Mechanisms of Microtubule Dynamic Turnover

微管动态周转机制

• 批准号: 6931050
• 负责人: Patricia Wadsworth
• 金额: $ 29.01万
• 依托单位: UNIVERSITY OF MASSACHUSETTS AMHERST
• 依托单位国家: 美国
• 财政年份: 2000
• 资助国家: 美国
• 起止时间: 2000-08-01 至 2008-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6931050
• 关键词: RNA interference; actins; cell cycle; cell line; centromere; centrosome; chromosome movement; confocal scanning microscopy; dynein ATPase; green fluorescent proteins; intracellular transport; kinesin; microtubules; mitotic spindle apparatus; nuclear membrane; photoactivation; tubulin

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.

描述（由申请人提供）：微管是有丝分裂、细胞器运动以及细胞形态的建立和维持所需的动态细胞骨架聚合物。每个细胞周期中微管细胞骨架都会发生显着重组：广泛的间期微管阵列被双极有丝分裂纺锤体取代，双极有丝分裂纺锤体负责在有丝分裂过程中将复制的染色体准确地分离到两个子细胞中。有丝分裂纺锤体在有丝分裂结束时被分解，间期微管阵列被重组。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表达带有光激活 GFP 变体标记的微管蛋白的细胞的局部光激活来创建。使用 GFPMicrotubules GFP 标记的动粒蛋白 CenpA 同时监测染色体运动。将表征前期光激活标记的运动并确定它们在核膜破裂中的作用。通过比较含有和缺乏中心体的细胞中的微管行为，可以检查中心体在纺锤体微管运动中的作用。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）使单个有丝分裂马达蛋白失活。将使用 Z 系列图像的 3D 重建在固定制剂中检查细胞骨架组织。这些结果将提供有关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促进有丝分裂过程中染色体运动的新信息。了解正常细胞如何构建有丝分裂纺锤体是了解癌症中缺陷细胞分裂的重要第一步。