Reconstitution and biophysical study of chromosome segregation machinery

项目总结 在细胞分裂过程中，复制的染色体被一种名为有丝分裂的精密分子机器分离。 纺锤形。我们的目标是通过重组主轴活动和应用来揭示这台机器是如何运行的 先进的生物物理工具，用于操纵和跟踪单个分子。我们专注于组件 对纺锤体功能最重要的有动点、微管和纺锤体极。动感驱动 通过保持与微管顶端持久的、承重的连接来进行染色体运动，即使在 尖端在它们的把手下组装和拆卸。运动家务也有某种意义，当它们是 错误地连接，如果是这样，它们分离并产生可扩散的等待信号，以延迟后期，直到 做了适当的附件。纺锤形微管由纺锤体组织成双极构型 两极，也必须维持力量，以支持染色体运动和纺锤体组装。在过去 在工作中，我们已经开发了运动分析，其中天然动粒或重组动粒亚复合体 附着在单个动态微管上。就像体内的动粒一样，孤立的动粒颗粒 即使在微管尖端组装和拆卸时，也要保持尖端结合--这种行为我们称之为“尖端耦合”。 我们还重组了微管和纺锤体之间的连接，酵母对应物 并首次对它们的机械强度进行了测量。总而言之，我们的重组 使我们能够在主轴功能的主要领域取得关键发现。通过扩展我们的方法，我们 现在可以解决有丝分裂中许多复杂的、长期存在的问题的本质，以直接的方式 在活细胞中是不可能的。今后五年，我们将重点解决几个重要问题：(1)如何做好 动点从它们的组成部分自发地自组装？(2)力是如何从 通过着丝粒中部的外部微管结合界面，最终到达着丝粒 DNA？(3)动轴和纺锤极的动态行为如何受力的影响？ 体验？(4)运动芯片如何避免产生错误的依恋？(5)未连接的或 错误连接的动粒会产生‘等待’信号来延迟细胞周期吗？我们的工作将继续使用 我们首创的先进的、反馈控制的激光陷阱用于测量动粒移动和 主轴磁极机械学。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，并监测个体内部的动态结构变化 动感家务。通过将激光捕获与荧光相结合，我们将直接测试 动轴和纺锤极的组成和结构会影响它们的功能。

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.