SKeletal Muscle - REgulation of FORce and Maturation through Tubulin isotypes

Objective

Microtubules are formed by polymerization of tubulin heterodimers. Tubulin monomers rise from 16 genes each with their own spatiotemporal signature, resulting in a patterned microtubule network. As tubulin dimers define binding microtubule associated proteins (MAPs) and occurring post-translational modifications (PTMs), I wonder whether microtubule properties and patterns are encoded/defined by the tubulin isotypes themselves. This proposal aims at 1-confirmation of the expressed tubulin isotypes in skeletal muscle, depending on developmental stages, sex, and fiber type. 2-Understanding of how tubulin isotypes contribute to macroscopic phenotypes such as myoblasts fusion, general fiber organization and muscle contraction. 3-Deciphering of the molecular mechanisms at play upon tubulin isotype loss-of-function. 4-Assessement of potential beneficial effects of tubulin loss-of-function in diseases such as Duchenne muscular Dystrophy (DMD), Centronuclear Myopathy (CNM), and myasthenia. Using innovative in vitro differentiation tools, animal models and state of the art microscopy, I will explore all potential effects of selected tubulin isotypes, from macroscopic phenotypes to molecular mechanisms, as well as in disease. Expected techniques to use are: RT-q-PCR, co-cultures motoneurons-myoblasts, CRISPR/Cas9 mediated KO, shRNA KD, gene transfer by AAV injection, force measurement in vivo and ex vivo, immunohisto- and cytochemistry, live-imaging with fluorescent probes, and image analysis. Microtubule and tubulin being involved in many types of diseases (cancer, development, degeneration…) this project will lead to many economic, social and therapeutic benefits.