Microtubule Dynamics and Chromosome Segregation

微管动力学和染色体分离

• 批准号: 8538415
• 负责人: Linda Wordeman
• 金额: $ 33.55万
• 依托单位: UNIVERSITY OF WASHINGTON
• 依托单位国家: 美国
• 财政年份: 2004
• 资助国家: 美国
• 起止时间: 2004-08-01 至 2016-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8538415
• 关键词: ATP Hydrolysis; Address; Affect; Aneuploidy; Appearance; Behavior; Biochemical; Biological; Biological Assay; Buffers; Cell division; Cells; Centrosome; Characteristics; Chromosomal Instability; Chromosomal Stability; Chromosome Segregation; Chromosomes; Data; Defect; Development; Drug Targeting; Event; Exhibits; Family; Family member; Fluorescent in Situ Hybridization; Genes; Goals; Image; In Vitro; Kinesin; Kinetochores; Knowledge; Lead; Learning; Length; Life; Link; Malignant Neoplasms; Measures; Microscopy; Microtubules; Mitosis; Mitotic; Mitotic spindle; Molecular; Molecular Motors; Motor; Motor Activity; Movement; Mutation; Normal Cell; Pharmaceutical Preparations; Pharmacotherapy; Phenotype; Physiological; Plus End of the Microtubule; Point Mutation; Polymers; Process; Proteins; Publishing; RNA Interference; Reading; Research; Resolution; Role; Small Interfering RNA; Temperature; Therapeutic Agents; Tissues; Tubular formation; Tubulin; Tumor Suppressor Genes; Tumor Suppressor Proteins; Visual; analog; arm; beta Tubulin; cancer cell; cancer therapy; cellular imaging; daughter cell; design; dimer; drug development; molecular transporter; mutant; programs; research study; screening; single molecule; tumor progression

DESCRIPTION (provided by applicant): The broad goal of this project is to understand how dynamic microtubules are controlled during cell division to build the mitotic spindle and to segregate chromosomes with high fidelity. This project is designed to investigate the mechanism by which mitotic microtubule motor proteins of the kinesin family regulate microtubule dynamics in order to facilitate mitotic spindle assembly, chromosome segregation and long-term stability of chromosome number. Our approach is interdisciplinary, ranging from single molecule biophysical analyses using total internal reflection microscopy (TIRF) and purified components to cellular approaches using high resolution quantitative live cell imaging, mutant constructs and siRNA depletions. We will use TIRF microscopy of live microtubules at physiological temperature and buffer conditions to evaluate the effect of mitotic kinesins on microtubule assembly and disassembly. Armed with an understanding of the activity the motor provides to microtubules in isolation we will transfer our studies to live mitotic cells. Using hig resolution imaging, selective depletions of kinesins and mutant analogs we will evaluate how kinesin motor activity is used within the mitotic spindle. This is essential basic knowledge which will guide the development of drugs targeted to mitotic kinesins for use in cancer treatment. Finally, we have preliminary evidence that small alterations in microtubule dynamics that do not have a profound impact on cell division in the short term have significant effects on long-term chromosome instability. Furthermore, known tumor suppressor genes which have not previously been linked to microtubules may exhibit unexpected direct or indirect effects on microtubule dynamics. We have a simple visual screen that will provide us with a panel of genes implicated in microtubule dynamics alterations and, by extension, chromosome instability. This will assist in molecular characterization of cancers and targeted drug development. Using these approaches we will learn how motile kinesins target to and regulate polymer assembly and disassembly at microtubule ends and what effect alterations in microtubule dynamics has on short-term spindle assembly and chromosome segregation. This project is designed to evaluate the effect that small changes in microtubule dynamics in the mitotic spindle has on chromosome instability and, in this way, understand the mechanistic forces underlying tumor progression.

描述（由申请人提供）：该项目的广泛目标是了解如何在细胞分裂过程中控制动态微管，以构建有丝分裂的纺锤体并用高富达隔离染色体。该项目旨在调查丝分裂微管运动蛋白调节微管动力学的机制，以促进有丝分裂纺锤体组件，染色体分离和染色体数的长期稳定性。我们的方法是跨学科的，范围从使用总内反射显微镜（TIRF）的单分子生物物理分析以及使用高分辨率定量活细胞成像，突变体构建体和siRNA耗竭的细胞方法。我们将在生理温度和缓冲液条件下使用活微管的TIRF显微镜来评估有丝分裂驱动素对微管组装和拆卸的影响。武装着电动机向微管分离提供的活动的活动，我们将研究将研究转移到活有丝分裂细胞。使用HIG分辨率成像，驱动蛋白和突变类似物的选择性耗竭，我们将评估如何在有丝分裂纺锤体内使用驱动蛋白运动活性。这是基本知识，它将指导针对有丝分裂驱动蛋白用于癌症治疗的药物的开发。最后，我们有初步的证据表明，在短期内对细胞分裂没有深远影响的微管动力学的微小变化对长期染色体不稳定性具有重大影响。此外，以前尚未与微管相关的已知肿瘤抑制基因可能会对微管动力学表现出意外的直接或间接影响。我们有一个简单的视觉屏幕，它将为我们提供与微管动力学改变有关的基因面板，并扩展染色体不稳定性。这将有助于癌症和靶向药物开发的分子表征。使用这些方法，我们将学习运动动力蛋白如何针对微管末端的聚合物组装和拆卸方式，以及微管动力学对短期纺锤体组装和染色体隔离的影响改变。该项目旨在评估有丝分裂纺锤体中微管动力学的小变化对染色体不稳定性的影响，并以这种方式了解肿瘤进展的机械力。