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）：着丝粒是一种多蛋白机器，通过利用微管尖端拆卸过程中释放的能量来产生力和运动，它们还确保有丝分裂的准确性，并且在未附着或未附着时释放不正确的微管附着物。如果附着不正确，它们还会招募产生“等待”信号的检查点蛋白，从而延迟后期并为正确附着的形成提供更多时间。这些重要功能发生后，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有关动粒功能的长期问题。具体来说，这在活细胞中是不可能的，我们将：（1）测量原丝从可拆卸尖端卷曲时产生力的能力，并确定这种“构象波”的贡献。可以强制着丝粒产生；（2）确定保守的微管聚合酶 Stu2 如何稳定着丝粒-微管附着以及其在着丝粒上的活性如何以张力依赖性方式调节；（3）直接观察个体检查点的关联；具有单个动粒的蛋白质，并区分它们的结合是否受到微管侧面的横向附着、末端附着或机械张力的直接抑制。将阐明动粒如何产生移动染色体的力，以及它们与纺锤体微管的附着如何受到调节，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削弱动粒的突变引起的。 -微管附着或破坏动粒调节也正在开发有前途的新化疗药物以针对动粒和纺锤体检查点成分，这些努力将对特定组件的作用及其运行机制的更全面的了解将带来巨大的好处。