Kinesin Force Production and Biomechanics of Division

驱动蛋白力的产生和分裂的生物力学

基本信息

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
项目类别：
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

项目摘要

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、交联的微管转换机械力被认为主要是由运动蛋白产生的。微管动力学——尽管在电机等领域进行了数十年的研究，但微管仍快速生长和收缩。经过一个多世纪的除法研究，电机机制仍然没有被完全理解，并且临界负载- 主轴的轴承元件尚未确定参与轴承载荷的分子。纺锤体可能是马达和其他纺锤体蛋白，但穿过这些分子的力尚未被改变。探究——在分裂过程中力如何在空间和时间上变化尚不清楚。将通过识别驱动蛋白马达的产力弹簧状元件并通过测量纺锤体中运动蛋白的负载对于果蝇的分裂至关重要 – 该马达产生使微管滑动的力，并通过其交联活性抵抗力。将设计和测试突变体，以及使电机机械和化学解耦的结构变化将分析新的 TsNcd FRET 张力传感器。创建并将在有丝分裂纺锤体中进行分析，以测量有丝分裂期间 Ncd 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结果。有关产生力的驱动蛋白马达的结构变化、马达承受的负载的信息主轴中的变化，以及电机产生的力和微管交联的变化如何影响负载。我们将测试 Ncd 电机主要通过交联在主轴中产生张力的假设微管，机械地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) 确定驱动蛋白中对于力产生至关重要的弹簧状元素检验中央 ß 片的弯曲或变形在过程中存储和释放自由能的假设机械化学循环，充当电机难以捉摸的弹簧状元件，以及 2) 测量由于使用新的 TsNcd 张力产生力和抵抗其他力，主轴中的电机负载由 kinesin-14 Ncd 电机和之前报道的 FRET 张力传感器创建的传感器，以及测定增加 Ncd 交联或同时交联和滑动的突变体。蛋白质，包括相反方向的马达，将被测试以识别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。