Toward a molecular mechanism of branching microtubule nucleation

Abstract

The microtubule cytoskeleton supports cell function by giving cells their shape, organizing their interior and segregating chromosomes. Microtubules are nucleated from specific locations at precise times in the cell cycle. However, the factors that constitute these microtubule nucleation pathways and their mode of action still need to be identified. Recently, it was uncovered that microtubules nucleate from pre-existing microtubules within the mitotic spindle, which requires the proteins TPX2, augmin and gamma-TuRC, but the molecular mechanism was unknown. Here, I investigate the role of TPX2 in branching microtubule nucleation, and via an in vitro reconstitution, demonstrate that the three factors TPX2, augmin and gamma-TuRC are sufficient to initiate this process.

The C-terminal region of TPX2 is the minimal domain capable of stimulating branching microtubule nucleation in Xenopus egg extract. It contains newly identified gamma-TuRC nucleation activator motifs that are necessary for the function of the protein. While it is dispensable for branching microtubule nucleation, the N-terminal region of TPX2 enhances the efficiency of the reaction. Furthermore, TPX2 has the unexpected ability to directly recruit gamma-TuRC and augmin to pre-existing microtubules. In an in vitro reconstitution, TPX2 and augmin enable gamma-TuRC-dependent microtubule nucleation at preferred branching angles of less than 90 degrees from regularly spaced patches along microtubules. This work elucidates how the microtubule nucleator gamma-TuRC is localized and activated to cause branching microtubule nucleation, a process that is critical for spindle assembly. I anticipate these results will help explain how other microtubule nucleation pathways give rise to specific microtubule architectures in the cell.