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Complementary activities of TPX2 and chTOG constitute an efficient importin-regulated microtubule nucleation module

Nature Cell Biology volume 17, pages 1422–1434 (2015)Cite this article

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A Corrigendum to this article was published on 30 October 2015

This article has been updated

Abstract

Spindle assembly and function require precise control of microtubule nucleation and dynamics. The chromatin-driven spindle assembly pathway exerts such control locally in the vicinity of chromosomes. One of the key targets of this pathway is TPX2. The molecular mechanism of how TPX2 stimulates microtubule nucleation is not understood. Using microscopy-based dynamic in vitro reconstitution assays with purified proteins, we find that human TPX2 directly stabilizes growing microtubule ends and stimulates microtubule nucleation by stabilizing early microtubule nucleation intermediates. Human microtubule polymerase chTOG (XMAP215/Msps/Stu2p/Dis1/Alp14 homologue) only weakly promotes nucleation, but acts synergistically with TPX2. Hence, a combination of distinct and complementary activities is sufficient for efficient microtubule formation in vitro. Importins control the efficiency of the microtubule nucleation by selectively blocking the interaction of TPX2 with microtubule nucleation intermediates. This in vitro reconstitution reveals the molecular mechanism of regulated microtubule formation by a minimal nucleation module essential for chromatin-dependent microtubule nucleation in cells.

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Figure 1: Human chTOG is a microtubule polymerase.
Figure 2: The central part of human TPX2 determines its binding preference for growing microtubule ends.
Figure 3: Effect of full-length TPX2 and TPX2mini on microtubule dynamic instability parameters.
Figure 4: TPX2 binds to a unique binding region at growing microtubule ends.
Figure 5: Surface-immobilized TPX2 arrests nucleation intermediates, but in combination with chTOG efficiently nucleates microtubules.
Figure 6: In solution TPX2 nucleates microtubules more efficiently than chTOG.
Figure 7: The central region of TPX2 is sufficient to stimulate microtubule nucleation.
Figure 8: Regulation of TPX2- and chTOG-stimulated microtubule nucleation by importins.

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  • 05 October 2015

    In the version of this Article originally published online there was an incorrect citation in the methods section. This sentence should have read “GMPCPP-stabilized biotinylated fluorescently labelled microtubule ‘seeds’ for assays with dynamic microtubules were prepared as described previously41 (containing 12% of either Atto647N- or Atto565-labelled tubulin)”. This error has been corrected.

References

  1. Helmke, K. J., Heald, R. & Wilbur, J. D. Interplay between spindle architecture and function. Int. Rev. Cell Mol. Biol. 306, 83–125 (2013).

    Article  CAS  PubMed  Google Scholar 

  2. Gard, D. L. & Kirschner, M. W. A microtubule-associated protein from Xenopus eggs that specifically promotes assembly at the plus-end. J. Cell. Biol. 105, 2203–2215 (1987).

    Article  CAS  PubMed  Google Scholar 

  3. Brouhard, G. J. et al. XMAP215 is a processive microtubule polymerase. Cell 132, 79–88 (2008).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  4. Kronja, I., Kruljac-Letunic, A., Caudron-Herger, M., Bieling, P. & Karsenti, E. XMAP215-EB1 interaction is required for proper spindle assembly and chromosome segregation in Xenopus egg extract. Mol. Biol. Cell 20, 2684–2696 (2009).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  5. Reber, S. B. et al. XMAP215 activity sets spindle length by controlling the total mass of spindle microtubules. Nat. Cell Biol. 15, 1116–1122 (2013).

    Article  CAS  PubMed  Google Scholar 

  6. Gergely, F., Draviam, V. M. & Raff, J. W. The ch-TOG/XMAP215 protein is essential for spindle pole organization in human somatic cells. Genes Dev. 17, 336–341 (2003).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  7. Cassimeris, L. & Morabito, J. TOGp, the human homolog of XMAP215/Dis1, is required for centrosome integrity, spindle pole organization, and bipolar spindle assembly. Mol. Biol. Cell 15, 1580–1590 (2004).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  8. Heald, R. et al. Self-organization of microtubules into bipolar spindles around artificial chromosomes in Xenopus egg extracts. Nature 382, 420–425 (1996).

    Article  CAS  PubMed  Google Scholar 

  9. Carazo-Salas, R. E. et al. Generation of GTP-bound Ran by RCC1 is required for chromatin-induced mitotic spindle formation. Nature 400, 178–181 (1999).

