X-chromosome inactivation (XCI) in female mammals is a classic example for epigenetic gene regulation. In mice, early preimplantation embryos and the placenta display imprinted XCI where the paternally inherited X is inactive. At the blastocyst stage, embryos reactivate their paternal X to undergo later random XCI where either the maternal or paternal X gets inactivated. In the proposed experiments I want to study the X-reactivation process, which is currently poorly understood. The noncoding RNA Xist is specifically expressed on the inactive X and crucial in the silencing process. On the active X, Xist is repressed by expression of its antisense partner Tsix. My first aim will be to test, if induced Tsix-expression is sufficient to cause Xist downregulation and X-reactivation at different stages of development. This will show, if X-reactivation is limited to the blastocyst-stage or if it is exclusively under control of Tsix-expression. In a complementary approach, I will use single-cell microarrays to screen for genes specifically linked to the X-reactivation process. This unbiased genome-wide screen will allow me to identify new factors involved in X-reactivation which can be further analyzed by functional assays routinely used in the Lee-lab. My scientific background lies in the fields of germ cell determination, maternal effect genes and neurobiology. In the proposed projects I will extensively widen my scientific horizon by gaining new experiences in chromatin biology, non-coding RNAs and dosage compensation. The new approaches I will learn will aid me significantly in my future scientific career.

Vesicle budding into the endosomal structure gives rise to multi-vesicular bodies (MVB). The protein machinery involved carries out a variety of cellular/pathological processes e.g. receptor down regulation and enveloped virus release. A number of studies led to the discovery that CHMP (charged MVB proteins) are implicated in the final step of membrane fission. However, our understanding of lipid and protein rearrangements involved in the fission process is extremely limited. The CHMP3 structure determined by the host laboratory revealed a flat helical arrangement with a basic surface required for membrane interaction and two homo-oligomerisation interfaces. I now aim to determine the structure of: (i) monomeric, cytosolic, full length CHMP(s) in their proposed closed conformation for understanding the molecular basis for activation and membrane targeting and (ii) heterodimeric CHMP complexes for identification of CHMP-CHMP and CHMP-membrane interaction interfaces. Membrane binding and deformation properties will be addressed on solid supported bilayers of defined composition: (i) I will quantify CHMP-bilayer and lateral CHMP-CHMP interactions on bilayer using surface sensitive label free and fluorescence techniques (ii) mechanistic basis of curvature generation in bilayer e.g. induced phase-separation and/or intercalation of side chains into the bilayer will be addressed by neutron reflectometry. My training in chemical biology and biophysics will be useful for understanding the protein and lipid rearrangements involved in the fission process. At the same time this proposal offers an excellent opportunity for learning structural biology.

The initiation of DNA replication is a complex and highly regulated process, which requires the ordered assembly of proteins at origins of DNA replication. The experiments proposed in this research project aim to dissect and order the processes, which start after assembly of the prereplicative complex (pre-RC) and lead to activation of the origin and thus the establishment of a replication fork. I will employ an in vitro-assay in which the pre-RC is assembled on origin DNA using extracts of Saccharomyces cerevisiae cells arrested in G1, which are additionally supplemented with pre-RC proteins. Using this assay I will study the recruitment of factors acting downstream of the pre-RC starting with the Mcm10 protein. Requirements for this association will be determined both within the pre-RC as well as in Mcm10. Analyzing the recruitment of proteins at different stages (e.g. binding to the Mcm10/ pre-RC assembly) will allow me to order the events that ultimately lead to the formation of a replication fork. In a second approach I will investigate a proposed role of polymerase epsilon in the initiation of DNA replication. To this end I will purify polymerase epsilon associated proteins from the initiation stage. Furthermore I will use the temperature-sensitive degron technique to downregulate polymerase epsilon in cells arrested in G1 and to assay for defects in replication initiation following the release from the arrest. These approaches should significantly increase our knowledge of how eukaryotic replication origins are activated.

