Direct observation of branching MT nucleation in living animal cells

Abstract

160 words) 1 Centrosome-mediated microtubule (MT) nucleation has been well-characterized; however, 2 numerous non-centrosomal MT nucleation mechanisms exist. The branching MT nucleation 3 pathway envisages that the γ-tubulin ring complex (γ-TuRC) is recruited to MTs by the augmin 4 complex to initiate nucleation of new MTs. While the pathway is well-conserved at a molecular 5 and functional level, branching MT nucleation by core constituents has never been directly 6 observed in animal cells. Here, multi-color TIRF microscopy was applied to visualize and 7 quantitatively define the entire process of branching MT nucleation in dividing Drosophila cells. 8 The stereotypical branching nucleation event entailed augmin first binding to a "mother" MT, 9 recruitment of γ-TuRC after 16s, followed by nucleation 15s later of a "daughter" MT at a 36° 10 branch angle. Daughters typically remained attached throughout their ~40s lifetime unless the 11 mother depolymerized past the branch point. Assembly of branched MT arrays, which did not 12 require D-TPX2 (Drosophila TPX2) or evident regulation by a RanGTP gradient, enhanced 13 localized RhoA activation during cytokinesis. 14 15

Kinetochore-mediated microtubule assembly and Augmin-dependent amplification drive k-fiber maturation in mammals

“…Strikingly, our live-cell superresolution tracking of EB3-comets in the vicinity of the kinetochore allowed us to follow with unprecedented spatiotemporal resolution microtubule growth events on individual k-fibers and revealed a wide angular dispersion relative to the kfiber axis. This is consistent with Augmin-mediated branched microtubule nucleation from pre-existing microtubules (Alfaro-Aco et al, 2020;Petry et al, 2013;Thawani et al, 2019;Verma and Maresca, 2019), but has been completely overlooked in previous models of k-fiber formation and maturation.…”

“…7B, C, Fig. S5E), consistent with Augmin-mediated microtubule branching (Alfaro-Aco et al, 2020;Hsia et al, 2014;Kamasaki et al, 2013;Liu et al, 2014;Petry et al, 2013;Tariq et al, 2020;Thawani et al, 2019;Verma and Maresca, 2019).…”

“…This is the case of k-fiber maturation, whose underlying molecular mechanism remained poorly understood. A popular, yet unproven, assumption is that k-fiber maturation in mammals relies on the Augmin complex, based on its established roles in microtubule amplification from pre-existing microtubules, both in vitro and in cells, including in the mitotic spindle (Kamasaki et al, 2013;Petry et al, 2013;Tariq et al, 2020;Thawani et al, 2019;Verma and Maresca, 2019). However, how Augmin mediates k-fiber maturation remained unclear due to two main reasons.…”

“…These microtubules are then captured and oriented with their plus-ends towards the kinetochore by CENP-E/kinesin-7 motors, and pre-formed k-fibers subsequently incorporated into the spindle by a Dynein-mediated interaction with non-kinetochore microtubules (Elting et al, 2014;Hueschen et al, 2017;Khodjakov et al, 2003;Maiato et al, 2004;Sikirzhytski et al, 2014;Sikirzhytski et al, 2018). Augmin, an octameric Y-shaped complex that recruits γ-tubulin to pre-existing microtubules, triggers branched microtubule nucleation, thereby contributing to rapid microtubule amplification in the spindle (Goshima et al, 2008;Goshima et al, 2007;Hsia et al, 2014;Kamasaki et al, 2013;Lawo et al, 2009;Petry et al, 2013;Petry et al, 2011;Uehara et al, 2009b;Verma and Maresca, 2019;Wainman et al, 2009). In particular, the Augmin complex has been previously implicated in kfiber formation (Goshima et al, 2008;Lawo et al, 2009;Uehara et al, 2009a;Zhu et al, 2008).…”

In vitro reconstitution of branched microtubule nucleation

“…For example, a clear role has been reported for the MT associated protein, TPX2, in stimulating Augmin-dependent branched MT nucleation in Xenopus meiotic extracts 9,28 . However, in support of our conclusions, Drosophila TPX2 has recently been shown to be dispensible for the phenomenon in vivo 29 . Our experiments also highlight the intriguing possibility that, in some cells, Augmin might recruit pre-existing γ -TuRC-containing MTs, nucleated elsewhere in the cell, anchoring then to specific sites and increasing local MT density.…”