During development, a few neural stem (NS) cells give rise to all the different cell types of the mammalian central nervous system. To do so, they can divide asymmetrically into another self-renewing NS cell and a lineage-committed sibling. Defective cell fate choices can cause aberrant neurogenesis, including microcephaly or tumor formation. The mechanisms regulating asymmetric cell division and lineage choice in NS cells are, however, only poorly understood. Here I propose the use of homogeneous NS cell cultures to explore cellular and molecular mechanisms regulating NS cell differentiation. I plan to establish an assay for asymmetric divisions of NS cells that will be used to genome-wide RNAi screening for genes regulating cell fate decisions. I furthermore plan to screen under selective culture conditions to identify genes that are required for differentiation of NS cells into neurons and astrocytes. Candidate genes will be further characterized and their in vivo function analyzed in cortical slice cultures. The principles gained from this system will provide insight into vertebrate neurogenesis and might reveal general strategies of somatic stem cell divisions.

Post-transcriptional regulation of gene expression in metazoa involves a complex cellular network. Many trans-acting factors, including microRNAs, influence mRNAs stability, translatability, or mRNA subcellular localization by binding to specific cis-acting sequences or structures at the 3’ and 5’ untranslated regions of a mRNA. These interactions are decisive for translation of mRNA into protein and are influenced by the cellular context in time and space. Deregulation of cellular mRNA homeostasis is associated with many human diseases. The cellular microenvironment is composed of an intricate blend of extracellular matrix components and numerous neighboring cell types, and influences cellular behavior and gene expression profile in vivo. However, the effect of cellular microenvironment on post-transcriptional regulation of gene expression has never been addressed in detail in metazoa. I will study the effects of cellular microenvironment on post-transcriptional gene regulatory networks in mammals. Multidimensional cell culture systems and arrays of microenvironments will be used for identification of extracellular factors regulating miRNA-dependent and miRNA-independent post-transcriptional gene regulation machineries in mammalian cells. Experiments will also be designed to study the microenvironmental effects on miRNA mediated suppression of genes in a mouse model. Understanding how multiple extra- and intracellular signals are integrated to determine the differential fate of a mRNA in human cells, will pave the path for future studies on the importance of altered stability and translatability of mRNAs during tumorogenesis or organogenesis.

The loss of axons and dendrites usually precedes the death of the cell bodies in many neurodegenerative diseases. Since the molecular pathways involved in the death of cell bodies are pretty well understood, those underlying the degeneration of the axons and dendrites remain to date largely elusive. In a work from the host lab currently under review, Galectin-1 is identified as a major effector of the neuronal processes degeneration following injury of the adult nervous system. The receptor p75NTR is expressed early during neurons development and is also known to be up-regulated following nervous system’s injury. Using neurons generated from embryonic stem cells, the authors compare the proteome of wild type cells with these of p75NTR over-expressing neurons, and observe a three-fold over-expression of Galectin-1. Its transcriptional regulation is under the control of p75NTR through c-Jun N-terminal kinase and RhoA signaling, but do not seem to depend on them for the induction of the degeneration mechanisms observed. The research project I propose will first focus on the identification of the receptor(s) of Galectin-1. Many candidates exist, and we propose to analyze their profile of expression, as well as to use a biochemical approach to identify cell surface receptors interacting with Galectin-1. Once identified we plan to deplete, using RNA interference tool, the transcript(s) of Galectin-1 receptor(s) in order to test its/their involvement in the degeneration process. A second aim of this project will be to study the precise cellular and molecular mechanisms involved in the Galectin-1 signaling, causing the degeneration of neuronal processes.

Since their discovery over 100 years ago, enzymes are a topic of intensive research. Recently, it was shown that enzymes are dynamic entities existing in different conformations. These conformations, which are represented by different wells in the energy landscape, exhibit different rate constants for the catalytic process. It is likely that, during enzyme evolution, the original activity and the newly evolving activity are linked with different conformations. The proposed project aims to analyze the presence and evolution of different conformations when an enzyme is subjected to directed in vitro evolution. It will combine two new and innovative approaches for the evolution of enzymes and for the characterization of the dynamics of single enzymes. The evolution of enzymes will be carried out in a process combining in vitro compartmentalization with microfluidics, allowing the systematic control of all evolutionary steps (mutation, recombination and selection). A well-known model protein for the analysis of the evolution of enzyme activity (evolved beta-galactosidase from E. coli) will either be evolved to exhibit a higher activity or a different substrate specificity. In either case, single molecule experiments will be performed to determine the distribution of the rate constants. The results provide information about the existence and distribution of different enzyme conformations and will allow testing to what extent a stabilisation of different conformations with different activities contributes to the evolution of enzymes with higher activity or new functions.

This proposal concerns the role of clathrin and actin in the invasion of host cells by the intracellular bacterium Listeria monocytogenes. Listeria uptake proceeds by a clathrin-mediated route distinct from the canonical clathrin coated-pit/coated vesicle pathway, and it thus provides a way to probe molecular mechanisms in the non-canonical mode of uptake. The live cell imaging methods developed in Dr. Kirchhausen’s laboratory are a powerful way to decipher mechanisms underlying clathrin-mediated traffic. (1) I will use these methods to study the dynamics of clathrin and actin recruitment during Listeria entry and hence to probe their relative functions. Subsequently, the pattern of recruitment of proteins involved in generation of membrane curvature and vesicle pinching from the donor membrane will be investigated. (2) I will dissect which properties of a cargo, its size or its association with a particular receptor, will determine its route of entry. For this purpose beads of varying sizes will be modified with (a) the Listeria surface proteins InlA and InlB necessary for invasion and (b) with transferrin and other ligands that rely on the canonical pathway mediated by coated pits and coated vesicles to serve as model cargoes for live cell imaging studies. Hereby I will capitalize on my background in surface chemistry to conjugate the proteins in a well-defined density and orientation to the beads. The proposed studies will allow me to investigate the general principles governing the uptake of cargo via canonical and non-canonical clathrin-mediated endocytosis.

