Dissection de la fonction du RCPG d'adhésion BAI3 dans la fusion des myoblastes

Myoblast fusion is a critical step in embryonic muscle development and muscle regeneration in adults. Myoblasts govern this process by establishing cell-cell contacts and fusing their membranes. Genetic studies in Drosophila have shown the importance of activating the small GTPase Rac. This event is possible thanks to its activator, guanine nucleotide exchange factor Myoblast City, and its scaffold protein ELMO. The degree of conservation of molecules involved in myoblast fusion between Drosophila and vertebrates was unknown. Until teams demonstrated that the components regulating the reorganization of the cytoskeleton in Drosophila were conserved in vertebrates. Thus Wasp, Rac and Dock180 (Myoblast City mammal orthologue) are all essential for the fusion of myoblasts in both systems. This machinery is activated by well-characterized surface receptors in Drosophila. Their equivalents remain unknown in vertebrates. The hypothesis to answer this question is that if a myoblast cell surface is able to interact with the ELMO / DOCK complex then it will be a potential candidate to be a regulator of myoblast fusion. The first objective of the thesis is to identify the surface receptor capable of regulating myoblast fusion in vertebrates. As ELMO / DOCK are important for fusion, then they will be able to directly bind the pro-fusion receptor. Thus, using ELMO as prey, the Adhesion G protein-coupled receptor BAI3 has been identified. It induces fusion by its interaction with the intracellular complex ELMO/DOCK/Rac. The involvement of this complex is well established in the fusion of myoblasts. Loss of Function studies of BAI3 and ELMO in C2C12 cells showed fusion blocking of myoblasts without affecting the differentiation program. Moreover, during the embryonic development, the presence of mutant BAI3, unable to bind to ELMO2, completely inhibits the fusion. In conclusion, this objective made it possible to identify BAI3, its role in fusion as well as the importance of its interaction with ELMO2 to complete the fusion of vertebrate myoblasts. While the role of BAI3 is defined in myoblast fusion, the second objective is to understand the regulation of BAI3. As myoblast fusion is a precisely regulated process, its regulator, BAI3, must be regulated as well. Since BAI3 is a GPCR then it must have ligand (s) iv regulating the activation of the receptor and the influence of this signal should affect the recruitment of ELMO2. Thus, two proteins that regulate the activation of BAI3 have been identified. The first is the small secreted protein C1qL4 that acts as an inhibitor of BAI3 during myoblast fusion. The second partner is the transmembrane receptor Stabilin-2. Thus Stabilin-2 interacts with the extracellular region of BAI3 and activates it. BAI3 transmits the signal by triggering the canonical mechanism of GPCRs which will allow the recruitment of the ELMO / DOCK complex at the membrane. In conclusion, this thesis identified the first surface receptor capable of regulating myoblast fusion, previously unknown in vertebrates. In addition, the regulatory mechanism of BAI3 was also characterized with the C1qL4 inhibitory function and the Stabilin-2 activator function. These new players and this mechanism undoubtedly represent new therapeutic avenues for the treatment of muscular dystrophies.

Keyphrases: Myogenesis, Myotube formation, GPCR signaling, cell-cell fusion, Model system, Formation de myotubes