Reconstitution and biophysical study of chromosome segregation machinery

染色体分离机制的重建和生物物理研究

基本信息

批准号：
10064632
负责人：
CHARLES ASBURY
金额：
$ 65.74万
依托单位：
UNIVERSITY OF WASHINGTON
依托单位国家：
美国
项目类别：
财政年份：
2020
资助国家：
美国
起止时间：
2020-01-01 至 2024-12-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10064632
关键词：
Affect Anaphase Architecture Area Behavior Binding Biological Assay Cell Cycle Cell division Cells Centrosome Chromosome Segregation Chromosomes Complex Coupling DNA Diffuse Drug Targeting Feedback Fluorescence Goals Individual Kinetochores Lasers Malignant Neoplasms Measurement Measures Mechanics Medical Microtubules Mitosis Mitotic spindle Molecular Machines Monitor Movement Nature Recombinants Signal Transduction Structure Techniques Testing Tubulin Weight-Bearing state Work Yeasts biophysical analysis biophysical tools cell motility chromosome movement experience fascinate grasp in vivo new therapeutic target particle reconstitution side effect single molecule spindle pole body

Affect Anaphase Architecture Area Behavior Binding Biological Assay Cell Cycle Cell division Cells Centrosome Chromosome Segregation Chromosomes Complex Coupling DNA Diffuse Drug Targeting Feedback Fluorescence Goals Individual Kinetochores Lasers Malignant Neoplasms Measurement Measures Mechanics Medical Microtubules Mitosis Mitotic spindle Molecular Machines Monitor Movement Nature Recombinants Signal Transduction Structure Techniques Testing Tubulin Weight-Bearing state Work Yeasts biophysical analysis biophysical tools cell motility chromosome movement experience fascinate grasp in vivo new therapeutic target particle reconstitution side effect single molecule spindle pole body

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项目摘要

Project summary During cell division, duplicated chromosomes are segregated by an exquisite molecular machine, the mitotic spindle. Our goal is to uncover how this machine operates by reconstituting spindle activities and applying advanced biophysical tools for manipulating and tracking individual molecules. We focus on the components most central to spindle function, kinetochores, microtubules, and spindle poles. Kinetochores drive chromosome movements by maintaining persistent, load-bearing attachments to microtubule tips, even as the tips assemble and disassemble under their grip. Kinetochores also somehow sense when they are erroneously attached and, if so, they detach and generate diffusible ‘wait’ signals to delay anaphase until proper attachments are made. Spindle microtubules are organized into a bipolar configuration by the spindle poles, which also must sustain forces to support chromosome movements and spindle assembly. In past work, we have developed motility assays where native kinetochores or recombinant kinetochore subcomplexes are attached to individual dynamic microtubules. Like kinetochores in vivo, the isolated kinetochore particles remain tip-bound even as the microtubule tips assemble and disassemble – a behavior we call ‘tip-coupling’. We have also reconstituted attachments between microtubules and spindle pole bodies, the yeast counterparts of centrosomes, and made the first measurements of their mechanical strength. Altogether our reconstitutions have enabled us to make key discoveries in major areas of spindle function. By expanding our approach, we can now attack the essence of many complex, long-standing problems in mitosis, in direct ways that would be impossible in living cells. Over the next five years, we will focus on several important questions: (1) How do kinetochores spontaneously self-assemble from their component parts? (2) How are forces transmitted from the outer microtubule-binding interface through the middle of the kinetochore and ultimately to the centromeric DNA? (3) How are dynamic behaviors at kinetochores and spindle poles affected by the forces they experience? (4) How do kinetochores avoid making erroneous attachments? (5) How do unattached or erroneously attached kinetochores generate ‘wait’ signals to delay the cell cycle? Our work will continue to use the advanced, feedback-controlled laser traps that we pioneered for measuring kinetochore movement and spindle pole mechanics. In addition, newly developed fluorescence techniques will allow us to observe kinetochore assembly at the single molecule level and to monitor dynamic structural changes within individual kinetochores. By combining laser trapping with fluorescence we will test directly how changes in the composition and architecture of kinetochores and spindle poles affect their function.

项目摘要在细胞分裂期间，重复的染色体被独家分子机隔离主轴。我们的目标是通过重新建立主轴活动并应用用于操纵和跟踪各个分子的高级生物物理工具。我们专注于组件纺锤功能，动力学，微管和主轴杆最重要的。动力学驱动器染色体运动通过维持微管尖端的持久载荷附件，即使提示在握把下组装并拆卸。动力学也以某种方式感知到它们错误地附着，如果是这样，它们会分离并生成可扩散的“等待”信号，以延迟后期直到进行适当的附件。主轴微管被组织为双极构型两极，也必须维持支持染色体运动和主轴组件的力。过去工作，我们开发了本地动物学或重组动物学子复合物的运动性测定连接到单个动态微管上。像体内动力学一样，分离的动力学颗粒即使微管尖端组装并拆卸 - 我们称之为“尖端耦合”的行为。我们还在微管和主轴杆体之间重组附件，酵母对应物中心体，并首先测量了它们的机械强度。总共我们的重组使我们能够在主轴功能的主要领域中进行关键发现。通过扩展我们的方法，我们现在可以以直接的方式攻击许多复杂，长期存在有丝分裂问题的本质活细胞不可能。在接下来的五年中，我们将重点关注几个重要问题：（1）动力学的组成部分自我组装自我组装？（2）力量如何从通过动力学的中间的微管结合界面，最终到达丝粒脱氧核糖核酸？（3）动态行为在动力学和主轴电线杆上如何受到力的影响经验？（4）动力学如何避免产生错误的附件？（5）如何无关或错误连接的动孔会产生“等待”信号以延迟细胞周期？我们的工作将继续使用我们开创了用于测量动力学运动和的高级反馈控制激光陷阱主轴杆力学。此外，新开发的荧光技术将使我们能够观察动力学组装在单分子水平上，并监视单个内部的动态结构变化动力学。通过将激光捕获与荧光相结合，我们将直接测试如何变化动力学和主轴杆的组成和结构会影响其功能。