Mechanisms of Microtubule Dynamic Turnover

微管动态周转机制

• 批准号: 6828124
• 负责人: Patricia Wadsworth
• 金额: $ 26.92万
• 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/6828124
• 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（由申请方提供）：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。微管细胞骨架在每个细胞周期都发生了显著的重组：广泛的间期微管阵列被双极有丝分裂纺锤体所取代，这是有丝分裂过程中复制的染色体准确分离成两个子细胞的原因。有丝分裂末期纺锤体解体，分裂间期微管阵列重新形成。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活细胞中微管行为的分析。在所提出的实验中，微管晶格上的标记将通过表达标记有可光活化的GFP变体的微管蛋白的细胞的局部光活化来产生。将使用GFP微管GFP标记的动粒蛋白CenpA同时监测染色体运动。在前期的光活化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微管的行为来研究。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或RNAi使单个有丝分裂马达蛋白失活。将使用Z系列图像的3-D重建在固定的制备物中检查细胞骨架组织。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在有丝分裂过程中促进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中有缺陷细胞分裂的重要第一步。