ROLE OF AUGMIN IN HUMAN MITOTIC SPINDLES

这个子项目是许多利用资源的研究子项目之一 由NIH/NCRR资助的中心拨款提供。子项目的主要支持 而子项目的主要调查员可能是由其他来源提供的， 包括其它NIH来源。 列出的子项目总成本可能 代表子项目使用的中心基础设施的估计数量， 而不是由NCRR赠款提供给子项目或子项目工作人员的直接资金。 有丝分裂纺锤体是一种双极微管阵列，在有丝分裂过程中染色体分离中起着关键作用。通常认为中心体是纺锤体中微管的主要成核中心。然而，通过实时成像和功能研究，包括我们在内的几个实验室最近提出了一种新的模型，其中许多纺锤体微管在纺锤体内部产生，因此，微管的负端分布在整个纺锤体中。此外，我们已经鉴定了负责纺锤体内微管成核的蛋白质复合物“augmin”，其中新的微管可能从现有微管成核（“分支成核”）（Goshima et al. 2007，2008; Uehara et al. 2009，2010）。然而，这些模型尚未得到证实，因为负末端的分布尚未通过电子显微镜在人类细胞中确定。在这项研究中，我们想测试的模型，通过直接可视化的负端纺锤体微管使用电子断层扫描。具体来说，我们希望进行1）三维数据的断层重建和人U2 OS细胞中期纺锤体的三维建模。2)对缺失augmin的U2 OS细胞进行相同的分析，这导致中期纺锤体中微管数量减少，并与对照细胞进行比较。

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. Primary support for the subproject and the subproject's principal investigator may have been provided by other sources, including other NIH sources. The Total Cost listed for the subproject likely represents the estimated amount of Center infrastructure utilized by the subproject, not direct funding provided by the NCRR grant to the subproject or subproject staff. The mitotic spindle, a bipolar microtubule array, plays a critical role in chromosome segregation during mitosis. Conventionally, the centrosome is considered to be th emajor microtubule nucleation center in the spindle. However, through live imaging and functional studies, several laboratories including ours have recently proposed a new model in which many spindle microtubules are generated inside the spindle, and therefore, minus-ends of the microtubules are distributed throughout the spindle. Furthermore, we have identified a protein complex 'augmin' that is responsible for the intra-spindle microtubule nucleation, in which new microtubules are possibly nucleated from the existing microtubules ("branched nucleation") (Goshima et al. 2007, 2008; Uehara et al. 2009, 2010). However, these models have not been proven, because the distribution of minus-ends have not been determined by electron microscopy in human cells. In this study, we would like to test the models through directly visualizing the minus-ends of spindle microtubules using electron tomography. Specifically, we wish to perform 1) Tomographic reconstruction of 3-D data and 3-D modeling for the metaphase spindle of human U2OS cells. 2) the same analysis for the U2OS cells depleted of augmin, which resulted in reduced microtubule numbers in the metaphase spindle, and compare with the control cells.