Biochemical reconstitution of branching microtubule nucleation

Abstract

16Microtubules are nucleated from specific locations at precise times in the cell cycle. However, the 17 factors that constitute these microtubule nucleation pathways still need to be identified along with 18 their mode of action. Here, using purified Xenopus laevis proteins we biochemically reconstitute 19 branching microtubule nucleation, a nucleation pathway where microtubules originate from pre-20 existing microtubules, which is essential for spindle assembly and chromosome segregation. We 21 found that besides the microtubule nucleator gamma-tubulin ring complex (g-TuRC), the two 22 branching effectors augmin and TPX2 are required to efficiently nucleate branched microtubules. 23Specifically, TPX2 generates regularly-spaced patches that recruit augmin and g-TuRC to 24 microtubules, which then nucleate new microtubules at preferred branching angles of less than 90 25 degrees. Our work demonstrates how g-TuRC is brought to its nucleation site for branching 26 microtubule nucleation. It provides a blueprint for other microtubule nucleation pathways and for 27 generating a particular microtubule architecture by regulating microtubule nucleation. 28 29

Phase separation of TPX2 enhances and spatially coordinates microtubule nucleation

“…7, graph). Indeed, recent reconstitution experiments have shown that sites of condensation may serve as branch points for new MTs 21 .…”

“…See also Supplementary Fig. 5e first be deposited on MTs for augmin to be recruited 21,22 . Furthermore, TPX2 interacts with the tetrameric kinesin Eg5 30,43 , which was recently demonstrated to promote MT nucleation in vitro 44 .…”

“…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 .…”

How Does SUMO Participate in Spindle Organization?

“…Compared to interphase, mitotic microtubules are shorter and more dynamic; they are nucleated from γ-tubulin complexes either at centrosomes or along pre-existing MTs [1]. Away from centrosomes, MT nucleation requires Ran-dependent liberation of spindle assembly factors such as TPX2, HURP, or NuSAP, together with augmin complexes that are assembled along preexisting microtubules [1,2,3,4,5]. At kinetochores, microtubules interact with several microtubule-associated proteins (MAPs, including microtubule-dependent motors), and appear as cold-resistant MT bundles called kinetochore-fibers (k-fibers) [6].…”

Microtubule-associated proteins promote microtubule generation in the absence of γ-tubulin in human colon cancer cells

“…The ringshaped g-tubulin complex (g-TuRC) serves as the structural template for the initial tubulin assembly, thereby accelerating the initial lag phase (Zheng et al, 1995). However, the g-2 TuRC alone is not an efficient MT nucleator, and efficient nucleation requires association with other proteins, such as CDK5RAP2, XMAP215/ch-TOG, TPX2, and augmin (Alfaro-Aco et al, 2020;Choi et al, 2010;Consolati et al, 2020;Flor-Parra et al, 2018;Tariq et al, 2020;Thawani et al, 2018). Some of these activators likely alter the conformation of g-TuRC to better fit the ends of MT protofilaments (Consolati et al, 2020;Liu et al, 2020;Wieczorek et al, 2020), whereas others cooperate with g-TuRC (Consolati et al, 2020;Flor-Parra et al, 2018;King et al, 2020;Thawani et al, 2018).…”