MOLECULAR ANALYSIS OF MITOTIC SPINDLE COMPONENTS

本提案的目的是确定和研究 有丝分裂纺锤体的分子结构。 虽然微管蛋白是纺锤体中最丰富的成分， 它主要起结构性作用。 微管的活性 很可能通过控制它们的分子来理解。 组装和相互作用与极点，染色体，中间体 以及可能的其他纺锤体结构。 该提案将重点关注 最近鉴定出的特定分子是 纺锤体。 通过与单克隆抗体反应测定 特异于磷酸化表位（MPM），这些表位的子集 已经显示，在有丝分裂时， 并在返回间期时去磷酸化。 的 特定纺锤体组分的磷酸化/去磷酸化 可能对纺锤体的形成起到重要的调节作用， 染色体运动和纺锤体解体。 这项建议 试图确定MPM反应性磷蛋白的组成 有丝分裂MTOC以及识别重要的非MPM 反应性成分;以生物化学方式定义磷酸化的 表位，以确定鉴定的表位的细胞分布。 纺锤体分子在脱磷酸以及磷酸形式， 间期和有丝分裂;以测定功能， 磷酸化/去磷酸化事件，通过测定 磷酸化对微管成核能力的影响 和在体内通过显微注射 磷酸化/去磷酸化抑制剂;测试功能 通过显微注射抗体， 开始纯化推定的蛋白激酶， 导致事件的磷蛋白磷酸酶;和 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揭示控制 对于理解生物化学缺陷的重要机制 导致癌细胞不受控制的分裂。

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.