Gene expression can be regulated through alterations in nuclear architecture providing control of genome function. Mechanical loading induces both nuclear distortion and alteration in gene expression in a variety of cell types. One putative transduction mechanism for this phenomenon involves alterations to nuclear architecture, resulting from the mechanical perturbation to the cell, to induce altered gene expression. Moreover, remodeling of the nucleus occurs during stem cell differentiation, causing alterations in nuclear stiffness and potentially influencing nuclear architecture-mediated mechano-regulation of transcription. These putative mechanotransduction processes have not been studied in detail before and form the basis of the current proposal. Accordingly the aims of this project are to test the following hypotheses:

1. Mechanically-induced nuclear distortion causes alterations to the nuclear architecture and chromosomal territories sufficient to affect gene transcription

2. Stem cell differentiation modulates the process set out in hypothesis 1, via differentiation-induced nuclear remodeling that affects the mechanical properties of the nucleus.

The successful realization of the aims necessitates collaboration between researchers with complementary expertise in bioengineering, biophysics and cell/molecular biology. Accordingly, we plan an ambitious and innovative program of research that benefits from a highly interdisciplinary approach, building on the existing strengths of the collaborators. The studies will utilize model systems involving human mesenchymal stem cells (MSC) and induced pluripotent stem cells (IPSC) in conjunction with state-of-the-art techniques for the mechanical perturbation of cells, functional analysis of nuclear organization and biomechanical analysis. Ultimately a mechanistic model for functional mechanoregulation of nuclear architecture will be developed to incorporate elements of the modeling of the mechanical properties of the nucleus in conjunction with functional genomic analysis.

Members of the dynamin superfamily are mechano-chemical enzymes which oligomerise around lipid templates and use the energy of nucleotide binding and hydrolysis to remodel membranes. The underlying mechanisms of membrane deformation and the molecular basis for the mechano-chemical function are not understood and will be addressed in this proposal. My group will explore structure, function and mechanism of the dynamin-related Mx GTPases which mediate cellular resistance against several RNA viruses. The structure of a full-length Mx member will be solved by X‑ray crystallography to reveal the general architecture of this family. The catalytic mechanism of assembly-stimulated GTP hydrolysis and the subsequent conformational changes essential for the mechanical function will be biochemically and structurally characterised. Cryo-electron microscopy reconstructions will clarify how Mx proteins oligomerise around lipid and viral templates. Furthermore, the molecular basis of antiviral activity will be elucidated by characterising the interaction of Mx proteins with lipids and viral structures. Results obtained for the Mx family will be compared to other membrane-remodelling GTPases to reveal general principles governing oligomerisation and membrane remodelling in this superfamily.

Increasing evidence indicates that small RNAs participate in a vast number of biological processes. This is also of vital importance to the nervous system which relies on complex gene expression regulatory mechanisms during development and adult functions. While several studies have found non-coding RNA to participate in nervous system development and function, a detailed analysis of the identity, abundance and targets of non-coding RNAs during these processes is missing. We will search for new candidate non-coding RNAs that may affect differentiation of embryonic stem cells towards neurons. We will profile small RNAs and search for candidate miRNA targets based on biochemical purification of RISC bound mRNAs. By using special culture conditions for differentiated neurons we will separate dendrites from cell bodies to profile dendrite-localized small RNAs and its possible targets. Our hope is to identify miRNAs or other small RNAs that regulate local protein synthesis at dendrites which is postulated to mediate synaptic tagging in the most persistent forms of synaptic plasticity and memory. Next, we will develop new strategies to measure rapid induction of dendritic translation for studying the role of miRNAs in local derepression of translation upon synaptic stimulation.
As a postdoctoral fellow I hope to learn from multidisciplinary interactions offered in the Friedrich Miescher Institute (FMI). This project will allow me to interact with researchers from different fields such as bioinformatics, neuroscience and cell biology. My bet is that learning to interact with specialists from different fields will enable me to pursue high quality research in the future.

Animal development is characterized by the amplification and differentiation of progenitor cells into tissues of characteristic shape and function. The expression of many differentiation factors is concomitant with- and required for- any series of morphological changes, giving rise to final tissue and organ shape. The mechanisms whereby these factors lead to a stereotyped remodeling of cell- and tissue architecture are largely unknown. The goal of this project is to understand how cells reorganize their content in response to the expression of key developmental transcription factors. Changes in cell shape and behavior must be regulated by remodeling the extensive network of protein-protein interactions underlying both cytoskeleton as well as membrane dynamics. Structural and regulatory proteins must be transported and localized to their site of action following specific routes. This project focuses on how tissue specific gene expression drives specific patterns of protein and membrane trafficking and how this trafficking regulates cell fate and behavior.

