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.

It has been demonstrated in some cases that not all cancer cells are the same. In some cancers, only a small proportion of cells is primarily responsible for the growth of the cancer. These tumorigenic cells have become known as ‘cancer stem cells’, are distinguishable by cell surface marker expression and able to give rise to other cancer stem cells as well as non-tumorigenic cells within the cancer. The existence of cancer stem cells has profound implications for the way that research into the causes and treatment of cancer is conducted, as long term control over this disease will likely require their specific targeting. My research proposal aims primarily to determine whether cancer stem cells exist in human melanoma. In order to do this, I will take fresh specimens removed from patients with melanoma and evaluate them for heterogeneous expression of cell surface markers. Phenotypically defined melanoma cell populations will be purified and evaluated for tumorigenicity by transplantation into immunocompromised mice. If tumorigenic and non-tumorigenic cancer cells co-exist, I will also test whether the properties of tumorigenic melanoma cells change during disease progression. In addition, I will also perform gene-expression profiling on tumorigenic and non-tumorigenic melanoma cell subpopulations to identify genes that correlate with tumorigenicity. Together, these approaches have the potential to reveal important new insights into the biology of melanoma that could enhance the management of this disease.

Mutations in Pkd1 and Pkd2 genes, encoding polycystin-1 (PC-1, a membrane receptor) and polycystin-2 (PC2, an ion channel) proteins respectively, are implicated in the development of skeletal diseases. The PC1 and PC2 complex is believed to form a mechanosensitive receptor-ion channel complex that is involved in flow detection in kidney epithelial cells and is essential to normal kidney physiology. Although recent evidence suggests the presence of PC1 and PC2 in osteocytes (the mechaonsensor in bone) may be important for their physiology, their role in the mechanosensitivity of these cells remains unknown. To fully address the functional role of the ciliary PCs in the mechanosensitivity of osteocyes, our research plan consists of three main steps, divided as follows: 1). Mechanosensitivity of the primary cilium in OB/OC cells. 2). Role of PCs in the mechanosensitivity of OB/OC cells. 3) Link between PC-stimulation and cellular activation of OB/OC cells. We believe these approaches will not only lead to a better understanding of the molecular mechanisms underlying the mechanosensitivity of bone cells, but will also shed light on mechanisms involved in the pathogenesis of skeletal diseases.

Cell to cell variability in gene expression and protein levels has recently become a subject of intense study. However, it is still unclear whether such variability has any bearing on cell fate in mammalian cells. This question is especially relevant to cell death, which has a central role in tumor suppression, the immune response, and host-pathogen interactions. Is cell death a deterministic process, in which genetically identical cells in a homogenous environment behave the same? Or is there an element of chance, where cells under the same conditions behave differently? To try to answer this question, I propose to challenge macrophage cells with diverse pro-apoptotic factors, alone and in combination with TNF, a pro-survival factor in macrophages. I will also examine variability in cell death after infection with macrophage tropic HIV. I will use time-lapse microscopy to determine whether the probability of cell death during a given time interval is the same or different between cells, and whether the extent of variability depends on the pathway involved. If significant variability in cell fate does exist, I will investigate how different cell fates are correlated to different temporal activation dynamics of the NF-kappaB pathway, a central pro-survival pathway in many cell types. Understanding variability in cell death may lead to better understanding of processes where cell death plays a key role, such as HIV infection, cancer therapy, and the immune response. This systems biology based understanding of immune cell physiology will be a new direction for me, but my background in systems biology and live cell imaging gives me the tools to carry out this project.

While considerable research has examined the neurophysiology of a single cognitive operation, much less is known on how we chain these basic operations into complex tasks. Understanding this important aspect of neuroscience is a main goal in my career and the research proposal which accompanies my CDA application constitutes a major step in this direction. I propose an empirical and theoretical investigation to understand how a cognitive task can be parsed in independent processing stages, and the factors that determine whether these stages can be carried out in parallel (simultaneously with other concurrent processes) or rather establish a serial processing bottleneck. A particular strength of this proposal is that it is guided by a theoretical framework -developed during the course of my HFSP postdoctoral fellowship- which guides neurophysiological experiments establishing very concrete predictions. I expect these investigations to feed back to the models which at this stage are simple mathematical schemes and remain to be determined at the neurophysiological level. Thus, the two principal goals of my CDA are 1) to parse, using human imaging and single-cell recordings in monkeys, the stream of activations of a cognitive task in discrete states, understand the temporal ordering of these states and their rearrangement when the main task is carried out with a concurrent interfering task and 2) to develop a biophysical (biologically plausible) model capable of explaining the key observations of different behavioral interference experiments, as a first step to develop realistic models of complex task execution.

