@article{1c167756e9434bcc81eaa129e8e8d4ac,
title = "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 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.",
author = "Raymundo Alfaro-Aco and Akanksha Thawani and Sabine Petry",
note = "Funding Information: We are grateful to Christiane Wiese for providing XenC antibodies, Jae-Geun Song and Brian Mahon for help in the expression and purification of augmin, Matt King for help in the expression and purification of TPX2, all members of the Petry laboratory for discussions, Thomas Surrey for sharing the detailed protocol for making biotin-PEG-functionalized coverslips, and James Wakefield for sharing unpublished data, critically reading this manuscript and for coordinating submissions of manuscripts to eLife. We thank Ron Vale with whom the original project vision was conceived. This work was supported by the HHMI Gilliam Fellowship and the NSF Graduate Research Fellowship (to RA), the American Heart Association predoctoral fellowship 17PRE33660328 (to AT), the NIH New Innovator Award, the Pew Scholars Program in the Biomedical Sciences, and the David and Lucile Packard Foundation (to SP). Funding Information: We are grateful to Christiane Wiese for providing XenC antibodies, Jae-Geun Song and Brian Mahon for help in the expression and purification of augmin, Matt King for help in the expression and purification of TPX2, all members of the Petry laboratory for discussions, Thomas Surrey for sharing the detailed protocol for making biotin-PEG-functionalized coverslips, and James Wakefield for sharing unpublished data, critically reading this manuscript and for coordinating submissions of manuscripts to eLife. We thank Ron Vale with whom the original project vision was conceived. This work was supported by the HHMI Gilliam Fellowship and the NSF Graduate Research Fellowship (to RA), the American Heart Association predoctoral fellowship 17PRE33660328 (to AT), the NIH New Innovator Award, the Pew Scholars Program in the Biomedical Sciences, and the David and Lucile Packard Foundation (to SP). National Institute of General Medical Sciences. T32GM007388. Raymundo Alfaro-Aco. Howard Hughes Medical Institute. Gilliam Fellowship. Raymundo Alfaro-Aco. National Science Foundation. Graduate Research Fellowship. Raymundo Alfaro-Aco. American Heart Association. 17PRE33660328. Akanksha Thawani. National Institute of General Medical Sciences. 1DP2GM123493-01. Sabine Petry. David and Lucile Packard Foundation. 2014-40376. Sabine Petry. Publisher Copyright: {\textcopyright} Alfaro-Aco et al.",
year = "2020",
month = jan,
doi = "10.7554/eLife.49797",
language = "English (US)",
volume = "9",
journal = "eLife",
issn = "2050-084X",
publisher = "eLife Sciences Publications",
}