MECHANISM AND CONTROL OF MICROTUBULE ASSEMBLY

微管是一种动态结构， 在间期细胞中的细胞质，并提供结构 有丝分裂中纺锤体的结构和可能的动力 细胞 本提案的主要目标是了解 微管动力学在体内和体外，并确定是否 选择的有丝分裂事件取决于动力学性质。 将使用各种实验方法，包括 蛋白质的荧光和半抗原标记，显微注射，半抗原， 介导的免疫细胞化学，荧光光漂白，和 数字成像显微镜 这些方法允许测定 微管蛋白亚基的添加位点从前 现有的微管。 它们还允许确定 微管网络的周转动力学。 后修饰的亚基将通过以下鉴定： 用肽特异性抗体进行间接免疫荧光。 的 主要的具体目标将是完成我们的分析， 后期动态;确定周转机制， 中期;以确定亚基添加和损失的网站， 前中期的集合体;进一步研究微管 成纤维细胞前板层的动态变化及其 与细胞运动的关系;获得直接证据， 细胞中心附近微管周转的机制;测试 两个翻译后修饰的作用， 乙酰化，对微管周转的影响;评价 微管在高度分化的细胞类型，PC-12 神经元细胞系;并直接测试结构假设 缓慢的轴突运输。 结果应该有助于阐明确切的性质， 微管不稳定性的意义 结果也应该是 与调节运动和分裂的机制有关， 正常细胞 对正常细胞运动的理解， 分裂可以帮助分析癌细胞， 限制分裂的机制是异常的。

Microtubules are dynamic structures that serve to organize the cytoplasm in interphase cells and to provide the structural framework and possibly the motive force for the spindle in mitotic cells. The broad objectives of this proposal are to understand microtubule dynamics in vivo and vitro and to determine whether selected mitotic events are dependent upon the dynamic properties. A variety of experimental approaches will be used including fluorescent and hapten tagging of proteins, microinjection, hapten- mediated immunocytochemistry, fluorescence photobleaching, and digital imaging microscopy. These methods allow determination of the sites of tubulin subunit addition into an loss from pre- existing microtubules. They also permit a determination of the kinetics of turnover of the microtubule network. Posttranslationally modified subunits will be identified by indirect immunofluorescence with peptide specific antibodies. The principal specific aims will be to complete our analysis of anaphase dynamics; to determine the mechanism of turnover in metaphase; to identify sites of subunit addition and loss during congression in prometaphase; to investigate further microtubule dynamics in the leading lamellum of fibroblasts and their relationship to cell motility; to obtain direct evidence for the mechanism of microtubule turnover near the cell center; to test the role of two posttranslational modification, detyrosination and acetylation, on microtubule turnover; to evaluate the stability of microtubules in a highly differentiated cell type, the PC-12 neuronal cell line; and to directly test the structural hypothesis of slow axonal transport. The results should help elucidate the precise nature and significance of microtubule lability. The results should also be of relevance for mechanisms regulating motility and division in normal cells. An understanding of normal cell motility and division may assist in the analysis of cancer cells where the mechanisms restricting division are aberrant.