Identification of new regulators of myonuclei positioning during skeletal muscle fibers development

Description

Skeletal muscle fibers are built from fusion of myoblasts allowing the formation of myotubes. Those syncytia contain hundreds of nuclei that undergo many movements, simultaneously with myotubes maturation process, until reaching a final peripheric localization in mature fibers (myofibers). Disorganization of nuclei disposal is always associated with myofibers misfunctioning (i.e.: sarcopenia, centronuclear myopathy). Peripheral nuclei positioning in mature fiber appears to be essential for their functionality. Understanding the process of nuclei positioning turned up as key point.

Involvement of molecular motors are known in spacing myonuclei in the first steps of muscle differentiation, however the role of microtubules associated proteins (Maps) is less described. Consequently, identify new Maps involved in nuclei positioning during muscle formation may 1) provide a better understanding of muscle differentiation; 2) bring to light new targets potentially involved in pathologies presenting nuclei mispositioning.

In this context, my thesis work consisted in identifying proteins linked to microtubules, directly or indirectly, and involved in the regulation of myonuclei positioning throughout skeletal muscle fibers differentiation. Proteomes associated to microtubules and differentially expressed during muscle differentiation were identified using a mass-spectrometry analysis. Based on this identification, 239 proteins from the total cellular extracts of myotubes and myofibers; and 240 proteins identified in proteomes linked to microtubules were selected. A siRNA screen was performed on primary myotubes in order to decipher the involvement of those selected targets in myonuclei positioning during the first steps of muscle differentiation. This siRNA screen allowed the identification of three proteins which roles on myonuclei positioning appeared to be crucial.

Unexpectedly, we identified a cytoplasmic role of the mitotic protein NuMA1 in muscle biology field. This protein progressively accumulates into the cytoplasm where it stabilizes microtubule network in differentiating myofibers. This cytoplasmic fraction of NuMA1 proteins, in association with the dynein, regulates microtubule network architecture and myonuclei spacing. Its role is also crucial in maintaining peripherized myonuclei in mature myofibers and in neuromuscular junction regions.