MOLECULAR ANALYSIS OF MICROTUBULE FUNCTION IN YEAST

真核细胞中的染色体分离是由有丝分裂相介导的。 纺锤体是一种由微管和相关的 proteins. 我们的长期目标是了解 纺锤体组装和染色体分离。我们已经确定了两个 微管结合蛋白在这些过程中发挥重要作用， 酿酒酵母中的过程。 Stu1p是一个组件 的有丝分裂纺锤体，并需要组装一个双极 纺锤体。 Stu2 p是主轴杆体的一个组成部分，并且是必需的 用于主轴伸长。此外，我们还鉴定了三种蛋白质， Spi6p、Bim1p和Bik1p，它们在体内与Stu2p相互作用。 的目的 本文拟回答以下问题：1.是什么 Stu2p复合物在酵母中的性质？ 我们将确定Stu2p， Spi6p、Bim1p和Bik1p共同存在于一个独特的复合体中，或者 Stu2p是否与这些蛋白质形成单独的复合物。 我们将 还确定了Stu2p复合物的完整亚基组成 并描述这些亚基之间的相互作用。 2. 是什么 Stu2p复合体的细胞作用 我们将创建 突变的基因编码这些蛋白质，并检查 含有这些突变的细胞的表型。 这些研究将 让我们确定每个突变如何影响纺锤体功能， 微管结构的组装，以及微管在 vivo. 3. Stu2p复合物是否受细胞周期调控？ 我们将 确定Stu2p复合物蛋白质的水平和 Stu2p复合物受细胞周期调节。 此外，我们将 检查Stu2p复合物蛋白是否被磷酸化，如果是， 磷酸化是否受细胞周期调控。 4.哪些蛋白质 与Stu1p互动？作为有丝分裂纺锤体的组成部分，Stu1p可能 与各种其他主轴部件相互作用。 的 这些蛋白质的鉴定和表征对于 了解Stu1p的功能。 我们将采用遗传和 生物化学方法来鉴定与Stu1p相互作用的蛋白质， 指定它们在spindle函数中角色。

Chromosome segregation in eukaryotic cells is mediated by the mitotic spindle, a complex machine comprised of microtubules and associated proteins. Our long term goal is to understand the molecular basis of spindle assembly and chromosome segregation. We have identified two microtubule-binding proteins that play essential roles in these processes in the yeast, Saccharomyces cerevisiae. Stu1p is a component of the mitotic spindle and is required for assembly of a bipolar spindle. Stu2p is a component of the spindle pole body and is required for spindle elongation. In addition, we identified three proteins, Spi6p, Bim1p and Bik1p, that interact with Stu2p in vivo. The aims of this proposal are to answer the following questions: 1. What is the nature of the Stu2p complex in yeast? We will determine whether Stu2p, Spi6p, Bim1p and Bik1p reside together in a single unique complex, or whether Stu2p forms separate complexes with these proteins. We will also determine the complete subunit composition of the Stu2p complexes and characterize the interactions among these subunits. 2. What are the cellular roles of Stu2p complex components? We will create mutations in the genes encoding these proteins and examine the phenotypes of cells containing these mutations. These studies will allow us to determine how each mutation affects spindle function, the assembly of microtubule structures, and the dynamics of microtubules in vivo. 3. Are the Stu2p complexes cell cycle regulated? We will determine whether levels of the Stu2p complex proteins and assembly of the Stu2p complexes are cell cycle regulated. In addition, we will examine whether Stu2p complex proteins are phosphorylated and, if so, whether phosphorylation is cell cycle regulated. 4. Which proteins interact with Stu1p? As a component of the mitotic spindle, Stu1p likely interacts with a variety of other spindle components. The identification and characterization of these proteins is essential to understand how Stu1p functions. We will employ both genetic and biochemical approaches to identify proteins that interact with Stu1p and specify their roles in spindle function.