Saccharomyces cerevisiae microtubule and kinetochore dynamics

项目摘要/摘要 为了将复制的染色体准确地分离到两个子细胞，微管(MT) 细胞骨架必须完全重塑，才能形成两极纺锤体。一种大型的、动态的蛋白质复合体 着丝粒将复制的染色体连接到从相反的纺锤体极发出的微管上。 正确的动粒-微管连接对于保持基因组的完整性和预防 癌症和先天缺陷。因此，有丝分裂纺锤体组装和动粒附着必须良好。 协调一致。对这些过程及其协调的理解是这样一个事实，即有丝分裂 纺锤体和动点是极其复杂的分子机器(动点含有60种蛋白质)， 它们是多种蛋白激酶调节磷酸化的靶标，并且它们拥有惊人的 生化活动。动粒活动包括：(1)MT的侧向结合，(2)沿MT的移位 点阵到正端，(3)从横向结合到末端结合，(4)与动态MT的关联结束，而 微管蛋白亚基被交换，以及(5)作为染色体和MT+之间的力偶联装置 在后期结束。了解动粒如何执行其各种功能，结构 这些活动的基础，以及这些活动是如何在翻译后得到规范的，远远不是 完成。巴恩斯实验室最近开发的一种生化细胞裂解物分析方法首次联合 TIME，微管动力学研究最有力的两种方法：生化提取物研究 和遗传学。单个微管和与这些微管相关的单个动点的动力学是 用高灵敏度全内反射荧光显微镜进行了揭示和定量分析。 与特定细胞周期阶段同步的细胞裂解物是由特定有丝分裂的芽酵母突变体制成的 蛋白质。对MT动力学和动粒活动的定量分析将确定这些参数如何 在细胞周期中被调节，这些研究将确定执行特定的 行为。由于有丝分裂是一个高度保守的过程，从这些研究中吸取的经验教训有望 适用范围广泛。与许多其他化验方法不同，这种化验方法只使用同源的微管蛋白和 相互作用的蛋白质，避免因物种不匹配不相容而产生的人工制品。建议的研究 建立在最近对微管动力学调节和动粒动态活动的独特观察基础上 这个裂解系统。本研究的目的是：(1)研究完整动粒的生化活性和它们的生物活性。 调节，并将动粒结构与功能联系起来；以及(2)研究微管动力学如何 通过细胞核和细胞质中的细胞周期进行调节，重点是Kar3和Kip3激动素。

Project Summary/Abstract For replicated chromosomes to be segregated to two daughter cells accurately, the microtubule (MT) cytoskeleton must be completely remodeled to form a bipolar spindle. A large, dynamic protein complex called the kinetochore attaches replicated chromosomes to microtubules emanating from opposite spindle poles. Proper kinetochore-microtubule attachment is vital for preservation of genomic integrity and prevention of cancer and birth defects. Therefore, mitotic spindle assembly and kinetochore attachment must be well coordinated. Challenging understanding of these processes and their coordination is the fact that mitotic spindles and kinetochores are extremely complex molecular machines (kinetochores contain >60 proteins), that they are targets of phosphoregulation by multiple protein kinases, and that they possess a striking range of biochemical activities. Kinetochore activities include: (1) lateral MT binding, (2) translocation along the MT lattice to the plus end, (3) conversion from lateral to end-binding, (4) association with dynamic MT ends while tubulin subunits are exchanged, and (5) serving as force-coupling devices between chromosomes and MT plus ends during anaphase A. Understanding how the kinetochore performs its various functions, the structural underpinnings of these activities, and how these activities are regulated post-translationally, is far from complete. A biochemical cell-lysate assay recently developed in the Barnes laboratory combines, for the first time, two of the most powerful approaches for studies of microtubule dynamics: biochemical extract studies and genetics. Dynamics of single microtubules and single kinetochores associated with these microtubules are revealed and quantitatively analyzed by highly sensitive Total Internal Reflection Fluorescence microscopy. Cell lysates synchronized to specific cell-cycle stages are made from budding yeast mutants of specific mitotic proteins. Quantitative analysis of MT dynamics and kinetochore activities will establish how these parameters are regulated in the cell cycle, and these studies will identify the specific proteins that carry out the specific behaviors. Since mitosis is a highly conserved process, lessons learned from these studies are expected to apply broadly. Unlike many other assays, this assay exclusively uses homologous sources of tubulin and interacting proteins, avoiding artefacts that arise from species mismatch incompatibilities. Proposed studies build upon recent unique observations of microtubule dynamics regulation and kinetochore dynamic activity in this lysate system. The objectives are: (1) To investigate biochemical activities of intact kinetochores and their regulation, and to relate kinetochore structure to function; and (2) To investigate how microtubule dynamics are regulated through the cell cycle in both the nucleus and the cytoplasm, focusing on Kar3 and Kip3 kinesins.