XMAP215 is a microtubule nucleation factor that functions synergistically with the γ-tubulin ring complex

Thawani, Akanksha; Kadzik, Rachel S.; Petry, Sabine

doi:10.1038/s41556-018-0091-6

Cited by 159 publications

(222 citation statements)

References 64 publications

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“…In the presence of human γTuRC, nucleation from surface-immobilized γTuRC is strongly promoted ( Fig. 4a-c, Movie 6), in agreement with previous reports using the budding yeast and Xenopus orthologues of these proteins (Gunzelmann et al, 2018;Thawani et al, 2018). We find here that chTOG stimulates microtubule nucleation by human γTuRC by a factor of up to 21-fold, with the stimulatory effect saturating in a physiological chTOG concentration range ( Fig.…”

Section: Chtog and Tpx2 Stimulate γTurc-mediated Microtubule Nucleationsupporting

confidence: 92%

“…Using mass spectroscopy, we identified all human γTuRC core subunits (γ-tubulin, GCP2-6), as well as MZT1, MZT2 and actin, which were co-purified in previous γTuRC purifications ( Fig. S2 and Supplementary Material) (Choi et al, 2010;Hutchins et al, 2010;Oegema et al, 1999;Teixido-Travesa et al, 2012;Thawani et al, 2018). Additionally, we verified most subunits also by western blot using specific antibodies ( Fig.…”

Section: Purification Of Biotinylated Human γTurcmentioning

confidence: 76%

“…chTOG and TPX2 were also reported to promote microtubule outgrowth from stabilised microtubule 'seeds', suggesting similarities between outgrowth from such seeds and templating a microtubule by γTuRC (Wieczorek et al, 2015). Interestingly, both chTOG and TPX2 have also been reported to directly interact with γTuRC, which may increase the efficiency of their action Thawani et al, 2018). Moreover, TPX2 is known to interact additionally with the Augmin/HAUS complex that promotes branched microtubule nucleation from pre-existing microtubules in mitotic or meiotic spindles during cell division (Alfaro-Aco and Petry, 2017).…”

Section: Discussionmentioning

confidence: 99%

“…The exact stoichiometry and subunit order of human γTuRC is not known. Several proteins have been implicated in activating γTuRC, among which are MZT1 (GCP9) and MZT2 (GCP8), also sometimes considered core components of the metazoan complex (Hutchins et al, 2010;Kollman et al, 2011;Lin et al, 2016;Teixido-Travesa et al, 2012), the recruitment factors CDK5Rap2 (functional homologue of budding yeast Spc110) (Choi et al, 2010;Lin et al, 2014;Muroyama et al, 2016), or microtubule dynamics regulators such as the microtubule polymerase chTOG (XMAP215) or the multifunctional, catastrophe suppressing protein TPX2 Scrofani et al, 2015;Thawani et al, 2018).…”

Section: Introductionmentioning

confidence: 99%

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Microtubule Nucleation by Single Human γTuRC in a Partly Open Asymmetric Conformation

Consolati

Locke

Roostalu

et al. 2019

Preprint

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The γ-tubulin ring complex (γTuRC) is the major microtubule nucleator in cells. However, the mechanism of its regulation is not understood. Here, we purified human γTuRC and quantitatively characterized its nucleation properties in a TIRF microscopy-based real-time nucleation assay. We find that microtubule nucleation by γTuRC is kinetically inhibited compared to microtubule elongation. Determining the cryo-EM structure of γTuRC at 4 Å resolution reveals an asymmetric conformation with only part of the complex in a 'closed' conformation matching the microtubule geometry. Several factors stabilise the closed conformation. One is actin in the core of the complex and others, likely MZT1 or MZT2, line the outer perimeter of the closed part of γTuRC. The opposed side of γTuRC is in an 'open', nucleation-incompetent conformation, leading to a structural asymmetry, explaining the kinetic inhibition of nucleation by human γTuRC. Our data suggest possible regulatory mechanisms for microtubule nucleation by γTuRC closure.

