CoChat
Sign Up
Cited by 17Microtubule and mitosis dynamicsCancer Treatment and PharmacologyGenetic and Kidney Cyst Diseases

Hydrolysis-deficient mosaic microtubules as faithful mimics of the GTP cap

Open access
Estévez-Gallego, Juan, Blum, Thorsten B., Ruhnow, Felix, Gili, María, Speroni, Silvia, García-Castellanos, Raquel, Steinmetz, Michel O., Surrey, Thomas·2025·Nature Communications
DOI 10.1038/s41467-025-57555-6PMC PMC11893814
Open this paper in CoChat — free account

Abstract

A critical feature of microtubules is their GTP cap, a stabilizing GTP-tubulin rich region at growing microtubule ends. Microtubules polymerized in the presence of GTP analogs or from GTP hydrolysis-deficient tubulin mutants have been used as GTP-cap mimics for structural and biochemical studies. However, these analogs and mutants generate microtubules with diverse biochemical properties and lattice structures, leaving it unclear what is the most faithful GTP mimic and hence the structure of the GTP cap. Here, we generate a hydrolysis-deficient human tubulin mutant, αE254Q, with the smallest possible modification. We show that αE254Q-microtubules are stable, but still exhibit mild mutation-induced growth abnormalities. However, mixing two GTP hydrolysis-deficient tubulin mutants, αE254Q and αE254N, at an optimized ratio eliminates growth and lattice abnormalities, indicating that these ‘mosaic microtubules’ are faithful GTP cap mimics. Their cryo-electron microscopy structure reveals that longitudinal lattice expansion, but not protofilament twist, is the primary structural feature distinguishing the GTP-tubulin containing cap from the GDP-tubulin containing microtubule shaft. However, alterations in protofilament twist may be transiently needed to allow lattice compaction and GTP hydrolysis. Together, our results provide insights into the structural origin of GTP cap stability, the pathway of GTP hydrolysis and hence microtubule dynamic instability.

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 · 10.1038/s41467-025-57555-6

Cites

2 of 2 references verified0 citation errors · 0 not checkable

Statistics

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

Claims other papers make about this one

Each is checked against this paper’s own text: does it say what the citing paper says it does?