Human embryonic stem cells (hESCs) offer a unique opportunity for studying early human development and at the same time, form an exciting potential source of cells for regenerative medicine. Central to both goals is to map out the hierarchy of emerging precursors, and understand the underlying mechanisms of lineage specification and the regulation of large-scale organization across an extended embryonic tissue. Accordingly, my goal is to study the spatio-temporal regulation of early human morphogenesis using models of hESC differentiation. My approach consists of 3 interconnected research directions: (i) Multi-layer analysis of the kinetics of differentiation in response to morphogenetic signals, (ii) identification and isolation of early hESC-derived precursors followed by analysis of their differentiation program, their developmental potential, and the interactions between different precursors, and (iii) Studying the large-scale organization of morphogenesis using 3d culture models allowing juxtaposition of distinct, hESC-derived precursors and their exposure to gradients of morphogenetic signals. Preliminary kinetic profiles of mRNA, miRNA, and ribosome density are already beginning to reveal interesting insights about the early developmental program of differentiating hESCs. A large screen of cell surface markers in collaboration with the Weissman lab (Stanford University) further identified distinct precursors that are either present in the undifferentiated culture or emerge during early stages of differentiation. Altogether these studies should lead to a significant progress in defining and characterizing unmarked, therapeutically relevant, hESC-derived cells.

The control of translation initiation is an important mechanism for the regulation of gene expression in eukaryotes, and is known to be involved in cell growth, cancer, and stress responses such as starvation, hypoxia, ER-stress, and viral infection. At least 12 essential initiation factors (eIFs) play central roles in facilitating initiation as well as in translational regulation. Although distinct functions of each eIF are so far been characterized genetically and biochemically, it remains unclear how they interact with one another on ribosome to exert the integrated functions. As most eIFs form intermediate complexes with ribosome, initiator Met-tRNAi, or mRNA at specific stage of initiation, it is important to analyze the structures of the initiation intermediates to understand basis of functional association of eIFs. The purpose of the research proposed here is to investigate the initiation muti-factor complex assembly and its relation with individual functions. The study is also focused on structure and function of the largest and yet most uncharacterized initiation factor eIF3 consisting of 13 different subunits. The proposal will potentially shed new light into the comprehensive understanding of initiation factor-driven initiation and its regulation. The focus of my PhD work was to reveal the structure of archaeal homologs of translational apparatuses by X-ray crystallography. Studying the eukaryotic initiation complex extends my scope into the larger and complicated system as well as expanding my repertoire of techniques to preparation of multi-subunit factors from eukaryotic cells, and structural analysis with cryo-electron microscopy reconstitution.

Ubiquitination is the posttranslational modification of selected proteins by the covalent attachment of one or more ubiquitin molecules. The biological importance of protein ubiquitination is reflected in the fact that disruption of the conjugation pathway leads to developmental aberrations, abnormal responses to stimuli and defects in cell division and growth. At the center of the conjugation cascade is the substrate recruiting E3 ligase, which largely governs specificity. Although the role of E3 ligases in selectively controlling protein abundance, localization and activity is well studied, many aspects of E3 ligase regulation needs to be elucidated. The aim of this proposal is to understand the mechanism of E3 ligase activity regulation in response to external stimuli via analysis of KEG, a novel Arabidopsis RING E3. KEG is active as a E3 ligase and kinase, capable of post-translationally modifying one or more proteins, including itself, by ubiquitination and/or phosphorylation. The precise roles that these modifications play in the regulation of KEG function will be investigated. KEG is a negative regulator of ABA signaling, a major phytohormone which plays a key role in plant development and survival under stressful conditions. A combination of in vivo and in vitro approaches will be used to define the mechanism by which ABA signaling modulates KEG E3 ligase and/or kinase activity to control protein ubiquitination and subsequent degradation by the 26S proteasome.

