Biophysical study of reconstituted kinetochore-microtubule attachments

动粒是多蛋白质细胞器，在有丝分裂期间协调染色体的运动。 它们最基本的活动是维持 有丝分裂纺锤体内微管的组装和拆卸尖端。这 “尖端耦合”行为允许动粒利用微管分解产生力。它还 是确保有丝分裂准确性的重要调节活动的基础。为了揭示 动粒执行这些重要的功能，我们正在重建动粒活动使用纯 组件和应用新的工具来操纵和跟踪单个分子。我们将使用一个 从芽殖酵母中分离的天然动粒颗粒的独特组合，纯重组 动粒亚复合体和最先进的生物物理工具。我们的体外方法允许长时间 关于动粒功能的长期问题将以直接的方式得到回答，这是不可能的， 活细胞具体而言，我们将：（1）确定核心微管结合的相对贡献 亚复合物，Ndc 8 O和Dami，对纯化的天然动粒颗粒之间的偶联 芽殖酵母和单个动态微管尖端;（2）测试是否存在着丝粒-微管偶联 依赖于与尖端特异性微管蛋白结构如GTP帽、卷曲原丝或 暴露微管蛋白二聚体的纵向、横向和管腔面;（3）确定张力是否 通过捕获直接稳定激动剂-微管附着，独立于磷酸调节 （4）确定两种激酶Ipli和Mpsl对蛋白质合成的相对贡献。 （5）确定磷酸化模拟是否 Ndc 8 O和Dami亚复合物内特定位点的突变促进动粒脱离 通过直接削弱连接界面，通过触发微管结合剂从连接界面的释放， 动粒，或通过触发附着的微管的分解。这项工作将有助于阐明如何 运动器和其他尖端耦合器保持对组件的强而动态的附接， 分解细胞骨架丝的尖端，以及如何调节这种附着。了解 这些功能的基础对于理解癌症进展是必不可少的，因为染色体丢失， 这在癌症中经常发生，可能是由于削弱微管运动的突变造成的。 附件的权限.正在开发有希望的新化疗药物，以靶向有丝分裂细胞的组分。 机器，这些努力将大大受益于更完整的知识的作用和 特定动粒蛋白的机制。

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, Ndc8O and Dami, 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, Ipli and Mpsl, to the regulation of kinetochore-microtubule attachment stability; (5) determine whether phospho-mimicking mutations at specific sites within the Ndc8O and Dami 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.