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.

抽象的 在分裂期间，染色体在主轴上分离，一系列重叠的交联微管 这会导致机械力。这些力被认为主要由运动蛋白和 微管动力学 - 快速微管生长和收缩。尽管在电动机上工作了几十年， 与一个世纪的分裂工作相比，运动机制仍未完全理解，关键负荷 - 尚未确定主轴的轴承元素。涉及轴承载荷的分子 主轴可能是电动机和其他纺锤体蛋白，但是这些分子之间的力尚未 探测 - 尚不清楚部队在分裂过程中如何在空间和临时变化。提出的研究 通过识别驱动器电动机的产生力的弹簧元素和通过 测量主轴运动蛋白的负载。驱动蛋白14 NCD对于果蝇的分裂至关重要 - 电动机会产生滑动微管的力，并通过其交联活动抵抗力。新的NCD 突变体将经过设计和测试，以及将电机机械和化学化学的结构变化 将分析循环，改变电动机机械输出。新的TSNCD FRET张力传感器已经 创建并将在有丝分裂纺锤体中分配，以测量有丝分裂过程中NCD承担的负载 影响其他纺锤体蛋白的突变体和突变体的影响。提出的研究将产生 有关产生力，电动机承受的载荷的动力电机的结构变化的信息 在主轴上，以及电动机产生的力和微管交联的变化如何影响负载。 我们将检验以下假设：NCD电机通过交联产生纺锤体的张力 微管，机械抵抗相反指导的滑动力，而不是其减去末端的运动性。 具体目的是1）确定通过 测试弯曲或扭曲中央ß-sheet商店并在期间释放自由能的假设 机械化学循环，作为电动机的弹性弹簧元素的作用，2）测量 使用新的TSNCD张力，由于力产生和对其他力的抵抗而导致纺锤体中的电动载荷 由驱动蛋白14 NCD电机和先前报道的FRET张力传感器创建的传感器，以及 测定增加NCD交联或交联和滑动的突变体。其他主轴的突变体 将测试蛋白质，包括相对指导的电动机，以鉴定其他承重的纺锤体分子。 这些研究将提供有关运动蛋白电动机如何产生力的新信息，并为 有丝分裂主轴中的机械力，阻止了导致先天缺陷的分裂误差。