In some non-human primate species, such as the pig-tailed (PT) macaque, infection by simian immunodeficiency virus (SIV) is pathogenic, resulting in high viral loads, chronic T cell activation, inflammation and destruction of hematolymphoid microenvironments. These manifestations of immunopathology are not observed in SIV-infected African green monkeys (AGM), even in the face of high plasma viral loads. A consistently observed phenotype distinguishing these two outcomes is the absence of an antibody response to the p27 Gag capsid protein in the setting of non-pathogenic infection. Low or undetectable anti-p27 Gag antibody responses in SIV-infected AGM suggests a state of relative tolerance against p27 Gag epitopes. My proposal aims to determine how such selective tolerance is induced, with the hypothesis that the absence of anti-p27 Gag antibodies in AGM is due to the expression of endogenous retroviral sequences encoding a homologous Gag transcript. I will screen tissues from AGM and PT macaques by RT-PCR, in situ hybridisation and microarrays for expression of Gag transcripts, and identify the cell subpopulations in which they reside. I will also determine the frequency and functional capacity of p27 Gag-specific T regulatory cells in AGM and PT macaques, before and after infection with SIV. Experiments to induce tolerance by expressing Gag epitopes in PT macaques will then address the causal nature of antibody responses to p27 Gag in SIV disease pathogenesis. These studies may promote future consideration of HIV vaccines that selectively suppress the induction of anti-p27 Gag antibodies and will help to elucidate the mechanisms resulting in nonpathogenic SIV infection.

My aim is to contribute to our understanding of how perception is achieved. In the past I worked on the electrophysiology of rat tactile perception. Now I would like to approach the problem of perception from a new perspective by studying reading capacity in humans and learn a new technique, fMRI. Writing was invented only ~5400 years ago. Our ability to recognize words might thus result from the recycling of already existing systems for object recognition. Areas of ventral occipitotemporal cortex activated by objects (faces, tools...) lie next to the Visual Word Form Area (VWFA), an area activated preferentially by written words. I plan to investigate the universal features that have predisposed the VWFA to become specialized in word recognition. Letters are composed of elements analogous to viewpoint-invariant object junctions present in natural environments (e.g. T, L, X). We propose that the area that becomes the VWFA has an intrinsic predisposition for such junctions relatively to other object features. To test this hypothesis I will measure fMRI activations for objects presented either as photographs or drawings with lines and junctions selectively removed. To determine what function does the VWFA serve before one learns to read I will extend my experiments to special subject populations: adult illiterates entering literacy class and children entering school. In parallel, I will use the unique opportunity offered by patients with drug-resistant epilepsies to carry out single-neuron human recordings which will probe the neural code underlying word recognition. These experiments should provide an important insight into the distinctly human gift of written language.

Protein misfolding has been implicated in the pathogenesis of many neurodegenerative disorders such as Alzheimer’s disease (AD), Parkinson’s disease (PD), and prion diseases. Despite distinctive symptoms, they are all characterized by deposition of amyloid fibers in or around the affected neurons. Amyloid deposits are often present in many cell types, yet they affect only a subset of cells, specific to the disease. Amyloid formation is therefore not toxic to all cells, and conversely, not all amyloids are toxic. Clearly, there must be both intrinsic and extrinsic factors that render a misfolded protein toxic to a given cell type. I propose to study the mechanism of amyloid toxicity in budding yeast by combining chemical biology and genomics in a high-throughput manner. I will systematically screen for yeast genes that suppress or enhance the toxicity of Rnq1, a yeast prion. As a complementary approach, I will use a novel method, SICLOPPS, to select for cyclic peptides that suppress Rnq1 toxicity. The two-pronged approach will elucidate both genes and protein domains mediating amyloid toxicity, and also facilitate cross-platform verification of hits. The positive hits will be further studied in vivo and in vitro with methods pioneered in the host laboratory. The results should increase our understanding of cellular factors contributing to amyloid toxicity and provide new tools to study amyloid biology.