Molecular Biomechanics of Mitotic Chromosome Segregation

有丝分裂染色体分离的分子生物力学

• 批准号: 9762138
• 负责人: Ekaterina L Grishchuk
• 金额: $ 31.87万
• 依托单位: UNIVERSITY OF PENNSYLVANIA
• 依托单位国家: 美国
• 财政年份: 2018
• 资助国家: 美国
• 起止时间: 2018-09-01 至 2022-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9762138
• 关键词: Address; Affect; Affinity; Behavior; Binding; Binding Proteins; Biological Assay; Biomechanics; Cell physiology; Cells; Chromosome Segregation; Chromosomes; Closure by clamp; Complex; Cytoskeleton; Dependence; Diffuse; Disease; Electrostatics; Exhibits; Fostering; Frequencies; Friction; GCM protein; Generations; Glues; Health; Human; In Vitro; Kinetochores; Knowledge; Mechanics; Metaphase; Microtubules; Minus End of the Microtubule; Mission; Mitosis; Mitotic; Mitotic Chromosome; Modeling; Molecular; Mutation; N-terminal; Neuronal Plasticity; Phosphorylation; Physiological; Plus End of the Microtubule; Point Mutation; Property; Proteins; Public Health; Reporting; Research; Resistance; Role; Site; Slide; Spectrum Analysis; Structure; Surface; Tail; Technology; Tertiary Protein Structure; Testing; Time; Toes; Tubulin; United States National Institutes of Health; calponin; cell motility; chromosome movement; experience; experimental study; foot; improved; insight; instrument; laser tweezer; mutant; novel; prevent; protein complex; reconstitution; recruit; single molecule

PROJECT SUMMARY “Molecular friction” between kinetochore proteins and microtubule walls sustains inter-kinetochore tension and prevents mitotic kinetochores from slipping from the microtubule ends. This phenomenon is particularly important in human cells during oscillations of metaphase chromosomes, which move repeatedly toward the plus- and minus-ends of the microtubules under significant forces. The microtubule wall–binding protein, Ndc80, is thought to serve as the primary molecular glue connecting the gliding kinetochores to microtubule surface. However, it remained unclear whether Ndc80 can form mobile diffusive bonds that are capable of generating frictional resistance. To bridge this gap in our knowledge, we developed a highly sensitive dual- trap, three-bead assay employing ultrafast force-clamp spectroscopy. Using this instrument, we pulled on the microtubule wall–bound Ndc80 protein complex in vitro, imitating the forces it experiences during metaphase chromosome oscillations. Strikingly, under dragging force, Ndc80 glides on the microtubule wall in both directions, but it generates stronger friction and exhibits catch-bond-like behavior only when pulled towards the microtubule plus-end. Thus, Ndc80 can serve as an intrinsic regulator of the direction-dependent molecular friction at mitotic kinetochores. To capitalize on this novel finding, in Aim 1 we will use this powerful technology in combination with targeted mutations in the calponin-homology domains of the Hec1 and Nuf2 subunits of the Ndc80 complex to investigate the specific molecular features that are responsible for direction- dependent friction generation at a single molecule level. In Aim 2, we will examine microtubule- wall gliding and friction generation by multiple Ndc80 molecules, characterizing their ensemble properties and emergent behaviors. With phosphomimetic substitutions in the disordered tail of the Hec1 subunit, we will determine how these behaviors are regulated by phosphorylation at the sites that strongly affect chromosome oscillations. In Aim 3 we will increase the molecular complexity of our reconstitutions to investigate how other kinetochore-associated microtubule- binding proteins modify friction generation by the gliding Ndc80. The results from these studies will provide an unprecedented molecular-mechanical insights into the key microtubule-binding components of human kinetochores, enabling us to construct a highly quantitative model of the frictional interface between microtubules and chromosomes in dividing human cells.

项目概要 着丝粒蛋白和微管壁之间的“分子摩擦”维持着丝粒间的相互作用 张力并防止有丝分裂动粒从微管末端滑落。这 这种现象在人体细胞中期振荡期间尤为重要 染色体，反复向微管的正端和负端移动 在巨大的力量之下。微管壁结合蛋白 Ndc80 被认为是 将滑动动粒连接到微管表面的主要分子胶。然而，它 目前尚不清楚 Ndc80 是否可以形成能够产生移动扩散键 摩擦阻力。为了弥补我们知识上的差距，我们开发了一种高度灵敏的双 采用超快力钳光谱的陷阱、三珠测定。使用这个仪器，我们 在体外拉动微管壁结合的 Ndc80 蛋白复合物，模拟其作用力 中期染色体振荡期间的经历。引人注目的是，在拖曳力作用下，Ndc80 在微管壁上双向滑动，但会产生更强的摩擦力并表现出 仅当拉向微管正端时才会出现类似捕获键的行为。因此，Ndc80 可以 作为有丝分裂动粒方向依赖性分子摩擦的内在调节器。 为了利用这一新颖的发现，在目标 1 中，我们将结合使用这项强大的技术 在 Hec1 和 Nuf2 亚基的钙调蛋白同源域中进行靶向突变 Ndc80 复合物用于研究负责方向的特定分子特征 - 单分子水平上的依赖摩擦力产生。在目标 2 中，我们将检查微管- 多个 Ndc80 分子产生的壁滑动和摩擦，表征它们的整体 属性和紧急行为。在无序的尾部进行磷模拟取代 Hec1 亚基，我们将确定这些行为是如何通过磷酸化来调节的 强烈影响染色体振荡的位点。在目标 3 中，我们将增加分子 我们重建的复杂性，以研究其他动粒相关的微管如何- 结合蛋白通过滑动 Ndc80 改变摩擦力的产生。这些研究的结果 将为关键的微管结合提供前所未有的分子力学见解 人类动粒的组成部分，使我们能够构建一个高度定量的模型 人类细胞分裂时微管和染色体之间的摩擦界面。