The microtubule cytoskeleton is an evolutionarily conserved macromolecular assembly with a large variety of functions in living cells. Adaptation to specific functions is likely controlled by the tubulin code, a molecular mechanism to diversify microtubules. Here we will develop a systems approach to determine the molecular mechanisms and biological role of the posttranslational modification polyglutamylation, which due to its complexity and dynamic nature generates many combinatory patterns on microtubules. We aim at demonstrating how specific polyglutamylation patterns determine the functions of microtubules in neurons in a locally and temporally controlled manner. To reach this goal, we will develop superresolution-microscopy based tools to visualize different polyglutamylation patterns in cells, identify their physiological roles in neurons, and use proteomics and in vitro reconstitution experiments to decipher by which molecular mechanisms polyglutamylation controls its biological functions.

Morphogen gradients are used by various organisms to establish polarity along embryonic axes or within organs. In these systems, it is assumed that positional information is provided by the concentration of the morphogen detected by each cell in the target tissue and responsible for the determination of cell identity. The extreme robustness of these processes ensures reproducibility of developmental patterns and emergence of properly proportioned individuals despite varying size and environmental conditions. Most descriptions of developmental regulation assume that highly reproducible transcription programs are directly controlling the mechanisms of differentiation. However, when studied at the single cell level, transcription is frequently observed to be an extremely noisy process, hardly suggestive of such precise control. Understanding how reproducible transcription patterns can robustly emerge from these smooth morphogen gradients given inherent stochastic transcription is an important challenge, and constitutes the general objective of this proposal. Recently, methods to observe the kinetics of the transcription process directly in living cells have been developed. These methodes combine fluorescent labeling of nascent mRNA with live-cell imaging at high spatial and temporal resolution. We have recently adapted these approaches to one of the best characterized model organism, the fruit fly embryo. Our goal was to better understand how cell identity is controlled by the Bicoid (Bcd) morphogen system along the antero-posterior (AP) axis of the embryo. Focusing on the expression of the main Bcd target, hunchback (hb), at the onset of zygotic transcription, we have successfully built an MS2 reporter reproducing endogenous expression of the gene. Despite high nuclei-to-nuclei variability in transcription kinetics, the hb promoter was able to establish a sharp expression boundary along to separate anterior expressing from posterior non expressing nuclei. Surprisingly, it only takes ~3 min at each interphase for the system to measure subtle differences in Bicoid concentration and produce a complete sharp boundary. If one assumes that the only driver for the hb transcription process is the Bcd gradient, and further that Bcd molecules reach their target sites by 3D diffusion, simple statistical mechanics considerations (combined with current estimates of Bcd concentration and mobility) show that a minimum of 25 min should be necessary for the system to produce the observed sharp boundary. The difference between predicted (25 min) and observed (~ 3 min) time-requirement is of 1 order of magnitude and calls for alternative explanations. We will explore the possibility of alternative mechanisms in an unbiased manner by combining genetics and optogenetics, live-imaging, advanced data analysis and modeling. Our goal is to identify mutant or natural variant contexts altering the dynamics of hb expression, characterize the contribution of spatial vs temporal correlations in the Bcd system to address the question of transcriptional memory, revisit the question of Bcd target search with the development of cutting-edge imaging to measure Bcd physical parameters (concentration/mobility) and use quantitative data driven modeling to shed light on the process and its robustness. The strength of our project, relies on a unique expertise in the MS2 approach combined with an established and productive collaboration between biologists, biophysicists and theoreticians.

Oligonucleotides containing runs of three or four adjacent guanines may spontaneously arrange into four-stranded DNA supramolecular structures called G-quadruplexes (G4s). These non-canonical structures are likely to form in G-rich regions throughout the genome suggesting possible functional roles in key biological processes. Currently, G4s are thought to represent a possible third level of genetic regulation and cumulative data suggest G4s to be linked with human diseases. Therefore, they have become objects of intense study. However, the dynamic nature of these secondary structures makes their identification in living cells extremely difficult and for this reason it is a challenge to definitely establish their biological relevance. The knowledge of G4 structures about their location in the genome and their dynamics in cells are important aspects for further exploring G4 functions and its significance in both biology and medicine. The aim of this project is to develop and apply techniques that overcome the current limitations for mapping G4s in cells and to identify loci-specific G4 structures at the genome-wide level. This project will allow to develop DNA structure/function elucidation techniques that couple potent small synthetic molecules (G4-ligands) with high-throughput DNA sequencing to directly and globally map G4 structures in cells and reveal their functional and regulatory roles in mammalian cells. These objectives will be achieved by the construction of specifically tagged G4-selective non-alkylating and alkylating molecules that will enable systematic genome-wide identification of G4 ligand binding sites and, as a consequence, identification of G-rich domains that are likely to fold into quadruplexes in living cells. This new ligands will allow the development of a new chemical-immunoprecipitation-sequencing methodology (Chem-IP-Seq) to map G4 ligand binding site at single-base resolution. Our strategy is expected to provide answers to the current questions concerning the consensus sequences forming G-quadruplexes in vivo, thereby contributing to a deeper understanding of the G4 accessibility, dynamic, and regulatory roles; in particular, their connection to mutational processes associated with diseases. Unprecedently, the mapping of G4 ligand binding sites at single-base resolution will allow to connect cellular response with the exact binding site of the ligand. In fine overarching aim of this work is to validate G4s as therapeutic targets and pave the way for targeted transcriptomic analysis that will allow identifying the biological responses induced by folding and unfolding of G4s on gene expression.

