NME7 is a functional component of the γ-tubulin ring complex

Liu, Pengfei; Choi, Yuk-Kwan; Qi, Robert Z.

doi:10.1091/mbc.e13-06-0339

Cited by 68 publications

(78 citation statements)

References 48 publications

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“…Our findings suggest that influencing γ/αβ-tubulin interaction favorably or unfavorably may underlie a dominant mechanism for regulating nucleation in the cell by other, yet unidentified nucleation factors. Additionally, γ-TuRC’s activity is further regulated via accessory proteins such as CDK5RAP2, and NME7 2, 20, 39, 40 . While the mechanisms of these additional regulation layers are yet to be defined, the insights on MT nucleation by γ-TuRC and XMAP215 provide an essential basis to build upon.…”

Section: Discussionmentioning

confidence: 99%

The transition state and regulation of γ-TuRC-mediated microtubule nucleation revealed by single molecule microscopy

Thawani

Rale

Coudray

et al. 2019

Preprint

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12Determining how microtubules (MTs) are nucleated is essential for understanding how the 13 cytoskeleton assembles. Yet, half a century after the discovery of MTs and ab-tubulin subunits 14 and decades after the identification of the γ-tubulin ring complex (γ-TuRC) as the universal MT 15 nucleator, the underlying mechanism largely remains a mystery. Using single molecule studies, 16we uncover that γ-TuRC nucleates a MT more efficiently than spontaneous assembly. The laterally 17 interacting array of γ-tubulins on γ-TuRC facilitates the lateral association of αβ-tubulins, while 18 longitudinal affinity between γ/αβ-tubulin is surprisingly weak. During nucleation, 3-4 αβ-tubulin 19 dimers bind stochastically to γ-TuRC on average until two of them create a lateral contact and 20 overcome the nucleation barrier. Although γ-TuRC defines the nucleus, XMAP215 significantly 21 increases reaction efficiency by facilitating ab-tubulin incorporation. In sum, we elucidate how 22 MT initiation occurs from γ-TuRC and determine how it is regulated. 23Results 49 50 Reconstituting and visualizing microtubule nucleation from γ-TuRC 51To study how γ-TuRC nucleates MTs (Fig. 1A), we purified endogenous γ-TuRC from Xenopus 52 egg extracts and biotinylated the complexes to immobilize them on functionalized glass ( Fig. S1A-53 C). Upon perfusing fluorescent αβ-tubulin, we visualized MT nucleation live with total internal 54 reflection fluorescence microscopy (TIRFM). Strikingly, MT nucleation events occurred 55 specifically from single γ-TuRC molecules ( Fig. 1B; Fig. S1D and Movie S1-2). Kymographs 56 revealed that attached γ-TuRC assembled ab-tubulin into a MT de novo starting from zero length 57 within the diffraction limit of light microscopy (Fig. 1C), ruling out an alternative model where 58MTs first spontaneously nucleate and then become stabilized via γ-TuRC. By observing the 59 fiduciary marks on the MT lattice ( Fig. 1C) and generating polarity-marked MTs from attached g-60 TuRC (Fig. S1E), we showed that γ-TuRC caps the MT minus-end, while only the plus-end 61 polymerizes. Altogether, our results show that γ-TuRC directly nucleates MTs. 62 63 Defining the microtubule nucleus on γ-TuRC 64To determine how γ-TuRC nucleates MTs, we measured the kinetics of MT nucleation for a 65 constant density of γ-TuRC and increasing αβ-tubulin concentration ( Fig. 1D and Movie S3). 66 Surprisingly, γ-TuRC nucleated MTs starting from 7 µM tubulin (Fig. 1D), which is higher than 67 the minimum tubulin concentration (C*) needed for growth at pre-formed MT plus-ends (C* = 1.4 68 µM, Fig. 1E). Furthermore, the number of MTs nucleated from γ-TuRC increased non-linearly 69 with tubulin concentration as opposed to the linear increase in MT's growth-speed with tubulin 70 concentration (Fig. 1E). By measuring the number of MTs nucleated over time with varying ab-71 interferometry, we compared the interaction of ab-tubulin dimers with themselves versus with g-118 tubulin. Specific interactions between probe-bound αβ-tubulin and increasing concentratio...

