Structure and function of Mis12 (Minichromosome Instability) centromere complex in mitosis

精确的染色体分离对于所有真核生物的基因组稳定性是必不可少的。有丝分裂检查点是确保准确染色体分离的故障安全机制。动粒（kT）是有丝分裂检查点蛋白停靠的结构平台，因此是有丝分裂检查点调节发生的基础。Mis 12（minichromosome instability，微小染色体不稳定性）是一种重要的着丝粒蛋白，它决定了kT和有丝分裂检查点蛋白的组装。着丝粒蛋白Mis 12作为蛋白质复合物存在，并指定CENP-A独立的着丝粒组装途径。我们假设Mis 12复合物对于着丝粒结构完整性以及有丝分裂检查点信号传导是重要的。为了了解Mis 12的功能机制，我们建议研究Mis 12的结构和功能。我们将通过筛选随机连接子扫描以及hMis 12缺失和截短突变体集合来定位hMis 12的着丝粒定位结构域。为了了解Mis 12复合物中亚基之间的相互作用，我们将通过酵母双杂交测定、免疫共沉淀和GST下拉实验筛选hMis 12突变体集合的蛋白质-蛋白质相互作用结构域。荧光共振能量转移技术将用于原位分析Mis 12复合物之间的蛋白质相互作用。将通过光漂白后荧光恢复技术分析hMis 12和hMis 12复合物的细胞周期特异性着丝粒动力学的调节。将分析特异性蛋白质-蛋白质相互作用缺陷的Mis 12突变体的体内功能后果（对有丝分裂检查点的影响、动粒-MT相互作用的稳定性和动粒结构的组装）。

Accurate chromosome segregation is essential for the genomic stability of all eukaryotes. The mitotic checkpoint is a failsafe mechanism that ensures accurate chromosome segregation. The kinetochore (kT) is the structural platform where mitotic checkpoint proteins dock and therefore, is the foundation of where mitotic checkpoint regulation occurs. Mis12 (minichromosome instability) is one of the key centromere proteins that specify kT and mitotic checkpoint protein assembly. The centromere protein Mis12 exists as a protein complex and specifies a CENP-A independent centromere assembly pathway. We hypothesize that the Mis12 complex is important for centromere structural integrity as well as mitotic checkpoint signaling. To understand the mechanism of Mis12 function, we propose to study the structure and function of Mis12. We will map the centromere localization domain of hMis12 by screening a random linker scanning as well as deletion and truncation mutant collection of hMis12. To understand the interaction between subunits in the Mis12 complex, we will screen the hMis12 mutant collection for protein-protein interaction domain(s), by yeast 2-hybrid assays, co-immunoprecipitation and GST pull-down experiments. Fluorescence Resonance Energy Transfer technique will be used to analysis protein interactions amongst the Mis12 complex in situ. The regulation of cell-cycle specific centromere dynamics of hMis12 and the hMis12 complex will be analyzed by the Fluorescent Recovery After Photobleaching technique. Mis12 mutants that are defective in specific protein-protein interaction will be analyzed for the functional consequence in vivo (effects on the mitotic checkpoint, the stability of kinetochore-MT interaction, and assembly of the kinetochore structure).