CoChat
Sign Up
PreprintCited by 38Microtubule and mitosis dynamicsGenetic and Kidney Cyst DiseasesCancer Treatment and Pharmacology

The DOI matches its Crossref record; 1 of the 1 references it cites were found in a registry or an index.

Structure of the native γ-Tubulin Ring Complex capping spindle microtubules

Open access
Barford, David, Dendooven, Tom, Yatskevich, Stanislau, Burt, Alister, Bellini, Dom, Kilmartin, John·2023·Nature Structural & Molecular Biology
DOI 10.21203/rs.3.rs-3481382/v1PMC PMC11257966

Preprint — published as 10.1038/s41594-024-01281-y

Open this paper in CoChat — free accountOpen PDF

Abstract

Microtubules (MTs) are fundamental to cellular architecture, function and organismal development 1. MT filaments assemble the mitotic spindle apparatus responsible for chromosome segregation, whereas the MT-based cytoskeletal network mediates dynein and kinesin-based intracellular transport. MTs are formed by the dynamic oligomerization and depolymerization of α/β-tubulin dimers in a head-to-tail fashion, with α-tubulin exposed at the ‘minus’ end of MTs and β-tubulin capping the more dynamic MT ‘plus’ end 2. In cells, the large and evolutionary conserved γ-Tubulin Ring Complex (γTuRC) templates efficient MT nucleation from their ‘minus’ end at MT-organizing centres (MTOCs)3–6. Because all known γTuRC structures are devoid of MTs and exhibit an ‘open’, inactive conformation, the molecular mechanism of γTuRC-mediated MT nucleation remains unknown. Here, we used cryo-electron tomography (cryo-ET) to determine the structure of the native γTuRC capping the minus end of a MT in the context of enriched yeast mitotic spindles. In our structure, γTuRC adopts an active closed conformation to function as a perfect geometric helical template presenting a ring of g-tubulin subunits to seed nucleation of exclusively 13-protofilament microtubules. Our cryo-ET reconstruction also revealed that a novel coiled-coil protein staples the first row of α/β-tubulin molecules directly to alternating positions along the γ-tubulin ring. This positioning of α/β-tubulin onto γTuRC reveals a role for the coiled-coil protein in augmenting γTuRC-mediated microtubule nucleation. Based on our results we describe a molecular model for γTuRC activation and MT nucleation.

Overview

The claims and statistics of this paper haven’t been checked yet.

Run the check — free, no account needed. It reads the open-access text and checks the sources it cites.

A few free checks a day · papers already verified open instantly · results are public

Integrity signals

Registry

Crossref · 10.21203/rs.3.rs-3481382/v1

Cites

1 of 1 references verified0 citation errors · 0 not checkable

Statistics

Verification

Claims the paper makes

The claims and statistics of this paper haven’t been checked yet.

Run the check — free, no account needed. It reads the open-access text and checks the sources it cites.

A few free checks a day · papers already verified open instantly · results are public

Self-check

Percentages, sums and reported differences re-computed from the paper’s own numbers.

The self-check needs the paper’s full text.

