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

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.

项目概要在细胞分裂过程中，复制的染色体被精密的分子机器（有丝分裂）分离我们的目标是通过重构主轴活动并应用主轴来揭示该机器的运行方式。用于操纵和跟踪单个分子的先进生物物理工具我们专注于组件。对纺锤体功能、动粒、微管和动粒驱动至关重要。通过微管尖端的持久、承重附着来维持染色体运动，即使动粒在它们的抓握下组装和拆卸也会以某种方式感知。错误地附着，如果是这样，它们会分离并产生可扩散的“等待”信号以延迟后期，直到纺锤体微管被纺锤体组织成双极结构。过去，两极还必须维持支持染色体运动和纺锤体组装的力。工作中，我们开发了运动测定法，其中天然着丝粒或重组着丝粒亚复合物与体内的动粒一样，分离的动粒颗粒附着在单个动态微管上。即使微管尖端组装和分解，仍保持尖端结合——我们称之为“尖端耦合”的行为。我们还重建了微管和纺锤体极体（酵母盟友）之间的附着物中心体，并对其机械强度进行了首次测量。通过扩展我们的方法，我们能够在主轴功能的主要领域取得重大发现。现在可以直接解决有丝分裂中许多复杂的、长期存在的问题的本质在接下来的五年里，我们将关注几个重要的问题：（1）如何做到这一点。 (2) 动粒从其组成部分自发地自组装？外部微管结合界面穿过着丝粒中部并最终到达着丝粒 DNA？（3）动粒和纺锤极的动态行为如何受到它们的作用力的影响？ (4) 动粒如何避免错误的附着？错误附着的动粒会产生“等待”信号来延迟细胞周期？我们首创的先进的反馈控制激光陷阱用于测量动粒运动此外，新开发的荧光技术将使我们能够观察。在单分子水平上进行动粒组装并监测个体内的动态结构变化通过将激光捕获与荧光相结合，我们将直接测试动粒的变化。动粒和纺锤极的组成和结构影响它们的功能。