Mechanism of Spindle Pole Body Duplication and Separation in Yeast Meiosis

酵母中心体，也称为纺锤体，组织包括细胞分裂在内的各种细胞过程所需的微管。在减数分裂期间，中心体在染色体复制时进行复制。在减数分裂的第一次分裂中，重复的中心体分离形成双极纺锤体，这是同源体分离所必需的。在第二次减数分裂中，中心体在没有染色体复制的情况下再次复制，为姐妹染色单体的分离建立两条纺锤体。真核生物减数分裂中中心体复制和分离与两条染色体分离周期的协调是维持基因组完整性的基础。本项目的目的是确定极光激酶Ipl1和polo样激酶Cdc5拮抗调控中心体复制和分离的分子机制。利用创造性酵母遗传学结合活细胞显微镜和生化方法，将确定Ipl1和Cdc5在中心体复制中的时间和空间要求。此外，它们的磷酸化底物将通过一种新的磷酸化蛋白质组方法进行鉴定，并表征它们在中心体动力学调节中的生化活性。这些研究将提供酵母中心体复制中Ipl1和Cdc5介导的蛋白磷酸化的机制观点，并对理解动物中心体动力学机制具有普遍意义。更广泛的影响该项目将培养遗传学和细胞生物学的研究生和本科生，并将为代表性不足和少数民族的学生提供独特的机会，让他们在课堂上设计和进行目标导向的实验。参加实验生物学课程（PI每年教授一次）的学生将用绿色荧光蛋白标记中心体成分，并筛选酵母中中心体的遗传异常。这项研究将为中心体复制的调控提供分子见解，并通过教育那些将成为我们未来工作人员的学者和科学家，为社会做出贡献。

Intellectual meritThe yeast centrosome, also called the spindle pole body, organizes microtubules required for a variety of cellular processes that include cell division. During meiosis, the centrosome is duplicated when chromosomes replicate. Duplicated centrosomes separate to form a bipolar spindle required for homolog separation during the first division of meiosis. Centrosomes duplicate again in the absence of chromosome replication to establish two spindles for sister chromatid separation in the second meiotic division. Coordination of centrosome duplication and separation with the two chromosome segregation cycles in meiosis is fundamental to maintain genome integrity in eukaryotes. The goal of this project is to determine the molecular mechanism by which the Aurora kinase Ipl1 and the Polo-like kinase Cdc5 act antagonistically to regulate centrosome duplication and separation. Using creative yeast genetics in combination with live-cell microscopy and biochemical methods, the temporal and spatial requirements of Ipl1 and Cdc5 in centrosome duplication will be determined. In addition, their phosphorylated substrates will be identified by a novel phosphoproteome approach and characterized for their biochemical activities in regulation of centrosome dynamics. These studies will provide a mechanistic view of protein phosphorylation mediated by Ipl1 and Cdc5 in yeast centrosome duplication and have general implications for understanding the mechanism of centrosome dynamics in animals.Broader impactsThis project will train graduate and undergraduate students in genetics and cell biology, and will also provide unique opportunities for underrepresented and minority students to design and conduct goal-oriented experiments in their classroom. Students enrolled in the Experimental Biology course, which the PI teaches once a year, will tag centrosome components with the green fluorescent protein and screen for genetic abnormalities of the centrosome in yeast. This research will provide molecular insights into the regulation of centrosome duplication and contribute to society by educating those who will be part of our future work force as scholars and scientists.