Molecular Biomechanics of Mitotic Chromosome Segregation

项目总结 动粒蛋白与微管壁之间的“分子摩擦”支撑着动粒间 张力，防止有丝分裂动粒从微管末端滑落。这 在人类细胞中，中期分裂相振荡的现象尤为重要 反复向微管正端和负端移动的染色体 在强大的力量下。微管壁结合蛋白Ndc80被认为是 将滑动的动点连接到微管表面的初级分子胶。然而，它 目前尚不清楚Ndc80是否能够形成能够产生 摩擦阻力。为了弥合我们知识上的差距，我们开发了一种高度敏感的双重- TRAP，三珠超快测力钳光谱分析。使用这个仪器，我们 在体外拉上微管壁结合的NDC80蛋白复合体，模拟它的受力 中期染色体振荡的经历。引人注目的是，在拖曳的力量下，Ndc80 在微管壁上双向滑动，但产生更强的摩擦并表现出 只有当被拉向微管正端时，才会出现捕捉键样行为。因此，NDC80可以 作为有丝分裂着丝点方向相关的分子摩擦的内在调节器。 为了利用这一新发现，在目标1中，我们将结合使用这项强大的技术 在Hec1和Nuf2亚单位的Calponin同源结构域具有靶向性突变 NDC80复合体，以研究负责方向的特定分子特征- 在单分子水平上的依赖摩擦产生。在目标2中，我们将研究微管- 多个Ndc80分子的壁面滑动和摩擦产生，表征了它们的系综 属性和突发行为。在无序的尾巴上有仿磷取代 Hec1亚单位，我们将确定这些行为是如何通过磷酸化在 强烈影响染色体振荡的位置。在目标3中，我们将增加分子 我们重组的复杂性，以研究其他动粒相关微管- 结合蛋白可以改变Ndc80滑行时产生的摩擦。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 人类运动中枢的组成部分，使我们能够构建一个高度定量的 人类细胞分裂过程中微管与染色体之间的摩擦界面。

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.