ANALYSIS OF SPINDLE MICROTUBULE DYNAMICS

有丝分裂过程中染色体运动的分子机制 仍然是细胞生物学中的一个重大悬而未决的问题。有丝分裂纺锤体 部分由微管(MT)的双极排列组成，该微管排列 从纺锤体两极向外辐射，并与中期板重叠。在一个 为了阐明MTS在染色体运动中的作用，技术人员 将荧光标记的微管蛋白引入活细胞 以监测稳定状态下的微管蛋白行为。在展示的实验中 在这里，微管蛋白亚单位的结合和解离的途径 纺锤形纤维MTS，特别是动粒纤维MTS，将是 通过测量荧光重分布的速度和模式进行检查 光漂白后(FRAP)。在这些FRAP实验中，微管蛋白 纺锤形纤维中的荧光在选定的区域被光漂白，并且 荧光的恢复，这发生在未漂白的亚基与 MTS中漂白的亚基，使用视频或光度学进行监测 技巧。主轴MT后期拆卸部位将 大纺锤体的FRAP实验确定 或者是精母细胞。相关微管的动力学特性 蛋白质，MAP，将通过准备荧光类似物来测量 几种不同的地图和测量FRAP。这些蛋白质的能力 为了调节微管蛋白的速率，还将确定FRAP。的作用 细胞ATP在染色体运动、微管蛋白和MAP动力学中的作用 下定决心。最后，维持MTS动态活动的条件 在体外将确定一个有丝分裂的功能模型 可以获得主轴。更全面地了解 有丝分裂可以改善许多疾病的治疗， 会发生不受控制的细胞增殖。有能力具体地 控制单元划分可以通过动态分析来实现 有丝分裂纺锤体组件的性质。

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.