Genetic response induced by physical cues is critical in cell-cell communication, differentiation, and development, however, the mechanisms by which cells initiate changes in gene expression due to mechanical signals remain elusive. The goal of the proposed research is to investigate the role of physical stimuli on gene expression within a population of single cells and to determine the contributions of specific components of cellular architecture to force transduction. To achieve this, I will develop a novel drop-based microfluidic device merged with cell biology techniques. Individual cells will be encapsulated in an extracellular matrix gel in a drop and flowed through constricted channels to induce physical stress. Gene expression levels will be measured for single cells and information on variations in gene expression within the population of cells will be obtained. This research is critical for an integrated understanding of how individual cells and populations of cells adapt and evolve in response to physical stimuli. My training in the physical sciences and previous exposure to cell culture will help me to pursue this project, yet the topic of my proposed research clearly differs from my background in biomembrane physics. This fellowship will enable me to develop my understanding of the dynamic nature of cellular architecture and gene expression as well as the biological tools that I can integrate into my research. Moreover, I will develop a unique microfluidic skill set that I will use in the future as a research group leader to bring new approaches to understanding the function of Nature’s design.

Transport of proteins and RNA through the nuclear membrane is achieved through the nuclear pore complex (NPC). NUP98, an NPC component, forms fusions with several genes as a consequence of cancer-associated chromosomal translocations. NUP98 fusion proteins (N98FPs) are implicated in the pathogenesis of various aggressive hematopoietic malignancies. The mechanisms by which N98FPs exert leukemogenic activities are elusive, however it is known that NUP98 acts predominantly in RNA export. Defective nucleocytoplasmic trafficking has been described in many cancer cells suggesting that a disrupted NPC plays a role in tumorigenesis. I hypothesize that displacement of Nup98 as consequence of gene fusion disrupts NPC function and leads to aberrant RNA export. Also, the 11p15.5 locus which contains NUP98, is frequently deleted suggesting that a tumor suppressor in this region is causative to the development of many tumor types. The impact of NUP98 loss on tumorigenesis has not been studied previously. To address the consequences its loss and identify a bona fide tumor suppressive role for NUP98, I have proposed both in vitro and in vivo experiments. Understanding how N98FPs affect the NPC will explain their leukemogenic potential and facilitate the characterization of NUP98 function. This project is a significant departure from my previous research in characterizing the role of apoptotic molecules in T-cell immunity. This experience will permit me to gain knowledge, skills and expertise to foster my development into a well-rounded research scientist. I believe that I can achieve these goals with the help of this fellowship, as it will support me during my studies outside of Canada.

The repair of damaged DNA is a process essential to life. Disruption of DNA repair pathways may alter genetic output and lead to cancer or cell death. Many molecular factors required for the cellular response to DNA damage have been identified and sophisticated mechanisms of repair have been defined. Lacking in our knowledge is a clear understanding as to how DNA damage response factors function in the context of the native genetic template, chromatin. A highly compact and generally inhibitory DNA-protein complex, chromatin represents a formidable barrier to all DNA-templated activities. Discerning how cells "mark" and access sites of DNA damage in a chromatin environment is essential to our understanding of DNA damage response. The ambition of my research program is to elucidate the mechanisms by which chromatin is regulated during the response to DNA damage. An essential component of this program is the integration of novel methodologies into my research plan. Specifically I wish to develop a combined biochemical and mass spectrometry approach to identify and characterize chromatin modifications that mark sites of DNA damage. The specific aim of this Career Development Award is to support the development of this cross-disciplinary approach. Application of the Career Development Award to my aim will broaden my investigative potential by laying the foundation to a new path of discovery.

Telomerase is an unusual reverse transcriptase consisting of a protein catalytic subunit (TERT) and a tightly associated RNA subunit (TR). TR encodes the template for the telomeric DNA repeat which is copied repetitively onto the 3’ end of chromosomes by TERT. The absence of telomerase activity limits the replicative potential of normal human somatic cells, whereas its reactivation is an essential step in over 90% of tumors during carcinogenesis. Given that no structural information on telomerase is available I propose to determine the structure of the human TERT/TR complex (hTERT/hTR) by X-ray crystallography. Since the production and the three dimensional analysis of large ribonucleoprotein complexes has proved to be challenging in the past, I will engage in two concurrent approaches: On one hand I will aim to develop a protocol to express larger amounts of full length hTERT/hTR complex and on the other hand produce functional hTERT/hTR subcomplexes. Reconstituted complexes will be analyzed structurally and biochemically. These insights will then be used to generate models of telomerase function which will be tested in vitro and in vivo. This fellowship will enrich my research experience greatly. It will allow me to leverage my strong background in biochemistry and gain insights into biophysics, which is a new discipline for me. Furthermore it will allow me to work with eukarya, a more complex model system than archaea, which were the focus of my prior research. This exposure will broaden my scientific perspective and enable me to answer complex structural and biological questions as an independent investigator.