    Article  CAS  PubMed  Google Scholar 

  10. Ohba, T., Nakamura, M., Nishitani, H. & Nishimoto, T. Self-organization of microtubule asters induced in Xenopus egg extracts by GTP-bound Ran. Science 284, 1356–1358 (1999).

    Article  CAS  PubMed  Google Scholar 

  11. Kalab, P., Pu, R. T. & Dasso, M. The ran GTPase regulates mitotic spindle assembly. Curr. Biol. 9, 481–484 (1999).

    Article  CAS  PubMed  Google Scholar 

  12. Wilde, A. & Zheng, Y. Stimulation of microtubule aster formation and spindle assembly by the small GTPase Ran. Science 284, 1359–1362 (1999).

    Article  CAS  PubMed  Google Scholar 

  13. Kalab, P., Weis, K. & Heald, R. Visualization of a Ran-GTP gradient in interphase and mitotic Xenopus egg extracts. Science 295, 2452–2456 (2002).

    Article  CAS  PubMed  Google Scholar 

  14. Kalab, P., Pralle, A., Isacoff, E. Y., Heald, R. & Weis, K. Analysis of a RanGTP-regulated gradient in mitotic somatic cells. Nature 440, 697–701 (2006).

    Article  CAS  PubMed  Google Scholar 

  15. Caudron, M., Bunt, G., Bastiaens, P. & Karsenti, E. Spatial coordination of spindle assembly by chromosome-mediated signaling gradients. Science 309, 1373–1376 (2005).

    Article  CAS  PubMed  Google Scholar 

  16. Goshima, G., Mayer, M., Zhang, N., Stuurman, N. & Vale, R. D. Augmin: a protein complex required for centrosome-independent microtubule generation within the spindle. J. Cell Biol. 181, 421–429 (2008).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  17. Petry, S., Groen, A. C., Ishihara, K., Mitchison, T. J. & Vale, R. D. Branching microtubule nucleation in Xenopus egg extracts mediated by augmin and TPX2. Cell 152, 768–777 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  18. Wittmann, T., Wilm, M., Karsenti, E. & Vernos, I. TPX2, a novel xenopus MAP involved in spindle pole organization. J. Cell Biol. 149, 1405–1418 (2000).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  19. Gruss, O. J. et al. Ran induces spindle assembly by reversing the inhibitory effect of importin α on TPX2 activity. Cell 104, 83–93 (2001).

    Article  CAS  PubMed  Google Scholar 

  20. Gruss, O. J. et al. Chromosome-induced microtubule assembly mediated by TPX2 is required for spindle formation in HeLa cells. Nat. Cell Biol. 4, 871–879 (2002).

    Article  CAS  PubMed  Google Scholar 

  21. Aguirre-Portoles, C. et al. Tpx2 controls spindle integrity, genome stability, and tumor development. Cancer Res. 72, 1518–1528 (2012).

    Article  CAS  PubMed  Google Scholar 

  22. Perez de Castro, I. & Malumbres, M. Mitotic stress and chromosomal instability in cancer: the case for TPX2. Genes Cancer 3, 721–730 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  23. Schatz, C. A. et al. Importin α-regulated nucleation of microtubules by TPX2. EMBO J. 22, 2060–2070 (2003).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  24. Giesecke, A. & Stewart, M. Novel binding of the mitotic regulator TPX2 (target protein for Xenopus kinesin-like protein 2) to importin-α. J. Biol. Chem. 285, 17628–17635 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  25. Trieselmann, N., Armstrong, S., Rauw, J. & Wilde, A. Ran modulates spindle assembly by regulating a subset of TPX2 and Kid activities including Aurora A activation. J. Cell Sci. 116, 4791–4798 (2003).

    Article  CAS  PubMed  Google Scholar 

  26. Brunet, S. et al. Characterization of the TPX2 domains involved in microtubule nucleation and spindle assembly in Xenopus egg extracts. Mol. Biol. Cell 15, 5318–5328 (2004).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  27. Tanenbaum, M. E. et al. Kif15 cooperates with eg5 to promote bipolar spindle assembly. Curr. Biol. 19, 1703–1711 (2009).

    Article  CAS  PubMed  Google Scholar 

  28. Vanneste, D., Takagi, M., Imamoto, N. & Vernos, I. The role of Hklp2 in the stabilization and maintenance of spindle bipolarity. Curr. Biol. 19, 1712–1717 (2009).

    Article  CAS  PubMed  Google Scholar 

  29. Koffa, M. D. et al. HURP is part of a Ran-dependent complex involved in spindle formation. Curr. Biol. 16, 743–754 (2006).

