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Structure of the γ-tubulin ring complex-capped microtubule

Nature Structural & Molecular Biology (2024)Cite this article

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Abstract

Microtubules are composed of α-tubulin and β-tubulin dimers positioned head-to-tail to form protofilaments that associate laterally in varying numbers. It is not known how cellular microtubules assemble with the canonical 13-protofilament architecture, resulting in micrometer-scale α/β-tubulin tracks for intracellular transport that align with, rather than spiral along, the long axis of the filament. We report that the human ~2.3 MDa γ-tubulin ring complex (γ-TuRC), an essential regulator of microtubule formation that contains 14 γ-tubulins, selectively nucleates 13-protofilament microtubules. Cryogenic electron microscopy reconstructions of γ-TuRC-capped microtubule minus ends reveal the extensive intra-domain and inter-domain motions of γ-TuRC subunits that accommodate luminal bridge components and establish lateral and longitudinal interactions between γ-tubulins and α-tubulins. Our structures suggest that γ-TuRC, an inefficient nucleation template owing to its splayed conformation, can transform into a compacted cap at the microtubule minus end and set the lattice architecture of cellular microtubules.

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Fig. 1: Cryo-EM of γ-TuRC-capped microtubules reveals selective nucleation of 13-protofilament microtubules.
Fig. 2: Longitudinal interactions along a protofilament and a GCP-spoke of γ-TuRC-capped microtubule minus end.
Fig. 3: Structural rearrangements of tubulins and GCPs at the seam.
Fig. 4: Lateral interactions established upon the compaction of γ-TuRC to cap the microtubule minus end.

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Data availability

The following cryo-EM maps have been deposited at the Electron Microscopy Data Bank: free recombinant γ-TuRC (EMD-43481), overall γ-TuRC-capped microtubule (EMD-43482), refined γ-TuRC-capped microtubule (EMD-43483), symmetry expanded map of γ-tubulins and α-tubulins (EMD-43085) and γ-TuRC nucleated microtubule protofilament (EMD-43519). The rigid-body fit models for free recombinant γ-TuRC, overall γ-TuRC-capped microtubule and the refined γ-TuRC-capped microtubule have been deposited in the Protein Data Bank (PDB 8VRD, PDB 8VRJ and PDB 8VRK, respectively). The refined models for the symmetry expanded map of γ-tubulins and α-tubulins and the γ-TuRC nucleated microtubule protofilament have been deposited in PDB (PDB 8VA2 and PDB 8VT7, respectively). Source data are provided with this paper.

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Acknowledgements

This work was funded by a National Institutes of Health grant (GM130234) to T.M.K. A.A. was supported in part by a Pels Family Center Postdoctoral Fellowship at The Rockefeller University. Some of this work was performed at the Simons Electron Microscopy Center (SEMC) at the New York Structural Biology Center, with major support from the Simons Foundation (SF349247). We are grateful to R. Roeder for the gift of HeLa cell extracts. We are grateful to J. Mendez, SEMC, for help with data acquisition. We are grateful to M. Ebrahim, J. Sotiris H. Ng and the Evelyn Gruss Lipper Cryo-Electron Microscopy Resource Center for cryo-EM support. We are also grateful to H. A. Pasolli at the Electron Microscopy Resource Center, both at The Rockefeller University, for EM support. We are grateful to Y. Niu and G. M. Alushin at The Rockefeller University for help with processing microtubule protofilament structure.

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  1. These authors contributed equally: Amol Aher, Linas Urnavicius.

Authors and Affiliations

  1. Laboratory of Chemistry and Cell Biology, The Rockefeller University, New York, NY, USA

    Amol Aher, Linas Urnavicius, Allen Xue & Tarun M. Kapoor

  2. Simons Electron Microscopy Center, New York Structural Biology Center, New York, NY, USA

    Kasahun Neselu

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Contributions

A.A., L.U. and T.M.K. conceived the study. A.A. and L.U. performed the experiments. A.A., L.U., A.X. and K.N. analyzed the data. T.M.K. supervised the study. A.A., L.U. and T.M.K. wrote the paper.

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Correspondence to Tarun M. Kapoor.

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T.M.K is a co-founder of and has an ownership interest in RADD Pharmaceuticals, Inc. All other authors declare no competing interests.

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Nature Structural & Molecular Biology thanks Rui Zhang and the other, anonymous, reviewer(s) for their contribution to the peer review of this work. Primary Handling Editor: Katarzyna Ciazynska, in collaboration with the Nature Structural & Molecular Biology team. Peer reviewer reports are available.

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Note added in proof: In a manuscript by Brito et al., Science 383, 870–876 (2024), the authors arrive at many similar conclusions regarding conformational changes from free to the microtubule bound form of γ-TuRC.

Extended data

Extended Data Fig. 1 TIRF-based optimization of γ-TuRC-dependent microtubule nucleation assay with slow GTP hydrolysing mutant of ɑ-tubulin, E254D.

