Biophysical study of reconstituted kinetochore-microtubule attachments

 描述(申请人提供)：动粒是一种多蛋白质机器，通过利用微管顶端分解过程中释放的能量产生力量和运动来驱动有丝分裂染色体分离。动点也保证了有丝分裂的准确性。它们感知并释放不正确的微管连接，当没有连接或不正确连接时，它们还招募产生‘等待’信号的检查点蛋白，延迟后期，给适当的连接形成更多的时间。为了揭示这些重要功能是如何发生的，我们正在使用纯组件重建动粒活动，并应用最先进的生物物理工具来操纵和跟踪单个分子。我们独特的体外方法使长期存在的关于动粒功能的问题可以直接得到回答，而这在活细胞中是不可能的。具体地说，我们将：(1)测量原细丝从拆卸尖端卷曲出来的力量产生能力，并确定这一“构象波”可对运动中心的强制生产作出的贡献；(2)确定保守的微管聚合酶Stu2如何稳定运动中心-微管连接，以及其在运动中心的活性如何以依赖于张力的方式调节；(3)直接观察单个检查点蛋白与单个运动中心的关联，并区分它们的结合是通过侧向附着在微管一侧、通过末端附着还是通过机械拉伸直接抑制。这项工作将阐明动点如何产生力量来移动染色体，以及它们与纺锤体微管的连接是如何受到调节的。了解这些动粒功能的基础对于了解癌症的进展是至关重要的，因为在癌症中频繁发生的染色体丢失可能是由于削弱动粒-微管连接或扰乱动粒调节的突变造成的。针对动粒和纺锤体检查点组件的有希望的新化疗药物也在开发中，这些努力将大大受益于对特定组件的作用和它们的作用机制的更全面的了解。

 DESCRIPTION (provided by applicant): Kinetochores are multiprotein machines that drive mitotic chromosome segregation by harnessing energy released during microtubule tip disassembly to generate force and movement. Kinetochores also ensure the accuracy of mitosis. They sense and release improper microtubule attachments and, when unattached or improperly attached, they also recruit checkpoint proteins that generate `wait' signals, delaying anaphase and giving more time for proper attachments to form. To uncover how these vital functions occur, we are reconstituting kinetochore activities using pure components and applying state-of-the-art biophysical tools for manipulating and tracking individual molecules. Our unique in vitro approach allows long-standing questions about kinetochore function to be answered in direct ways that would be impossible in living cells. Specifically, we will: (1) measure the force-generating capacity of protofilaments as they curl out from a disassembling tip and determine the contribution that this `conformational wave' can make to force production at kinetochores; (2) determine how the conserved microtubule polymerase, Stu2, stabilizes kinetochore-microtubule attachments and how its activity at kinetochores is regulated in a tension-dependent manner; (3) directly observe the association of individual checkpoint proteins with single kinetochores and distinguish whether their binding is directly inhibited by lateral attachment to the side of a microtubule, by end-attachment, or by mechanical tension. Together this work will elucidate how kinetochores generate force to move chromosomes, and how their attachments to spindle microtubules are regulated. Understanding the basis for these kinetochore functions is essential for understanding cancer progression because chromosome loss, which occurs frequently in cancer, can result from mutations that weaken kinetochore-microtubule attachments or disrupt kinetochore regulation. Promising new chemotherapeutics are also being developed to target kinetochore and spindle checkpoint components, and these efforts will benefit substantially from a more complete knowledge of the roles of specific components and the mechanisms by which they operate.