Biochemical reconstitution of branching microtubule nucleation. Author Raymundo Alfaro-Aco, Akanksha Thawani, Sabine Petry Publication Year 2020 Type Journal Article Abstract 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 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. Keywords Animals, Escherichia coli, Humans, Gene Expression Regulation, Microscopy, Fluorescence, Multiprotein Complexes, Brain, Microtubule-Associated Proteins, Cell Cycle Proteins, Xenopus Proteins, Xenopus laevis, Microtubules, Spindle Apparatus, Mitosis, Microtubule Proteins, Cattle, Tubulin Journal Elife Volume 9 Date Published 2020 Jan 14 ISSN Number 2050-084X DOI 10.7554/eLife.49797 Alternate Journal Elife PMCID PMC6959992 PMID 31933480 PubMedPubMed CentralGoogle ScholarBibTeXEndNote X3 XML