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Section: Chtog and Tpx2 Stimulate γTurc-mediated Microtubule Nucleationsupporting

confidence: 92%

Section: Purification Of Biotinylated Human γTurcmentioning

confidence: 76%

Section: Discussionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

See 2 more Smart Citations

Microtubule Nucleation by Single Human γTuRC in a Partly Open Asymmetric Conformation

Consolati

Locke

Roostalu

et al. 2019

Preprint

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“…In cells, MTs are nucleated at tubulin concentrations significantly lower than those required for nucleation in vitro 5 . The efficiency of nucleation is increased by the template of the γ-tubulin ring complex (γTuRC) and other cellular factors [6][7][8][9][10][11][12][13][14][15] , but the molecular mechanism allowing highly efficient nucleation is unknown. Despite its high efficiency, the kinetics of MT assembly show a time lag between the onset of the reaction and the start of MT growth from the template 6,7 , suggesting that even in the presence of a template, the initial phase of assembly is thermodynamically unfavourable until the nucleation intermediate reaches a critical size.…”

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confidence: 99%

GTP-dependent formation of straight oligomers leads to nucleation of microtubules

Ayukawa

Iwata

Imai

et al. 2020

Preprint

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Microtubule (MT) nucleation is essential for cellular activities, but its mechanism is not known because of the difficulty involved in capturing rare stochastic events in the early stage of polymerization. In cells, MTs are nucleated at tubulin concentrations significantly lower than those required for spontaneous nucleation in vitro. The high efficiency of nucleation is due to the synergistic effects of various cellular factors, but the underlying mechanism has not been clarified yet. Here, combining negative stain electron microscopy and kinetic analysis, we demonstrate that the formation of single-stranded straight oligomers with critical size is essential for nucleation in vitro. While the single-stranded oligomers of GTP-tubulin that form prior to MT nucleation show variable curvatures including a few straight oligomers, only curved oligomers are observed among the GDP-bound counterparts. The Y222F mutation in β-tubulin increases the proportion of straight oligomers and drastically accelerates MT nucleation. Our results support a model in which GTP binding causes a small shift in the distribution of oligomer curvature, generating a minor population of straight oligomers compatible with lateral association and further growth to MTs.Our study suggests that cellular factors involved in nucleation promote it via stabilization of straight oligomers.

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The gamma‐tubulin ring complex: Deciphering the molecular organization and assembly mechanism of a major vertebrate microtubule nucleator

et al. 2021

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Microtubules are protein cylinders with functions in cell motility, signal sensing, cell organization, intracellular transport, and chromosome segregation. One of the key properties of microtubules is their dynamic architecture, allowing them to grow and shrink in length by adding or removing copies of their basic subunit, the heterodimer αβ-tubulin. In higher eukaryotes, de novo assembly of microtubules from αβ-tubulin is initiated by a 2 MDa multi-subunit complex, the gamma-tubulin ring complex (γ-TuRC). For many years, the structure of the γ-TuRC and the function of its subunits remained enigmatic, although structural data from the much simpler yeast counterpart, the γ-tubulin small complex (γ-TuSC), were available. Two recent breakthroughs in the field, high-resolution structural analysis and recombinant reconstitution of the complex, have revolutionized our knowledge about the architecture and function of the γ-TuRC and will form the basis for addressing outstanding questions about biogenesis and regulation of this essential microtubule organizer. K E Y W O R D Sgamma-tubulin ring complex, gamma-tubulin small complex, gamma-tubulin, microtubule nucleation, microtubules, recombinant gamma-tubulin ring complex Abbreviations: AAA+, adenosine triphosphatases associated with various cellular activities;Arps, actin-related proteins; CDK5RAP2, CDK5 regulatory subunit-associated protein 2; CEP192, centrosomal protein of 192 kDa; ch-TOG, colonic hepatic tumor-overexpressed gene; CM1, centrosomin motif 1; GCP, γ-TuC component protein; γ-TuCs, γ-tubulin complexes; γ-TuRC, γ-tubulin ring complex; γ-TuSC, γ-tubulin small complex; GRIP1/2, γ-tubulin ring protein 1/2; HSP90, heat shock protein 90; INO80, inositol-requiring mutant 80 complex; MZT1/2, mitotic spindle organizing protein 1/2; NEDD1, neural precursor cell expressed, developmentally down-regulated 1; NME7, nucleotide diphosphate kinase 7; PCM, pericentriolar material; PIH1D1, PIH1 domain containing 1; R2TP, Rvb1-Rvb2-Tah1-Pih1; RPAP3, RNA polymerase II associated protein 3; RUVBL1/2, RuvB-like 1/2; SPB, spindle pole body; SWR1, SWI2/SNF2-related 1 complex; XMAP215, Xenopus microtubule assembly protein with 215 kDThis is an open access article under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited.

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XMAP215 is a microtubule nucleation factor that functions synergistically with the γ-tubulin ring complex

Cited by 159 publications

References 64 publications

Microtubule Nucleation by Single Human γTuRC in a Partly Open Asymmetric Conformation

Microtubule Nucleation by Single Human γTuRC in a Partly Open Asymmetric Conformation

GTP-dependent formation of straight oligomers leads to nucleation of microtubules

The gamma‐tubulin ring complex: Deciphering the molecular organization and assembly mechanism of a major vertebrate microtubule nucleator

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