The mammalian cerebral cortex has been subject to a dramatic expansion in surface area along the evolutionary scale. This process is accompanied by the appearance of stereotyped folds and fissures, and is recapitulated during normal development in the majority of higher mammals, including carnivores and primates. The development of the cerebral cortex involves three major steps: production of neurons, migration of neurons along radial glia, and alignment of neurons into discrete layers. Recent studies in mice have shown that certain alterations in the rates of proliferation and cell death of cortical progenitors result in an overproduction of cells, and an artificial expansion and folding of the mouse cerebral cortex. However, virtually nothing is known about the mechanisms determining the formation of stereotyped folding patterns in the cerebral cortex of higher mammals, and even less is known about the mechanisms that choreograph the processes of increased proliferation, radial migration, and layering, that give rise to a gyrated cerebral cortex in those species. To this aim, we propose to use a combination of in vivo and in vitro approaches in both ferrets and mice to achieve three specific objectives: i) to unravel the changes occurring in cortical progenitors related to cortical expansion in a naturally expanded cerebral cortex ii) to understand the mechanisms that ensure the existence of a radial glial scaffold for neuron migration during cortical growth iii) to determine the mechanisms controlling that CR cells (which orchestrate neuronal migration and layer formation) are present throughout the marginal zone of the tangentially expanding cerebral cortex.

The neocortex decodes sensory information from the external world. Cortical neurons are organized in columns, each tuned to one specific sensory stimulus. A growing body of evidences suggests that the neocortex directly participates in associative learning too. A stimulus that is initially neutral to a mouse, like the deflection of a whisker, acquires the emotional value of another sensory stimulus, like a tail shock, when the two are presented together. After conditioning, the mouse freezes at the deflection of that whisker alone. This learning correlates with changes in the barrel cortex, the region that contains the somatotopic representation of whiskers: more neurons become responsive to the conditioned whisker, regardless of their column. In the wild, this mechanism could “let the animal know” what specific signal to look for in order to adopt the behavior that might save its life, like freezing at the sound of a rattlesnake. Still, the specific cortical circuits involved in this phenomenon remain largely unknown. I will combine behavioral studies with electrophysiological recordings to unveil them. I will use the technique of glutamate uncaging to map the neuronal connectivity in brain slices. I will be able to identify the connections that are changed by associative learning and describe the cellular mechanisms that changed them. I will also compare the ontogeny of associative learning with the maturation of cortical connections. Finally, the comparison of normal mice with models of mental retardation will further reveal the role of cortical networks in learning. The carrier development award will help me to establish this new line of research in my home country.

miRNAs play fundamental role in shaping gene expression. Yet, the mechanisms by which miRNAs control gene expression remain largely unknown. The miRNA-mediated repression complex was shown to localize to so-called P bodies, cytoplasmic foci containing non-translated mRNAs and degradation enzymes. Moreover, some of P body components were shown to play a role in miRNA pathway. Hence, thorough analysis of P body content and structural organization would help to elucidate the mechanisms of miRNA-directed repression. I propose to analyze the composition of P bodies and examine the role of newly identified P body components in the regulation of gene expression by miRNAs. First, I propose to isolate P bodies from mammalian cell culture using affinity tags fused to various known components of P bodies and analyze the content of the purified complexes by mass spectrometry and microarray analysis. Second, in parallel with the biochemical technique, I propose to do biocomputational search for novel component of P bodies. Third, the novel components of P bodies identified during this study will be analyzed for their role in miRNA pathway, using reporter system recapitulating miRNA-directed repression in mammalian cell culture. Fourth, based on the data about intermolecular interactions within P bodies, I will be able to determine distinct compartments within P bodies. The analysis of mRNA distribution between such compartments may lead to better understanding of the role of P bodies in differential stability of miRNA-targeted mRNAs. As a result, thorough analysis of P body composition would provide more insight into the mechanisms underlying miRNAs function.

EMSY is a recently identified transcriptional regulator that localises to sites of repair following DNA damage and associates with proteins involved in chromatin remodelling. The EMSY gene is amplified in sporadic breast cancer and can silence the transcriptional activation potential of the breast cancer susceptibility gene BRCA2, a tumour suppressor involved in chromatin remodelling and DNA repair. However, the precise mechanism of action of EMSY in transcriptional regulation remains unclear. Preliminary studies show that EMSY binds an internal, alternative promoter within the human tumour suppressor gene p53 and suggest that it may have a role in the control of cellular behaviour through repression of transcription of p53 isoforms. The fact that both EMSY and the various p53 isoforms are differentially expressed between breast tumours and normal breast tissue suggests the existence of a mechanism by which the above process may determine cell fate. The specific aim of this proposal is to decipher the mechanisms through which EMSY regulates transcription. The proposed investigation will employ a series of basic biochemical, molecular and cell biological approaches and use the regulation of transcription of p53 isoforms as the central biological process through which to study the mechanism of interest. The importance of transcriptional regulation by EMSY in the development of breast cancer will be investigated and the roles of BRCA2 and certain chromatin remodelling enzymes in this mechanism directly addressed. This work may define a novel mechanism of transcriptional regulation and provide a deeper understanding of the events involved in the development of breast cancer.