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

肌纤维是由成肌细胞在肌发生过程中融合形成的长多核细胞。细胞核沿肌纤维周围有规律地排列。正确的核定位涉及核膜蛋白、中心体蛋白和运动蛋白之间的相互作用。异常定位，如核聚集与肌肉营养不良有关，这表明正确的核定位对形成健康功能的骨骼肌很重要。然而，分子机制的细节和参与核定位的蛋白质的确切作用尚不清楚。在肌母细胞中，微管组织中心（MTOC）的蛋白质位于中心体。研究表明，在分化数小时后，MTOC的标记物从中心体中释放出来，转而定位于核周表面。这些蛋白保留了它们作为微管成核位点的作用。我们的假设是中心体蛋白通过与nesprin1结合定位到核周表面，nesprin1是一种核膜蛋白，是LINC复合物的一部分，物理上将细胞核与细胞骨架成分连接起来。LINC复合组分的突变可引起核聚类，这一事实支持了这一点。一种解释可能是由于MTOC蛋白向核膜的募集和随后的微管重组受到损害。由于运动蛋白使用微管运输货物，核向细胞周围的运动受到影响。该项目将验证我们的假设，从而全面了解中心体蛋白在肌发生过程中微管成核中的作用。我们将确定参与微管成核和核定位的特定蛋白质的功能，并使用一系列细胞生物学和结构技术定义蛋白质相互作用。这将建立在我们目前的肌生成分子模型的基础上。

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.