Mechanisms of Microtubule Dynamic Turnover

描述(申请人提供)：微管是一种动态的细胞骨架聚合物，是有丝分裂、细胞器运动以及细胞形态建立和维持所必需的。微管细胞骨架在每个细胞周期都会发生戏剧性的重组：广泛的间期微管阵列被两极有丝分裂纺锤体取代，负责在有丝分裂过程中将复制的染色体准确地分离成两个子细胞。有丝分裂结束时，有丝分裂纺锤体解体，间期微管排列重塑。我们工作的长期目标是了解细胞如何在每个细胞周期中完成微管阵列的重塑。我们实验方法的一个关键特征是分析活细胞中的微管行为。在拟议的实验中，微管晶格上的标记将通过局部光激活表达带有可光激活的GFP变体的微管蛋白的细胞来创建。染色体运动将同时使用GFP微管GFP标记的动粒蛋白CenpA进行监测。将对前期光活化标记的移动进行表征，并确定它们在核包膜破裂中的作用。中心体在纺锤体微管运动中的作用将通过比较含有中心体和缺乏中心体的细胞中的微管行为来检验。为了确定负责微管运动的分子马达蛋白，将使用转录后基因沉默(RNAi)来灭活单个有丝分裂马达蛋白。细胞骨架组织将通过Z系列图像的3D重建在固定的准备中进行检查。这些结果将提供关于纺锤体组装过程中微管运动的分子基础以及这些运动如何在有丝分裂过程中对染色体运动做出贡献的新信息。了解正常细胞如何构建有丝分裂纺锤体是了解癌症中有缺陷的细胞分裂的重要第一步。

DESCRIPTION (provided by applicant): Microtubules are dynamic cytoskeletal polymers that are required for mitosis, organelle motility and the establishment and maintenance of cell form. The microtubule cytoskeleton is dramatically reorganized each cell cycle: the extensive interphase microtubule array is replaced with a bipolar mitotic spindle, which is responsible for the accurate segregation of the duplicated chromosomes into two daughter cells during mitosis. The mitotic spindle is disassembled at the end of mitosis and the interphase microtubule array reformed. The long-term goal of our work is to understand how cells accomplish the remodeling of the microtubule array during each cell cycle. A key feature of our experimental approach is the analysis of microtubule behavior in living cells. In the proposed experiments, marks on the microtubule lattice will be created by local photoactivation of cells expressing tubulin tagged with a photoactivatible GFP variant. Chromosome motion will be monitored simultaneously using a GFPMicrotubules GFP-tagged kinetochore protein, CenpA. The movement of the photoactivated marks at prophase will be characterized and their role in nuclear envelope breakdown determined. The role of the centrosome in spindle microtubule movements will be examined by comparison of microtubule behavior in cells containing and lacking centrosomes. To determine the molecular motor proteins that are responsible for microtubule movements, individual mitotic motor proteins will be inactivated using post-transcriptional gene silencing, or RNAi. Cytoskeletal organization will be examined in fixed preparations using 3-D reconstructions of Z-series of images. The results will provide new information about the molecular basis for microtubule motions during spindle assembly and how these motions contribute to chromosome motion during mitosis. Learning how normal cells construct a mitotic spindle is an important first step towards understanding defective cell division in cancer.