Self-check

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

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

Cites

Flagged first, then by citations

Research works printed but not linked: 78

  1. [1]Brouhard, G. J. & Rice, L. M. Microtubule dynamics: an interplay of biochemistry and mechanics. Nat. Rev. Mol. Cell Biol. 19, 451–463 (2018). 29674711 PMC6019280 · DOI
  2. [2]Petry, S. Mechanisms of mitotic spindle assembly. Annu. Rev. Biochem. 85, 659–683 (2016). 27145846 PMC5016079 · DOI
  3. [3]Franker, M. A. & Hoogenraad, C. C. Microtubule-based transport—basic mechanisms, traffic rules and role in neurological pathogenesis. J. Cell Sci. 126, 2319–2329 (2013). 23729742
  4. [4]Aher, A. & Akhmanova, A. Tipping microtubule dynamics, one protofilament at a time. Curr. Opin. Cell Biol. 50, 86–93 (2018). 29573640 · DOI
  5. [5]Mitchison, T. & Kirschner, M. Dynamic instability of microtubule growth. Nature 312, 237–242 (1984). 6504138 · DOI · Paper page
  6. [6]Carlier, M. F. Guanosine-5’-triphosphate hydrolysis and tubulin polymerization. Review article. Mol. Cell Biochem 47, 97–113 (1982). 6755216 · DOI
  7. [7]Walker, R. A., Pryer, N. K. & Salmon, E. D. Dilution of individual microtubules observed in real time in vitro: evidence that cap size is small and independent of elongation rate. J. Cell Biol. 114, 73–81 (1991). 2050742 PMC2289054 · DOI
  8. [8]Nogales, E. An electron microscopy journey in the study of microtubule structure and dynamics. Protein Sci. 24, 1912–1919 (2015). 26401895 PMC4815221 · DOI
  9. [9]Maurer, S. P., Bieling, P., Cope, J., Hoenger, A. & Surrey, T. GTPgammaS microtubules mimic the growing microtubule end structure recognized by end-binding proteins (EBs). Proc. Natl. Acad. Sci. USA 108, 3988–3993 (2011). 21368119 PMC3053978 · DOI
  10. [10]Liao, M., Kuo, Y. W. & Howard, J. Counting fluorescently labeled proteins in tissues in the spinning-disk microscope using single-molecule calibrations. Mol. Biol. Cell 33, ar48 (2022). 35323029 PMC9265152 · DOI
  11. [11]Mustyatsa, V. V. et al. Fine structure and dynamics of EB3 binding zones on microtubules in fibroblast cells. Mol. Biol. Cell 30, 2105–2114 (2019). 31141458 PMC6743451 · DOI
  12. [12]Seetapun, D., Castle, B. T., McIntyre, A. J., Tran, P. T. & Odde, D. J. Estimating the microtubule GTP cap size in vivo. Curr. Biol. 22, 1681–1687 (2012). 22902755 PMC3461128 · DOI
  13. [13]Dixit, R. et al. Microtubule plus-end tracking by CLIP-170 requires EB1. Proc. Natl. Acad. Sci. USA 106, 492–497 (2009). 19126680 PMC2626730 · DOI
  14. [14]Komarova, Y. et al. Mammalian end binding proteins control persistent microtubule growth. J. Cell Biol. 184, 691–706 (2009). 19255245 PMC2686402 · DOI · Paper page
  15. [15]Bieling, P. et al. Reconstitution of a microtubule plus-end tracking system in vitro. Nature 450, 1100–1105 (2007). 18059460 · DOI · Paper page
  16. [16]Maurer, S. P. et al. EB1 accelerates two conformational transitions important for microtubule maturation and dynamics. Curr. Biol. 24, 372–384 (2014). 24508171 PMC3969257 · DOI
  17. [17]Li, F. et al. Cryo-EM structure of VASH1-SVBP bound to microtubules. eLife9, e58157 (2020). PMC7449697 32773040
  18. [18]Benoit, M., Asenjo, A. B. & Sosa, H. Cryo-EM reveals the structural basis of microtubule depolymerization by kinesin-13s. Nat. Commun. 9, 1662 (2018). 29695795 PMC5916938 · DOI
  19. [19]Manka, S. W. & Moores, C. A. The role of tubulin-tubulin lattice contacts in the mechanism of microtubule dynamic instability. Nat. Struct. Mol. Biol. 25, 607–615 (2018). 29967541 PMC6201834 · DOI · Paper page
  20. [20]Zhang, R., LaFrance, B. & Nogales, E. Separating the effects of nucleotide and EB binding on microtubule structure. Proc. Natl. Acad. Sci. USA 115, E6191–E6200 (2018). 29915050 PMC6142192
