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（由申请人提供）：动粒是多蛋白机器，通过利用微管尖端拆卸期间释放的能量产生力和运动来驱动有丝分裂染色体分离。动粒也确保有丝分裂的准确性。它们感知并释放不正确的微管附着，当未附着或不正确附着时，它们还招募产生“等待”信号的检查点蛋白，延迟后期并为正确附着的形成提供更多时间。为了揭示这些重要功能是如何发生的，我们正在使用纯成分重建动粒活动，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可以直接回答关于动粒功能的长期问题，这在活细胞中是不可能的。具体而言，我们将：（1）测量原丝从分解尖端卷曲时产生力的能力，并确定这种"构象波"对着丝粒产生力的贡献;（2）确定保守的微管聚合酶Stu 2如何稳定着丝粒-微管附着，以及它在着丝粒的活性如何以张力依赖的方式调节;（3）直接观察单个检查点蛋白与单个动粒的结合，并区分它们的结合是否被微管侧面的侧向附着、末端附着或机械张力直接抑制。这项工作将阐明动粒如何产生力来移动染色体，以及它们与纺锤体微管的连接是如何受到调节的。了解这些动粒功能的基础对于了解癌症进展至关重要，因为癌症中经常发生的染色体丢失可能是由于削弱动粒-微管附着或破坏动粒调节的突变引起的。也正在开发有前途的新化疗药物，以靶向动粒和纺锤体检查点组件，这些努力将大大受益于对特定组件的作用及其运作机制的更完整的了解。