Schizophrenia is a devasting mental disease causing psychotic episodes and social and cognitive impairments. Dopamine dependent brain circuits between prefrontal cortex and striatum are known to play a key role but their complexity prevented the disentanglement of their contributions to the disorder. The proposed project is aimed at examining the functional relationship between the elements of these circuits and schizophrenia-associated symptoms. The study will be based on optical control of the excitability of dopaminergic neurons. I will functionally integrate the fast light-responsive microbial opsins halorhodopsins (NpHR) to suppress spiking, and channelrhodopsins (ChR2) to enhance spiking of dopaminergic neurons, thus, making the neurons controllable by light. A combination of transgenic founder selection, viral delivery methods and the usage of the selective impact of fibers travelling between brain areas will be used to restrict the expression of the opsins to populations of dopaminergic neurons in the mice and monkey brain. I will use behavioral studies, optogenetic manipulations, fiber optic approaches, voltametry, and single-unit recordings to investigate the relationship among alterations in the dopaminergic circuits and the elucidation of putative schizophrenia-like endophenotypes. In contrast to existing pharmacological monkey models [1, 2] and knockout mouse models [3, 4] of schizophrenia, the optogenetic models will allow for locally highly restricted circuit manipulations on a millisecond time scale. This promises a detailed understanding of the causal dopamine-dependent factors of the disorder with clinical relevance particularly due to the monkey model.

Calcium-calmodulin-dependent protein kinase II (CaMKII) and the NMDA receptor are both necessary components of the cellular machinery underlying learning and memory. Growing evidence suggests that the interaction between theses two proteins is essential for Long-Term-Potentiation (LTP), a prominent cellular model of memory formation. The goal of this research project is to study the dynamics of the interaction between CaMKII and the NMDA receptor in the context of synaptic plasticity. To address this question, we will use FRET-FLIM imaging in living hippocampal neurons expressing GFP-tagged NMDARs and mCherry-tagged CaMKIIs. We will induce and measure LTP in the spines of theses organotypic slices by the means of theta-burst stimulation, electrophysiology recordings and confocal imaging of a diffusible volume marker. During this summer, I will learn several techniques associated with living slices, as well as doing experiments that will allow us to better understand the molecular basis of memory. The short-term fellowship will help to make this project possible; which will be a great opportunity for my scientific career.

Temperature and nutrient availability are crucial environmental parameters that restrict bacterial growth. To locate the sites of optimal nutrient availability and optimal temperature, bacteria perform chemotaxis (following the gradient of nutrient concentration) and thermotaxis (directing movement according to temperature). In E. coli, the chemotactic response is virtually temperature-independent. In contrast, thermotaxis is explicitly temperature-dependent. Despite these strongly contrasting functions, both chemotaxis and thermotaxis exploit the same signaling network. Our work aims at a quantitative characterization of the thermal properties of this network that enable both robust chemotaxis and thermotaxis over a wide temperature range.

We propose to exploit single-tethered-cell measurements and fluorescence resonance energy transfer (FRET) to elucidate the mechanisms underlying robust chemotaxis of bacteria. To this end, experimental data on the temperature influence on the network is being collected by means of FRET and tethered-cell measurements, that will serve as inputs for our theoretical modeling. Our study shall address the following questions. How does one signaling network achieve both thermotaxis and chemotaxis despite their apparently contrasting requirements? What are the mechanisms, that underlie the robustness of chemotaxis over a broad temperature range? How do multiple sensory stimuli such as attractants/repellents and temperature changes interact?