(a) Transmission EM micrograph of negatively stained native γ-TuRC (representative image from 441 micrographs). (b) 2D class-averages showing two orientations of native γ-TuRC particles, top view (upper panel, 1182 particles) and side-view (bottom panel, 1048 particles) from negative stain EM. (c,d) 2D class averages for native γ-TuRC-capped minus-ends (177 ends, 125 from which were used for protofilament number analysis) (c) and for uncapped ends from spontaneously nucleated microtubules (183 ends) from cryo-EM (d). (e) SDS-PAGE analysis (Coomassie stained) of recombinant γ-TuRC-GFP following sucrose density gradient centrifugation. Expected positions for the components of the complex are indicated. (f,g) Images from a TIRF-microscopy sequence showing the γ-TuRC (green), tubulin (magenta) and merge channels after 2 minutes 30 s in the presence of chTOG (100 nM) and either wild type tubulin (10 µM) (f) or E254D (TUBA1B-E254D, TUBB3) tubulin (10 µM) (g). (h) Plot of the percentage (mean ± s.d.) of microtubules associated with a γ-TuRC-GFP puncta or not associated with a γ-TuRC-GFP puncta at 50 s post start of nucleation in the presence of E254D tubulin (10 µM) and chTOG (100 nM), total 95 microtubules, n = 3 independent experiments for each condition. (i) Plot of the cumulative number of microtubules (mean ± s.d.) nucleated by γ-TuRC in the presence of chTOG (100 nM) and either wild type tubulin or E254D tubulin (10 µM) over time, is shown. Data were fitted using linear regression, E254D tubulin (red line, total 193 microtubules) and wild type tubulin (blue line, no microtubules were detected). n = 4 independent experiments for each condition.

Source data

Extended Data Fig. 2 Cryo-EM data processing workflow for γ-TuRC-capped microtubule minus-end and free γ-TuRC.

A multi-step workflow is schematized.

Extended Data Fig. 3 Estimates of resolution and projection angle distribution for the overall γ-TuRC-capped microtubule minus-end and symmetry expanded α-α and γ-γ-tubulin density maps.

(a-d) Two views showing the Euler angle distribution (a,b), gold-standard Fourier shell correlation (FSC) curve (c) and directional FSC as estimated using the 3DFSC server62 (d) for the overall γ-TuRC-capped microtubule end density map presented in Figs. 1 and 2. (e-h) Two views showing the Euler angle distribution (e,f), gold-standard Fourier shell correlation (FSC) curve (g) and directional FSC as estimated using the 3DFSC server62 (h) for the symmetry expanded ɑ-ɑ and γ-γ-tubulin density map presented in Fig. 4.

Extended Data Fig. 4 Cryo-EM reconstruction of γ-TuRC-nucleated microtubule protofilament reveals a compacted lattice.

(a) Schematic for γ-TuRC-nucleated microtubule used for cryo-EM reconstruction (b, c) Cryo-EM density map for γ-TuRC-nucleated microtubule protofilament showing a view of the ɑ/β-tubulin dimer from the outside (b) and from within the lumen (c) (ɑ-tubulin: lighter green; β-tubulin: darker green). (d-e) Densities (mesh) for the nucleotide bound to ɑ-tubulin (GTP) (d) and β-tubulin (GDP) (e). (f-g) Models for 2 tubulin heterodimers from a γ-TuRC-nucleated microtubule protofilament and a protofilament from GDP-microtubule (PDB ID:6DPV) (f) or GMPCPP-microtubule (PDB ID:6DPU) (g) aligned at the first ɑ-tubulin. Distances between Cɑ atoms for 10 residues across 2 successive ɑ-tubulins were measured and averaged. (h) Four tubulin models rigid-body fitted into γ-TuRC-nucleated microtubule map at position 7 were used to measure distances between Cɑ atoms for 10 residues across two successive ɑ-tubulins.

Extended Data Fig. 5 Cryo-EM data processing workflow for a γ-TuRC-nucleated microtubule protofilament.

A multi-step workflow is schematized.

Extended Data Fig. 6 Estimates of resolution and projection angle distribution for γ-TuRC-nucleated microtubule protofilament.

(a-c) Gold-standard Fourier shell correlation (FSC) curve (a), directional FSC as estimated by the 3DFSC server62 (b) and euler angle distribution (c) for the γ-TuRC-nucleated microtubule protofilament density map presented in Extended Data Fig. 4.

Extended Data Fig. 7 Estimates of resolution and projection angle distribution for the refined γ-TuRC-capped microtubule minus-end map and free γ-TuRC map.

(a-d) Two views showing the Euler angle distribution (a,b), gold-standard Fourier shell correlation (FSC) curve (c) and directional FSC as estimated by the 3DFSC server62 (d) for the refined γ-TuRC-capped microtubule end density map presented in Fig. 3. (e-h) Two views showing the Euler angle distribution (e,f), gold-standard Fourier shell correlation (FSC) curve (g) and directional FSC as estimated using the 3DFSC server62 (h) for the free recombinant γ-TuRC density map, corresponding to Extended Data Figure 8.

Extended Data Fig. 8 Comparison of γ-TuRC in its free and microtubule bound form and analyses of lateral α-α and β-β tubulin interactions.

(a, b) Comparison of the rigid-body fitted models of native γ-TuRC (gray) and recombinant γ-TuRC (colored, GCPs in pink, γ-tubulins in purple) showing top (a) and side views (b). (c, d) Comparison of the rigid-body fitted models of recombinant γ-TuRC (gray) and microtubule bound recombinant γ-TuRC (colored, GCPs in pink, γ-tubulins in purple) showing top (c) and side views (d). (e-h) Lateral interactions at the ɑ-tubulin-ɑ-tubulin interface (e,g) and β-tubulin-β-tubulin interface (ribbons) (f,h).

Extended Data Table 1 Model building summary for the γ-TuRC nucleated microtubule end
Full size table
Extended Data Table 2 Model building summary for γ-TuRC structure
Full size table

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Aher, A., Urnavicius, L., Xue, A. et al. Structure of the γ-tubulin ring complex-capped microtubule. Nat Struct Mol Biol (2024). https://doi.org/10.1038/s41594-024-01264-z

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