the function, by gene targeting, of MrgB4/B5 members of the GPCR family of Mrgs, which is believed to be involved in nociception. Since the MrgD member is specifically expressed in cutaneous afferents, it is implied that the other Mrgs might be characteristic for nociceptor subsets innervating different organs, including viscera. Additionally, we will conduct a screen that will be based on the comparison of the gene expression profile of single cells from cutaneous nociceptors (marked by MrgD) and nociceptor afferents enriched by other markers for visceral targets. The outlined research will introduce me to the problems, methods and logic of the new field of neurobiology, giving me the chance to devise novel and creative ways, in which I can bring my previous training in mouse molecular genetics to bear on solving these problems.

A fundamental question in vertebrate development is how regional identity is specified in the developing embryo. For instance, the developing hindbrain is transiently subdivided into seven contiguous segments, termed rhombomeres, a highly conserved process in vertebrate development. The regional domains defined by the boundaries of each rhombomere form the scaffold upon which the subsequent specialization of neural function in the brain is built. Zebrafish provide a powerful model to genetically dissect the molecular mechanisms involved in this process. Each rhombomere has a unique identity based on segment-specific expression of Hox genes Hox gene function is crucial for rhombomeric identity. Recent work in Drosophila has shown that the zinc-finger proteins Teashirt, Disco, and Disco-related behave genetically as Hox cofactors. We have cloned zebrafish homologs of Teashirt, and have found that some of these are expressed in Hox-like patterns during zebrafish development, as would be predicted of a Hox cofactor. I propose to determine whether Teashirt genes are required for Hox gene function in the developing hindbrain of zebrafish. I will identify Tsh mutants in zebrafish and study their effects on known Hox-dependent processes. Second, I will investigate whether these Teashirt proteins interact physically with Hox proteins or other components of the Hox DNA binding complex. Lastly, I will clone zebrafish disco homologs and study their expression and function. Ultimately, this research will help our understanding of how neural patterning is established and maintained in vertebrates, an important first step in understanding higher neural function.