Genes, the DNA sequences that are copied into RNA to specify proteins, carry the primary genetic information. The long DNA molecules in cells carry many genes and these are interspersed with other DNA sequences that provide a critical second level of information, controlling whether or not each gene is expressed within a specific cell at a specific time. These gene control sequences are recognized and bound by proteins. A gene's expression is controlled not only by protein-DNA complexes near its start (the promoter) but also by the specific pairing of that complex with proteins bound to other control sequences that are often far away on the same DNA molecule (an enhancer), causing the DNA in between to form a loop. These long-range DNA-looping interactions are not well understood and it is a mystery how the promoters of different genes on the DNA link up with the correct enhancers in order to control the genes properly. This integrated multidisciplinary project aims to test the idea that different protein-mediated DNA loops can interfere with each other, thus restricting this web of possible interactions. To do this, we will set up a model system that uses two well-understood proteins, each known to form simple DNA loops. Using different arrangements of the protein binding sites on the DNA, we will use a range of techniques to measure how readily each loop forms in the presence or absence of the other loop. Measurements will be made on DNA inside living bacterial cells by assaying the effect of the loops on gene expression. Outside cells, protein-induced DNA looping will also be measured with instruments that can hold, stretch and twist individual molecules of DNA, and will be visualised with specialised microscopes. Computer simulations that reproduce the movement, bending and twisting of naked DNA and DNA bound by proteins will be used to try to reproduce the experimental results. This combination of approaches will show just how strongly DNA loops interfere with each other and help us understand how.

How cells control the mutually exclusive expression of a single member of multigene families is a fascinating but unresolved question. For example, individual sensory neurons express a single odorant receptor out of a repertoire of about 1300 receptor genes. Similarly, the major variant surface antigen of the human malaria parasite Plasmodium falciparum, PfEMP1 (P. falciparum erythrocytic membrane protein 1), is encoded by the large, polymorphic var gene family whose ca. 60 members are expressed in a mutually exclusive manner. Monoallelic expression of odorant receptors is regulated in parts by a unique enhancer element that activates a single odorant receptor promoter via interchromosomal interactions. This project aims to determine whether a similar mechanism controls singular var gene activation. Specifically, I will use chromosome conformation capture techniques and large-scale sequencing (Solexa) to identify chromosomal elements associating with active var genes and analyse their role in mutually exclusive var gene expression through knock-in and knock-out studies. Furthermore, I aim to develop in vivo DNA pull-down techniques to identify proteins associating with var regulatory elements. These studies should provide new insights into the regulation of expression of a major virulence factor of this important human pathogen.

Intrinsically disordered proteins are most important for the eukaryotic cell, but many aspects of this recently discovered “class” of proteins are yet to be investigated. The main objectives of this proposal is to define a new single-molecule nanoanalytical technology which will enable the efficient detection of non-covalent molecular interactions with microsecond resolution in order to answer fundamental questions about these proteins, for example, what comes first, folding or binding? Importantly, we also aim to build a strong interdisciplinary collaboration based on our diverse backgrounds. This will hopefully lead to further collaborations not only within our team but with other fellow scientists. The fundamental role intrinsically unstructured proteins play in the cell has therefore only recently been appreciated. These proteins are not obscure peculiarities; up to 30% of the different proteins expressed in eukaryotic cells may be partly or completely unstructured in absence of their ligand. A number of reasons have been suggested as to why proteins are intrinsically unstructured which are as follows: 1. It is a way of decoupling affinity and specificity; i.e., a high specificity can be combined with a low affinity. 2. It increases the association rate with the ligand. 3. It allows for increased plasticity with regard to the ligand in the cell. 4. A large interaction surface area is provided in a short amino acid sequence as the protein folds around its ligand. (A short protein requires less energy when it is synthesized.) 5. Rapid turnover in the cell as unfolded proteins are normally short-lived. Numbers one and two are perhaps contradictory, as an increased association rate would lead to higher affinity, and not lower. The contradiction reflects the scarcity of data in the field and highlights the need for more experiments to be performed. What is clear is that intrinsically disordered proteins play key roles in many different cellular events such as transcription.

The function of the mammalian target of rapamycin (mTOR) protein is altered in metabolic diseases and cancer. mTOR coordinates nutrients availability with the complex signalling network necessary for cell growth and cell division. mTOR is activated by the presence of certain growth factors, insulin, high levels of ATP and amino acids. Interestingly, the molecular mediators of amino acid singling, the Rag family of GTPases, have only recently been characterized.Aberrant activation of mTOR by the insulin-dependent branch is known to take place in metabolic disorders as diabetes. However, the relevance of the amino acid-dependent mTOR activation is still not clear in vivo. The project I propose consists in defining the role of Rag GTPases in vivo, by means of using a knock-in (KI) mouse model for RagB GTPase. The endogenous RagB gene will be replaced by an artificial gene encoding a permanently-active mutant protein by substitution of the glutamine in amino acidic position 128 with leucine, under endogenous regulation. The KI will also contain a translation STOP cassette flanked by loxP sites, so we will be able to restrict temporally and spatially the expression of the transgene by means of different Cre recombinase expression systems. We hypothesize that permanent mTOR activation by RagB KI will lead to severe diabetic-like metabolic alterations and that these alterations play an important role in the clinic.My research expertise is based on cancer studies centred on the p53 gene. For my work as a postdoctoral fellow I will turn my research focus to the study of mTORC1 signalling in vivo and its consequences in metabolic diseases as diabetes.