Citations

Cites

Flagged first, then by citations

Research works printed but not linked: 59

  1. Akhmanova, A. & Steinmetz, M. O. Control of microtubule organization and dynamics: two ends in the limelight. Nat Rev Mol Cell Biol 16, 711–726, doi:10.1038/nrm4084 (2015). · DOI · Paper page
  2. Brouhard, G. J. & Rice, L. M. Microtubule dynamics: an interplay of biochemistry and mechanics. Nat Rev Mol Cell Biol 19, 451–463, doi:10.1038/s41580-018-0009-y (2018). · DOI
  3. Zheng, Y., Wong, M. L., Alberts, B. & Mitchison, T. Nucleation of microtubule assembly by a gamma-tubulin-containing ring complex. Nature 378, 578–583, doi:10.1038/378578a0 (1995). · DOI
  4. Thawani, A. & Petry, S. Molecular insight into how gamma-TuRC makes microtubules. J Cell Sci 134, doi:10.1242/jcs.245464 (2021). · DOI
  5. Liu, P., Wurtz, M., Zupa, E., Pfeffer, S. & Schiebel, E. Microtubule nucleation: The waltz between gamma-tubulin ring complex and associated proteins. Curr Opin Cell Biol 68, 124–131, doi:10.1016/j.ceb.2020.10.004 (2021). · DOI
  6. Paz, J. & Luders, J. Microtubule-Organizing Centers: Towards a Minimal Parts List. Trends Cell Biol 28, 176–187, doi:10.1016/j.tcb.2017.10.005 (2018). · DOI
  7. Voter, W. A. & Erickson, H. P. The kinetics of microtubule assembly. Evidence for a two-stage nucleation mechanism. J Biol Chem 259, 10430–10438 (1984). · DOI
  8. Kuchnir Fygenson, D., Flyvbjerg, H., Sneppen, K., Libchaber, A. & Leibler, S. Spontaneous nucleation of microtubules. Phys Rev E Stat Phys Plasmas Fluids Relat Interdiscip Topics 51, 5058–5063, doi:10.1103/physreve.51.5058 (1995). · DOI
  9. Roostalu, J. & Surrey, T. Microtubule nucleation: beyond the template. Nat Rev Mol Cell Biol 18, 702–710, doi:10.1038/nrm.2017.75 (2017). · DOI
  10. Liu, P. et al. Insights into the assembly and activation of the microtubule nucleator gamma-TuRC. Nature 578, 467–471, doi:10.1038/s41586-019-1896-6 (2020). · DOI
  11. Wieczorek, M. et al. Asymmetric Molecular Architecture of the Human gamma-Tubulin Ring Complex. Cell 180, 165–175 e116, doi:10.1016/j.cell.2019.12.007 (2020). · DOI · Paper page
  12. Consolati, T. et al. Microtubule Nucleation Properties of Single Human gammaTuRCs Explained by Their Cryo-EM Structure. Dev Cell 53, 603–617 e608, doi:10.1016/j.devcel.2020.04.019 (2020). · DOI
  13. Wieczorek, M. et al. Biochemical reconstitutions reveal principles of human gamma-TuRC assembly and function. J Cell Biol 220, doi:10.1083/jcb.202009146 (2021). · DOI
  14. Wurtz, M. et al. Reconstitution of the recombinant human gamma-tubulin ring complex. Open Biol 11, 200325, doi:10.1098/rsob.200325 (2021). · DOI
  15. Zimmermann, F. et al. Assembly of the asymmetric human gamma-tubulin ring complex by RUVBL1-RUVBL2 AAA ATPase. Sci Adv 6, 1–20, doi:10.1126/sciadv.abe0894 (2020). · DOI
  16. Kollman, J. M., Polka, J. K., Zelter, A., Davis, T. N. & Agard, D. A. Microtubule nucleating gamma-TuSC assembles structures with 13-fold microtubule-like symmetry. Nature 466, 879–882, doi:10.1038/nature09207 (2010). · DOI
  17. Kollman, J. M. et al. Ring closure activates yeast gammaTuRC for species-specific microtubule nucleation. Nat Struct Mol Biol 22, 132–137, doi:10.1038/nsmb.2953 (2015). · DOI
  18. Brilot, A. F. et al. CM1-driven assembly and activation of yeast gamma-tubulin small complex underlies microtubule nucleation. Elife 10, 1–35, doi:10.7554/eLife.65168 (2021). · DOI
  19. Zupa, E. et al. The cryo-EM structure of a gamma-TuSC elucidates architecture and regulation of minimal microtubule nucleation systems. Nat Commun 11, 5705, doi:10.1038/s41467-020-19456-8 (2020). · DOI
  20. Lyon, A. S. et al. Higher-order oligomerization of Spc110p drives gamma-tubulin ring complex assembly. Mol Biol Cell 27, 2245–2258, doi:10.1091/mbc.E16-02-0072 (2016). · DOI
  21. Lin, T. C. et al. Cell-cycle dependent phosphorylation of yeast pericentrin regulates gamma-TuSC-mediated microtubule nucleation. Elife 3, e02208, doi:10.7554/eLife.02208 (2014). · DOI
