Identification of proteins involved in the establishment of the centrosome-independent, polarized microtubule cytoskeleton during differentiation of skeletal muscle cells

许多依赖微管的过程需要将微管排列成合适的阵列。产生微管模式涉及成核的控制以及微管两端的调节稳定。典型的组织培养细胞含有从中心体发出的放射状微管阵列。但是微管组装的成核并不局限于中心体，并且在植物、真菌和分化的动物细胞中经常独立于主要的MTOC而发生。然而，非中心体微管成核和锚定的详细机制知之甚少。在拟议的项目中，微管重排的机制将在分化肌细胞中进行研究。在肌形成过程中，中心体被消除，而微管在核表面成核。对于几种蛋白质，从中心体到核周边的搬迁被证明，但其招聘机制仍有待发现。此外，先前等长的成肌细胞在分化过程中早期伸长，并且在发育中的肌管的两极之间形成平行的微管束，这很可能需要将正端锚定在细胞两极以及沿着它们的长度进行广泛的微管稳定化沿着以促进长微管聚合物的生长。该项目的主要目的是详细描述肌形成过程中的微管重组，并鉴定和表征参与建立非中心体成核位点和稳定平行微管到达细胞两极的蛋白质。这将有助于我们理解在肌细胞分化过程中细胞形态的调节和一般的中心体独立微管模式的产生所必需的机制。

Many microtubule-dependent processes require the arrangement of microtubules into suitable arrays. Generating microtubule patterns involves control of nucleation as well as regulated stabilization ob both microtubule ends. Typical tissue culture cells contain a radial array of microtubules emanating from the centrosome. But nucleation of microtubule assembly is not restricted to the centrosome and occurs frequently independent of a major MTOC in plants, fungi and differentiated animal cells. However, detailed mechanisms of non-centrosomal microtubule nucleation and anchorage are poorly understood. In the proposed project, mechanisms underlying microtubule rearrangements will be investigated in differentiating muscle cells. During myogenesis the centrosomes are eliminated while microtubules are nucleated on the nuclear surface. For several proteins, a relocation from the centrosome to the nuclear periphery was shown, but the mechanism of their recruitment remain to be discovered. Furthermore, previously isometric myoblasts elongate early during differentiation and parallel bundles of microtubules are formed between the poles of the developing myotubes, which most likely requires anchoring of plus ends at the cell poles as well as extensive microtubule stabilization along their length to promote the growth of long microtubule polymers. The major aim of this project is to describe the microtubule reorganization during myogenesis in detail and to identify and characterize proteins involved in the establishment of non-centrosomal nucleation sites and in the stabilization of parallel microtubules reaching the cell poles. This will help us to understand the mechanisms that are essential for the modulation of cell morphology during muscle cell differentiation and for the generation of centrosome-independent microtubule patterns in general.