Cytokinesis, the final step of cell division, has two crucial processes to achieve: equal sorting of cellular components between the daughter cells, and membrane ingression

and closure. Both processes strongly rely on membrane components and properties. It is known that some lipids localize to the cleavage site and that membrane transport is required to relieve cortical tension during cytokinesis, necessary for the physical separation of cells. However, the precise role and mechanism of this essential membrane retrieval are unknown, as is the nature of the lipids and proteins involved, in part because it has been difficult to study the role of lipids in membrane organization due to a lack of appropriate tools. The goal of this multi-disciplinary proposal is to understand which lipids participate in cytokinesis, how they are involved in membrane rupture, and how cells regulate their participation. Specifically, we aim to i) synthesize and validate probes to detect different lipids, ii) investigate which lipids are associated with cytokinesis and how they are spatially and temporally regulated during the completion of cytokinesis and iii) understand how mechanical parameters can influence the completion of cytokinesis.Unifying the scientific expertise of our three teams, we will use chemistry and chemical biology to design lipid probes, cell biology to use these probes to investigate the origin and role of lipids involved in cytokinesis and biophysics/modeling to address the functional importance of membranes during the mechanism of cytokinesis. The innovation of this proposal does not only lie in its unique question, but also in the combination of diverse approaches. Our comprehensive and systematic investigation will provide new insights into the important question of how cells divide, which will not only be important for basic biology, but also for understanding how cell division is mis-regulated in cancer.

Nonsense-mediated mRNA decay (NMD) is a surveillance pathway that eliminates transcripts lacking complete open reading frames, thereby ensuring that potentially toxic polypeptide fragments do not accumulate. NMD is triggered by premature translation termination, a response that stands in striking contrast to stop codon recognition by the ribosome at the end of each round of normal translation. In this proposal, we seek to define the mechanistic differences between premature and normal translation termination, and to use that information to understand how only the former leads to NMD. Our plan considers four important aspects of this problem, namely that: the “core” components of termination and NMD have been identified (the eRFs, Pab1, the Upf proteins, and the ribosome); structural information is available for some components, but not for most of their complexes; the precise roles of the different factors and complexes in both termination and NMD is unknown; and there is a high likelihood that there is a large set of hitherto unidentified and/or uncharacterized regulators of termination and NMD that must play important roles in these processes. Accordingly, we have organized a France-Sweden-U.S. collaboration that brings to bear complementary sets of expertise in structural biology, biochemistry, and molecular genetics in pursuit of three aims. Specifically, it is our intent to: i) Determine 3D structures for the interactions of the Pab1, eRF, and Upf proteins; ii) Define the kinetics of the interactions between these proteins and the ribosome; and iii) Identify and characterize additional regulators of NMD and termination. To address these aims, we have planned a highly synergistic work flow, where the success of any one group will facilitate and enhance the success of the other two. The three complementary groups will provide each other with purified proteins and high resolution structures (France), detailed and dynamic biochemical parameters (Sweden), and mutants defective in specific steps of termination/NMD and new regulators of both processes (U.S.).

Spatial regulation of translation within the cytoplasm results in the accumulation of newly synthesized proteins in discrete locations of the cell. mRNA localization mechanism is one aspect of the post-transcriptional control of gene expression fundamental to many cell types. The Zipcode Binding protein 1 (ZBP1) is a pivotal molecule in cellular morphology, physiology, development and cancer progression. ZBP1 is essential for localizing and regulating the translation of ß-actin mRNA near the leading edge in several cell types such as fibroblasts, or the growth cone of neurons. It binds to a short sequence in the ß-actin 3’UTR, the zipcode motif. Due to other mRNAs encoding for cell-contact related proteins also contain putative zipcode motif in their 3’UTRs, I propose that they may be coordinately localized and translated in a mechanism similar to ß-actin mRNA. Accordingly, I hypothesize that cell-contact sites represent a cellular microdomain where ZBP1 mediates localization and translation of these specific mRNAs in order to locally produce the factors required for the consolidation of these structures. Therefore, the cell may respond to extra-cellular stimuli regulating translation initiation of functionally related mRNAs at the correct time and place.

