Department of Cell and Tissue Biology, University of California, San Francisco, San Francisco, CA 94143, USA.
Department of Bioengineering, Stanford University, Stanford, CA 94305, USA.
Curr Biol. 2017 Jul 24;27(14):2112-2122.e5. doi: 10.1016/j.cub.2017.06.018. Epub 2017 Jul 6.
Active forces generated at kinetochores move chromosomes, and the dynamic spindle must robustly anchor kinetochore fibers (k-fibers) to bear this load. The mammalian spindle bears the load of chromosome movement far from poles, but we do not know where and how-physically and molecularly-this load distributes across the spindle. In part, this is because probing spindle mechanics in live cells is difficult. Yet answering this question is key to understanding how the spindle generates and responds to force and performs its diverse mechanical functions. Here, we map load-bearing across the mammalian spindle in space-time and dissect local anchorage mechanics and mechanism. To do so, we laser-ablate single k-fibers at different spindle locations and in different molecular backgrounds and quantify the immediate relaxation of chromosomes, k-fibers, and microtubule speckles. We find that load redistribution is locally confined in all directions: along the first 3-4 μm from kinetochores, scaling with k-fiber length, and laterally within ∼2 μm of k-fiber sides, without detectable load sharing between neighboring k-fibers. A phenomenological model suggests that dense, transient crosslinks to the spindle along k-fibers bear the load of chromosome movement but that these connections do not limit the timescale of spindle reorganization. The microtubule crosslinker NuMA is needed for the local load-bearing observed, whereas Eg5 and PRC1 are not detectably required, suggesting specialization in mechanical function. Together, the data and model suggest that NuMA-mediated crosslinks locally bear load, providing mechanical isolation and redundancy while allowing spindle fluidity. These features are well suited to support robust chromosome segregation.
动粒产生的力使染色体移动，而动态纺锤体必须牢固地锚定动粒纤维（k 纤维）以承受这种负载。哺乳动物纺锤体承受着远离两极的染色体运动的负载，但我们不知道这种负载在哪里以及如何——从物理和分子上——分布在纺锤体上。部分原因是在活细胞中探测纺锤体力学很困难。然而，回答这个问题是理解纺锤体如何产生和响应力以及执行其多样化的机械功能的关键。在这里，我们在时空上绘制了哺乳动物纺锤体的承载图，并剖析了局部锚固力学和机制。为此，我们在不同的纺锤体位置和不同的分子背景下用激光消融单根 k 纤维，并定量测量染色体、k 纤维和微管斑点的即时松弛。我们发现，负载再分配在各个方向上都是局部受限的：在距动粒的前 3-4μm 范围内，与 k 纤维长度成比例，在 k 纤维侧面的 2μm 范围内横向受限，相邻 k 纤维之间没有可检测到的负载共享。一个现象学模型表明，k 纤维上与纺锤体紧密、瞬时交联的蛋白承受着染色体运动的负载，但这些连接并不限制纺锤体重组的时间尺度。观察到的局部承载需要微管交联蛋白 NuMA，而 Eg5 和 PRC1 则不需要，这表明在机械功能上存在专业化。总的来说，数据和模型表明，NuMA 介导的交联蛋白局部承载负载，提供机械隔离和冗余，同时允许纺锤体流动性。这些特性非常适合支持强大的染色体分离。