This proposal addresses the important issue of how the DNA damage response takes place in the context of chromatin, the physiological substrate for DNA repair and checkpoint signalling machineries in eukaryotic cells. In response to DNA double-strand breaks, the phosphorylation of the histone variant H2AX has been widely documented. The phosphorylated species, referred to as gammaH2AX, specifically binds chromatin modifiers and DNA damage response proteins. However, how these factors work together in a coordinated manner to promote an efficient damage response is still unclear. I propose to pursue two complementary approaches to address this issue in mammalian cells. First, I will investigate the chromatin-associated function of damaged chromatin interactors with a particular focus on the checkpoint mediator MDC1 (Mediator of DNA Damage Checkpoint 1), a well characterised gammaH2AX binding factor. I will search for chromatin modifying activities associating with MDC1 in vivo and of relevance to the DNA damage response. Second, I will analyse the dynamic binding of chromatin modifiers to damaged chromatin, focusing on Poly(ADP-Ribose) Polymerase 1 (PARP1) binding and FACT (Facilitates Chromatin Transcription) release upon H2AX phosphorylation. Through this work, I expect to gain mechanistic insights into how molecular partners of gammaH2AX operate, both in the DNA damage response and on chromatin structure. Not only does this project build on my previous experience in the chromatin field, but it will also allow me to develop novel and complementary skills in the field of the DNA damage response, which will greatly benefit me in my future career as an independent scientist.

The influence of parasites on the evolution of living organisms is a central topic in all disciplines in evolutionary biology since all organisms are at risk from parasitism. Even if some studies provide evidence that parasites are able to alter the host’s phenotype to enhance their fitness, modification of host nutrient intake by parasites has never been evoked as an adaptive strategy to enhance parasites’ growth and survival at least once inside the host. Recent advances in nutritional theory offer an opportunity, for the first time, to quantify the relationship between parasitism, nutritional state and immune responses, and to explore the extent to which hosts can self-medicate through altering their diet choice to fight infection, or instead are manipulated to enhance parasite’s development. To tackle such questions I will investigate the prediction that the host’s nutritional behaviour is expected to change with the nutritional burden provided by the parasite. In so doing, I will monitor and compare the feeding behaviour and the immunity responses of a generalist leaf-feeding caterpillar, Helicoverpa armigera infected by either a macro- and/or a microparasite with regard to its ability to self-select diets containing a protective nutrient balance or a protective toxin. Diets will include differentially radio-labelled compounds to establish the extent to which nutrients ingested by the host are diverted to parasite’s growth. This novel and interdisciplinary approach will have considerable impact on our understanding of the evolution of hosts’ life history traits and important implications in the emerging field of Darwinian medicine.

How acquired memories are stored and retrieved in functional neuronal networks in living animals is a fundamental question that is still largely unresolved. The current proposal offers an approach to gain insight into this question with two complementary strategies. The first is to develop and optimize a learning assay for larval zebrafish in which the animals learn to associate two distinct but intrinsically neutral visual stimulus patterns with danger and safety respectively. This is achieved by pairing the presence of the fish in a visually distinct area of the tank with the delivery of mild electroshocks. The second proposed strategy is to develop an in-vivo calcium imaging assay in those larvae to allow the monitoring of activity in large populations of neurons in response to these specific visual stimuli. The small size of the larval zebrafish brain in combination with the translucence of the preparation permits functional in-vivo imaging of any chosen part of the central nervous system. Initially, the well established methods of single cell electroporation of dextran dyes and the injection of calcium indicators in the form of AM-esters will be used. In addition, the established role of the zebrafish as a genetic model system will also allow targeting of specific populations of neurons for the expression of genetically encoded calcium indicators. The function and reliability of all these calcium indicators is analyzed by simultaneous in vivo patch clamp recordings of the labeled neurons. The general aim of these experiments is to seek and describe specific neuronal populations that respond differentially to correlates of acquired information in the vertebrate brain.

