Biochemical reconstitution of branching microtubule nucleation.

Raymundo Alfaro-Aco,Sabine Petry,Akanksha Thawani

doi:10.7554/elife.49797

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.

Highlights

Microtubules are nucleated from specific locations in the cell, and several of these microtubule nucleation pathways converge to form a particular cytoskeletal architecture (Kollman et al, 2011; Lin et al, 2015; Luders and Stearns, 2007)
The eight-subunit protein complex augmin is required for branching microtubule nucleation in plant, human and Drosophila cells, and meiotic Xenopus egg extract, where its depletion leads to reduced spindle microtubule density, less kinetochore fiber tension, metaphase arrest, and cytokinesis failure (David et al, 2019; Decker et al, 2018; Goshima et al, 2008; Hayward et al, 2014; Ho et al, 2011; Kamasaki et al, 2013; Lawo et al, 2009; Nakaoka et al, 2012; Petry et al, 2011; Uehara et al, 2009)
Using multicolor time-lapse total internal reflection (TIRF) microscopy, we first confirmed that an endogenous, pre-existing microtubule can serve as a template for branching microtubule nucleation when exposed to Ransupplemented extract that releases branching factors (Figure 1A and Figure 1—video 1)

Summary

Introduction

Microtubules are nucleated from specific locations in the cell, and several of these microtubule nucleation pathways converge to form a particular cytoskeletal architecture (Kollman et al, 2011; Lin et al, 2015; Luders and Stearns, 2007). The eight-subunit protein complex augmin is required for branching microtubule nucleation in plant, human and Drosophila cells, and meiotic Xenopus egg extract, where its depletion leads to reduced spindle microtubule density, less kinetochore fiber tension, metaphase arrest, and cytokinesis failure (David et al, 2019; Decker et al, 2018; Goshima et al, 2008; Hayward et al, 2014; Ho et al, 2011; Kamasaki et al, 2013; Lawo et al, 2009; Nakaoka et al, 2012; Petry et al, 2011; Uehara et al, 2009). We use biochemical reconstitution of its purified components to mechanistically dissect branching microtubule nucleation

Results and discussion

Materials and methods

Funding Funder