Würtz M et al. (2021), Reconstitution of the recombinant human γ-tubul...

XB-ART-57771

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.

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Species referenced: Xenopus laevis
Genes referenced: actb golga4 myc tubgcp3 tubgcp5
GO keywords: gamma-tubulin ring complex

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	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.

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