Spatiotemporal organization of branched microtubule networks

Abstract

To understand how chromosomes are segregated, it is necessary to explain the precise spatiotemporal organization of microtubules (MTs) in the mitotic spindle. We useXenopusegg extracts to study the nucleation and dynamics of MTs in branched networks, a process that is critical for spindle assembly. Surprisingly, new branched MTs preferentially originate near the minus-ends of pre-existing MTs. A sequential reaction model, consisting of deposition of nucleation sites on an existing MT, followed by rate-limiting nucleation of branches, reproduces the measured spatial profile of nucleation, the distribution of MT plus-ends and tubulin intensity. By regulating the availability of the branching effectors TPX2, augmin and γ-TuRC, combined with single-molecule observations, we show that first TPX2 is deposited on pre-existing MTs, followed by binding of augmin/γ-TuRC to result in the nucleation of branched MTs. In sum, regulating the localization and kinetics of nucleation effectors governs the architecture of branched MT networks.Impact StatementA sequential reaction pathway involving TPX2, augmin and γ-TuRC governs the assembly and architecture of branched microtubule networks.

Phase separation of TPX2 enhances and spatially coordinates microtubule nucleation

“…Interestingly, tubulin lowers the concentration at which TPX2 phase separates from~200 nM to~50 nM, which, compellingly, corresponds to the estimated concentration range for endogenous TPX2 in X. laevis egg cytosol of 25-100 nM ( Supplementary Fig. 3a-c) 22,33 . Surprisingly, TPX2 localizes to MTs at the much lower concentration of 1 nM ( Fig.…”

“…We observed that TPX2 binds to the first emerging MT and then exclusively associates with the growing MT network ( Fig. 2b and Supplementary Movie 2) 22 . Based on these results, we hypothesized that TPX2-tubulin co-condensates form preferentially on MTs, and that condensates in solution would only form in the absence of MTs.…”

“…Branching MT nucleation requires the C-terminal 480-716aa of TPX2, which may function through an interaction with γ-TuRC [22][23][24] and its co-nucleation factor XMAP215 7,[17][18][19]25 . Moreover, branching MT nucleation requires direct localization of γ-TuRC to pre-existing MTs via the protein complex augmin 20 .…”

“…Branching MT nucleation requires the universal MT nucleator module, consisting of the γ-Tubulin Ring Complex (γ-TuRC) 15,16 and its recently discovered co-factor XMAP215 [17][18][19] , as well as the protein complex augmin that directly recruits γ-TuRC along the length of a pre-existing MT 20 . Branching MT nucleation is initiated by the MT-associated protein TPX2 10 , which has been proposed to both recruit augmin to MTs 21,22 and activate γ-TuRC via TPX2's C-terminal domain 23,24 . In vitro, TPX2 can directly generate MTs from tubulin via its N-terminal domain 25,26 , but this domain is dispensable for MT nucleation in the cytosol 23,27 .…”

In vitro reconstitution of branched microtubule nucleation

“…It does not, however, exclude the possibility that in vivo , at least in some biological systems, other proteins tune branched MT nucleation to the individual needs of the cell. 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 .…”