    Article  CAS  PubMed  Google Scholar 

  30. Wittmann, T., Boleti, H., Antony, C., Karsenti, E. & Vernos, I. Localization of the kinesin-like protein Xklp2 to spindle poles requires a leucine zipper, a microtubule-associated protein, and dynein. J. Cell Biol. 143, 673–685 (1998).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  31. Ma, N., Titus, J., Gable, A., Ross, J. L. & Wadsworth, P. TPX2 regulates the localization and activity of Eg5 in the mammalian mitotic spindle. J. Cell Biol. 195, 87–98 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  32. Helmke, K. J. & Heald, R. TPX2 levels modulate meiotic spindle size and architecture in Xenopus egg extracts. J. Cell Biol. 206, 385–393 (2014).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  33. Tsai, M. Y. et al. A Ran signalling pathway mediated by the mitotic kinase Aurora A in spindle assembly. Nat. Cell Biol. 5, 242–248 (2003).

    Article  CAS  PubMed  Google Scholar 

  34. Bayliss, R., Sardon, T., Vernos, I. & Conti, E. Structural basis of Aurora-A activation by TPX2 at the mitotic spindle. Mol. Cell 12, 851–862 (2003).

    Article  CAS  PubMed  Google Scholar 

  35. Kufer, T. A. et al. Human TPX2 is required for targeting Aurora-A kinase to the spindle. J. Cell Biol. 158, 617–623 (2002).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  36. Brunet, S. et al. Meiotic regulation of TPX2 protein levels governs cell cycle progression in mouse oocytes. PLoS ONE 3, e3338 (2008).

    Article  PubMed  PubMed Central  Google Scholar 

  37. Groen, A. C., Maresca, T. J., Gatlin, J. C., Salmon, E. D. & Mitchison, T. J. Functional overlap of microtubule assembly factors in chromatin-promoted spindle assembly. Mol. Biol. Cell 20, 2766–2773 (2009).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  38. Bird, A. W. & Hyman, A. A. Building a spindle of the correct length in human cells requires the interaction between TPX2 and Aurora A. J. Cell Biol. 182, 289–300 (2008).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  39. Scrofani, J., Sardon, T., Meunier, S. & Vernos, I. Microtubule nucleation in mitosis by a RanGTP-dependent protein complex. Curr. Biol. 25, 131–140 (2015).

    Article  CAS  PubMed  Google Scholar 

  40. Kollman, J. M., Merdes, A., Mourey, L. & Agard, D. A. Microtubule nucleation by γ-tubulin complexes. Nat. Rev. Mol. Cell Biol. 12, 709–721 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  41. 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).

    Article  CAS  PubMed  Google Scholar 

  42. Al-Bassam, J. et al. Fission yeast Alp14 is a dose-dependent plus end-tracking microtubule polymerase. Mol. Biol. Cell 23, 2878–2890 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  43. Podolski, M., Mahamdeh, M. & Howard, J. Stu2, the budding yeast XMAP215/Dis1 homolog, promotes assembly of yeast microtubules by increasing growth rate and decreasing catastrophe frequency. J. Biol. Chem. 289, 28087–28093 (2014).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  44. Li, W. et al. EB1 promotes microtubule dynamics by recruiting Sentin in Drosophila cells. J. Cell Biol. 193, 973–983 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  45. Wieczorek, M., Bechstedt, S., Chaaban, S. & Brouhard, G. J. Microtubule-associated proteins control the kinetics of microtubule nucleation. Nat. Cell Biol. 17, 907–916 (2015).

    Article  CAS  PubMed  Google Scholar 

  46. Bieling, P. et al. CLIP-170 tracks growing microtubule ends by dynamically recognizing composite EB1/tubulin-binding sites. J. Cell Biol. 183, 1223–1233 (2008).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  47. Bieling, P. et al. Reconstitution of a microtubule plus-end tracking system in vitro. Nature 450, 1100–1105 (2007).

    Article  CAS  PubMed  Google Scholar 

  48. Maurer, S. P. et al. EB1 accelerates two conformational transitions important for microtubule maturation and dynamics. Curr. Biol. 24, 372–384 (2014).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  49. 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).

    Article  CAS  PubMed  Google Scholar 

  50. Maurer, S. P., Bieling, P., Cope, J., Hoenger, A. & Surrey, T. GTPγS microtubules mimic the growing microtubule end structure recognized by end-binding proteins (EBs). Proc. Natl Acad. Sci. USA 108, 3988–3993 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  51. Ghosh, S., Hentrich, C. & Surrey, T. Micropattern-controlled local microtubule nucleation, transport, and mesoscale organization. ACS Chem. Biol. 8, 673–678 (2013).

