How cellular structures self-assemble is one of the most exciting questions in biology, with potential implications ranging from nanoscience to medicine. In eukaryotes many self-assembly processes are mediated by cytoskeleton proteins such as actin. In rod-shaped prokaryotes the actin-homolog MreB, an essential protein involved in cell shape maintenance, forms a helical pattern along the bacterial axis. This research aims to elucidate the biochemical and biophysical origin of MreB self-assembly by introducing new techniques into this field. Cellular structures in micron-sized organisms are hard to study because of optical diffraction limits. Using the recently developed method of fluorescent photoactivatable localization microscopy, MreB structure will be determined in-vivo with sub-diffraction resolution. Statistical analysis of different cytoskeleton characteristics, such as number of helical turns and helical pitch, will be measured at various steady-state and kinetic conditions. These results will be compared to theories of MreB polymerization and to a new numerical method. To further elucidate the affects of polymerization on membranes' shape, MreB cytoskeleton will be reconstituted in-vitro within lipid vesicles. This reconstituted system will allow measurement of forces applied by MreB cytoskeleton on the cell walls. Liposomes confined within polydimethylsiloxane microchannels will be used to assess how geometry affects cytoskeleton structure. The combination of in-vivo, in-vitro and in-silico approaches will shed new light on the fascinating question of self-assembly in bacteria.

The brain is a highly dynamic system that can change at different time scales, from very fast changes within fraction of a second to long processes of weeks and years. Understanding of brain-behavior coupling and its dynamics is at the heart of modern brain research. It also has practical implications, as we suggest here in relation to sight restoration efforts using sensory substitution devices (SSDs) that convert visual information into auditory input. Blind children might adapt and utilize SSDs better, as their brain is more plastic. Thus, teaching them to use SSDs for detailed artificial vision could prove beneficial. Surprisingly, no one has systematically attempted this before. Here we plan to adapt a method (we developed during my HFSP fellowship Amedi et al, Nature Neuroscience 2007) to teach artificial vision to blind children and adults and study brain dynamics while learning this new crossmodal transformations using safe advanced neuroimaging and electrophysiological methods. Specifically, we will test for the development of specialized functional architecture in the visual cortex (spatial topography, visual streams and object categories) using SSDs. On top of advancing sight restoration and yielding new insights in human-machine interfacing, it will contribute to knowledge of brain plasticity. By comparing blind to sighted controls we will also be able to gain insights on the development and dynamics of multisensory integration. The CDA will help to concretize multimodal approaches, fund demanding longitudinal studies and collaborate with neuroscientists, ophthalmologists (sight restoration using prostheses) and engineers (implementing new SSD algorithms).

Abstract The function of dendritic cells (DC) is to recognize specific pathogens associated molecules (PAMPs) and prime an immune response by presenting the information to dedicated professional immune cells. The networks responsible for DC to process information from recognition of specific pathogenic antigen, to the precise activation, of the DC, promoting its migration into lymph nodes, to activate T helper cells, are unclear. The focus of my post-doctoral research is to elucidate the mechanism controlling the genetic and epigenetic modifications that underlie the specific response of dendritic cells to PAMPs. Specifically, I will address a model whereby the information of a specific PAMP pattern is dynamically stored in modular components of signaling and transcription machinery, that along with changes in chromatin accessibility and microRNAs underlie the fine-tuned change in expression in each target gene leading to the desired cellular function. Utilizing mRNA and microRNA microarrays, along with chip-chip arrays for different chromatin modifications I will establish the dynamics of the signaling and transcriptional events induced by challenging DC with PAMPs. Computational methods will then be used to reconstruct a predictive model of the regulatory networks and chromatin state that underlie the response of DCs. Finally RNAi libraries will allow us to both test our predicted models and improve their function. This project will involve integration of biochemical and molecular proficiency I gained in my Ph.D. along with immunological, genetic and computational approaches I will acquire with the help of Drs Regev and Hacohen at the Broad institute.

It has long been postulated that long lasting changes in synaptic strength, known as synaptic plasticity, is the cellular basis underlying learning and memory. Synaptic strength is determined in major part by the number of AMPA receptors in the postsynaptic membrane. Recent studies have shown that the AMPA receptor auxiliary subunit, stargazin and its binding partner PSD95 play an important role in the trafficking and targeting of AMPA receptors to synapses. Stargazin is the best studied member of transmembrane AMPA receptor regulatory proteins (TARPs), a subfamily of the calcium channel gamma subunit gene family. While it is known that stargazin is phosphorylated in vivo, it is still not well characterized. I propose to define the molecular mechanisms of how different TARPs, together with their phosphorylation status, regulate AMPA receptor maturation, trafficking and function in synaptic plasticity in vivo. I will identify novel interacting proteins and phosphorylation-regulated proteins for each TARP by taking advantage of my previous biochemical skills. Next, I will investigate the functional roles of the TARPs-interacting proteins and the cellular dynamics of TARPs by taking advantage of the host laboratory’s experience in neurobiological and neuroelectrophysiological approaches, which are indispensable for the success of the proposed project. This postdoctoral investigation will allow me to develop my expertise in live imaging, electrophysiology, production of knockout mice and ultimately in behavioral analyses. The knowledge I will gain from these studies will better prepare me to direct my own research to understand the cellular basis of brain function.

