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Open Biol
2021 Feb 01;112:200325. doi: 10.1098/rsob.200325.
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Reconstitution of the recombinant human γ-tubulin ring complex.
Würtz M
,
Böhler A
,
Neuner A
,
Zupa E
,
Rohland L
,
Liu P
,
Vermeulen BJA
,
Pfeffer S
,
Eustermann S
,
Schiebel E
.
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Cryo-electron microscopy recently resolved the structure of the vertebrate γ-tubulin ring complex (γ-TuRC) purified from Xenopus laevis egg extract and human cells to near-atomic resolution. These studies clarified the arrangement and stoichiometry of γ-TuRC components and revealed that one molecule of actin and the small protein MZT1 are embedded into the complex. Based on this structural census of γ-TuRC core components, we developed a recombinant expression system for the reconstitution and purification of human γ-TuRC from insect cells. The recombinant γ-TuRC recapitulates the structure of purified native γ-TuRC and has similar functional properties in terms of microtubule nucleation and minus end capping. This recombinant system is a central step towards deciphering the activation mechanisms of the γ-TuRC and the function of individual γ-TuRC core components.
Figure 1. A modularized expression system enables purification of 2xFLAG-tagged recombinant γ-TuRC. (a) Schematic representation of the 14-spoke vertebrate γ-TuRC. Colours: GCP2 (light blue), GCP3 (dark blue), γ-tubulin (orange/yellow), GCP4 (brown), GCP5 (green), GCP6 (purple), actin (red) and luminal bridge (pink). (b) Cloning strategy for the recombinant γ-TuRC using the MultiBac vectors pACEBac1, PIDC, pIDK and pIDS with polyhedrin (polH) expression cassette. Human genes of the γ-TuRC were inserted via Infusion cloning. The ‘modules', plasmids with one or two genes of interest, were combined via subsequent Cre-recombination. Construct 1 for γ-TuRC expression consists of 2xFLAG-GCP5, GCP6, GCP4, TUBG1 and ACTB. This construct was used for bacmid and virus production and for protein expression. It was complemented with construct 2 coding for MZT1, GCP2 and GCP3. (c) Recombinant γ-TuRC was isolated via FLAG affinity purification and FLAG peptide elution in a single-step protocol and used for subsequent characterization using negative stain EM as well as biochemical approaches. Scheme of the γ-TuRC indicating the approximate position of the 2xFLAG tag at the N-terminus of GCP5. Colours as in (a). (d) Section of a negative stain EM micrograph of human recombinant γ-TuRC after FLAG affinity purification. Yellow boxes indicate exemplary γ-TuRC particles. Scale bar: 100 nm. (e) Section of Coomassie Blue-stained SDS-PAGE gel of γ-TuRC elution after FLAG affinity purification. (f) Cell lysate and FLAG affinity-purified recombinant γ-TuRC (FLAG elution) were probed using immunoblotting against the indicated antibodies. See electronic supplementary material, figure S2 for uncropped images.
Figure 2. Negative stain EM analysis of recombinant human and native X. laevis γ-TuRCs. (a) Comparison of representative two-dimensional classes from the native X. laevis (left) and recombinant human γ-TuRC (right) in two different views, as indicated in the cartoons. Number of particles in each class is given. Scale bar: 10 nm. (b) Three-dimensional EM densities of (i,iii) the native X. laevis (2490 particles) and (ii,iv) recombinant human γ-TuRC (2064 particles) in two different views. The atomic model of the human γ-TuRC (PDB-6V5 V) [9] was docked as a rigid body and superposed to the EM densities. Colouring as in figure 1a. (c) Zooms focused on the luminal bridge density of the native X. laevis (i) and recombinant human γ-TuRC (ii). The same colouring as in figure 1a.
Figure 3. Size exclusion chromatography of recombinant human γ-TuRC. (a) For SEC experiments, construct 1 (2xFLAG-GCP5, GCP6, GCP4, TUBG1 and ACTB) and construct 2 (MZT1, GCP2 and GCP3) were co-expressed with C-terminal Myc-His tagged γ-tubulin and purified via FLAG purification. (b) Chromatogram of a SEC run with ‘Superose 6 Increase (10/300) GL' column. Peak fractions (i (γ-TuRC peak), ii, iii) were analysed via immunoblotting (c,d). Red, brown and blue markers on x-axes (b) indicate borders of analysed fractions (i, ii, iii) in (d). Size markers thyroglobulin 669 kDa (Vt) and aldolase 158 kDa (Va) are indicated. (c) FLAG elution left and ‘γ-TuRC peak' fraction (i, b) were probed by immunoblotting against the indicated antibodies. (d) Immunoblot analysis of ethanol precipitated peak fractions (i, ii, iii) from SEC experiment shown in (b). Equal amount of fraction volumes was loaded. See electronic supplementary material, figure S2 for uncropped images.