The neurophysiological investigation of the hippocampus offers a compelling opportunity to unravel the neural mechanisms underlying memory and learning. In rats, hippocampal neurons and neurons projecting from the medial entorhinal cortex (MEC) have been long known to fire in correlation with the animal’s specific location in the environment, hence their name “place cells”. The recently discovered “grid cells” have an even more striking spatial response: the multiple firing locations of the same cell are the vertices of a grid regularly tessellating the environment. In this project we will first investigate whether the place cells observed in MEC are actually grid cells operating at a coarse spatial scale, as suggested by the grids’ topographical organization. Second, we will test current hypotheses concerning the interaction and roles of self-motion estimation and external cues perception in the construction of the grid representation. For these two aims, we will conduct specifically designed experiments based on the simultaneous recording of populations of well-isolated neurons in behaving animals. Third, we will interpret the experimental data in anatomically and physiologically realistic computational simulations of the hippocampus’ activity, in order to explain how the information encoded in input is processed and transformed into the place-cell representation in output. By this research plan, the fellowship would greatly help me through the dramatic change of research field from theoretical artificial intelligence to experimental neurobiology, motivated by my personal interest in cognitive neuroscience that arose during my previous research experiences.

Despite rapid progress in our understanding of how changes in cell morphology drive neuronal development, surprisingly little is known about how changes in cell shape underlie the remarkable plasticity of the adult brain. Tantalizing clues are found in human congenital mental retardation syndromes, many of which involve loss-of-function in genes regulating the actin cytoskeleton. Drosophila long-term memory formation is an ideal system with which to shed more light on this observation. The fly brain, like that of humans, exhibits distinct, interdependent memory phases, which may be assayed simply and rapidly. It is also now possible to manipulate gene expression in Drosophila with precision in both time and space. I aim to test how actin-mediated changes in cell morphology regulate long-term memory formation, using a gene silencing approach to knock down a large number of cytoskeletal regulatory proteins, specifically in the memory centres of the adult Drosophila brain. I will also use single-cell labelling techniques to characterise these morphological changes directly. This project will take full advantage of my previous experiences, studying the molecular interactions that stimulate actin polymerisation in mammalian cells, but will extend my scope dramatically, allowing me to apply this knowledge to a complex, biological system within an intact organism. In doing so, I hope to discover how signal transduction networks that regulate cell shape control neural plasticity, while at the same time uncovering processes that may represent a physical basis for the storage of long-term memory within the brain.

Long-lasting changes in synaptic strength (“long-term plasticity”), which underlie higher cognitive functions such as learning and memory, require ongoing protein synthesis. The local translation of pre-existing mRNAs that are targeted to synaptic sites on dendrites contributes in an important way to certain forms of long-term plasticity. However, little is known about the specific mRNAs that are translated at synapses during plasticity, and the molecular mechanisms that are involved in the stimulus-induced translational control of these mRNAs are largely unknown. We recently discovered that a member of the microRNA family of small non-coding RNAs, miR-134, is involved both in the repression of the dendritic Limk1 mRNA prior to stimulation and in brain-derived neurotrophic factor (BDNF)-induced stimulation of local Limk1 translation. MiR-134 mediated repression of Limk1 translation restricts the growth of dendritic spines, sites of excitatory synaptic transmission, implicating miR-134 in the control of synaptic function. I now propose to study the role of miR-134 in synaptic plasticity in vivo using conditional inactivation of the miR-134 gene in mice. Phenotypic characterization of the miR-134 mutant mice will involve cell biological, electrophysiological and behavioral assays. I further plan to perform a genome-wide screen for the identification of additional microRNAs that are enriched at synapses and function during synaptic development. Together, the proposed research promises to provide a better understanding of microRNA-dependent translational control mechanisms in synaptic development and their role in higher cognitive function.