  22. Leong, S. L. et al. Reconstitution of Microtubule Nucleation In Vitro Reveals Novel Roles for Mzt1. Curr Biol 29, 2199–2207 e2110, doi:10.1016/j.cub.2019.05.058 (2019). · DOI
  23. Knop, M. & Schiebel, E. Receptors determine the cellular localization of a gamma-tubulin complex and thereby the site of microtubule formation. EMBO J 17, 3952–3967, doi:10.1093/emboj/17.14.3952 (1998). · DOI
  24. Wieczorek, M., Bechstedt, S., Chaaban, S. & Brouhard, G. J. Microtubule-associated proteins control the kinetics of microtubule nucleation. Nat Cell Biol 17, 907–916, doi:10.1038/ncb3188 (2015). · DOI
  25. Thawani, A. et al. The transition state and regulation of gamma-TuRC-mediated microtubule nucleation revealed by single molecule microscopy. Elife 9, 1–34, doi:10.7554/eLife.54253 (2020). · DOI
  26. Thawani, A., Kadzik, R. S. & Petry, S. XMAP215 is a microtubule nucleation factor that functions synergistically with the gamma-tubulin ring complex. Nat Cell Biol 20, 575–585, doi:10.1038/s41556-018-0091-6 (2018). · DOI
  27. Ali, A., Vineethakumari, C., Lacasa, C. & Luders, J. Microtubule nucleation and gammaTuRC centrosome localization in interphase cells require ch-TOG. Nat Commun 14, 289, doi:10.1038/s41467-023-35955-w (2023). · DOI
  28. Bullitt, E., Rout, M. P., Kilmartin, J. V. & Akey, C. W. The yeast spindle pole body is assembled around a central crystal of Spc42p. Cell 89, 1077–1086, doi:Doi 10.1016/S0092-8674(00)80295-0 (1997). · DOI
  29. Rout, M. P. & Kilmartin, J. V. Components of the yeast spindle and spindle pole body. J Cell Biol 111, 1913–1927, doi:10.1083/jcb.111.5.1913 (1990). · DOI
  30. Winey, M. et al. Three-dimensional ultrastructural analysis of the Saccharomyces cerevisiae mitotic spindle. J Cell Biol 129, 1601–1615, doi:10.1083/jcb.129.6.1601 (1995). · DOI
  31. Zupa, E., Liu, P., Wurtz, M., Schiebel, E. & Pfeffer, S. The structure of the gamma-TuRC: a 25-years-old molecular puzzle. Curr Opin Struct Biol 66, 15–21, doi:10.1016/j.sbi.2020.08.008 (2021). · DOI
  32. Howes, S. C. et al. Structural differences between yeast and mammalian microtubules revealed by cryo-EM. J Cell Biol 216, 2669–2677, doi:10.1083/jcb.201612195 (2017). · DOI · Paper page
  33. Khodjakov, A. & Rieder, C. L. The sudden recruitment of gamma-tubulin to the centrosome at the onset of mitosis and its dynamic exchange throughout the cell cycle, do not require microtubules. J Cell Biol 146, 585–596, doi:10.1083/jcb.146.3.585 (1999). · DOI
  34. Aldaz, H., Rice, L. M., Stearns, T. & Agard, D. A. Insights into microtubule nucleation from the crystal structure of human gamma-tubulin. Nature 435, 523–527, doi:10.1038/nature03586 (2005). · DOI
  35. Kilmartin, J. V. Purification of yeast tubulin by self-assembly in vitro. Biochemistry 20, 3629–3633, doi:10.1021/bi00515a050 (1981). · DOI
  36. Wieczorek, M., Huang, T. L., Urnavicius, L., Hsia, K. C. & Kapoor, T. M. MZT Proteins Form Multi-Faceted Structural Modules in the gamma-Tubulin Ring Complex. Cell Rep 31, 107791, doi:10.1016/j.celrep.2020.107791 (2020). · DOI
  37. Huang, T. L., Wang, H. J., Chang, Y. C., Wang, S. W. & Hsia, K. C. Promiscuous Binding of Microprotein Mozart1 to gamma-Tubulin Complex Mediates Specific Subcellular Targeting to Control Microtubule Array Formation. Cell Rep 31, 107836, doi:10.1016/j.celrep.2020.107836 (2020). · DOI
  38. Cota, R. R. et al. MZT1 regulates microtubule nucleation by linking gammaTuRC assembly to adapter-mediated targeting and activation. J Cell Sci 130, 406–419, doi:10.1242/jcs.195321 (2017). · DOI
  39. Akram, Z. et al. Yeast as a Model to Understand Actin-Mediated Cellular Functions in Mammals-Illustrated with Four Actin Cytoskeleton Proteins. Cells 9, doi:10.3390/cells9030672 (2020). · DOI
  40. Lin, T. C. et al. MOZART1 and gamma-tubulin complex receptors are both required to turn gamma-TuSC into an active microtubule nucleation template. J Cell Biol 215, 823–840, doi:10.1083/jcb.201606092 (2016). · DOI