Neocortical development requires tightly regulated processes, and perturbations lead to malformations (MCDs). Our groups have demonstrated the importance of the cytoskeleton during these key developmental steps. Recently, Cytoplasmic dynein 1, heavy chain 1 (DYNC1H1) mutations were identified in MCDs, as well as in motor neuron degeneration, referred to as « Dyneinopathies ». The wide spectrum of these disorders, together with dynein’s pleiomorphic cellular functions, raise fundamental questions about the effect of mutations on different cellular partners and processes. In this transversal project, we unite molecular and cellular neurobiologists, with clinicians and geneticists. This project will address crucial elements concerning the dyneinopathies, i.e. to find out the various molecular and cellular mechanisms by which disease-related dynein mutations disrupt cellular functions. We will question how distinct mutations perturb dynein’s behavior in patient-derived cells, as well as in mouse progenitors and post-mitotic neurons. Individual genotype-phenotype correlations will advance comprehension of these severe and variable disorders. Our objectives are: · To refine the phenotypic spectrum and natural history of dyneinopathies ranging from fœtus to adulthood, and to understand how DYNC1H1 mutations generate a broad spectrum of phenotypes by correlating phenotype, genotype and protein modelling of mutations, in order to provide clues concerning key and distinct mechanisms perturbed in these disorders. Related to this, we will search for mutations in other genes involved in dynein-dependent pathways. · To assess the biochemical consequences of DYNC1H1 mutations on dynein complexes and to identify their cellular consequences and effects on major dynein-dependent processes in patient-derived fibroblasts, as well as in genome-edited cell lines with different versions of mutant DYNC1H1. · To characterize the effects of selected DYNC1H1 mutations in neuronal progenitors and post-mitotic neurons in the developing mouse cortex. To this end, we will begin by studying a new Dync1h1 knock-in mouse mutant carrying the p.Lys3334Asn mutation responsible for human MCD, identifying perturbed cellular mechanisms during cortical development. These data will be compared with an existing mouse mutant model (Legs at odd angles (Dync1h1 +/Loa) that shows subtle cortical defects and a slow motor neuron loss similar to « peripheral dyneinopathies ». We will thus shed light on perturbed mechanisms specifically affecting cortical development and leading to MCDs, versus other mutations affecting motoneuron survival and potentially disturbing different dynein functions. Importantly, we will determine whether individual mutations may lead to loss of function effects while others may lead to gain of function or dominant negative effects. These experiments will provide major information on the molecular mechanisms leading to the different pathologies as well as strongly increase our knowledge on the in vivo regulation of the dynein motor. We will characterize the critical roles of mutated DYNC1H1 during proliferation, migration and differentiation of neurons in the cortex. Such studies may distinguish MCD from peripheral (SMA-LED) phenotypes and help determine pharmacological interventions, necessary in each case. In addition to exploration of the proposed pathophysiological mechanisms, these studies could have diagnostic implications, especially to assess the pathogenic effect of some rare variants for which conclusions are difficult to draw. This information will be important to better classify patients and identify those requiring early specialized care. In addition, this work will pinpoint different facets of dynein function, involving its numerous partners and the microtubule cytoskeleton, in cortical development.

Intracellular traffic is a fundamental biological process in which 10 to 20% of the proteins present in a eukaryotic cell have been estimated to, directly or indirectly, participate. Among them, small GTPases of the Rab family (over sixty proteins in humans) play a central regulatory role. Localized on the external side of all cellular compartments, they cycle between an inactive (GDP-bound) and active (GTP-bound) form. The GDP/GTP cycle is tightly coupled to a membrane association/dissociation process. Rab proteins are involved in many aspects of the life of transport intermediates (vesicles or tubules) that shuttle between compartments, such as budding from donor membranes, movement along components of the cytoskeleton, and docking/fusion events. Another important function of Rab GTPases is to regulate the dynamic formation of membrane domains on organelles. To perform their multiple tasks, Rab GTPases interact with a wide variety of effectors. Rab effectors include scaffolding proteins, adaptor proteins, lipid kinases, lipid phosphatases and tethering factors. This proposal will focus on one important class of Rab effectors, i.e. molecular motors. Three motor protein superfamilies are present in mammalian cells (kinesin, dynein and myosin) and members of all of them have been involved in a wide variety of transport events. Our group was the first to identify a direct interaction between a Rab GTPase (Rab6) and a kinesin-like protein (Rabkinesin-6, also named MKlP2 or KIF20A). Since then, several motors have been shown to be specific effectors of many Rab GTPases. It is generally thought that Rab GTPases are involved in the recruitment of molecular motors to specific membranes and/or in the regulation of motor activity. However, direct evidence for such a role for Rab GTPases is still lacking. The general objective of the present proposal is to use a combination of cell biology, biochemical and biophysical approaches to assemble a comprehensive model for the functional basis of the interactions between three Rab GTPase (Rab6, Rab8 and Rab11) with their molecular motor partners throughout their functional cycle during intracellular traffic. It involves two groups of the UMR 144 (Institut Curie, Paris) who have strong expertise in their respective fields, Rab GTPases (Bruno Goud) and 3D structure of molecular motors (Anne Houdusse). We propose to perform extensive structural-functional characterization of a range of complexes between several Rab GTPases and their interacting motor proteins to reveal the molecular basis of their selectivity.