Biophysical study of reconstituted kinetochore-microtubule attachments

重建动粒-微管附件的生物物理学研究

• 批准号: 8338863
• 负责人: CHARLES ASBURY
• 金额: $ 37.92万
• 依托单位: UNIVERSITY OF WASHINGTON
• 依托单位国家: 美国
• 财政年份: 2006
• 资助国家: 美国
• 起止时间: 2006-09-29 至 2015-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8338863
• 关键词: Affect; Behavior; Binding; Cell division; Cells; Chromosome Segregation; Chromosomes; Coupling; Cytoskeletal Filaments; Drug Delivery Systems; Ensure; Face; Filament; Guanosine Triphosphate; In Vitro; Individual; Kinetochores; Knowledge; Lateral; Left; Life; Malignant Neoplasms; Measures; Microtubules; Mitosis; Mitotic; Mitotic spindle; Modeling; Molecular; Molecular Machines; Mutation; Organelles; Phosphorylation; Phosphotransferases; Proteins; Recombinants; Regulation; Relative (related person); Role; Rupture; Saccharomycetales; Side; Sister; Site; Structure; Testing; Tubulin; Weight-Bearing state; Work; Yeasts; aurora B kinase; base; chromosome loss; chromosome movement; design; dimer; man; mutant; nanomachine; particle; reconstitution; scaffold; tool; tumor progression

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.

描述（由申请人提供）：动力学是多蛋白细胞器，可在有丝分裂过程中策划染色体的运动。他们最基本的活动是在染色体之间保持持久的承重附件以及丝裂纺锤体中微管的组装和拆卸尖端。这种“尖端耦合”行为允许运动学可利用微管拆卸产生力。它还构成了重要的监管活动，通过这些调节活动确保有丝分裂的准确性。为了揭示动力学如何执行这些重要功能，我们正在使用纯组件重新建立动力学活动，并应用新工具来操纵和跟踪单个分子。我们将使用从萌芽的酵母，纯重组的动物学子复合物和最先进的生物物理工具中分离出的天然动力学颗粒的独特组合。我们的体外方法允许以直接的方式以直接的方式回答有关活细胞在活细胞中不可能的长期问题。具体而言，我们将：（1）确定核心微管结合子复合物，NDC80和DAM1的相对贡献，对从萌芽的酵母和个体动态微管尖端纯化的天然动力学颗粒之间的耦合； （2）测试动力学 - 微管耦合是否依赖于与小管蛋白的尖端小管蛋白结构的相互作用，例如小管蛋白二聚体； （3）确定张力是否通过捕获键样机制直接稳定了动物学微管的附件，独立于磷酸盐调节； （4）确定两种激酶IPL1和MPS1对动物学微管附着稳定性的相对贡献； （5）确定张力是否抑制了磷酸化触发的脱离，并测试了如何发生这种情况的候选模型； （6）确定在NDC80中的特定位点模拟磷酸化的突变和DAM1亚复合物是否是通过直接削弱附着界面来促进动力学分离，通过触发从动力学的微管绑定器释放，或通过触发附着的微管群体触发拆卸的微管的隔离。这项工作将有助于阐明动力学和其他尖端耦合器如何保持强大但动态的附件，以对细胞骨架细丝的组装和拆卸尖端以及如何调节这些附件。理解这些功能的基础对于理解癌症的进展至关重要，因为染色体损失经常发生在癌症中，这可能是由于弱化的动型微管附着的突变而引起的。正在开发有希望的新化学治疗剂来靶向有丝分裂机械的组成部分，这些努力将从更完整的知识中受益于特定的动型蛋白的作用和机制。