The human innate immune system represents an effective first line of defense against pathogens. Therefore, drugs that enhance innate responses might prove extraordinarily useful in the battle against infectious diseases. However, our current understanding of the innate immune system is limited and many of the molecules, mechanisms and cascades that provide resistance against microorganisms remain to be elucidated. Salmonella enterica and enterohaemorrhagic E. coli (EHEC) use secretion systems to inject effector proteins into host cells. These effectors play a central role in pathogenesis. Therefore, it is my hypothesis that many effectors interact directly with components of the host innate immune system and can be used as baits to identify novel molecules involved in host innate responses. Hence, I aim at identifying host cell interaction partners of S. enterica and EHEC effector proteins using a mass spectrometry based quantitative proteomics technique. I plan to characterize further one or a few of the effectors that interact with known or suspected host innate immunity molecules in order to understand how these interactions lead to disease. Hence, this study will provide insight in the pathogenic mechanisms employed by Salmonella and E. coli and will further our understanding of human innate immunity.

Collagen fibrils are the principal source of tensile strength in animal tissues and define the shape and form of tissues in which they occur. Type I collagen is the major component of fibrils in tendon, bone and skin. Collagen is synthesized as Type I procollagen (PC-1) in a precursor that is co-translationally translocated into the endoplasmic reticulum (ER) (1). Most lethal cases of Osteogenesis Imperfecta (OI) are caused by mutations in PC-1 that yield improperly folded PC-1 molecules which are retained in the ER (2). The mechanism by which 300nm long fibril of PC-1 destined for secretion from the cell is transported from the ER through the Golgi to the plasma membrane has long been of challenge to cell biologists (3). It has been proposed from previous work that PC-1 trafficking is not mediated by typical COPII vesicles which are usually 60-70nm in diameter, although PC-1 transport depends on COPII components (4-7). However, recent work done by the Schekman group has provided evidence that Sec23A (COPII component) is involved in collagen transport as evidenced by accumulation of collagen in the ER of fibroblasts from patients having Cranio-lenticulo-sutural dysplasia (8). In an attempt to resolve this issue and get clear insights into this fundamental problem of trafficking of large secretory molecules, I would like to investigate PC-1 transport from the ER using human fibroblasts cells and an in vitro vesicle budding reaction (9). This study will provide a platform to explore the effects of different lethal OI associated PC-1 mutations on its folding and export from the ER and to unravel the mechanism of protein misfolding defects associated with human diseases.

Vertebrae, ribs and most skeletal muscles, derive from somites, which are transient embryonic segments, and mutations affecting somite formation are responsible for human congenital scoliosis. The segmentation of the presomitic mesoderm (PSM) into somites is a unique example of a temporally and spatially repeated embryonic process and the favored hypothesis to explain this process is the “clock and wavefront” model. The “segmentation clock” generates coordinated oscillations of gene expression in the PSM while the “wavefront” determines the site where these oscillations arrest and where the next segment boundary is positioned. The canonical Wnt signaling pathway is thought to be involved in both clock and wavefront. However, direct evidence is lacking because of the effects of this pathway on posterior embryonic outgrowth and early specification of the mesoderm from which somites originate. For this fellowship, I will use inducible transgenic zebrafish lines to transiently modulate Wnt signaling after mesoderm has been specified and before tail outgrowth is affected. Our preliminary results clearly show that this transient disruption of Wnt specifically affects somite patterning. I therefore propose to use a mathematical model side by side with an exhaustive experimental study to elucidate the parameters of the segmentation process that are affected. In addition to molecular markers for both clock and wave front, I will use a new quantitative method to measure the rate of somite formation in the live embryo. This project will thus take advantage of the zebrafish model to investigate the spatial and temporal functions of Wnt signaling in somitogenesis.

The main objective of this project is to study the evolution signaling pathways in yeast in order to understand how genotypic variability impacts on cellular interaction and how these in turn affect phenotypic properties. Quantitative epistatic interactions of S. cerevisiae and S. pombe environmental response pathway components will be determined under different environmental conditions. In parallel to the experimental work, a computational analysis of these pathways will be set-up to determine the extent of sequence and structural changes. This will include (1) determining ortholog/paralogs in currently sequenced yeast genomes to calculate rates of change, sequence divergence and gene gain/loss (3) determining by structural analysis the divergence of binding specificities, and finally (4) reconstructing ancestral states of the pathway components of interest at different evolutionary times. The inferred sequences together with available structural information will permit me to study the mutational pathways that could underlie differences in cellular interactions and phenotypes detected in the experimental analysis. This will allow linking divergence in environmental responses with changes in cellular interactions (genetic interactions, pathways and complexes) and the underlying mutations. Finally, the ancestral network states of diverged pathways will be reconstructed by phylogenetic analysis and recombined into extant species for detailed analysis.

