Biophysical study of reconstituted kinetochore-microtubule attachments

描述(申请人提供)：动点是在有丝分裂过程中协调染色体运动的多蛋白质细胞器。它们最基本的活动是维持染色体和有丝分裂纺锤体内微管的组装和拆卸尖端之间持久的承重连接。这种“尖端耦合”的行为允许运动中枢利用微管解体来产生力量。它也是重要的调控活动的基础，通过这些活动，它们可以确保有丝分裂的准确性。为了揭示动点是如何执行这些重要功能的，我们正在使用纯成分重建动点活动，并应用新的工具来操纵和跟踪单个分子。我们将使用从发芽酵母中分离出来的天然动粒颗粒、纯重组动粒亚复合体和最先进的生物物理工具的独特组合。我们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)确定核心微管结合亚复合体Ndc80和Dam1对从萌芽酵母中纯化的天然动粒颗粒与单个动态微管末端之间的耦合的相对贡献；(2)测试动粒-微管耦合是否依赖于与末端特异的微管结构的相互作用，如GTP帽、卷曲的原细丝或暴露的微管二聚体的纵面、侧面和管腔面；(3)确定张力是否通过类似Catch键的机制独立于光调节直接稳定动粒-微管附着；(4)确定两个激酶Ipl1和Mps1在动粒-微管附着稳定性调节中的相对贡献；(5)确定张力是否抑制磷酸化触发的分离，并测试可能如何发生的候选模型；(6)确定Ndc80和DAM1亚复合体中特定位点的模拟磷酸突变是否通过直接削弱附着界面、通过触发微管结合物从动粒释放或通过触发附着的微管的分解来促进动粒脱离。这项工作将有助于阐明动芯片和其他末端偶联器如何保持对细胞骨架细丝的组装和拆卸末端的强烈而动态的附着，以及这种附着是如何调节的。了解这些功能的基础对于了解癌症的进展是至关重要的，因为在癌症中经常发生的染色体丢失可能是由于削弱动粒-微管连接的突变造成的。有希望的新的化疗药物正在开发，以靶向有丝分裂机制的组件，这些努力将大大受益于对特定动粒蛋白的作用和机制的更全面的了解。

DESCRIPTION (provided by applicant): Kinetochores are multiprotein organelles that orchestrate the movement of chromosomes during mitosis. Their most fundamental activity is maintaining persistent, load-bearing attachments between the chromosomes and the assembling and disassembling tips of microtubules within the mitotic spindle. This 'tip-coupling' behavior allows kinetochores to harness microtubule disassembly to produce force. It also underlies vital regulatory activities by which they ensure the accuracy of mitosis. To uncover how kinetochores perform these important functions, we are reconstituting kinetochore activities using pure components and applying new tools for manipulating and tracking individual molecules. We will use a unique combination of native kinetochore particles isolated from budding yeast, pure recombinant kinetochore subcomplexes, and state-of-the-art biophysical tools. Our 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) determine the relative contributions of the core microtubule-binding subcomplexes, Ndc80 and Dam1, to the coupling between native kinetochore particles purified from budding yeast and individual dynamic microtubule tips; (2) test whether kinetochore-microtubule coupling relies on interactions with tip-specific tubulin structures such as GTP caps, curled protofilaments, or exposed longitudinal, lateral, and luminal faces of tubulin dimers; (3) determine whether tension stabilizes kinetochore-microtubule attachments directly, independently of phosphoregulation, via a catch bond-like mechanism; (4) determine the relative contributions of two kinases, Ipl1 and Mps1, to the regulation of kinetochore-microtubule attachment stability; (5) determine whether tension suppresses phosphorylation-triggered detachment, and test candidate models for how this may occur; (6) determine whether phospho-mimicking mutations at specific sites within the Ndc80 and Dam1 subcomplexes promotes kinetochore detachment by directly weakening the attachment interface, by triggering the release of microtubule-binders from the kinetochore, or by triggering disassembly of attached microtubules. This work will help elucidate how kinetochores and other tip-couplers maintain strong yet dynamic attachments to the assembling and disassembling tips of cytoskeletal filaments, and how such attachments are regulated. Understanding the basis for these functions is essential for understanding cancer progression because chromosome loss, which occurs frequently in cancer, can result from mutations that weaken kinetochore- microtubule attachments. Promising new chemotherapeutics are being developed to target components of the mitotic machinery, and these efforts will benefit substantially from a more complete knowledge of the roles and mechanisms of specific kinetochore proteins.