MOLECULAR ANALYSIS OF MITOTIC SPINDLE COMPONENTS

这项建议的目的是确定和研究 在分子水平上对有丝分裂纺锤体的成分进行分析。 尽管微管蛋白是纺锤体中含量最丰富的成分， 它主要起到结构性作用。微管的活性 很可能通过支配它们的分子被理解 与极点、染色体、中体的组装和相互作用 也许还有其他的纺锤结构。这项提案将重点放在 最近发现的特定分子是 纺锤形。通过与单抗反应来确定 针对磷酸化表位(MPM)，这些表位的子集 已经证明在有丝分裂时成分会被磷酸化。 并在返回到相间时去磷酸化。这个 特定纺锤体组件的磷酸化/去磷酸化 可能对纺锤体的形成起重要的调节作用， 染色体运动和纺锤体解体。这项建议 寻求确定MPM反应性磷蛋白的组成 有丝分裂的MTOCs以及识别重要的非MPM 活性成分；用生物化学方法定义磷酸化的 表位，以确定已鉴定的细胞分布 纺锤体分子中的脱磷和磷化形式 间期和有丝分裂；以分析 体外磷酸化/去磷酸化事件的检测 磷酸化对微管成核能力的影响 中心体和体内的微量注射 磷酸化/去磷酸化抑制剂；测试其功能 通过微量注射抗体对选定的纺锤体组件进行检测； 开始提纯可能的蛋白激酶(S)和 负责这些事件的磷酸蛋白磷酸酶(S)；以及 最后，尝试鉴定和克隆所选纺锤体的基因 分子。这些关于细胞分裂的基础研究可能会揭示控制 了解生化缺陷的重要机制 这会导致癌细胞不受控制地分裂。

The purpose of this proposal is to identify and study the function of components of the mitotic spindle at the molecular level. Although tubulin is the most abundant component of the spindle, it serves primarily a structural role. The activity of microtubules is likely to be understood through the molecules that govern their assembly and interaction with poles, chromosomes, the midbody and perhaps other spindle structures. This proposal will focus on specific molecules recently identified as components of the spindle. As determined by reaction with monoclonal antibodies specific for a phosphorylated epitope (MPM), a subset of these components has been shown to become phosphorylated at mitosis and dephosphorylated upon the return to interphase. The phosphorylation/dephosphorylation of specific spindle components may serve important regulatory functions for spindle formation, chromosome movement and spindle disassembly. This proposal seeks to determine the MPM-reactive phosphoprotein composition of mitotic MTOCs as well as to identify important non-MPM reactive components; to define biochemically the phosphorylated epitope, to determine the cellular distribution of identified spindle molecules in the dephospho as well as phospho forms in interphase as well as mitosis; to assay the function of phosphorylation/dephosphorylation events in vitro by assaying the effect of phosphorylation on the microtubule nucleating capacity of centrosomes and in vivo by microinjection of phosphorylation/dephosphorylation inhibitors; to test the function of selected spindle components by microinjection of antibodies; to begin the purification of putative protein kinase(s) and phosphorprotein phosphatase(s) responsible for the events; and finally, to attempt to identify and clone genes for selected spindle molecules. These basic studies on cell division may reveal control mechanisms important for understanding the biochemical defects which result in the uncontrolled division of cancer cells.