MECHANISMS OF MITOTIC SPINDLE ASSEMBLY AND FUNCTION

该项目的总体目标是确定分子和 调节微管（Mt）组装和移动结构机制 染色体 因为染色体的运动是紧密结合的， MT组装的动态，我们的方法继续专注于 建立微管组装的通路和调节。 一 我们的研究工作的主要力量是，开发和 新技术在定量光学显微镜中的应用， 荧光模拟细胞化学，笼状化合物，激光光阱， 视频增强对比度和数字图像处理和分析， 测量活细胞中MT相关过程的动力学， 体外重构制剂。 主要的具体目标是：（1）确定动力学机制 Mt正负端的不稳定性（a）使用Mt的视频测定 体外复溶制剂和细胞提取物中的动力学 在细胞周期的间期和有丝分裂期获得，和（B）通过 在海胆卵和蛤卵母细胞中鉴定细胞质因子 提高拉伸速度，调节转变频率， 的动态不稳定性和核生长，以建立体外 能够支持类生命纺锤体组装和染色体的条件 运动;（2）阐明动粒在运动过程中的功能 染色体运动（a）通过做详细的动力学研究， 体内动粒运动，（B）使用笼状的光活化 荧光微管蛋白标记动粒微管区域， 分辨率视频显微镜跟踪荧光微管蛋白和动粒 运动性，（c）分析MT对动粒的附着和动态 附着部位的不稳定性，以及（d）使用显微操作， 激光光学陷阱，以测试如何在张力的变化， 动粒可以调节动粒Mts的动态不稳定性;（3） 来识别和表征分子马达， 减数分裂和有丝分裂过程（a）通过继续我们的视频分析， 与Sharyn Endow合作研究果蝇的功能域 分离马达，如红葡萄酒，当在细菌中表达时， (b)使用我们的视频分析来测试着丝粒的体外运动性， Kerry Bloom制作的酵母人工染色体区域。

The overall objective of this project is to determine the molecular and structural mechanisms which regulate microtubule (Mt) assembly and move chromosomes. Because chromosome movement is tightly coupled to the dynamics of Mt assembly, our approach continues to be focused on establishing the pathways and regulation of microtubule assembly. A major strength of our research effort is, the development and application of new techniques in quantitative optical microscopy, fluorescence analog cytochemistry, caged compounds, laser optical traps, video-enhanced contrast and digital image processing and analysis to measure the dynamics of Mt-associated processes in living cells and reconstituted preparations in vitro. The major specific aims are: (1) to determine the mechanism of dynamic instability of plus and minus ends of Mts (a) using video assays of Mt dynamics in reconstituted preparations in vitro and in extracts of cells obtained in interphase and mitotic phases of the cell cycle, and (b) by identifying cytoplasmic factors in sea urchin eggs and clam oocytes which enhance elongation velocity, regulate the transition frequencies of dynamic instability, and nucleate growth to establish the in vitro conditions which can support life-like spindle assembly and chromosome movement; (2) to clarify the functioning of kinetochores during chromosome movements (a) by doing detailed kinetic studies of kinetochore movements in vivo, (b) using photoactivation of caged fluorescent tubulin to mark regions of kinetochore microtubules and high resolution video microscopy to track fluorescent tubulin and kinetochore motility, (c) analyzing Mt attachment to kinetochores and dynamic instability at the attachment site, and (d) using micromanipulation and laser optical traps to test how changes in tension forces at the kinetochore may modulate dynamic instability of kinetochore Mts; and (3) to identify and characterize molecular motors which participate in meiotic and mitotic processes (a) by continuing our video assays in collaboration with Sharyn Endow of domain functions of Drosopholia segregation motors like the claret and when expressed in bacteria, and (b) using our video assays to test for in vitro motility of centromeric regions of yeast artificial chromosomes generated by Kerry Bloom.