The goal of this project is to investigate the potential use of the human embryonic stem cells(hESC)-derived cerebellar progenitors as a therapeutic strategy in rat model of cerebellar lesions. Recently we developed the first protocol for generation the cerebellar cells from hESC (Erceg et al., 2009, submitted). These cells could be useful in the treatment of different types of cerebellar disorders. The next step will be to evaluate the in vivo function. The proposed project will be to transplant generated cells in a rat model of cerebellar lesion to asses whether these cells are capable to restore already lost motor function in cerebellar lesions. The experiments and short-term stay will be performed in the Laboratory of Prof. Lura Petrosini, Dept.of Psychology, University "Sapienza" di Roma, Italy. In her Lab we will learn and perform the surgical lesions in the rat. This research project requires cerebellar lesions in adult rats. These lesions may involve the surgical ablation of a hemicerebellum with or without the ablation of the deep cerebellar nuclei of the same side. They may involve the surgical ablation of an entire hemicerebellum, or of a cerebellar hemisphere or of the vermian region. After the lesion the animals will be tested for their motor symptomatology. Testing for cognitive symptomatology will performed when no change in the motor symptomatology is observed (about three weeks after the surgical lesion). Animals will submitted to behavioral testing only if they exhibit stable motor symptomatology consistent with a cerebellar lesion.

Brain function depends on the precise assembly of neuronal networks during development, however the mechanisms underlying this process are poorly understood. During spinal cord development, inputs to motor neurons are established sequentially, starting with local interneuron circuits, followed by peripheral sensory input, and ending with the assembly of connections from the motor cortex that are responsible for accurate voluntary movement. I hypothesize that during development each step in the sequence of circuit connectivity influences later events. Specifically, I propose to address whether functional proprioceptive connectivity is required for later development of cortical input to spinal motor neurons. I will first characterize the time course with which corticospinal projections and synapses develop in mice, with a special focus on direct monosynaptic connections from motor cortex to spinal motor neurons. I will anterogradely label corticospinal projections using transgenic mice that express GFP only in the forebrain and I will retrogradely label motor neuron monosynaptic partners by a modified rabies virus expressing GFP or RFP that is able to jump just one synapse. I will also study the topographic distribution of cortical motor neurons projecting to specific motor neuron pools. Secondly, I will determine when established connections become functional combining viral expression of channel rhodopsin in corticospinal projections with intracellular and/or voltage dye recordings of motor neurons. Finally, I will compare the obtained wild-type results with the ones from transgenic mice in which proprioceptive connectivity to motor neurons is ablated.

The question of the origin of life is one of the most fascinating in science. It is commonly believed that, prior to our form of life, there was on Earth another form of life based on RNA, the so-called “RNA world”. Research is ongoing to build a laboratory system that resembles the RNA world in order to study how it might operate. Such a system requires RNA enzymes that have the ability to self-replicate and that have sufficient functional complexity to evolve in a Darwinian manner. The Joyce group has pursued a strategy to create such a system, based on the R3C ligase ribozyme, which they developed previously using in vitro evolution. A complex network is being constructed in which many different RNA enzymes cross-replicate, in the absence of proteins or any other biological entity. Competition and the possibility of occasional mutations should give the system the features of Darwinian evolution. The aim of the proposed research is to develop a kinetic model that could describe such a system. The model will be validated based on comparison with experimental results. Computer simulations will be performed, with the aim of helping to design new experiments that provide a deeper understanding of the system, operating in the context of serial transfer experiments performed within a microfluidic device. The model will describe the time-dependent behavior of the system under various conditions, thereby providing an informative picture of the self-sustained Darwinian evolution of RNA.