Formation of synapses is a central issue in neurobiological research. Since synapses in the brain are small and difficult to study, many players involved in synaptogenesis were originally identified in neuromuscular junctions (NMJ). These specialized synapses are about a hundred times bigger than those in the brain, with complex topology that can be easily evaluated by microscopy. Importantly, it was demonstrated recently that muscle cells are able to form autonomously complex postsynaptic specialization, without contacting a neuronal cell. During my post-doc, I will rely on my experience from yeast genome-wide visual screening, to set-up a high-through-put system to screen for novel regulators of the postsynaptic apparatus in muscle cells. In this project, I will use shRNA library that combines viral-based gene transfer with RNAi-mediated gene silencing and will allow me to affect expression of selected genes. I will start my investigation with 500 genes whose transcripts localize to the synapses. These genes are mostly expressed from the specialized synaptic nuclei, localized directly underneath the postsynaptic structures and are expected to be enriched in synaptic proteins. The ultimate goal of this project is to identify genes that regulate the formation of the NMJ so that I can extend the investigation to the less accessible synapses of the brain. I hope that this work will eventually be focus of my own laboratory.

Bird song is a stereotyped learned behavior that provides an experimentally tractable system for elucidating the neural mechanisms of motor learning. Models of motor learning require variability for exploration of different motor outputs and the selective reinforcement of those that match the learning target. Consistent with these models, song variability has been observed and appears to be important for song learning. In adult birds, song variability is modulated by social contexts, i.e. song variability is higher when the bird sings alone (undirected song) as compared to when the bird sings to another bird (directed song). Electrophysiological recordings and immediate early gene expression studies show social context dependent variability in the activity of neurons in a specialized pallial-basal ganglia circuit called the anterior forebrain pathway (AFP). Lesions of the output nucleus of the AFP eliminate social context dependent differences in song variability. Although these experiments suggest that the AFP is essential for generating song variation, they do not answer important questions concerning the source and control of neuronal variability. I propose to use a combination of single unit electrophysiological and pharmacological techniques to study the origin of and mechanisms underlying neuronal variability in the AFP. More generally, I hope to gain insights into brain mechanisms of motor learning. This grant will provide me with the opportunity to learn new techniques and explore bird song, a wonderful model for studying the neural basis of learning and memory.

Infectious viruses can enter the cell through several endocytic routes. These routes consist of a complex system of organelles and intracellular trafficking pathways. An integrated and comparative analysis of these routes is currently not available. The host laboratory is examining the role of the druggable human genome (7,000 genes) by RNA interference in five different infectious entry routes. At the moment, we are determining the role of these genes in particular steps in the routes by using quantitative light microscopy, fluorescent ligands, and marker proteins. The research goal is to gain a systems view on different endocytic pathways using infectious virus entries. We will develop data-analysis and graph theoretic methods for discovering which viruses hijack the same endocytic routes and groups of genes whose silencing leads to similar phenotypes. In addition, we will develop methods for integrating the measurement data with protein-protein and genetic interaction networks that are available in databases. We will assign the network nodes labels indicating in which sub-cellular structure they are active and study the features such networks with spatial information. Finally, we will examine in detail the genetic regulatory network of one endocytic pathway by silencing each gene of the pathway and measuring the altered expression profile of all other genes. The proposed research highly differs from my theoretical background in signal processing and complex systems. I will gain knowledge in cellular biology, laboratory work, and new measuring techniques. This combination of skills will be very important for my future scientific career in systems biology.

