MOLECULAR ANALYSIS OF MITOTIC SPINDLE COMPONENTS

本提案的目的是确定和研究 有丝分裂纺锤体的组成部分在分子水平。 虽然微管蛋白 是纺锤体中最丰富的成分，它主要用于 结构作用。 微管的活动很可能被理解为 通过控制其组装和相互作用的分子 两极、染色体和其他纺锤体结构。 这项建议 将集中在最近确定的特定分子的成分， 主轴和识别组件迄今未知。 最近 鉴定的组分是微管相关蛋白（MAP）， 210，000和125，000的分子量，我们首次从癌细胞中分离到 细胞系HeLa。 目前未知的成分将由一个 新技术和旧技术的结合。 微管亲和方法将 用于从培养的细胞制备MAP。 细胞分级分离将是 用于制备有丝分裂纺锤体、中心体和中间体 培养细胞 将使用单克隆抗体技术制备 MAPs抗体和鸟枪抗体用于其他有丝分裂纺锤体成分。 用快速间接免疫荧光法筛选克隆的杂交瘤细胞， 用于分泌梭形阳性抗体免疫荧光方法。 将对可能的纺锤体组分抗体进行特异性表征 和交叉反应性。 抗原将通过光和电子的结合定位在细胞中 显微免疫细胞化学 地图的功能和其他确定的 将通过使用抗体来研究纺锤体成分 抑制适当的体外测定。 对于MAP，检测试剂盒将检测 用于抑制与微管的结合，以确定特异于 分子的功能结构域。 对于中心粒周围的分子 云，该测定将测试微管成核的抑制 由微管蛋白亚基形成。 还将通过以下方式测试功能： 确定将选定的抗体显微注射到活体中的效果 细胞 将通过光学显微镜分析选定的活细胞， 随后，将通过免疫细胞化学和/或 电子显微镜检查以确定抗体的作用。 这些基本 对细胞分裂的研究可能揭示了对细胞分裂的重要控制机制。 了解导致不受控制的 癌细胞的分裂。

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 and perhaps other spindle structures. This proposal will focus on specific molecules recently identified as components of the spindle and on identifying components hitherto unknown. The recently identified components are the microtuble-associated proteins (MAPs) of 210,000 and 125,000 molecule weight first isolated by us from the carcinoma cell line, HeLa. The as yet unknown components will be sought by a combination of new and old technologies. Microtubule affinity methods will be used to prepare MAPs from cultured cells. Cell fractionation will be used to prepared mitotic spindlese, centrosomes and mid-bodies from cultured cells. Monoclonal antibody techniques will be used to prepare antibodies to MAPs and to shotgun for other mitotic spindle components. The cloned hybridomas will be screened by a rapid indirect immunofluorescence method for secretion of spindle-positive antibodies. Likely spindle component antibodies will be characterized for specificity and cross-reactivity by a gel binding assay and by immunoprecipitation. Antigens will be localized in cells by a combination of light and electron microscopic immunocytochemistry. The function of MAPs and other identified spindle components will be investigated through the use of antibody inhibition of appropriate in vitro assays. For MAPs, the assays will test for inhibition of binding to microtubles to define reagents specific for functional domains of the molecules. For molecules of the pericentriolar cloud, the assays will test for inhibition of nucleation of microtuble formation from tubulin subunits. Function will also be tested by determining the effects of microinjecting selected antibodies into living cells. Selected living cells will be analyzed by light microscopy and subsequently the same cell will be analyzed by immunocytochemical and/or electron microcopy to define the action of the antibody. 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.