Canonically, material internalized by endocytosis traffics through the endocytic pathway on route to the lysosome. Sorting of cargo is dependent on the formation of intralumenal vesicles that are characteristic of all endosomes. These vesicles form by inward invagination from the limiting membrane of the late endosome. Although much has been learned regarding the machinery responsible for the sorting of cargo into these intralumenal vesicles, the proximal factors regulating multivesicular membrane dynamics remain ill defined. For example, these intralumenal membranes can form internal vesicles, but also onion-like sheets of membranes (multilamellar structures), and essentially nothing is known about the biochemical and biophysical parameters that control the organization and dynamics of these membranes. I propose to investigate the contribution of the ionic composition of the late endosome in controlling multivesicular structure formation and dynamics. Specifically, I will look at the effects of the known regulators of invagination, Alix and LBPA, have on ion flux and how changes in lumenal ion composition affect lipid dynamics. In contrast to my previous research efforts that were focused on specific aspects of leukocyte biology on a cellular scale, this project will elucidate molecular mechanisms of intralumenal body formation, a process fundamental to cellular function. I will be investigating the biophysical control of lipid dynamics using techniques novel to me. Together, these experiments will provide for a greater understanding of the regulation of multivesicular structure formation, and better equip me to direct my own research program in the future.

Summary: cysteine string protein (CSP) and its associated chaperones (e.g. Hsp90 and Hsc70) likely maintain protein conformation in the neurons. Misfolding can lead to protein aggregation, cytotoxicity and neurodegeneration, as in Alzheimer's or Parkinson's diseases. Interestingly, in the absence of CSP, as shown by fruit fly and mouse experiments, accelerated neurodegeneration leads to neuromuscular symptoms and early death, reminiscent of multiple human disorders. This suggests that CSP may have an essential role in suppressing pathogenic misfolding. I will dissect the role of CSP and its complexes in the inhibition of protein aggregation and neuronal death with in vitro and animal experiments. Since chaperones inhibit aggregation at an early pre-pathological stage, this study may reveal a preventative approach to neurodegeneration. Departure from previous research: My motivation for joining the host lab is to learn the collection of methods that would extend my skills toward neuronal study in mammals. Transition to this field would include, a) adapting familiar areas of biochemistry and cell biology to the neuronal paradigm, and importantly b) learning the novel techniques of transgenic mouse generation, histopathology, neuronal culture and electrophysiology. How the fellowship will serve my professional goals: My aspirations in the study of neuronal function depend on learning to generate, breed and investigate animal-models. The proposed project is an opportunity to extend my molecular cell biology background toward in-depth neuronal studies in knock-out and transgenic mice. This will broaden my scope toward interdisciplinary investigation of molecular phenomena.

Although it is widely accepted that neurons in the neocortex have wide variety of morphological and physiological characteristics, the mechanism underlying diversity of cortical neurons remains largely unknown. The host laboratory has developed a method for transcriptome analysis of specific neuronal type and constructed a database. The objective of this proposal is to reveal mechanisms of transcriptional regulation in maintenance of specific characteristics of cortical neurons. To achieve the goal, I will attempt to identify transcriptional factors (TFs) that are responsible for characteristic neuronal phenotypes. I will screen TFs which are differentially expressed among neuronal types from the database. To examine the roles of TFs in the maintenance of neuronal characters, I will silence or overexpress them and examine morphological and physiological phenotypes in specific type of neurons. And then I will look for downstream genes of TFs by transcriptome analysis and address what genes make actual characteristics. I expect this proposed project will provide first detailed mechanism for neuronal diversity, which are indispensable for understanding cortical microcircuits and analysis of mouse model of brain disorder. Through this project I will learn neurophysiology and genome informatics that I have never experienced in the previous field, molecular and cellular biology. I believe integrative approaches of physiology, molecular biology, and informatics will provide new insights in neuroscience in post-genomic era. This fellowship will give me a chance of acquiring those techniques, which will be very beneficial for establishment of my own research in near future.