  21. [21]McIntosh, J. R. et al. Microtubules grow by the addition of bent guanosine triphosphate tubulin to the tips of curved protofilaments. J. Cell Biol. 217, 2691–2708 (2018). 29794031 PMC6080942 · DOI
  22. [22]Chretien, D., Fuller, S. D. & Karsenti, E. Structure of growing microtubule ends: two-dimensional sheets close into tubes at variable rates. J. Cell Biol. 129, 1311–1328 (1995). 7775577 PMC2120473 · DOI
  23. [23]Rickman, J., Duellberg, C., Cade, N. I., Griffin, L. D. & Surrey, T. Steady-state EB cap size fluctuations are determined by stochastic microtubule growth and maturation. Proc. Natl. Acad. Sci. USA 114, 3427–3432 (2017). 28280102 PMC5380103 · DOI
  24. [24]Duellberg, C., Cade, N. I., Holmes, D. & Surrey, T. The size of the EB cap determines instantaneous microtubule stability. eLife5, e13470 (2016). PMC4829430 27050486
  25. [25]Hyman, A. A., Salser, S., Drechsel, D. N., Unwin, N. & Mitchison, T. J. Role of GTP hydrolysis in microtubule dynamics: information from a slowly hydrolyzable analogue, GMPCPP. Mol. Biol. cell 3, 1155–1167 (1992). 1421572 PMC275679 · DOI
  26. [26]Alushin, G. M. et al. High-resolution microtubule structures reveal the structural transitions in alphabeta-tubulin upon GTP hydrolysis. Cell 157, 1117–1129 (2014). 24855948 PMC4054694 · DOI · Paper page
  27. [27]Hyman, A. A., Chretien, D., Arnal, I. & Wade, R. H. Structural changes accompanying GTP hydrolysis in microtubules: information from a slowly hydrolyzable analogue guanylyl-(alpha,beta)-methylene-diphosphonate. J. Cell Biol. 128, 117–125 (1995). 7822409 PMC2120325 · DOI · Paper page
  28. [28]Roth, D., Fitton, B. P., Chmel, N. P., Wasiluk, N. & Straube, A. Spatial positioning of EB family proteins at microtubule tips involves distinct nucleotide-dependent binding properties. J. Cell Sci. 132, jcs219550 (2018). PMC6398475 30262468
  29. [29]Estevez-Gallego, J. et al. Structural model for differential cap maturation at growing microtubule ends. eLife9, e50155 (2020). PMC7064335 32151315
  30. [30]Zhang, R., Alushin, G. M., Brown, A. & Nogales, E. Mechanistic origin of microtubule dynamic instability and its modulation by EB proteins. Cell 162, 849–859 (2015). 26234155 PMC4537847 · DOI · Paper page
  31. [31]Roostalu, J. et al. The speed of GTP hydrolysis determines GTP cap size and controls microtubule stability. eLife9, e51992 (2020). PMC7018511 32053491
  32. [32]LaFrance, B. J. et al. Structural transitions in the GTP cap visualized by cryo-electron microscopy of catalytically inactive microtubules. Proc. Natl. Acad. Sci. USA119, e2114994119 (2022). PMC8764682 34996871
  33. [33]Kuo, Y. W. & Howard, J. In vitro reconstitution of microtubule dynamics and severing imaged by label-free interference-reflection microscopy. Methods Mol. Biol. 2430, 73–91 (2022). 35476326 PMC9131738 · DOI
  34. [34]Hirst, W. G., Kiefer, C., Abdosamadi, M. K., Schaffer, E. & Reber, S. In vitro reconstitution and imaging of microtubule dynamics by fluorescence and label-free microscopy. STAR Protoc. 1, 100177 (2020). 33377071 PMC7757405 · DOI
  35. [35]Gardner, M. K. et al. Rapid microtubule self-assembly kinetics. Cell 146, 582–592 (2011). 21854983 PMC3171214 · DOI
  36. [37]Oosawa, F. Size distribution of protein polymers. J. Theor. Biol. 27, 69–86 (1970). 5419909 · DOI
  37. [38]Cleary, J. M. et al. Measurements and simulations of microtubule growth imply strong longitudinal interactions and reveal a role for GDP on the elongating end. eLife11, e75931 (2022). PMC9064298 35420545
  38. [39]Ayukawa, R. et al. GTP-dependent formation of straight tubulin oligomers leads to microtubule nucleation. J. Cell Biol. 220, e202007033 (2021). PMC7871348 33544140
  39. [40]Wisanpitayakorn, P., Mickolajczyk, K. J., Hancock, W. O., Vidali, L. & Tuzel, E. Measurement of the persistence length of cytoskeletal filaments using curvature distributions. Biophys. J. 121, 1813–1822 (2022). 35450824 PMC9199094 · DOI
