Division of Biological Science, Graduate School of Science, Nagoya University, Nagoya, Japan.
Division of Molecular Cell Engineering, National Institute of Genetics, Research Organization of Information and Systems, Shizuoka, Japan.
Elife. 2018 May 31;7:e36559. doi: 10.7554/eLife.36559.
To position the mitotic spindle within the cell, dynamic plus ends of astral microtubules are pulled by membrane-associated cortical force-generating machinery. However, in contrast to the chromosome-bound kinetochore structure, how the diffusion-prone cortical machinery is organized to generate large spindle-pulling forces remains poorly understood. Here, we develop a light-induced reconstitution system in human cells. We find that induced cortical targeting of NuMA, but not dynein, is sufficient for spindle pulling. This spindle-pulling activity requires dynein-dynactin recruitment by NuMA's N-terminal long arm, dynein-based astral microtubule gliding, and NuMA's direct microtubule-binding activities. Importantly, we demonstrate that cortical NuMA assembles specialized focal structures that cluster multiple force-generating modules to generate cooperative spindle-pulling forces. This clustering activity of NuMA is required for spindle positioning, but not for spindle-pole focusing. We propose that cortical Dynein-Dynactin-NuMA (DDN) clusters act as the core force-generating machinery that organizes a multi-arm ensemble reminiscent of the kinetochore.
为了将有丝分裂纺锤体定位在细胞内，星体微管的动态正极被膜相关的皮质力发生机制拉动。然而，与染色体结合的动粒结构不同，扩散倾向的皮质机制如何组织起来产生大的纺锤体拉力仍知之甚少。在这里，我们在人细胞中开发了一种光诱导的重组系统。我们发现，诱导皮质靶向 NuMA，但不是动力蛋白，足以拉动纺锤体。这种纺锤体拉动活性需要 NuMA 的 N 端长臂招募动力蛋白-动力蛋白复合物，基于动力蛋白的星体微管滑行，以及 NuMA 的直接微管结合活性。重要的是，我们证明皮质 NuMA 组装专门的焦点结构，将多个力生成模块聚类在一起，以产生协同的纺锤体拉力。NuMA 的这种聚类活性对于纺锤体定位是必需的，但对于纺锤体极聚焦不是必需的。我们提出，皮质 Dynein-Dynactin-NuMA (DDN) 聚类作为核心力生成机制，它组织了一个多臂的集合，类似于动粒。