(1) My proposed project: Oocyte maturation in Drosophila is supported by nurse cells, which supply the oocyte with nutrients through cytoplasmic bridges. Maternal components are sorted for transport in a temporal-specific manner, but the underlying mechanism is largely unknown. The objective of this project is to elucidate the molecular basis of intercellular transport between nurse cells and the oocyte. To achieve the goal, I will dissect the transport events by using several transport reporters available in my host lab. Imaging of cultured egg chambers, inhibitor experiments, and domain analysis allow us to examine transport dependence on cytoskeletal organization or novel sequence motifs. Moreover, I propose a genetic screen to identify mutants showing transport defects I will mutagenize flies carrying a GFP reporter transgene, and isolate mutants that have abnormal GFP localizations. After I identify responsible genes of those mutants, I will examine their functions and dissect the spatiotemporal regulation of the transport machinery. (2) How it differs from my previous research experience and how this will serve my professional growth: I have been studying planar cell polarity in wing. In the proposed research, I will study intercellular transport in oogenesis by using forward genetics, which is completely new to me. Besides, I will be working with germ-line cells that are substantially different from somatic cells. This training will provide me an opportunity to study new features of cells and signaling pathways. The genetic screen will form the foundation for my future when I establish my own research program and educate students as an independent investigator.

MicroRNAs (miRNAs) are small regulatory non-coding RNAs, which destabilize or inhibit translation of protein-coding target mRNAs. Hundreds of miRNAs and thousands of their regulatory targets have recently been identified or predicted, suggesting that miRNAs are among the most abundant gene regulators in animals. High evolutionary conservation and tissue-specific expression of miRNAs suggest their importance, but their mode of action and their precise role in development remain unclear. Little is known about the temporal and spatial relationship between miRNAs and their targets, i.e. whether they are preferentially co-expressed or anti-correlated. Further, the regulation of miRNA transcription and effects of perturbed miRNA expression are largely unknown. To address these questions, I will study zebrafish brain, since zebrafish provides many advantages to study vertebrate development and 1/3 of all identified fish miRNAs are expressed in brain. I plan to perform genome-wide expression profiles, double in situ analyses, and analyses of miRNA reporters that I will generate. Using transgenic fish and transplantation experiments, I plan to study miRNA gain- and loss-of-function situations. I aim to place brain miRNAs downstream of known signaling pathways by analyzing miRNA expression in zebrafish mutants with brain defects. For my postdoc, I want to redirect my research to more global thinking about regulatory networks, where many genes are regulated or influenced by miRNAs. I will become familiar with numerous miRNA-related techniques, which are new for me. I also will have the opportunity to study vertebrate brain development, an area that I have not yet been exposed to.

Learning and memory require plastic neural circuits. However, stability is required to preserve these induced changes. Therefore, the brain must tightly regulate plasticity and stability of its circuitry, lest a constant barrage of new, irrelevant experiences dilute old memories or unalterable networks ignore new, salient experiences. Neuromodulators are a likely mechanism for regulating plasticity. And two key ones are acetylcholine and noradrenaline which, according to a recently proposed theory, may work in concert and encode expected and unexpected uncertainty to guide behavior. A cohesive picture of how neuromodulators effect neural circuits has remained elusive. We propose to use newly developed tools and techniques to determine how neuromodulators affect synaptic, cellular, and network physiology in vivo with stimulated release of these neuromodulators. We will make extensive use of 2-photon in vivo imaging to monitor changes in dendritic spines, calcium transients in single cells, and activity-associated calcium signals in populations of neurons in this study. We will patch neurons in vivo to examine effects on synaptic transmission and intrinsic properties and use a specially developed electrode array to control local circuit activity. By examining this problem on different levels in an intact preparation with quantitative tools, we hope to illuminate the synaptic, cellular and network mechanisms which underlie the effects of neuromodulators. The training this proposal represents will complement my previous work and help prepare me to run a multidisciplinary neuroscience research lab that explores neural plasticity using a wide range of in vivo and in vitro tools.