  41. Moritz, M., Braunfeld, M. B., Sedat, J. W., Alberts, B. & Agard, D. A. Microtubule nucleation by gamma-tubulin-containing rings in the centrosome. Nature 378, 638–640, doi:10.1038/378638a0 (1995). · DOI
  42. Mickolajczyk, K. J., Geyer, E. A., Kim, T., Rice, L. M. & Hancock, W. O. Direct observation of individual tubulin dimers binding to growing microtubules. Proc Natl Acad Sci U S A 116, 7314–7322, doi:10.1073/pnas.1815823116 (2019). · DOI
  43. Wigge, P. A. et al. Analysis of the Saccharomyces spindle pole by matrix-assisted laser desorption/ionization (MALDI) mass spectrometry. J Cell Biol 141, 967–977, doi:10.1083/jcb.141.4.967 (1998). · DOI
  44. Higuchi, R., Krummel, B. & Saiki, R. K. A general method of in vitro preparation and specific mutagenesis of DNA fragments: study of protein and DNA interactions. Nucleic Acids Res 16, 7351–7367, doi:10.1093/nar/16.15.7351 (1988). · DOI
  45. Taxis, C. & Knop, M. System of centromeric, episomal, and integrative vectors based on drug resistance markers for Saccharomyces cerevisiae. Biotechniques 40, 73–78, doi:10.2144/000112040 (2006). · DOI
  46. Battye, T. G., Kontogiannis, L., Johnson, O., Powell, H. R. & Leslie, A. G. iMOSFLM: a new graphical interface for diffraction-image processing with MOSFLM. Acta Crystallogr D Biol Crystallogr 67, 271–281, doi:10.1107/S0907444910048675 (2011). · DOI
  47. Evans, P. R. & Murshudov, G. N. How good are my data and what is the resolution? Acta Crystallogr D Biol Crystallogr 69, 1204–1214, doi:10.1107/S0907444913000061 (2013). · DOI
  48. McCoy, A. J. et al. Phaser crystallographic software. J Appl Crystallogr 40, 658–674, doi:10.1107/S0021889807021206 (2007). · DOI · Paper page
  49. Afonine, P. V. et al. Towards automated crystallographic structure refinement with phenix.refine. Acta Crystallogr D Biol Crystallogr 68, 352–367, doi:10.1107/S0907444912001308 (2012). · DOI · Paper page
  50. Tegunov, D. & Cramer, P. Real-time cryo-electron microscopy data preprocessing with Warp. Nat Methods 16, 1146–1152, doi:10.1038/s41592-019-0580-y (2019). · DOI
  51. Burt, A., Gaifas, L., Dendooven, T. & Gutsche, I. A flexible framework for multi-particle refinement in cryo-electron tomography. PLoS Biol 19, e3001319, doi:10.1371/journal.pbio.3001319 (2021). · DOI
  52. Castano-Diez, D., Kudryashev, M., Arheit, M. & Stahlberg, H. Dynamo: a flexible, user-friendly development tool for subtomogram averaging of cryo-EM data in high-performance computing environments. J Struct Biol 178, 139–151, doi:10.1016/j.jsb.2011.12.017 (2012). · DOI
  53. Bharat, T. A. & Scheres, S. H. Resolving macromolecular structures from electron cryo-tomography data using subtomogram averaging in RELION. Nat Protoc 11, 2054–2065, doi:10.1038/nprot.2016.124 (2016). · DOI
  54. Scheres, S. H. RELION: implementation of a Bayesian approach to cryo-EM structure determination. J Struct Biol 180, 519–530, doi:10.1016/j.jsb.2012.09.006 (2012). · DOI · Paper page
  55. Tegunov, D., Xue, L., Dienemann, C., Cramer, P. & Mahamid, J. Multi-particle cryo-EM refinement with M visualizes ribosome-antibiotic complex at 3.5 A in cells. Nat Methods 18, 186–193, doi:10.1038/s41592-020-01054-7 (2021). · DOI
  56. Emsley, P., Lohkamp, B., Scott, W. G. & Cowtan, K. Features and development of Coot. Acta Crystallogr D Biol Crystallogr 66, 486–501, doi:10.1107/S0907444910007493 (2010). · DOI · Paper page
  57. Meng, E. C. et al. UCSF ChimeraX: Tools for Structure Building and Analysis. Protein Sci, e4792, doi:10.1002/pro.4792 (2023). · DOI
  58. Forsberg, B. O., Shah, P. N. M. & Burt, A. A robust normalized local filter to estimate compositional heterogeneity directly from cryo-EM maps. Nat Commun 14, 5802, doi:10.1038/s41467-023-41478-1 (2023). · DOI
  59. Cook, A. D., Manka, S. W., Wang, S., Moores, C. A. & Atherton, J. A microtubule RELION-based pipeline for cryo-EM image processing. J Struct Biol 209, 107402, doi:10.1016/j.jsb.2019.10.004 (2020). · DOI · Paper page

