Protein translation consists in translating the genetic information carried by the messenger RNA (mRNA) to amino acids. This process is performed by the ribosome that is universally conserved in all cells. However, its structure and assembly present significant differences between bacteria and eukaryotes. On the structural level, the eukaryotic ribosome encloses more ribosomal proteins and ribosomal RNA (rRNA). In addition some of its key catalytic sites can structurally differ from their bacterial counterparts, such as the decoding center in the A-site. Partly because of these differences, the bacterial ribosome can be targeted specifically by a number of antibiotics without hindering the translation process in the eukaryotic host cells. However, the relative conservation of the ribosome among eukaryotes complicates substantially the search for specific drugs against eukaryotic pathogens such as certain protozoa like Plasmodia and kinetoplastids. Our previous work along with other studies demonstrates the existence of significant structural differences between ribosomes of certain pathogenic protozoa and mammals. Using Cryogenic electron microscopy (cryo EM), we endeavor to investigate such ribosomal structural differences. It is anticipated that, because of their location on the ribosome, these structural differences will affect some of the vital steps of protein translation especially the initiation process. Indeed this expectation is strongly suggested by the existence of large rRNA insertions, called expansion segments (ESs), at a highly conserved site involved in protein translation initiation on the ribosomes of kinetoplastids. In this proposal, using cryo EM, we will 1. focus on the structural differences between the ribosomes of certain pathogenic protozoa and mammals and their implication in translation initiation (i) by characterizing for the first time initiation complexes from kinetoplastids and compare them to their mammalian counterparts and (ii) by following up on our previous work on solving the structure of various mammalian initiation complexes. 2. We will focus on the structure of protozoa-specific ribosomal features and we will (i) draw an inventory of structures of such specific features and (ii) attempt to fish for any molecules they interact with. 3. We will investigate the structure of Plasmodial ribosomes at different stages of the parasite life cycle, as the structure of their rRNAs vary according to the latter. Cryo EM was marked the past few of years by substantial technological development on both the hardware and software levels, making it a method of choice for the structural investigation of large macromolecular assemblies at near-atomic, and possibly atomic, resolutions. Our results will unravel the structures of protein translation initiation complexes in both protozoa and mammals and will represent a framework for the design of future experiments aiming at better understanding the function of various initiation factors. Most importantly, our results will significantly enhance our knowledge of protein translation in protozoa specifically and will represent a promising step in the research for more efficient treatments against dangerous eukaryotic pathogens.

While early morphogenesis of skeletal muscles has been extensively studied in amniotes, many basic questions on later events of muscle patterning are still unknown. Based on exciting preliminary data collected by the Maire and Marcelle teams, we propose to use the chicken and the mouse models to decipher the role of so-called "primary and secondary myotubes" in the building of functional skeletal muscles. Anatomists studying muscle formation in vertebrate embryos half a century ago have identified two distinct waves of myogenesis. The first, taking place during the embryonic phase of development, generates primary myotubes. The second, observed in fetal life, results in the emergence of secondary myotubes. It is suspected that primary myotubes serve as scaffolds for the morphogenesis of the secondary lineage. Primary myotubes form before motoneurons reach the muscle masses. Myoblasts present in muscle masses during the emergence of primary and secondary myotubes (named embryonic and fetal myoblasts, respectively) display distinct morphological and biochemical characteristics in vitro. By addressing the embryonic origin and functional importance of primary and secondary myogenesis, our multi-level, multi-species project will result in key advances in our understanding of the cellular and molecular mechanisms regulating late events of myogenesis and that a comprehensive model describing muscle formation from the earliest signs of its incipience to late events of fiber type patterning will emerge from this study. We will use state of the art approaches including mouse genetic models, lineage tracing in the mouse embryo, recently developed snRNA-seq approaches, live high resolution imaging in the chick embryo to tackle the origin of myogenic progenitors’ diversity and their role in building the neuromuscular system. We will further characterize how primary myogenesis instruct secondary myogenesis and later muscle growth.