Mechanism and Regulation of Kinetochore assembly in budding yeast

细胞分裂过程中的基因组分离需要染色体和微管聚合物之间动态的、受调节的相互作用。为此目的，每条染色体在着丝粒处组装成一个大的多亚基蛋白复合体--动粒。包含超过40个结构亚基，动粒一方面作为传感器，监测染色体微管相互作用的质量，支持它们的校正，并协调后期开始与细胞周期机制。另一方面，动粒以能够驱动染色体运动的方式提供与动态微管加端的偶联。着丝粒的分子结构如何允许机械化学信号和加端偶联是关键的未回答的问题。在这里，我们建议调查的动粒结构和功能的芽殖酵母酿酒酵母，使用生化重组实验和遗传分析相结合。基于我们以前的分析，我们专注于这一建议的两个方面的动粒组装：首先，我们将调查的保守亚基Mif 2/CENP-C在组装过程中的作用。其次，我们将分析有丝分裂激酶如何控制着丝粒COMA复合体的组装。总之，我们的分析将提供新的机械见解的基本染色体分离机制的细胞。

Genome segregation during cell division requires dynamic, regulated interactions between chromosomes and microtubule polymers. For this purpose each chromosome assembles at the centromere a large multisubunit protein complex - the kinetochore. Containing more than 40 structural subunits, kinetochores on the one hand act as sensors that monitor the quality of chromosome-microtubule interactions, support their correction, and coordinate anaphase onset with the cell cycle machinery. On the other hand, kinetochores provide coupling to dynamic microtubule plus-ends in a manner that can drive chromosome motion. How the molecular construction of kinetochores allows mechanochemical signalling and plus-end coupling are key unanswered question. Here we propose to investigate the kinetochore architecture and function in the budding yeast Saccharomyces cerevisiae, using a combination of biochemical reconstitution experiments and genetic analysis. Building on our previous analysis we focus in this proposal on two aspects of kinetochore assembly: First, we will investigate the role of the conserved subunit Mif2/CENP-C during assembly. Second, we will analyze how mitotic kinases control assembly of the COMA complex at kinetochores. Taken together our analysis will provide new mechanistic insights into the fundamental chromosome segregation machinery of the cell.