Biochemical reconstitution of branching microtubule nucleation

Abstract Microtubules are nucleated from specific locations at precise times in the cell cycle. However, the factors that constitute these microtubule nucleation pathways still need to be identified along with their mode of action. Here, using purified Xenopus laevis proteins we biochemically reconstitute branching microtubule nucleation, a nucleation pathway where microtubules originate from pre-existing microtubules, which is essential for spindle assembly and chromosome segregation. We found that besides the microtubule nucleator gamma-tubulin ring complex (γ-TuRC), the two branching effectors augmin and TPX2 are required to efficiently nucleate branched microtubules. Specifically, TPX2 generates regularly-spaced patches that recruit augmin and γ-TuRC to microtubules, which then nucleate new microtubules at preferred branching angles of less than 90 degrees. Our work demonstrates how γ-TuRC is brought to its nucleation site for branching microtubule nucleation. It provides a blueprint for other microtubule nucleation pathways and for generating a particular microtubule architecture by regulating microtubule nucleation.