Abstract
To understand how chromosomes are segregated, it is necessary to explain the precise spatiotemporal organization of microtubules (MTs) in the mitotic spindle. We use Xenopus egg 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.
Highlights
The mitotic spindle is a complex and dynamic macromolecular machine that is responsible for segregating chromosomes during cell division (McIntosh et al, 2012; Prosser and Pelletier, 2017)
Nucleation events and MT growth were resolved by time-lapse imaging of fluorescently-labeled tubulin and end-binding protein 1 (EB1), which labels the growing MT plus-ends (Fig. 1A; Movie S1)
The pattern of MT nucleation that gives rise to the mitotic spindle in vivo remains to be determined because individual MTs cannot be resolved due to their high density in many regions of the cell (Brugues et al, 2012; Burbank et al, 2006; Needleman et al, 2010)
Summary
The mitotic spindle is a complex and dynamic macromolecular machine that is responsible for segregating chromosomes during cell division (McIntosh et al, 2012; Prosser and Pelletier, 2017). Because of the high density of MTs resulting from these nucleation pathways, resolving MT nucleation events in the spindle is challenging (Brugues et al, 2012; Burbank et al, 2006; Decker et al, 2018; Kaye et al, 2017; Needleman et al, 2010; Oh et al, 2016; Prosser and Pelletier, 2017; Redemann et al, 2017; Tulu et al, 2006; Yang et al, 2007).
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