Neural stem cells are self-renewing multipotent progenitors which can be used to replace neurons in degenerated or diseased nervous systems. However, many questions must be answered before neuronal replacement therapies using endogenous precursors become a reality. In particular, how the stem cell integrates the multiple signals from the environment that are responsible for its division, differentiation and survival is still unknown. The existence of the primary cilium as a common feature of neural stem cells throughout life, and its location at the interface between the cytoplasm and the cerebral ventricle makes it a key organelle for the reception of such signals. Recent findings reveal that it is an antenna displaying specific receptors and relaying signals from these receptors to the cell body. Our goal is to understand the putative functions of neural stem cell primary cilium during neurogenesis. We will consider the following questions: First, is the primary cilium motile in the ventricle fluid? If it is, what is its function? Then, how is it related to cell proliferation or cell death? Finally, can it modulate cell specification and differentiation, and if it does, how? The Career Development Award will allow me to develop my primary independent research direction. This study should yield new insights into the cell biology of neurogenesis and contribute to the development of new repair therapies.

Although our knowledge about genomes and the functional elements they encode has grown considerably in the past years, we are still lacking a comprehensive picture how these elements are regulated in coordination during development. Here, I propose a global study of tissue specific regulation of gene expression in Drosophila. I plan to identify regulatory motifs by comparative genomics and combine them with global patterns from large-scale expression datasets across diverse tissues. I will combine pre- and post-transcriptional regulation at the level of transcription, alternative splicing, and translational control. I expect to find a coupling of these processes that might improve the predictions of each individual process. I aim to assign trans-acting factors and to place most genes into the four dimensional regulatory network of developmental gene expression. Using such a comprehensive approach, I hope to uncover fundamental principles how these networks specify developmental programs in the fly, and how these principles carry over to vertebrate models. This plan is significantly different from my previous work on protein structures and microRNA target prediction. I will use new methods for the annotation of eukaryotic genomes, comparative genomics, prediction of transcription factor binding sites, and motif discovery. I will become skilled at handling and using large-scale datasets such as binding and expression data. I plan to study available methods and develop new approaches to describe and analyze regulatory networks. Further, I will get familiar with experimental tools in molecular biology and biochemistry to validate my predictions in the Gelbart lab at Harvard.

Cells seem to leave nothing to chance, including the quality control at all steps of information transfer. RNA maturation involves extensive processing that has a relatively high error rate. Cells have developed an RNA quality control mechanism which identifies improperly processed RNAs and subsequently degrades them. This mechanism ensures that only properly processed RNAs are engaged in protein synthesis. The exosome and Trf4 complex are the central molecular machines in this process. The Trf4 complex recognizes and polyadenylates the aberrant RNAs, whereas the exosome is responsible for their degradation. I propose here to use multidimensional NMR spectroscopy to determine the three-dimensional structure of several protein-RNA complexes that are involved in RNA quality control. The proposed project will reveal the mechanism by which this RNA surveillance complex selects defective RNA for degradation and what structural features it recognizes. It is very likely that new structural features or mode of recognition will be discovered in this project which may have a considerable impact in several areas of biology. The CDA will enable me to establish an independent scientific career in my home country, where financial support for young scientists who wish to come back from abroad is still limited. It will give me the opportunity to conduct research in a very competitive area of structural biology, and allow me to use my interdisciplinary expertise to study the above mentioned subject. Finally, my future career will undoubtedly benefit from the prestige associated with the HFSP CDA.

Jumping is a ubiquitous behavior that involves large accelerations in short times, requiring specializations of the nervous system, muscles, and joints which can only be understood through study of all of these elements. Because both insects and vertebrates use musculo-skeletal structures, the mechanisms that insects have evolved for jumping are likely to be similar to those in vertebrates. To analyze these mechanisms, I propose to study two insects that have different mechanical structures for jumping, the froghopper (Circopis vulnerata), and the locust, (Schistocerca gregaria). To determine the differences and similarities between jumping in these two insects, I will combine an engineering and a neurobiological approach to answer seven questions: 1) how does energy utilization lead to differences in leg design? 2) What control strategies are used by the nervous system? 3) What are the internal muscle forces in each system? 4) How does wind resistance affect each insect’s energy budget? 5) How do muscle properties interact with lever arms? 6) How do muscle properties interact with elastic storage mechanisms? and 7) What are the methods by which these insects ensure that legs extend synchronously?. Preliminary work has yielded initial answers to these questions, demonstrating the feasibility of this project. This fellowship would change my research focus from control of slow musculo-hydrostatic structures to evolutionary design and control of fast musculo-skeletal structures. Engineering skills combined with the skills learned from a laboratory specialized in the neurobiological basis of movement would enable me to study a broad range of neuromechanical systems.