ANALYSIS OF SPINDLE MICROTUBULE DYNAMICS

有丝分裂中染色体运动的分子机制 仍然是细胞生物学中一个悬而未决的问题。 有丝分裂纺锤体 部分由微管（MT）的双极排列组成， 从纺锤体两极放射状排列，在中期板处重叠。 中 为了阐明MT在染色体运动中的作用，技术已经 被开发用于将荧光染料标记的微管蛋白引入活细胞 来监测稳定状态下微管蛋白的行为。 在实验中， 在这里，微管蛋白亚基的缔合和解离的途径， 纺锤体纤维MT，特别是动粒纤维MT， 通过测量荧光再分布的速率和模式来检查 光漂白后（FRAP）。 在这些FRAP实验中， 纺锤体纤维中的荧光在选定区域被光漂白， 荧光的恢复，这发生在未漂白的亚基与 使用视频或光度计监测MT中的漂白亚基 技术. 后期纺锤体MT解体的部位 通过FRAP实验对Haemanthus胚乳的大纺锤体进行测定 或精母细胞。 微管相关的动力学性质 蛋白质（MAP）将通过制备蛋白质的荧光类似物来测量 几种不同的地图和测量FRAP。 这些蛋白质 调节微管蛋白FRAP的速率。 的作用 细胞ATP在染色体运动，微管蛋白和MAP动力学将是 测定 最后，维持MT的动态活动的条件 因此，将确定体外有丝分裂的功能模型， 可以得到纺锤体。 更全面地了解 有丝分裂可能导致许多疾病的治疗的改进， 发生不受控制的细胞增殖。 具体来说， 控制细胞分裂可以通过分析细胞分裂的动力学过程来实现。 有丝分裂纺锤体组分的性质。

The molecular mechanism responsible for chromosome movement during mitosis remains a major unanswered question in cell biology. The mitotic spindle is composed, in part, of a bipolar arrangement of microtubules (MTs) which radiate from the spindle poles and overlap at the metaphase plate. In an effort to elucidate the role of MTs in chromosome motion, techniques have been developed to introduce fluorochrome labeled tubulin into living cells to monitor tubulin behavior at steady-state. In the experiments presented here, the pathways of tubulin subunit association and dissociation with spindle fiber MTs, in particular the kinetochore fiber MTs, will be examined by measuring the rate and pattern of fluorescence redistribution after photobleaching (FRAP). In these FRAP experiments, tubulin fluorescence in spindle fibers is photobleached in a selected area and recovery of fluorescence, which occurs as unbleached subunits exchange with bleached subunits in MTs, is monitored using video or photometric techniques. The site or sites of spindle MT disassembly at anaphase will be determined by FRAP experiments on large spindles of Haemanthus endosperm or spermatocytes. The dynamic properties of microtubule associated proteins, MAPs, will be measured by preparing fluorescent analogs of several different MAPs and measuring FRAP. The ability of these proteins to modulate the rate of tubulin FRAP will also be determined. The role of cellular ATP in chromosome motion, tubulin and MAP dynamics will be determined. Finally, conditions which maintain the dynamic activity of MTs in vitro will be determined so that a functional model of the mitotic spindle can be obtained. A more complete understanding of the process of mitosis may lead to improvements in the treatment of many diseases where uncontrolled cell proliferation occurs. The ability to specifically control cell division may be realized through analysis of the dynamic properties of the components of the mitotic spindle.