Kinesin Force Production and Biomechanics of Division

驱动蛋白力的产生和分裂的生物力学

• 批准号: 10452616
• 负责人: Sharyn A. Endow
• 金额: $ 9.23万
• 依托单位: DUKE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-07-17 至 2023-10-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10452616
• 关键词: ATP Hydrolysis; ATP phosphohydrolase; Affect; Affinity; Aneuploidy; Binding; Biological Assay; Biomechanics; Cell division; Cells; Chemicals; Chromosome Positioning; Chromosomes; Congenital Abnormality; Coupling; Crystallization; Defect; Drosophila genus; Elements; Fluorescence Resonance Energy Transfer; Free Energy; Goals; Growth; Kinesin; Knowledge; Laboratories; Lead; Light; Malignant Neoplasms; Maps; Measures; Mechanics; Microtubules; Mitosis; Mitotic; Mitotic spindle; Molecular; Molecular Conformation; Motor; Mutate; Nucleotides; Output; Photobleaching; Power stroke; Production; Proteins; Reporting; Resistance; Role; Signal Transduction; Slide; Structure; Testing; Uncertainty; Weight-Bearing state; Work; beta pleated sheet; cell motility; crosslink; design; indexing; mechanical force; mutant; offspring; prevent; protein crosslink; sensor

Abstract During division, chromosomes segregate on the spindle, a large array of overlapping, crosslinked microtubules that transduces mechanical forces. The forces are thought to be produced primarily by motor proteins and microtubule dynamics – rapid microtubule growth and shrinking. Despite decades of work on motors and more than a century of work on division, the motor mechanism is still not fully understood and the critical load- bearing elements of the spindle have not been identified. The molecules involved in bearing loads in the spindle are probably motors and other spindle proteins, but the forces across these molecules have not been probed – how the forces change spatially and temporally during division is not known. The proposed studies will begin to fill this gap by identifying the force-producing spring-like element of the kinesin motors and by measuring loads across a motor protein in the spindle. Kinesin-14 Ncd is essential for division in Drosophila – the motor produces force to slide microtubules and resists forces through its crosslinking activity. New Ncd mutants will be designed and tested, and structural changes that decouple the motor mechanical and chemical cycles, altering motor mechanical output, will be analyzed. New TsNcd FRET tension sensors have been created and will be assayed in mitotic spindles to measure loads borne by Ncd during mitosis and determine effects of uncoupling mutants and mutants that affect other spindle proteins. The proposed studies will yield information about the structural changes in the kinesin motors that produce force, the loads borne by a motor in the spindle, and how changes in force and microtubule crosslinking produced by the motor affect the loads. We will test the hypothesis that the Ncd motor produces tension in spindles primarily by crosslinking microtubules, mechanically resisting oppositely-directed sliding forces, rather than by its minus-end motility. Specific aims are to 1) Identify the spring-like element of the kinesins essential for force production by testing the hypothesis that bending or distortion of the central ß-sheet stores and releases free energy during the mechanochemical cycle, functioning as the elusive spring-like element of the motor, and 2) Measure motor loads in spindles due to force production and resistance to other forces using new TsNcd tension sensors created from the kinesin-14 Ncd motor and a previously reported FRET tension sensor, and by assaying mutants that increase Ncd crosslinking or both crosslinking and sliding. Mutants in other spindle proteins, including oppositely-directed motors, will be tested to identify other load-bearing spindle molecules. These studies will provide new information about how kinesin motors produce force and contribute to mechanical forces in the mitotic spindle, preventing division errors that lead to birth defects.

摘要 在分裂过程中，染色体在纺锤体上分离，纺锤体是一大排重叠的、交联的微管 它能传递机械力。这些力量被认为主要是由马达蛋白和 微管动力学--微管快速生长和收缩。尽管在马达和更多领域工作了几十年 经过一个多世纪的除法工作，人们对电机的机理仍不完全了解，临界载荷-- 主轴的轴承部件尚未确定。在人体内承担负荷的分子 纺锤体可能是马达和其他纺锤体蛋白质，但这些分子之间的作用力并不是 已探索-在除法过程中，力如何在空间和时间上变化尚不清楚。建议进行的研究 将开始填补这一空白，通过识别动力马达的产生力的弹簧式元件和通过 测量纺锤体中的马达蛋白的负载。Kinesin-14 NCD对果蝇的分裂是必不可少的- 马达产生滑动微管的力，并通过其交联性活动抵抗力。新的非传染性疾病 将设计和测试变种，以及使发动机机械和化学分离的结构变化 将分析改变电机机械输出的循环。新的TsNcd FRET张力传感器已经 并将在有丝分裂纺锤体中进行检测，以测量NCD在有丝分裂期间所承担的负荷，并确定 解偶联突变体和影响其他纺锤体蛋白的突变体的影响。拟议的研究将产生 关于产生力的马达的结构变化的信息，即马达所承受的负荷 以及力的变化和由电机产生的微管交联度如何影响负载。 我们将检验NCD马达主要通过交联产生主轴张力的假设 微管，机械地抵抗相反方向的滑移力，而不是通过它的负端运动。 具体的目标是：1)通过以下方式确定动蛋白的弹力式元件 检验假设中心的弯曲或扭曲储存和释放自由能在 机械力化学循环，起到马达难以捉摸的弹簧式元件的作用；2)测量 使用新的TsNcd张力时，由于力的产生和对其他力的阻力，主轴中的电机负载 由Kinesin-14 NCD马达和之前报道的FRET张力传感器创建的传感器，以及 检测增加NCD交联度或同时增加交联度和滑移率的突变体。其他纺锤体中的突变体 蛋白质，包括反向马达，将被测试以识别其他承载纺锤体的分子。 这些研究将提供有关运动蛋白马达如何产生力和促进 有丝分裂纺锤体中的机械力，防止导致出生缺陷的分裂错误。