Biophysical study of reconstituted kinetochore-microtubule attachments

描述（由申请人提供）：着丝点是在有丝分裂过程中协调染色体运动的多蛋白细胞器。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负重附着。这种“尖端耦合”行为允许着丝点利用微管分解来产生力。它也是确保有丝分裂准确性的重要调控活动的基础。为了揭示着丝点是如何执行这些重要功能的，我们正在使用纯组分重构着丝点的活动，并应用新的工具来操纵和跟踪单个分子。我们将使用从出芽酵母中分离的天然着丝粒颗粒，纯重组着丝粒亚复合物和最先进的生物物理工具的独特组合。我们的体外方法可以直接回答关于着丝点功能的长期存在的问题，这在活细胞中是不可能的。具体来说，我们将：(1)确定核心微管结合亚复合物Ndc80和Dam1对从出芽酵母中纯化的天然着丝点颗粒与单个动态微管尖端之间耦合的相对贡献；(2)测试着丝点-微管耦合是否依赖于与尖端特异性微管结构（如GTP帽、卷曲的原丝或暴露的微管二聚体的纵向、横向和管腔面）的相互作用；(3)确定张力是否直接稳定着丝蛋白-微管的附着，独立于磷酸化调节，通过类似捕集键的机制；(4)确定Ipl1和Mps1两种激酶对着丝酶-微管附着稳定性调控的相对贡献；(5)确定张力是否抑制磷酸化触发的脱离，并测试候选模型以了解这种情况如何发生；(6)确定Ndc80和Dam1亚复合物内特定位点的磷酸化模拟突变是否通过直接削弱附着界面、触发微管结合物从着丝点释放或触发附着微管的解体来促进着丝点脱离。这项工作将有助于阐明着丝点和其他尖端偶联子如何保持对细胞骨架细丝的组装和拆卸尖端的强大而动态的附着物，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进展至关重要，因为染色体丢失，在癌症中经常发生，可能是由于削弱着丝点-微管附着物的突变造成的。有前途的新化疗药物正在开发针对有丝分裂机制的组成部分，这些努力将大大受益于对特定着丝点蛋白的作用和机制的更完整的了解。

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.