γ-TuRC asymmetry induces local protofilament mismatch at the RanGTP-stimulated microtubule minus end

The γ-tubulin ring complex (γ-TuRC) is a structural template for de novo microtubule assembly from α/β-tubulin units. The isolated vertebrate γ-TuRC assumes an asymmetric, open structure deviating from microtubule geometry, suggesting that γ-TuRC closure may underlie regulation of microtubule nucleation. Here, we isolate native γ-TuRC-capped microtubules from Xenopus laevis egg extract nucleated through the RanGTP-induced pathway for spindle assembly and determine their cryo-EM structure. Intriguingly, the microtubule minus end-bound γ-TuRC is only partially closed and consequently, the emanating microtubule is locally misaligned with the γ-TuRC and asymmetric. In the partially closed conformation of the γ-TuRC, the actin-containing lumenal bridge is locally destabilised, suggesting lumenal bridge modulation in microtubule nucleation. The microtubule-binding protein CAMSAP2 specifically binds the minus end of γ-TuRC-capped microtubules, indicating that the asymmetric minus end structure may underlie recruitment of microtubule-modulating factors for γ-TuRC release. Collectively, we reveal a surprisingly asymmetric microtubule minus end protofilament organisation diverging from the regular microtubule structure, with direct implications for the kinetics and regulation of nucleation and subsequent modulation of microtubules during spindle assembly.


Appendix Figure S4 -Detailed structural analysis of the γ-TuRC at the MT minus end.
A-C Rotation around the MT axis (A), translation towards the MT axis (B) and downward change in helical pitch (C) required to convert the isolated γ-TuRC (dark grey) or the γ-TuRC at the MT minus end (orange) to the hypothetical, fully closed γ-TuRC for each spoke.Inset schematics represent the quantified parameters.Parameters measured from the center of mass of γ-tubulin.D,E Comparison of the γ-TuRC at the MT minus end with the isolated γ-TuRC (PDB 6TF9 (Liu et al, 2020), (D)) and the hypothetical fully closed γ-TuRC (E), generated from EMD 2799 (Kollman et al, 2015) (see Methods).Lumenal bridge components are omitted.Molecular surfaces generated from atomic models using the molmap function in UCSF ChimeraX (Goddard et al, 2018).F Zoom-in of the lumenal bridge in the reconstruction of the isolated γ-TuRC (left panel) and the γ-TuRC at the MT minus end (right), both superposed with the atomic model of the isolated X. laevis γ-TuRC (PDB 6TF9) (Liu et al., 2020).Difference density (red, middle panel) superposed with the reconstruction of the γ-TuRC at the MT minus end (grey) highlights that the MZT1/GCP6 N and MZT1/GCP3 N modules are well resolved, while density for actin is significantly reduced.All reconstructions were low-pass filtered to 17 Å.Colouring as indicated; components outside the lumenal bridge are shown in grey.Source data is available for this figure.*** p<0.001, n.s.non-significant.Significance determined using a one-tailed Welch's t-test.D, E Immunogold labelling of γ-TuRC-capped MTs isolated from X. laevis egg extract incubated with GFP-CAMSAP2.GFP-CAMSAP2 and γ-tubulin both localise to the same end of γ-TuRC-capped MTs, which is not observed in samples where GFP-CAMSAP2 is absent (E).Red (CAMSAP2) and yellow (γ-TuRC) asterisks indicate gold beads at the MT minus end.Additional density at MT ends may be attributed to staining of antibodies as well as potential accumulation of condensates of CAMSAP2 (Imasaki et al, 2022).
F γ-TuRC-capped MT minus ends isolated from X. laevis egg extract were incubated with GFP-CAMSAP2 and labelled with single gold conjugate: anti-GFP antibody labelled with 10 nm Protein A gold conjugated to rabbit anti-goat antibody (left), anti-γ-tubulin antibody labelled with rabbit antimouse antibody that was subsequently labelled with 15 nm Protein A gold (middle) and labelling of only rabbit anti-mouse antibody with 15 nm Protein A gold (right).Red (CAMSAP2) and yellow (γ-TuRC) asterisks indicate gold beads at the MT minus end.Dots in legend indicate approximate observed size of respective gold beads.Scale bar in (D), (E) and (F) represents 25 nm.Source data is available for this figure .population of γ-TuRC with 12 or fewer spokes.Atomic model of the recombinant γ-TuRC (PDB 7QJC) is fit.Actin is displayed in red, MZT1 in pink, otherwise, colouring as in Fig. 2B.Spoke numbers are indicated.D Normalised average intensity of CAMSAP2 (red), γ-TuRC (yellow) and MT signals (blue) along the length of γ-TuRC-capped MTs observed by multi-colour fluorescence microscopy (n=65).Curves were normalised to peak at 1.Only MTs longer than 5 μm were considered.Data were combined from three biological replicates.E Gallery of examples illustrating CAMSAP2 (red) colocalising with the γ-TuRC (yellow) and binding to the lattice of in vitro nucleated MTs (blue).Scale bar represents 2 µm.F Average intensity of CAMSAP2 signal within 0.75 μm of the γ-TuRC signal peak (n=795 from 159 MTs, of which 48, 53 and 58 for each respective biological replicate) or further away (n=3068 from 159 MTs).Intensities were normalised to the mean CAMSAP2 intensity on the MT lattice for each biological replicate.p=4.0x10 -49 .G Fraction of γ-TuRC-capped MTs with CAMSAP2 binding to the minus end (within 1 μm of the MT end; n=159).H Fraction of γ-TuRC-capped MTs and uncapped MTs with CAMSAP2 binding to either end (within 1 μm of the MT end; n=159 in both conditions, p=8.63x10 -7 ).I Fraction of γ-TuRC-capped MTs and uncapped MTs with CAMSAP2 binding to the MT lattice (i.e., >1 μm from either MT end; n=159 in both conditions, p=0.05806).Dots in (F), (G), (H) and (I) indicate mean values of the three biological replicates; data are shown as mean with 95% confidence interval for all individual data points.*** p<0.001, n.s.non-significant.Significance determined using a one-tailed Welch's t-test.Source data is available for this figure.

