Biophysical study of reconstituted kinetochore-microtubule attachments

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。 他们最基本的活动是在 染色体和有丝分裂纺锤体内微管的组装和拆解尖端。这 “末端耦合”行为允许运动中枢利用微管解体产生力量。它还 这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。要揭示如何 动点执行这些重要的功能，我们正在用纯粹的 组件，并应用新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 关于动粒功能的问题将以直接的方式得到回答，这在 活细胞。具体地说，我们将：(1)确定核心微管结合的相对贡献 亚络合物NdC8O和Dami对纯化的天然动粒颗粒之间的偶联 发芽酵母和个体动态微管尖端；(2)检测动粒-微管偶联 依赖于与末端特定的微管蛋白结构的相互作用，例如GTP帽、卷曲的原丝或 暴露的微管蛋白二聚体的纵面、侧面和管腔面；(3)确定张力 通过卡环直接稳定动粒-微管附着物，而不依赖于磷调节 键状机制；(4)确定两种激酶IplI和Mps1对蛋白质的相对贡献。 动粒-微管附着稳定性的调节；(5)确定是否模仿磷酸化 Ndc8O和Dami亚复合体中特定位点的突变促进着丝粒脱离 通过直接削弱附着界面，通过触发微管结合剂从 动粒，或通过触发附着的微管的解体。这项工作将有助于阐明 动轴和其他顶端联轴器保持对装配和 细胞骨架细丝的分解尖端，以及这种附着物是如何调节的。了解 这些功能的基础对于了解癌症的进展是必不可少的，因为染色体丢失， 它经常出现在癌症中，可能是由削弱动粒微管的突变引起的 附属品。正在开发有希望的新的化疗药物来靶向有丝分裂的成分 这些努力将大大受益于更全面地了解这些角色和 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.