miRNA, the endogenous 21nt long non-coding RNA, repress gene expression by binding to the 3’ untranslated region (UTR) of a mRNA and inducing translation repression and/or degradation of the target messages in metazoa. In human cells, miRNA repressed mRNAs are localized in a specialized cytoplasmic structure named RNA processing body or P-body. P-bodies, originally discovered in yeast as the cytoplasmic sites of mRNA degradation, could also serve as storage sites for translationally repressed mRNAs. Cationic amino acid transporter 1 (CAT-1) mRNA, encoding a high affinity transporter protein for cationic amino acids, is repressed by a liver specific miRNA, miR-122, in human hepatic cells and is stored in P-bodies. Interestingly, this miRNA-repressed mRNA get relocalized from P-bodies and translated into protein in metabolically stressed cells. Stress-induced relief of miRNA mediated repression of CAT-1 mRNA requires the RNA binding protein HuR, which relocalizes from the nucleus under stress condition and binds CAT-1 mRNA in the cytoplasm. HuR binding to a specific sequence within the 3’ UTR, is essential for stress induced relocalization and derepression of the miRNA-repressed CAT-1 mRNA. The objective of our research is to understand the molecular mechanism of HuR mediated derepression of miRNAs action in human cells. We will investigate, both using in vivo and in vitro assays, whether binding of HuR to mRNA prevents repressive function of miRNPs or displaces miRNAs from the mRNA and thereby prevent miRNA-mediated repression.

The varied arrangement and interconnectivity of collagen fibers, the primary structural protein in the body, give rise to a tremendous array of macroscopic tissue behaviors from stiff tendons to compliant skin. Interactions between fibers determine how external forces are transferred to cells, which in turn regulates the cell behavior. Quantitative knowledge of structure-function relationships of these fibrous tissues is critical for elucidating complex mechanisms of cell interactions with the surrounding matrix in health and disease. Although the mechanical properties and arrangement of collagen fibers have been studied extensively, surprisingly little is known about their interconnectivity (crosslinking, electrostatic forces, friction, etc.)
The objectives of this proposal are to learn the specialized techniques to measure the mechanical properties of individual nanometer- and micron-scale collagen fibers utilizing atomic force microscopy (AFM) developed at U Twente, and to develop novel methods for investigating the interactions between electrospun collagen fibers using AFM.
Working hands-on with AFM and nanoscale biofiber composites would catalyze my research program by providing powerful tools to investigate tissues at the cell/fiber level and to measure cell-ECM interactions. These studies will establish an international collaboration in a new area of research and will aid in obtaining pilot results for national and international grants. Without the proposed international exchange, it would be unlikely that the aims of the proposed research could be achieved.

Cell polarity, asymmetry in both geometry and concentration of proteins, is ubiquitous throughout eukaryotic cells, serving a host of purposes, including contributing to cell differentiation. The core mechanisms for establishment of cell polarity are conserved, being driven largely by differentiation of regions of the cortex. The proposed research seeks to elucidate the essential physical principles governing this process in the C. elegans zygote, a well-characterized model organism. For this system, the initial establishment of polarity is largely governed by the actomyosin cortex alone, in response to a local disruption of myosin activity near the sperm-donated MTOC, making understanding the mechanisms for polarity generation a largely physical (as opposed to biochemical) problem. As such, I will develop analytical physical models of the cortex to investigate the minimal local regulation necessary for the otherwise globally-regulated actomyosin cortex (responding to a local down-regulation of myosin activity near the sperm MTOC) to establish a phase boundary between contractile and noncontractile phases. The model will capture the essential features of the cortex, including dynamics of myosin foci, formation of a phase boundary, and furrow dynamics. The model will be informed by published results in its qualitative construction and by quantitative experiments in its parametrization. I will develop automated image processing tools to quantify RNAi rundown of proteins regulating myosin activity. Thus, I will combine theoretical physical models with quantitative measurements to understand the underlying physics of cellular polarity establishment.

The goal of the proposed work is to analyze the gut microbiota of Drosophila melanogaster, the effect of this microbiota on host immune function, and the impact of host immunity on gut microbial colonization and microbiota composition. This study will identify specific gut microbes of both lab colony and wild-caught populations of D. melanogaster and determine the stability of these associations by examining the effect of diet and source on composition. The proposed work will determine whether the host innate immune system responds to microbial colonization of the gut by comparing the local and systemic immune response of axenic Drosophila to those with a gut microbiota. To determine if the gut microbiota mediates host response to pathogens, Drosophila with and without an indigenous community will be challenged with natural pathogens that are known to affect both local and systemic host immune responses. The impact of host immunity on the persistence and composition of gut-associated microbes will be examined by comparing gut-associated microbes of Drosophila lines deficient in key innate immune functions affecting gut physiology, gut immunity, and systemic immunity. Together these results will identify interactions between Drosophila host immunity and its associated microbiota.