Reconstitution and biophysical study of chromosome segregation machinery

项目摘要 在细胞分裂过程中，复制的染色体被一个精致的分子机器--有丝分裂素--分离。 纺锤体。我们的目标是通过重构主轴活动并应用 先进的生物物理工具来操纵和跟踪单个分子。我们专注于组件 最核心的纺锤体功能，动粒，微管，和纺锤体极。动粒驱动 染色体运动通过维持持久的，承重附件微管提示，即使作为 尖端在其抓握下组装和拆卸。动粒也能以某种方式感觉到它们什么时候 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， 进行适当的连接。纺锤体微管被纺锤体组织成双极结构 两极，也必须承受力，以支持染色体运动和纺锤体组装。过去 工作，我们已经开发了运动测定，其中天然动粒或重组动粒亚复合物 附着在单独的动态微管上。像体内的动粒一样，分离的动粒颗粒 即使在微管尖端组装和拆卸时也保持尖端结合-我们称之为“尖端耦合”的行为。 我们还重建了微管和纺锤体（酵母对应物）之间的附着 并首次测量了它们的机械强度。我们所有的重建 使我们在纺锤体功能的主要领域有了重大发现。通过扩展我们的方法，我们 现在可以直接攻击有丝分裂中许多复杂的、长期存在的问题的本质， 在活细胞中是不可能的未来五年，我们将重点研究几个重要问题：（1）如何 动粒会自发地从它们的组成部分自我组装起来(2)力是如何从 外部微管结合界面穿过着丝粒的中间，并最终到达着丝粒 DNA？(3)动粒和纺锤体极点的动力学行为是如何受到它们所受的力的影响的？ 经验？(4)Kinetochores如何避免产生错误的附件？(5)单身或 错误附着的动粒产生“等待”信号来延迟细胞周期？我们的工作将继续使用 先进的，反馈控制的激光陷阱，我们率先测量动粒运动， 主轴杆力学此外，新开发的荧光技术将使我们能够观察 动粒组装在单分子水平，并监测动态结构的变化， 动粒通过将激光捕获与荧光结合，我们将直接测试 着丝粒和纺锤体极的组成和结构影响它们的功能。

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.