Mechanisms of Microtubule Dynamic Turnover

描述（由申请人提供）：微管是动态的细胞骨架聚合物，是有丝分裂、细胞器运动和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发生显著的重组：广泛的间期微管阵列被双极性有丝分裂纺锤体所取代，在有丝分裂过程中，双极性有丝分裂纺锤体负责将复制的染色体精确分离成两个子细胞。有丝分裂结束时纺锤体解体，间期微管重组。我们工作的长期目标是了解细胞在每个细胞周期中如何完成微管阵列的重塑。我们实验方法的一个关键特征是对活细胞中的微管行为进行分析。在提出的实验中，微管晶格上的标记将通过表达带有光激活性GFP变体标记的微管蛋白的细胞的局部光激活而产生。染色体运动将使用gfp微管gfp标记的着丝点蛋白（CenpA）同时监测。前期光激活标记的运动将被表征，并确定它们在核膜破裂中的作用。中心体在纺锤体微管运动中的作用将通过比较含有和缺乏中心体的细胞中的微管行为来研究。为了确定负责微管运动的分子运动蛋白，将使用转录后基因沉默或RNAi使单个有丝分裂运动蛋白失活。细胞骨架组织将使用z系列图像的三维重建来检查固定制剂。这些结果将为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影响有丝分裂过程中的染色体运动提供新的信息。了解正常细胞如何构建有丝分裂纺锤体是了解癌症细胞分裂缺陷的重要第一步。

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.