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

准确的染色体分离对所有真核生物的基因组稳定性是至关重要的。有丝分裂检查点是一种故障安全机制，可以确保准确的染色体分离。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控发生的基础。MIS12(微小染色体不稳定性)是决定KT和有丝分裂检查点蛋白组装的关键着丝粒蛋白之一。着丝粒蛋白Mis12以蛋白质复合体的形式存在，并指定CENP-A独立的着丝粒组装途径。我们假设Mis12复合体对于着丝粒结构的完整性以及有丝分裂检查点信号是重要的。为了了解Mis12的功能机制，我们建议对Mis12的结构和功能进行研究。我们将通过筛选hMis12的随机连接子扫描以及缺失和截断突变集合来定位hMis12的着丝粒定位区域。为了了解MIS12复合体中亚基之间的相互作用，我们将通过酵母双杂交试验、免疫共沉淀和GST下拉实验筛选蛋白质-蛋白质相互作用结构域的hMIS12突变体集合(S)。荧光共振能量转移技术将用于原位分析Mis12复合体之间的蛋白质相互作用。HMis12和hMis12复合体对细胞周期特异着丝粒动力学的调节将通过光漂白后的荧光恢复来分析。将分析在特定蛋白质-蛋白质相互作用中存在缺陷的MIS12个突变体在体内的功能后果(对有丝分裂检查点的影响，动粒-MT相互作用的稳定性，以及动粒结构的组装)。

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).