Defining the role and essential interactions of the centrosomal protein pericentrin in microtubule formation during myogenesis

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Muscle fibers are long multinucleated cells formed from the fusion of myoblasts in a process called myogenesis. Nuclei are arranged regularly along the periphery of the myofiber. Correct nuclear positioning involves interactions between nuclear envelope proteins, centrosomal proteins and motor proteins. Aberrant positioning such as nuclear clustering is associated with muscular dystrophies, suggesting that correct nuclear postioning is important for the formation of healthy functioning skeletal muscle. Yet, the details of the molecular mechanisms and the precise roles of proteins involved in nuclear positioning are unclear. In myoblasts, proteins of the microtubule organising centre (MTOC) are localised at the centrosome. It has been shown that just hours after differentiation, markers of the MTOC are released from the centrosome and instead localise at the perinuclear surface. These proteins retain their role as a microtubule nucleation site. Our hypothesis is that centrosomal proteins localise to the perinuclear surface by binding to nesprin-1, a nuclear envelope protein that is part of the LINC complex that physically links the nucleus to cytoskeletal components. This is supported by the fact that mutations in LINC complex components can cause nuclear clustering. An explanation could be because recruitment of MTOC proteins to the nuclear envelope and subsequent microtubule reorganisation becomes impaired. Since motor proteins use microtubules to move cargo, nuclear movement to the cell periphery becomes affected. This project will test our hypothesis, leading to a comprehensive understanding of the roles of centrosomal proteins in microtubule nucleation during myogenesis. We will identify the function of specific protein involved in microtubule nucleation and nuclear positioning, and define the protein interactions using a range of cell biology and structural techniques. This will build upon our current molecular model of myogenesis.