Metadata

Authors
Barford, D., Dendooven, T., Yatskevich, S., Burt, A., Bellini, D., & Kilmartin, J.
Affiliations
MRC Laboratory of Molecular Biology (GB)
Corresponding author
  • Barford, David
  • Dendooven, Tom
  • Yatskevich, Stanislau
  • Kilmartin, John
ORCID
Venue
Nature Structural & Molecular Biology
Publisher
Springer Science and Business Media LLC
Published
2023-11-16
Volume
31
Issue
7
Pages
1134-1144
ISSN
1545-9985 (ISSN-L)
DOI
10.21203/rs.3.rs-3481382/v1
PMC
PMC11257966
OpenAlex
W4394753000
Cited by
38
References
1
Citations by year
2026: 13 · 2025: 10 · 2024: 14 · 2023: 1
FWCI
8.32
Type
journal article
Language
en
Open access
Licence
Locations
  • Nature Structural & Molecular Biology (publishedVersion, cc-by)
  • Research Square (acceptedVersion, cc-by)
  • PubMed Central (publishedVersion, cc-by)
  • PubMed (publishedVersion)
  • Apollo (University of Cambridge) (acceptedVersion, cc-by)
  • Edinburgh Research Explorer (University of Edinburgh) (submittedVersion, cc-by)
Funders
  • Wellcome Trust (203149)
  • UK Research and Innovation (MC_UP_1201/6, MC_U105178783)
  • Cancer Research UK
  • Deutsche Forschungsgemeinschaft (390540038, 203149, EXC 2008)
  • Technische Universität Berlin
  • Medical Research Council (MC_U105178783, MC_UP_1201/6, 203149)
Topics
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Cell Biology › Microtubule and mitosis dynamics
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Genetics › Genetic and Kidney Cyst Diseases
  • Health Sciences › Medicine › Oncology › Cancer Treatment and Pharmacology
Keywords
γ-tubulin ring complex, microtubule nucleation, spindle microtubules, Saccharomyces cerevisiae, coiled-coil proteins, microtubule organizing center, cryo-electron tomography
MeSH terms
Microtubule-Associated Proteins, Microtubules, Spindle Apparatus, Models, Molecular, Protein Conformation, Saccharomyces cerevisiae, Tubulin, Cryoelectron Microscopy, Saccharomyces cerevisiae Proteins, Electron Microscope Tomography

About these checks

Registry, retraction, Cites and statistics results are computed, not judged, with one exception: a printed reference that no index lists is read once by a model before it counts as a citation error. Claims are extracted by a model and kept only when their quote appears verbatim in the text; each cited source is then checked, with a model’s help, for whether it says what the claim relies on. Those verdicts can be wrong — read the claim’s quote and the cited source before relying on one.

Statistics and arithmetic are recomputed, not judged.

Confidence bands are shown only for fields calibrated on our annotated test set. None is calibrated yet, so no band is shown.

What counts as a claim ›How we measure confidence ›How we check this ›

Are you an author, or do you see a problem with this page? Request a review