Mechanism and Regulation of Kinetochore assembly in budding yeast

在细胞分裂过程中，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上组装一个大的多亚基蛋白质复合物-着丝点。着丝点含有40多个结构亚基，一方面充当传感器，监测染色体与微管相互作用的质量，支持它们的纠正，并协调细胞周期机制的后期开始。另一方面，着丝点以驱动染色体运动的方式为动态微管正端提供耦合。着丝点的分子结构如何允许机械化学信号和正端偶联是关键的未解之谜。本文拟采用生物化学重组实验和遗传分析相结合的方法，对出芽酵母酿酒酵母的着丝粒结构和功能进行研究。基于我们之前的分析，我们在本提案中关注着丝点组装的两个方面：首先，我们将研究保守亚基Mif2/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点上COMA复合体的组装。综上所述，我们的分析将为细胞的基本染色体分离机制提供新的机制见解。

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.