  40. [41]Manka, S. W. & Moores, C. A. Microtubule structure by cryo-EM: snapshots of dynamic instability. Essays Biochem. 62, 737–751 (2018). 30315096 PMC6281474 · DOI · Paper page
  41. [42]Cuveillier, C. et al. MAP6 is an intraluminal protein that induces neuronal microtubules to coil. Sci. Adv. 6, eaaz4344 (2020). 32270043 PMC7112752 · DOI
  42. [44]Vitre, B. et al. EB1 regulates microtubule dynamics and tubulin sheet closure in vitro. Nat. Cell Biol. 10, 415–421 (2008). 18364701 · DOI
  43. [45]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 (2020). 31610239 PMC6961209 · DOI · Paper page
  44. [46]Rai, A. et al. Taxanes convert regions of perturbed microtubule growth into rescue sites. Nat. Mater. 19, 355–365 (2020). 31819210 · DOI
  45. [47]Melki, R., Fievez, S. & Carlier, M. F. Continuous monitoring of Pi release following nucleotide hydrolysis in actin or tubulin assembly using 2-amino-6-mercapto-7-methylpurine ribonucleoside and purine-nucleoside phosphorylase as an enzyme-linked assay. Biochemistry 35, 12038–12045 (1996). 8810908 · DOI
  46. [48]Beckett, D. & Voth, G. A. Unveiling the catalytic mechanism of GTP hydrolysis in microtubules. Proc. Natl. Acad. Sci. USA 120, e2305899120 (2023). 37364095 PMC10319017 · DOI
  47. [49]Chew, Y. M. & Cross, R. A. Taxol acts differently on different tubulin isotypes. Commun. Biol. 6, 946 (2023). 37717119 PMC10505170 · DOI
  48. [50]Feng, W. et al. SUMOylation of alpha-tubulin is a novel modification regulating microtubule dynamics. J. Mol. Cell Biol. 13, 91–103 (2021). 33394042 PMC8104938 · DOI
  49. [51]Pamula, M. C., Ti, S. C. & Kapoor, T. M. The structured core of human beta tubulin confers isotype-specific polymerization properties. J. Cell Biol. 213, 425–433 (2016). 27185835 PMC4878094 · DOI
  50. [52]Vemu, A., Atherton, J., Spector, J. O., Moores, C. A. & Roll-Mecak, A. Tubulin isoform composition tunes microtubule dynamics. Mol. Biol. cell 28, 3564–3572 (2017). 29021343 PMC5706985 · DOI
  51. [53]Fu, G. et al. Integrated regulation of tubulin tyrosination and microtubule stability by human alpha-tubulin isotypes. Cell Rep. 42, 112653 (2023). 37379209 · DOI
  52. [54]Chen, J. et al. alpha-tubulin tail modifications regulate microtubule stability through selective effector recruitment, not changes in intrinsic polymer dynamics. Dev. Cell 56, 2016–2028.e2014 (2021). 34022132 PMC8476856 · DOI
  53. [55]Wasilko, D. J. et al. The titerless infected-cells preservation and scale-up (TIPS) method for large-scale production of NO-sensitive human soluble guanylate cyclase (sGC) from insect cells infected with recombinant baculovirus. Protein Expr. Purif. 65, 122–132 (2009). 19174191 · DOI
  54. [56]Ti, S. C., Wieczorek, M. & Kapoor, T. M. Purification of affinity tag-free recombinant tubulin from insect cells. STAR Protoc.1, 100011 (2020). PMC7416841 32783031
  55. [57]Castoldi, M. & Popov, A. V. Purification of brain tubulin through two cycles of polymerization-depolymerization in a high-molarity buffer. Protein Expr. Purif. 32, 83–88 (2003). 14680943 · DOI
  56. [58]Hyman, A. et al. Preparation of modified tubulins. Methods Enzymol. 196, 478–485 (1991). 2034137 · DOI
  57. [59]Smith, S. J. & Rittinger, K. Preparation of GTPases for structural and biophysical analysis. Methods Mol. Biol. 189, 13–24 (2002). 12094582
  58. [60]Seckler, R., Wu, G. M. & Timasheff, S. N. Interactions of tubulin with guanylyl-(beta-gamma-methylene)diphosphonate. Formation and assembly of a stoichiometric complex. J. Biol. Chem. 265, 7655–7661 (1990). 2332445 · DOI
  59. [61]Bieling, P., Telley, I. A., Hentrich, C., Piehler, J. & Surrey, T. Fluorescence microscopy assays on chemically functionalized surfaces for quantitative imaging of microtubule, motor, and +TIP dynamics. Methods Cell Biol. 95, 555–580 (2010). 20466153 · DOI