Appendix Table S1 -Mass spectrometry analysis of γ-TuRC-capped MTs isolated from X. laevis egg extracts.
α/ β-tubulin, γ-TuRC proteins and known interactors (Bohler et al, 2021) are listed according to abundance as ranked by total intensity.Due to genome duplication in X. laevis, some proteins are detected as their S and L isoform, corresponding to the long and short chromosomes of homoeologous pairs (Matsuda et al, 2015).Source data is available for this table.

Rank
Protein (and isoforms thereof)

Appendix Table S2 -Cross-correlation of the atomic models of open, partially closed and closed γ-TuRCs indicates that particle supplementation does not bias reconstruction of the MT-capping γ-TuRC, Paclitaxel-stabilised particles only.
We repeated the analysis in Table EV1 for only the set of particles that were stabilised using Paclitaxel, yielding comparable results that underline that particle supplementation does not bias reconstruction of the MT-capping γ-TuRC.Appendix Table S3 -Cross-correlation of the atomic models of open, partially closed and closed γ-TuRCs indicates that particle supplementation does not bias reconstruction of the MT-capping γ-TuRC, DTX-stabilised particles only.
We repeated the analysis in Table EV1 for only the set of particles that were stabilised using DTX, yielding comparable results that underline that particle supplementation does not bias reconstruction of the MT-capping γ-TuRC.We repeated the analysis in Table EV1 for only the set of particles that were not subjected to the shortening procedure during purification, yielding comparable results that underline that particle supplementation does not bias reconstruction of the MT-capping γ-TuRC.
Appendix Figure S7 -Binding of CAMSAP2 to the lattice and γ-TuRC-capped minus ends of MTs nucleated through the RanGTP pathway in X. laevis egg extract.A Fraction of γ-TuRC-capped MTs with CAMSAP2 binding to the minus end observed by multi-colour fluorescence microscopy (within 1 μm of the MT end; n=69, three biological replicates).Example fluorescence microscopy images are shown in Figure 4A.B Fraction of γ-TuRC-capped MTs and uncapped MTs with CAMSAP2 binding to either end observed by multi-colour fluorescence microscopy (within 1 μm of the MT end; n=69 for capped MTs, n=60 for uncapped MTs, p=0.000418, three biological replicates).C Fraction of γ-TuRC-capped MTs and uncapped MTs with CAMSAP2 binding to the MT lattice observed by multi-colour fluorescence microscopy (i.e., >1 μm from either MT end; n=69 for capped MTs, n=60 for uncapped MTs, p=0.3181).Dots in (A), (B) and (C) indicate mean values of the three biological replicates; data are shown as mean with 95% confidence interval for all individual data points.
of γ-TuRC after supplementation of open particles, followed by local refinement focused on spoke 1of γ-TuRC after refinement with supplementation of open particles (B), followed by refinement with global sampling without particle supplementation 0 of γ-TuRC after supplementation of open particles, followed by local refinement focused on spoke 1of γ-TuRC after refinement with supplementation of open particles (B), followed by refinement with global sampling without particle supplementation 0