Soft lithography represents a set of techniques that exploit elastomeric polymers such as poly(dimethylsiloxane) (PDMS) for the fabrication of various structures in the micro-scale. Microcontact printing, one of soft lithography methods, enables the patterning of mammalian cells using PDMS stamps. In this process the stamp is first "inked" with the cells and then pressed onto the surface to be patterned. The soft stamp enables a good contact with the surface and the cells are rapidly transferred from the stamp to the surface. This method allows the control over cell micron-scale spatial organization and can be used as a tool in various fields such as tissue engineering and cell biology research. An alternative material for stamp fabrication that was recently suggested is agarose hydrogel. It was demonstrated that by using agarose stamp, proteins and bacteria can be transferred to a surface while the stamp is providing an ideal "wet" environment. Moreover, it was shown that osteoblasts cells could be transferred onto porous scaffold for tissue engineering applications. The aim of this study is to establish the use of agarose stamps as a generic method for patterning various types of cells. In addition, other biocompatible hydrogels will be examined for this application. This will be followed by using hydrogel stamps to pattern cells inside micro-channels in order to explore the cells interaction with their environment.

The Mediator is a giant multi-subunit complex that is responsible for conveying regulatory signals from control elements to gene promoters in all eukaryotes from yeast to human. The Mediator is required for basal and regulated expression of nearly all Pol II- regulated genes and can therefore be regarded as a general transcription factor. Currently, the lack of detailed structural information on the Mediator prevents a deep understanding of its regulatory functions. The main purpose of this proposal is to determine the 3D structure of Mediator at high resolution. I will build upon an existing protocol in the Kornberg lab for the purification of milligram quantities of Mediator from S. cerevisiae. Suitable preparations of Mediator will be used for high-resolution structural analysis using both 2D and 3D crystallography techniques. Furthermore, I propose to investigate the structural and biochemical nature of the interaction between Mediator and transcriptional activators. A detailed understanding of Mediator structure should provide insights into the mechanisms underlying the transmission of regulatory information to the RNA Pol II and its associated factors. Previously, I characterised a number of fundamental structural properties of the 30nm chromatin fibre using EM. The proposed project will broaden my knowledge of the transcription field and provide training in a number of new techniques such as protein expression, the isolation and purification of large multi-subunit complexes and both 2D and 3D protein crystallography. In combination these techniques will provide powerful tools for future structural and biochemical studies of multi-subunit transcriptional complexes.

The Ski complex is essential in all the functions that are carried out by the cytoplasmic exosome, one of the major 3’-5’ degradation machinery in the cell. The principal aim of my work will be to determine the structure of the Ski complex. It is composed of 3 proteins, one of which containing an RNA helicase domain and two containing predicted protein-protein interaction domains. I also want to study structurally Ski7, an additional component that is likely to bridge the interaction between the Ski complex and the exosome. The structural information will be used to design specific mutations and test hypothesis on how it works in vivo in yeast. Finally, in the long term, it would be interesting to study the association of the Ski complex with Ski7 and the exosome in order to determine how all these different components are positioned and how the Ski complex can help the exosome in degrading mRNA.

Hace1 is a tumor suppressor, E3-ligase that is involved in protein folding and targeting misfolded proteins for degradation. The unfolded protein response (UPR) is proposed to be the link between the involvement of Hace1 in protein folding and cancer as tumor cells suffer from high unfolded protein load. Upon UPR activation, chaperons are up-regulated and misfolded proteins are either refolded or marked for degradation by the ubiquitin-proteosome system. UPR leads to apoptosis of untransformed cells and is growth promoting in transformed cells, hence a paradox. I propose to determine whether the tumor suppressor activity of Hace1 is related to reduction of misfolded protein load and reduced activation of the UPR. Two approaches will be taken to address this question: 1. Unfolded protein load will be induced in transformed and untransformed cells and the role of Hace1 in misfolded protein handling and cell survival will be determined. 2. Unfolded protein loads in live transformed an 'normal' cells will be determined by the diffusion parameters of RFP and the role of Hace1 in handling unfolded proteins in these cells will be studied. In my PhD thesis I studied the micro- and macro-localization of Ras in the cell using biophysical methods. My studies provided me with a strong basis in using a biophysical approach to study protein dynamics in live cells, which will contribute to the development of the novel method needed to determine unfolded protein load in live cells. By studding Hace1 I will have the opportunity to apply my skills to study how cells cope with physiological challenges and get acquainted with new approaches necessary to precede with my academic career.