Myosin molecules are proteins that function as motors inside cells. These molecular motors play crucial, dynamic roles in most cellular processes, including contraction, movement, and shape change. A variety of diseases owe their origins to defects in myosins, like DFNA22 syndrome, which is characterized by deafness. Myosins transport other biomolecules by hauling them along the actin cytoskeleton. This transport consumes energy at the ‘head’ domain of the motor, while its ‘tail’ domain is traditionally considered more structural and mainly required for dimerisation, since it is rather rigid. Until recently, coiled coils in mechano-sensory proteins like myosins were thought to be relatively straight and geometrically ideal. Detailed investigations of the implications of structural deviations from a strong coiled coil motif on biological function of myosins are just emerging. For instance, myosin VI in both Drosophila and humans contains a peculiar distribution of charged residues in the tail that Jim Spudich’s group has shown, by biophysical approaches, to be a ~70-residue long stable, fairly rigid single α-helix, not the coiled coil that was predicted earlier. Ramanathan Sowdhamini’s lab has developed computer algorithms for the analysis of coiled coil interfaces through energy calculations. Jim Spudich’s lab has a long-standing expertise in biophysical and cellular experiments on myosins. Henrik Flyvbjerg’s lab leads efforts to properly analyze stochastic processes and data in biophysics, such as those manifested in molecular motor studies and in motility assays. The three groups together will make a unique team by uniting their complementary tools of bioinformatics, biophysical studies, and mathematical modeling to obtain important structural and functional understandings of the relatively ignored, but vital, tail domains of the myosin family of molecular motors.

The knowledge of the structures of biological macromolecules is invaluable to elucidate the mechanistic details of their function. Complexes that are associated to lipid membranes carry out many important cellular functions, but their structures are largely elusive to date because these types of macromolecules are extremely difficult to assess by established experimental techniques, such as X-ray crystallography, single-particle cryo-EM, and NMR spectroscopy. I propose to unravel the structures of membrane-associated complexes using cryoelectron tomography (CET) and subsequent modeling. CET is currently the only generally applicable three-dimensional imaging method to retrieve the density of a given complex in its close-to-native state, and the technique is therefore uniquely suited to provide density maps of membrane-associated complexes. Subsequent modeling allows determining those structures, which are compatible with the densities, but also all other available information on the complex such as biochemical interactions as determined by proteomics studies and atomic structures of constituents. The developed methodology will be applied to the machinery that governs endoplasmic reticulum mediated decay of proteins (ERAD). Removal of erroneous proteins from the endoplasmic reticulum and subsequent degradation via ERAD is essential for cellular function. Malfunctions of protein degradation have been implicated in diseases such as Alzheimer and Parkison’s disease. The structure of the ERAD machinery is almost completely uncharted and even low-resolution structures will greatly elevate our rudimentary understanding of ERAD.

The process of myelination, in which myelin sheaths are formed around axons, represents one of the most spectacular processes in biology. Myelination of axons is required for the efficient transmission of nerve impulses and maintainance of axonal integrity in the central nervous system. As well as being of intrinsic biological interest, this process is relevant to our understanding of the regenerative process of remyelination following myelin diseases such as multiple sclerosis. Critical to this process is the mechanism by which a population of neural stem/precursor cells called oligodendrocyte precursor cells (OPCs) differentiates into myelin-forming oligodendrocytes, cells that migrate to and wrap their processes around axons to form the multiple layers of compacted membrane that constitute myelin. Thus far, only the biochemical stimulation of OPC differentiation has been studied. However, OPCs are also subjected to various kinds of mechanical strain during the processes of cell migration, axon engagement, and neuron growth, and it remains entirely unknown how such physical stimuli may regulate or contribute to OPC differentiation. In this project we seek to clarify the importance and modes of mechanical stimuli that impact OPC differentiation, by integrating international expertise in OPC biology, novel biophysical experiments, and material mechanics. Using well-established protocols for growing, differentiating, and characterizing OPCs in vitro used in the Franklin lab, we will systematically consider effects of global, local, and receptor-mediated strain on OPC morphology, molecular markers, and myelination potential. These strains will be applied using optical, scanning-nanoprobe, and substrate-based cell deformation techniques being developed by Guck and Van Vliet, in order to identify the key modes and magnitudes of mechanical stimuli that contribute to OPC differentiation. The conversion of effective mechanical stimulus to intracellular biochemical signaling pathways will then be identified by RNA profiling (including microarray profiling and post-hoc bioinformatic pathway analyis tools) and validated by ICC and Western blotting. In summary, the proposed research will develop and apply innovative experimental approaches to improve our understanding of the process and stimulation of axon myelination, and to identify potential new targets for treating demyelinating diseases. More generally, these systematic studies on a key precursor cell type will serve as a model to clarify the general mechanisms involved in mechanotransduction in the maintenance and differentiation of stem and precursor cells.