Biochemical reconstitution of branching microtubule nucleation

Author:

Alfaro-Aco Raymundo1ORCID,Thawani Akanksha2ORCID,Petry Sabine1ORCID

Affiliation:

1. Department of Molecular Biology, Princeton University, Princeton, United States

2. Department of Chemical and Biological Engineering, Princeton University, Princeton, United States

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 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.

Funder

National Institute of General Medical Sciences

Howard Hughes Medical Institute

National Science Foundation

American Heart Association

David and Lucile Packard Foundation

Publisher

eLife Sciences Publications, Ltd

Subject

General Immunology and Microbiology,General Biochemistry, Genetics and Molecular Biology,General Medicine,General Neuroscience

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