Figure 4. The recombinant human γ-TuRC has microtubule nucleation and minus end capping activity. (a) Representative negative stain EM images from microtubules nucleated by native X. laevis γ-TuRC; (i; XLγ-TuRC) or the recombinant human γ-TuRC (ii-vi, rHγ-TuRC). For microtubules nucleated by the recombinant human γ-TuRC, the capped microtubule minus ends (ii–vi; MT-) and the flared or sheet microtubule plus ends (ii'–vi'; +MT) are shown. A schematic representation of a γ-TuRC capped microtubule is shown in the green box. Scale bars, 25 nm. (b,c) Tubulin polymerization assay where the increase in fluorescence intensity over time represents αβ-tubulin polymerization into microtubules. Shown are error bars for the standard deviation of the mean of three (b) and four (c) technical replicates. (b) The recombinant human γ-TuRC (construct 1 and construct 2 in figure 1, rHγ-TuRC) was analysed together with elution buffer without recombinant γ-TuRC as negative control, and 3 µM Paclitaxel as positive control. (c) Recombinant human γ-TuRC (construct 1, construct 2 and C-terminal Myc-His6 tagged γ-tubulin) was purified by FLAG affinity purification and subsequent SEC (figure 3b i). The γ-tubulin content of ‘γ-TuRC peak’ fraction (rHγ-TuRC SEC) and native X. laevis γ-TuRC (XLγ-TuRC) was determined by immunoblotting. Samples were diluted in SEC buffer to equal γ-tubulin concentrations and were then used for the microtubule nucleation assay with SEC buffer as a negative control and 3 µM Paclitaxel as positive control.
Aldaz,
Insights into microtubule nucleation from the crystal structure of human gamma-tubulin.
2005, Pubmed
Aldaz,
Insights into microtubule nucleation from the crystal structure of human gamma-tubulin.
2005,
Pubmed
Bahtz,
GCP6 is a substrate of Plk4 and required for centriole duplication.
2012,
Pubmed
Brouhard,
Microtubule dynamics: an interplay of biochemistry and mechanics.
2018,
Pubmed
Consolati,
Microtubule Nucleation Properties of Single Human γTuRCs Explained by Their Cryo-EM Structure.
2020,
Pubmed
Cota,
MZT1 regulates microtubule nucleation by linking γTuRC assembly to adapter-mediated targeting and activation.
2017,
Pubmed
Eustermann,
Structural basis for ATP-dependent chromatin remodelling by the INO80 complex.
2018,
Pubmed
Fitzgerald,
Protein complex expression by using multigene baculoviral vectors.
2006,
Pubmed
Guillet,
Crystal structure of γ-tubulin complex protein GCP4 provides insight into microtubule nucleation.
2011,
Pubmed
Haren,
A stable sub-complex between GCP4, GCP5 and GCP6 promotes the assembly of γ-tubulin ring complexes.
2020,
Pubmed
Kollman,
Microtubule nucleating gamma-TuSC assembles structures with 13-fold microtubule-like symmetry.
2010,
Pubmed
Kollman,
Microtubule nucleation by γ-tubulin complexes.
2011,
Pubmed
Leong,
Reconstitution of Microtubule Nucleation In Vitro Reveals Novel Roles for Mzt1.
2019,
Pubmed
Lin,
MOZART1 and γ-tubulin complex receptors are both required to turn γ-TuSC into an active microtubule nucleation template.
2016,
Pubmed
Liu,
NME7 is a functional component of the γ-tubulin ring complex.
2014,
Pubmed
Liu,
Insights into the assembly and activation of the microtubule nucleator γ-TuRC.
2020,
Pubmed
,
Xenbase
Lüders,
GCP-WD is a gamma-tubulin targeting factor required for centrosomal and chromatin-mediated microtubule nucleation.
2006,
Pubmed
Mandelkow,
Microtubule dynamics and microtubule caps: a time-resolved cryo-electron microscopy study.
1991,
Pubmed
Moritz,
Structure of the gamma-tubulin ring complex: a template for microtubule nucleation.
2000,
Pubmed
Murphy,
The mammalian gamma-tubulin complex contains homologues of the yeast spindle pole body components spc97p and spc98p.
1998,
Pubmed
,
Xenbase
Nogales,
Structural insights into microtubule function.
2000,
Pubmed
Nogales,
Structural intermediates in microtubule assembly and disassembly: how and why?
2006,
Pubmed
Pereira,
Centrosome-microtubule nucleation.
1997,
Pubmed
,
Xenbase
Petry,
Microtubule nucleation at the centrosome and beyond.
2015,
Pubmed
Teixidó-Travesa,
The gammaTuRC revisited: a comparative analysis of interphase and mitotic human gammaTuRC redefines the set of core components and identifies the novel subunit GCP8.
2010,
Pubmed
,
Xenbase
Thawani,
The transition state and regulation of γ-TuRC-mediated microtubule nucleation revealed by single molecule microscopy.
2020,
Pubmed
,
Xenbase
Thawani,
XMAP215 is a microtubule nucleation factor that functions synergistically with the γ-tubulin ring complex.
2018,
Pubmed
,
Xenbase
Tovey,
Microtubule nucleation by γ-tubulin complexes and beyond.
2018,
Pubmed
Wieczorek,
MZT Proteins Form Multi-Faceted Structural Modules in the γ-Tubulin Ring Complex.
2020,
Pubmed
Wieczorek,
Asymmetric Molecular Architecture of the Human γ-Tubulin Ring Complex.
2020,
Pubmed
Wiese,
A new function for the gamma-tubulin ring complex as a microtubule minus-end cap.
2000,
Pubmed
,
Xenbase
Zhang,
Gctf: Real-time CTF determination and correction.
2016,
Pubmed
Zheng,
Nucleation of microtubule assembly by a gamma-tubulin-containing ring complex.
1995,
Pubmed
,
Xenbase
Zivanov,
New tools for automated high-resolution cryo-EM structure determination in RELION-3.
2018,
Pubmed
Zupa,
The cryo-EM structure of a γ-TuSC elucidates architecture and regulation of minimal microtubule nucleation systems.
2020,
Pubmed
Zupa,
The structure of the γ-TuRC: a 25-years-old molecular puzzle.
2021,
Pubmed
