Shining light on spindle positioning

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.
eLife digest Almost every time a cell divides, it must share copies of its genetic material between two new daughter cells. A large molecular machine called the mitotic spindle makes this happen. The spindle is made of protein filaments known as microtubules that radiate out from two points at opposite ends of the cell. Some of these filaments attach to the genetic material in the center of the cell; some extend in the other direction and anchor the spindle to the cell membrane. The anchoring filaments – also known as astral microtubules – can position the mitotic spindle, which controls whether the cell splits straight down the middle (to give two identically sized cells) or off-center (which gives cells of different sizes). The force required to move the spindle comes from complexes of proteins under the cell membrane that contain a molecular motor called dynein, its partner dynactin, and three other proteins – including one called NuMA. The astral microtubules interact with this force-generating machinery, but it was unclear how these proteins are arranged at the membrane. One way to explore interactions in a protein complex is to use a light-induced reconstitution system. This technique involves molecules that will bind together whenever a light shines on them. Fusing these molecules with different proteins means that experimenters can control exactly where, and when, those proteins interact. Okumura et al. have now used a light-induced reconstitution system to understand how the force-generating machinery positions the spindle in human cells. One of the system’s molecules was fused to a protein located at the cell membrane; the other was fused to either the dynein motor or NuMA protein. Using light to move dynein around on the membrane did not move the spindle. Yet, changing the position of NuMA, by moving the light, was enough to rotate the spindle inside the cell. Understanding how these complexes of proteins work increases our understanding of how cells divide. Using the light-induced system to move the spindle could also reveal more about the role of symmetric and asymmetric cell division in organizing tissues. In particular, being able to manipulate the position and size of daughter cells will provide insight into how cell division shapes and maintains tissues during animal development.