  60. [62]Ustinova, K., Ruhnow, F., Gili, M. & Surrey, T. Microtubule binding of the human augmin complex is directly controlled by importins and Ran-GTP. J. Cell Sci. 136, jcs261096 (2023). PMC10323247 37357828
  61. [63]Gell, C. et al. Microtubule dynamics reconstituted in vitro and imaged by single-molecule fluorescence microscopy. Methods Cell Biol. 95, 221–245 (2010). 20466138 · DOI
  62. [64]Simmert, S., Abdosamadi, M. K., Hermsdorf, G. & Schaffer, E. LED-based interference-reflection microscopy combined with optical tweezers for quantitative three-dimensional microtubule imaging. Opt. Express 26, 14499–14513 (2018). 29877486 · DOI
  63. [65]Ruhnow, F., Zwicker, D. & Diez, S. Tracking single particles and elongated filaments with nanometer precision. Biophys. J. 100, 2820–2828 (2011). 21641328 PMC3117161 · DOI
  64. [66]Alves-Vale, C. et al. Expression of NORAD correlates with breast cancer aggressiveness and protects breast cancer cells from chemotherapy. Mol. Ther. Nucleic Acids 33, 910–924 (2023). 37680988 PMC10480464 · DOI
  65. [67]Perkins, D. N., Pappin, D. J., Creasy, D. M. & Cottrell, J. S. Probability-based protein identification by searching sequence databases using mass spectrometry data. Electrophoresis 20, 3551–3567 (1999). 10612281 · DOI
  66. [68]Chiva, C. et al. QCloud: a cloud-based quality control system for mass spectrometry-based proteomics laboratories. PLoS ONE 13, e0189209 (2018). 29324744 PMC5764250 · DOI
  67. [69]Beer, L. A., Liu, P., Ky, B., Barnhart, K. T. & Speicher, D. W. Efficient quantitative comparisons of plasma proteomes using label-free analysis with MaxQuant. Methods Mol. Biol. 1619, 339–352 (2017). 28674895 PMC5575765 · DOI
  68. [70]MacLean, B. et al. Skyline: an open source document editor for creating and analyzing targeted proteomics experiments. Bioinformatics 26, 966–968 (2010). 20147306 PMC2844992 · DOI · Paper page
  69. [71]Perez-Riverol, Y. et al. The PRIDE database resources in 2022: a hub for mass spectrometry-based proteomics evidences. Nucleic Acids Res. 50, D543–D552 (2022). 34723319 PMC8728295 · DOI · Paper page
  70. [72]Ku, S., Heichette, C., Duchesne, L. & Chretien, D. Microtubule seeded-assembly in the presence of poorly nucleating nucleotide analogues. Bio Protoc. 10, e3714 (2020). 33659378 PMC7842541
  71. [73]Ramirez-Rios, S. et al. VASH1-SVBP and VASH2-SVBP generate different detyrosination profiles on microtubules. J. Cell Biol. 222, e202205096 (2023). PMC9750192 36512346
  72. [74]Zivanov, J., Nakane, T. & Scheres, S. H. W. Estimation of high-order aberrations and anisotropic magnification from cryo-EM data sets in RELION-3.1. IUCrJ 7, 253–267 (2020). 32148853 PMC7055373 · DOI
  73. [75]Zheng, S. Q. et al. MotionCor2: anisotropic correction of beam-induced motion for improved cryo-electron microscopy. Nat. Methods 14, 331–332 (2017). 28250466 PMC5494038 · DOI
  74. [76]Zhang, K. Gctf: Real-time CTF determination and correction. J. Struct. Biol. 193, 1–12 (2016). 26592709 PMC4711343 · DOI
  75. [77]Emsley, P., Lohkamp, B., Scott, W. G. & Cowtan, K. Features and development of Coot. Acta Crystallogr. D. Biol. Crystallogr. 66, 486–501 (2010). 20383002 PMC2852313 · DOI
  76. [78]Afonine, P. V. et al. Real-space refinement in PHENIX for cryo-EM and crystallography. Acta Crystallogr. D. Struct. Biol. 74, 531–544 (2018). 29872004 PMC6096492 · DOI
  77. [79]Torices, R. & Munoz-Pajares, A. J. PHENIX: an R package to estimate a size-controlled phenotypic integration index. Appl. Plant Sci. 3, 1400104 (2015). PMC4435464 25995974
  78. [80]Meng, E. C. et al. UCSF ChimeraX: tools for structure building and analysis. Protein Sci. 32, e4792 (2023). 37774136 PMC10588335 · DOI