The extracellular signal-regulated kinase (ERK) pathway regulates neuronal cell growth and differentiation, and contributes to activity-dependent neuronal events, such as synaptic plasticity and long-term potentiation. Downstream of this signaling cascade lies the RSK family of protein kinases, which comprises four evolutionarily conserved isoforms (RSK1-4) that have been genetically linked to neurological disorders in humans. Despite their involvement in the nervous system, very little is known about the neuronal function of the RSK isoforms, their targets, and how they contribute to the cellular processes regulated by ERK signaling. THE MAIN OBJECTIVES OF THIS RESEARCH PROGRAM ARE TO DEFINE THE ROLES OF THE RSK FAMILY IN NEURONS AND TO IDENTIFY NOVEL BRAIN-SPECIFIC SUBSTRATES OF THESE PROTEIN KINASES. In aim 1, we will examine the role of the RSK isoforms in primary neuron growth and differentiation using gain- and loss-of-function approaches. ERK signaling is required for activity-dependent protein synthesis and we have recently found that RSK regulates several components of the translational machinery. We hypothesize that the RSK isoforms are major effectors of neuronal protein synthesis and will test this possibility in Aim 2. In Aim 3, we will use phosphoproteomics tools that we have recently developed to identify RSK substrates in neurons, which will help understand how these kinases work at the cellular level. Together, these approaches will provide fundamental insights concerning the role of the RSK family in the developing nervous system, and will help build my career as an independent researcher.

Despite the importance of biomolecular recognition at the membrane boundary of organelles and cells, our understanding of the folding, function and specific interactions of transmembrane (TM) proteins lags far behind that of their soluble counterparts. For example, integrins are activated via modular hetero-dimerization and homo-oligomerization of their TM helices in a manner still enigmatic. Integrins regulate adhesion-related processes ranging from the immune response to metastasis formation. I will study integrin TM helices and their association. First, I will computationally characterize TM protein crystal structures to identify motifs and common key interactions involved in TM helix association. Representative patterns and TM helix oligomerization states will be identified and simulated using molecular dynamics so as to better understand their structures and dynamical properties in molecular detail. Second, I will computationally design helices that bind specifically to the TM-domain of immune-related integrin drug targets, VLA-4 (beta-1-class) and LFA-1 (beta-2-class). This effort leverages recent advances at Penn involving the design of peptides that bind and modulate the function of TM helices, including of beta-3-class integrins. To explore specificity and affinity, positive- and negative-design schemes will be implemented. Lastly, the DeGrado group is expert in the study of TM proteins, and I will characterize the designed peptides in-vitro and in-vivo, optimizing the design scheme in a feedback loop. The project will yield new quantitative understanding of TM protein specific recognition and energetics, opening a new field in integrin and TM protein research.

Transcriptional gene silencing is brought about by the formation of heterochromatin at specific chromosomal loci. Important chromosomal substructures such as the centromere depend on heterochromatin and play a crucial role in genome stability, cell division and regulation of gene expression. Consequently, uncontrolled formation and maintenance of heterochromatin are closely linked to diseases such as cancer and developmental disorders. It has been quite surprising to find that centromeres are established and maintained by a pathway closely linked to RNA interference (RNAi). In Schizosaccharomyces pombe three major protein complexes have been identified which are RITS - the siRNA carrying effector complex - an RNA dependent RNA polymerase complex (RDRC) as well as protein complexes based on the protein Rik1. My research will focus on the functional and structural characterization of RITS and Rik1. Production of the target complexes will be approached by recombinant heterologuous expression in the Baculo virus expression system with subsequent structural and functional investigation of the complexes aimed at elucidating their role in the process of RNAi mediated transcriptional gene silencing. Expression of protein complexes in eukaryotic cell cultures and challenging functional as well as high resolution structural analysis will favourably add to my previous experience in low resolution structural work. With this fellowship, I will get an excellent opportunity to develop my career towards an independent researcher in the very stimulating and highly interdisciplinary environment of the Cold Spring Harbor Laboratories.

