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染色体与有丝分裂纺锤体内微管组装和拆卸尖端之间持久的承重连接。这种“尖端耦合”行为允许动粒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节活动的基础。为了揭示动粒是如何执行这些重要功能的，我们正在使用纯成分重建动粒活动，并应用新工具来操纵和跟踪单个分子。我们将使用从芽殖酵母中分离的天然动粒颗粒、纯重组动粒亚复合物和最先进的生物物理工具的独特组合。我们的体外方法允许以直接的方式回答关于动粒功能的长期存在的问题，这在活细胞中是不可能的。具体而言，我们将：（1）确定核心微管结合亚复合物Ndc 80和Dam 1对从芽殖酵母中纯化的天然动粒颗粒与单个动态微管尖端之间偶联的相对贡献;（2）测试动粒-微管偶联是否依赖于与尖端特异性微管蛋白结构（例如GTP帽、卷曲的原丝或微管蛋白二聚体的暴露的纵向、横向和腔面）的相互作用;（3）确定张力是否通过类似捕获键的机制直接稳定激动剂-微管附着，而不依赖于磷酸调节;（4）确定两种激酶Ipl 1和Mps 1对激动剂-微管附着稳定性调节的相对贡献;（5）确定张力是否抑制磷酸化触发的脱离，并测试候选模型如何发生;（6）确定Ndc 80和Dam 1亚复合物内特定位点处的磷酸模拟突变是否通过直接削弱附着界面、通过触发微管结合剂从动粒释放或通过触发附着微管的分解来促进动粒脱离。这项工作将有助于阐明动粒和其他tip-coupler如何保持强大而动态的附件的组装和拆卸的细胞骨架丝的提示，以及如何调节这种附件。了解这些功能的基础对于了解癌症进展至关重要，因为在癌症中经常发生的染色体丢失可能是由削弱动粒-微管附着的突变引起的。正在开发有前途的新化疗药物，以靶向有丝分裂机制的组成部分，这些努力将大大受益于特定动粒蛋白的作用和机制的更完整的知识。