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: 10302986
• 负责人: Sharyn A. Endow
• 金额: $ 20.13万
• 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 2023-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10302986
• 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，纺锤体是大量重叠、交联的微管 它能转换机械力这些力被认为主要由马达蛋白产生， 微管动力学-快速微管生长和收缩。尽管几十年来一直致力于发动机等 比世纪的工作分工，电机机制仍然没有完全理解和临界负荷- 主轴的轴承元件尚未确定。分子参与轴承负荷在 纺锤体可能是马达和其他纺锤体蛋白质，但这些分子之间的力还没有被发现。 探索-如何在空间和时间上的力量变化分裂期间是未知的。拟议的研究 将开始通过识别驱动蛋白马达中产生力的弹簧样元件， 测量纺锤体中马达蛋白的负载。驱动蛋白-14 Ncd是果蝇分裂所必需的- 马达产生使微管滑动的力并通过其交联活性抵抗力。新NCD 突变体将被设计和测试，结构变化，解耦电机机械和化学 循环，改变电机机械输出，将进行分析。新的TsNcd FRET张力传感器已被 在有丝分裂纺锤体中测定，以测量有丝分裂期间Ncd所承受的负荷，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 关于产生力的驱动蛋白马达的结构变化的信息，马达所承受的负载 以及马达产生的力和微管交联的变化如何影响负载。 我们将测试的假设，即Ncd电机产生的张力，主要是通过交联锭子 微管，机械抵抗相反方向的滑动力，而不是通过其负端运动。 具体目标是：1）通过以下方法确定力产生所必需的驱动蛋白的弹簧样元件： 测试的假设，弯曲或扭曲的中心薄板储存和释放自由能， 机械化学循环，作为电动机的难以捉摸的弹簧元件，以及2）测量 使用新TsNcd张力时，由于力的产生和对其他力的抵抗，主轴中的电机负载 由驱动蛋白-14 Ncd马达和先前报道的FRET张力传感器创建的传感器，以及由 测定增加Ncd交联或交联和滑动两者的突变体。其他纺锤体突变体 蛋白质，包括相反方向的马达，将被测试，以确定其他承重纺锤体分子。 这些研究将提供关于驱动蛋白马达如何产生力并有助于 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。