Alzheimer’s disease (AD) is a prominent incurable neurodegenerative disease characterized by deposition of beta-amyloid plaques and neurofibrillary tangles in the brain. Both pathologies result from deregulation or mutations in key proteins essential for maintaining proper brain function. Deregulation of one such protein, the amyloid precursor protein (APP), results in the formation of beta-amyloid plaques. However, little is known about the physiological role of APP, its role in the pathogenesis of AD or how it is regulated. A substantial portion of post-transcriptional gene regulation is controlled by microRNA (miRNA) networks. It is not surprising therefore, that alterations in the expression, sequence or target sites of these small non-coding RNA molecules may be a significant but so far poorly recognized source of human genetic disease. I propose to investigate the impact of miRNAs in AD and how they affect neuronal function paying particular attention to the role of miRNAs in APP regulation. Through collaborative research and the use of techniques such as miRNA profiling, RNA affinity purification and manipulation of miRNA levels in our vast collection of AD mouse models I hope to reduce behavioral problems and brain pathology in APP transgenic mice and understand the impact of miRNA gene regulation on adult brain function. Where my previous work focused on epigenetic gene regulation and flowering time in plants, this fellowship will enrich my scientific experience, broaden my knowledge, enhance my skills and enable me elucidate the impact of miRNAs in neurodegenerative diseases using mouse models.

How eukaryotic cells adjust the abundance of their organelles according to need is a fundamental, yet largely unanswered biological question. We will address this question using the size control of the endoplasmic reticulum (ER) in yeast as a model. Under certain stress conditions, yeast cells activate the unfolded protein response, which brings about a massive expansion of the ER. Recent work in the host lab has shown that this expansion is followed by the selective sequestration of ER membranes into large autophagosome-like bodies (ALBs). Thus, an unusual form of autophagy, or cellular self-eating, counteracts the stress-induced ER expansion by selectively removing excess ER that is no longer needed. We now aim to elucidate the molecular mechanisms involved in the sequestration of ER membranes into ALBs. Specifically, we will first use electron microscopy to dissect the formation of ALBs into morphologically distinct steps. We will then biochemically reconstitute these steps in vitro. In parallel, we will use yeast genetics to identify the molecular machinery required to generate ALBs. The precise functions of the various parts of this machinery will subsequently be analyzed in the in vitro system. This project promises to shed more light on how cells balance membrane biosynthesis and degradation to maintain overall organelle homeostasis. Complementing my background in membrane trafficking in mammalian cells, I will acquire electron microscopy, genetics and biochemical in vitro reconstitution as new methods and yeast as a new model system. This project will enable me to extend my interest in cellular organization from cell membranes to the level of cell organelles.

Stem cells can generate differentiating and self-renewing daughter cells at the same time. How this balance is regulated is unclear. Currently, in mammals only very little is known about the factors that drive a stem cell to differentiate into a neuron. A whole-genome RNA interference (RNAi) screen in Drosophila melanogaster for genes that show defects in cell fate specification will highlight a set of new genes with functions during this process. It is the intent of the here proposed project to characterize the function of the mammalian orthologs of those proteins in asymmetric cell division and fate specification of neural stem cells in the developing embryonic mouse brain. To study their function, a novel technique for gene transfer into neural stem cells, of a living mouse embryo, the so called in utero electroporation will be used. The fellowship will give me the opportunity to conduct research in the field of neural stem cell biology, which will be different from my previous research experience where I never worked with stem cells. Furthermore, I will get insights in Drosophila genomics and bioinformatics whats also completely new to me, because previously I only worked with mammalian model systems. During the fellowship period I will be exposed, to a variety of ideas and techniques that are unique to the "stem cell world" (both in Drosophila and in Mouse) and this will for sure enhance my professional growth. Furthermore, the RNAi screen will point out a couple of new genes, during the fellowship I will be able to investigate just a subset of them. Therefore the resutlts from this screen are an excellent basis to start as an independent investigator later on.