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: 9103625
• 负责人: CHARLES ASBURY
• 金额: $ 39.66万
• 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 2020-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9103625
• 关键词: Address; Affect; Anaphase; Aneuploidy; Behavior; Binding; Biochemical; Biological Assay; Cell Cycle Progression; Cell division; Cells; Chromosome Segregation; Chromosomes; Complex; Congenital Abnormality; Coupling; Drug Targeting; Ensure; Eukaryota; Family; Fluorescence; Generations; Genetic Materials; Genomic Instability; In Vitro; Individual; Kinetochores; Knowledge; Lateral; Life; Malignant Neoplasms; Measures; Mechanics; Methods; Microtubules; Mitosis; Mitotic Chromosome; Mitotic spindle; Modeling; Molecular; Molecular Machines; Motor; Movement; Mutation; Polymerase; Production; Property; Proteins; Recruitment Activity; Regulation; Role; Side; Signal Transduction; Sister Chromatid; Structure; System; Techniques; Testing; Time; Tubulin; Weight-Bearing state; Work; Yeasts; base; biophysical analysis; biophysical tools; cell motility; chromosome loss; design; dynamic system; man; mutant; nanomachine; public health relevance; reconstitution; research study; segregation; tumor; tumor progression

 DESCRIPTION (provided by applicant): Kinetochores are multiprotein machines that drive mitotic chromosome segregation by harnessing energy released during microtubule tip disassembly to generate force and movement. Kinetochores also ensure the accuracy of mitosis. They sense and release improper microtubule attachments and, when unattached or improperly attached, they also recruit checkpoint proteins that generate `wait' signals, delaying anaphase and giving more time for proper attachments to form. To uncover how these vital functions occur, we are reconstituting kinetochore activities using pure components and applying state-of-the-art biophysical tools for manipulating and tracking individual molecules. Our unique 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) measure the force-generating capacity of protofilaments as they curl out from a disassembling tip and determine the contribution that this `conformational wave' can make to force production at kinetochores; (2) determine how the conserved microtubule polymerase, Stu2, stabilizes kinetochore-microtubule attachments and how its activity at kinetochores is regulated in a tension-dependent manner; (3) directly observe the association of individual checkpoint proteins with single kinetochores and distinguish whether their binding is directly inhibited by lateral attachment to the side of a microtubule, by end-attachment, or by mechanical tension. Together this work will elucidate how kinetochores generate force to move chromosomes, and how their attachments to spindle microtubules are regulated. Understanding the basis for these kinetochore functions is essential for understanding cancer progression because chromosome loss, which occurs frequently in cancer, can result from mutations that weaken kinetochore-microtubule attachments or disrupt kinetochore regulation. Promising new chemotherapeutics are also being developed to target kinetochore and spindle checkpoint components, and these efforts will benefit substantially from a more complete knowledge of the roles of specific components and the mechanisms by which they operate.

 描述（由适用提供）：动力学是多蛋白机，通过利用在微管尖端拆卸过程中释放的能量来驱动有丝分裂染色体的隔离以产生力和运动。动力学还确保有丝分裂的准确性。他们感知并释放不当的微管附件，并且在未连接或不正确的附件时，它们还募集了产生“等待”信号的检查点蛋白质，延迟了后期，并为形式适当的附件提供了更多时间。为了揭示这些重要功能的发生方式，我们正在使用纯组分并应用最先进的生物物理工具来操纵和跟踪单个分子。我们独特的体外方法允许以直接的方式回答有关活细胞不可能回答的有关动物功能的长期问题。具体而言，我们将：（1）测量原丝从拆卸尖端卷曲时的力产生能力，并确定这种“构象波”可以迫使动物学上产生的贡献； （2）确定配置的微管聚合酶stu2如何稳定动力学 - 微管附件，以及如何以张力依赖性方式调节其在动力学上的活性； （3）直接观察单个检查点蛋白与单个动物学的缔合，并区分其结合是否通过横向附着一起抑制，这项工作将阐明动力学如何产生力以移动染色体，以及如何调节其与纺锤体微管的附着。理解这些动力学功能的基础对于理解癌症进展至关重要，因为染色体损失经常在癌症中发生，可能是由于弱化的动型微氨基纤维附着或破坏动力学调节的突变而导致的。还开发了有希望的新化学治疗剂来瞄准动力学和主轴检查点组件，这些努力将从更完整的特定组件及其操作机制的作用中更加完整地了解。