Structural analysis of the role of TPX2 in branching microtubule nucleation

The mitotic spindle consists of microtubules (MTs), which are nucleated by the gamma-tubulin ring complex (gamma-TuRC). How the gamma-TuRC gets activated at the right time and location remains elusive. Recently, it was uncovered that MTs nucleate from preexisting MTs within the mitotic spindle, which requires the protein TPX2, but the mechanism basis for TPX2 action is unknown. Here, we investigate the role of TPX2 in branching MT nucleation. We establish the domain organization of Xenopus laevis TPX2 and define the minimal TPX2 version that stimulates branching MT nucleation, which we find is unrelated to TPX2's ability to nucleate MTs in vitro. Several domains of TPX2 contribute to its MT-binding and bundling activities. However, the property necessary for TPX2 to induce branching MT nucleation is contained within newly identified gamma-TuRC nucleation activator motifs. Separation-of-function mutations leave the binding of TPX2 to gamma-TuRC intact, whereas branching MT nucleation is abolished, suggesting that TPX2 may activate gamma-TuRC to promote branching MT nucleation.