Cited by

Sorted by citations

Authors
Estévez-Gallego, J., Blum, T. B., Ruhnow, F., Gili, M., Speroni, S., García-Castellanos, R., Steinmetz, M. O., & Surrey, T.
Affiliations
  • Paul Scherrer Institute (CH)
  • Barcelona Institute of Science and Technology (ES)
  • University of Basel (CH)
  • Institució Catalana de Recerca i Estudis Avançats (ES)
Corresponding author
Surrey, Thomas
ORCID
Venue
Nature Communications
Publisher
Springer Science and Business Media LLC
Published
2025-03-10
Volume
16
Issue
1
Pages
2396-2396
ISSN
2041-1723 (ISSN-L)
DOI
10.1038/s41467-025-57555-6
PMC
PMC11893814
OpenAlex
W4408286643
Cited by
17
References
2
Citations by year
2026: 15 · 2025: 2
FWCI
7.65
Type
journal article
Language
en
Open access
Licence
Locations
  • Nature Communications (publishedVersion, cc-by)
  • Europe PMC (PubMed Central) (publishedVersion, cc-by)
  • PubMed Central (publishedVersion, cc-by)
  • PubMed (publishedVersion)
  • Repositori digital de la UPF (Universitat Pompeu Fabra) (publishedVersion, cc-by)
  • DORA PSI (Paul Scherrer Institute) (submittedVersion, cc-by)
  • RePEc: Research Papers in Economics (submittedVersion)
  • DOAJ (DOAJ: Directory of Open Access Journals) (submittedVersion, cc-by-sa)
Funders
  • Schweizerischer Nationalfonds zur Förderung der Wissenschaftlichen Forschung (192566, 310030)
  • Ministerio de Educación, Cultura y Deporte (PID2019-108415GB-I00, FJC2020-043857-I)
  • Ministerio de Ciencia e Innovación (CEX2020-001049-S)
  • Agencia Estatal de Investigación (CEX2020-001049-S)
Topics
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Cell Biology › Microtubule and mitosis dynamics
  • Health Sciences › Medicine › Oncology › Cancer Treatment and Pharmacology
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Genetics › Genetic and Kidney Cyst Diseases
Keywords
microtubule growth, microtubule dynamic instability
MeSH terms
Guanosine Diphosphate, Guanosine Triphosphate, Humans, Hydrolysis, Microtubules, Mutation, Tubulin, Cryoelectron Microscopy

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