How microtubules (MT) are generated in the cell is a major question in understanding how the cytoskeleton is assembled. For several decades, γ-tubulin has been accepted as the cell’s universal MT nucleator. Although there is evidence that γ-tubulin complexes are not the sole MT nucleators, identification of other nucleation factors has proven difficult. Here, we report that the well-characterized MT polymerase XMAP215 (chTOG/Msps/Stu2p/Alp14/Dis1 homologue) is essential for MT nucleation in Xenopus egg extracts. The concentration of XMAP215 determines the extent of MT nucleation. Even though XMAP215 and γ-tubulin ring complex (γ-TuRC) possess minimal nucleation activity individually, together these factors synergistically stimulate MT nucleation in vitro. The N-terminal TOG domains 1–5 of XMAP215 bind αβ-tubulin and promote MT polymerization, while the conserved C-terminus is required for efficient MT nucleation and directly binds γ-tubulin. In sum, XMAP215 and γ-TuRC together function as the principal nucleation module that generates MTs in cells.

TPX2 is required for microtubule nucleation in mitosis, but the mechanism underlying its function is unclear. Alfaro-Aco et al. analyze the domains of TPX2 necessary for its activity and identify the minimal region required for branching microtubule nucleation.

Microtubules (MTs) must be generated from precise locations to form the structural frameworks required for cell shape and function. MTs are nucleated by the γ-tubulin ring complex (γ-TuRC), but it remains unclear how γ-TuRC gets to the right location. Augmin has been suggested to be a γ-TuRC targeting factor and is required for MT nucleation from preexisting MTs. To determine augmin's architecture and function, we purified augmin from insect cells. We demonstrate that augmin is sufficient to target γ-TuRC to MTs by in vitro reconstitution. Augmin is composed of two functional parts. One module (tetramer-II) is necessary for MT binding, whereas the other (tetramer-III) interacts with γ-TuRC. Negative-stain electron microscopy reveals that both tetramers fit into the Y-shape of augmin, and MT branching assays reveal that both are necessary for MT nucleation. The finding that augmin can directly bridge MTs with γ-TuRC via these two tetramers adds to our mechanistic understanding of how MTs can be nucleated from preexisting MTs.

Determining how microtubules (MTs) are nucleated is essential for understanding how the cytoskeleton assembles. While the MT nucleator, γ-tubulin ring complex (γ-TuRC) has been identified, precisely how γ-TuRC nucleates a MT remains poorly understood. Here, we developed a single molecule assay to directly visualize nucleation of a MT from purified Xenopus laevis γ-TuRC. We reveal a high γ-/αβ-tubulin affinity, which facilitates assembly of a MT from γ-TuRC. Whereas spontaneous nucleation requires assembly of 8 αβ-tubulins, nucleation from γ-TuRC occurs efficiently with a cooperativity of 4 αβ-tubulin dimers. This is distinct from pre-assembled MT seeds, where a single dimer is sufficient to initiate growth. A computational model predicts our kinetic measurements and reveals the rate-limiting transition where laterally associated αβ-tubulins drive γ-TuRC into a closed conformation. NME7, TPX2, and the putative activation domain of CDK5RAP2 do not enhance γ-TuRC-mediated nucleation, while XMAP215 drastically increases the nucleation efficiency by strengthening the longitudinal γ-/αβ-tubulin interaction.

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. Using purified Xenopus laevis proteins we biochemically reconstitute branching microtubule nucleation, which is critical for chromosome segregation. We found that besides the microtubule nucleator gamma-tubulin ring complex (γ-TuRC), the branching effectors augmin and TPX2 are required to efficiently nucleate microtubules from pre-existing microtubules. TPX2 has the unexpected capacity to directly recruit γ-TuRC as well as augmin, which in turn targets more γ-TuRC along the microtubule lattice. TPX2 and augmin enable γ-TuRC-dependent microtubule nucleation at preferred branching angles of less than 90 degrees from regularly-spaced patches along microtubules. This work provides a blueprint for other microtubule nucleation pathways and helps explain how microtubules are generated in the spindle.