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Section: Discussionmentioning

confidence: 99%

The transition state and regulation of γ-TuRC-mediated microtubule nucleation revealed by single molecule microscopy

Thawani

Rale

Coudray

et al. 2019

Preprint

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“…DYN1, DYN2 and OPA1) for membrane remodeling (Fig 2) [23] and this may be true for other GTPases. NME7 localizes to centrosomes (Fig 2) and is part of the gamma-tubulin ring complex (γTuRC), and its kinase activity is required for nucleation of microtubules (Table I) [30]. The observation that NDPK contains a reactive phospho-intermediate was originally made in 1965 [31] and it was subsequently demonstrated to be 1-pHis [32].…”

Section: Introductionmentioning

confidence: 99%

pHisphorylation: the emergence of histidine phosphorylation as a reversible regulatory modification

Fuhs

Hunter

2017

Current Opinion in Cell Biology

145

106

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Histidine phosphorylation is crucial for prokaryotic signal transduction and as an intermediate for several metabolic enzymes, yet its role in mammalian cells remains largely uncharted. This is primarily due to difficulties in studying histidine phosphorylation because of the relative instability of phosphohistidine (pHis) and lack of specific antibodies and methods to preserve and detect it. The recent synthesis of stable pHis analogs has enabled development of pHis-specific antibodies and their use has started to shed light onto this important, yet enigmatic posttranslational modification. We are beginning to understand that pHis has broader roles in protein and cellular function including; cell cycle regulation, phagocytosis, regulation of ion channel activity and metal ion coordination. Two mammalian histidine kinases (NME1 and NME2), two pHis phosphatases (PHPT1 and LHPP), and a handful of substrates were previously identified. These new tools have already led to the discovery of an additional phosphatase (PGAM5) and hundreds of putative substrates. New methodologies are also being developed to probe the pHis phosphoproteome and determine functional consequences, including negative ion mode mass spectroscopy and unnatural amino acid incorporation. These new tools and strategies have the potential overcome the unique challenges that have been holding back our understanding of pHis in cell biology.

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“…It was also found in this complex by interaction with CDK5RAP2, a human microcephaly protein that binds the complex and is involved in centrosomal attachment of γ‐tubulin [Choi et al., ]. Finally, NME7 interacts with the γTuRC through both NDK domains, with Arg‐322 in the second NDK domain being crucial to the binding [Liu et al., ]. The 34 amino acids predicted to be deleted by the mutation we identified, including Arg at position 322, are missing in the mutated protein (Fig.…”

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confidence: 85%

A HomozygousNme7Mutation Is Associated withSitus Inversus Totalis

et al. 2016

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We investigated the cause of situs inversus totalis (SIT) in two siblings from a consanguineous family. Genotyping and whole-exome analysis revealed a homozygous change in NME7, resulting in deletion of an exon causing an in-frame deletion of 34 amino acids located in the second NDK domain of the protein and segregated with the defective lateralization in the family. NME7 is an important developmental gene, and NME7 protein is a component of the γ-tubulin ring complex. This mutation is predicted to affect the interaction of NME7 protein with this complex as it deletes the amino acids crucial for the binding. SIT associated with homozygous deletion in our patients is in line with Nme7−/− mutant mice phenotypes consisting of congenital hydrocephalus and SIT, indicating a novel human laterality patterning role for NME7. Further cases are required to elaborate the full human phenotype associated with NME7 mutations. Hum Mutat 0:1–5, 2016.

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NME7 is a functional component of the γ-tubulin ring complex

Abstract: The γ-tubulin ring complex (γTuRC) is the primary microtubule nucleator in animal cells. NME7 possesses an intrinsic kinase activity that is involved in the stimulation of the γTuRC.

Cited by 68 publications

References 48 publications

The transition state and regulation of γ-TuRC-mediated microtubule nucleation revealed by single molecule microscopy

The transition state and regulation of γ-TuRC-mediated microtubule nucleation revealed by single molecule microscopy

pHisphorylation: the emergence of histidine phosphorylation as a reversible regulatory modification

A HomozygousNme7Mutation Is Associated withSitus Inversus Totalis

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