    Article  CAS  PubMed  Google Scholar 

  52. 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).

    CAS  PubMed  Google Scholar 

  53. Wang, H. W., Long, S., Finley, K. R. & Nogales, E. Assembly of GMPCPP-bound tubulin into helical ribbons and tubes and effect of colchicine. Cell Cycle 4, 1157–1160 (2005).

    Article  CAS  PubMed  Google Scholar 

  54. Mozziconacci, J., Sandblad, L., Wachsmuth, M., Brunner, D. & Karsenti, E. Tubulin dimers oligomerize before their incorporation into microtubules. PLoS ONE 3, e3821 (2008).

    Article  PubMed  PubMed Central  Google Scholar 

  55. van Breugel, M., Drechsel, D. & Hyman, A. Stu2p, the budding yeast member of the conserved Dis1/XMAP215 family of microtubule-associated proteins is a plus end-binding microtubule destabilizer. J. Cell Biol. 161, 359–369 (2003).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  56. 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).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  57. Hyman, A. A., Chretien, D., Arnal, I. & Wade, R. H. Structural changes accompanying GTP hydrolysis in microtubules: information from a slowly hydrolyzable analogue guanylyl-(α,β)-methylene-diphosphonate. J. Cell Biol. 128, 117–125 (1995).

    Article  CAS  PubMed  Google Scholar 

  58. Alushin, G. M. et al. High-resolution microtubule structures reveal the structural transitions in αβ-tubulin upon GTP hydrolysis. Cell 157, 1117–1129 (2014).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  59. Bechstedt, S., Lu, K. & Brouhard, G. J. Doublecortin recognizes the longitudinal curvature of the microtubule end and lattice. Curr. Biol. 24, 2366–2375 (2014).

    Article  CAS  PubMed  Google Scholar 

  60. Ayaz, P., Ye, X., Huddleston, P., Brautigam, C. A. & Rice, L. M. A TOG:αβ-tubulin complex structure reveals conformation-based mechanisms for a microtubule polymerase. Science 337, 857–860 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  61. Coombes, C. E., Yamamoto, A., Kenzie, M. R., Odde, D. J. & Gardner, M. K. Evolving tip structures can explain age-dependent microtubule catastrophe. Curr. Biol. 23, 1342–1348 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  62. Tsai, M. Y. & Zheng, Y. Aurora A kinase-coated beads function as microtubule-organizing centers and enhance RanGTP-induced spindle assembly. Curr. Biol. 15, 2156–2163 (2005).

    Article  CAS  PubMed  Google Scholar 

  63. Zacharias, D. A., Violin, J. D., Newton, A. C. & Tsien, R. Y. Partitioning of lipid-modified monomeric GFPs into membrane microdomains of live cells. Science 296, 913–916 (2002).

    Article  CAS  PubMed  Google Scholar 

  64. Snapp, E. L. et al. Formation of stacked ER cisternae by low affinity protein interactions. J. Cell Biol. 163, 257–269 (2003).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  65. Duellberg, C. et al. Reconstitution of a hierarchical +TIP interaction network controlling microtubule end tracking of dynein. Nat. Cell Biol. 16, 804–811 (2014).

    Article  CAS  PubMed  Google Scholar 

  66. 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).

    Article  CAS  PubMed  Google Scholar 

  67. Hyman, A. et al. Preparation of modified tubulins. Methods Enzymol. 196, 478–485 (1991).

    Article  CAS  PubMed  Google Scholar 

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Acknowledgements

We thank I. Lüke and C. Thomas for insect cell culture maintenance; C. Thomas for help with protein expression, and cloning and purification of the biotinylated Kin1rigor construct; C. Duellberg (The Francis Crick Institute, UK) for a partially purified MonoQ fraction of the untagged human EB1 protein. We are grateful to R. Heald (University of California at Berkeley, USA), D. Görlich (Max Planck Institute for Biophysical Chemistry, Germany), S. Royle (Warwick Medical School, UK), G. Stier (European Molecular Biology Laboratory, Germany) and I. Vernos (Centre for Genomic Regulation, Spain) for providing various plasmids. We thank all the members of the Surrey laboratory for discussions, and F. Fourniol and C. Duellberg for critical reading of the manuscript. T.S. acknowledges the ERC (Project 323042) and Cancer Research UK for funding; J.R. was supported by a Cancer Research UK postdoctoral fellowship, an EMBO Long-Term Fellowship (LTF-615-2012), and a Sir Henry Wellcome Postdoctoral Fellowship (100145/Z/12/Z).

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  1. The Francis Crick Institute, 44 Lincoln’s Inn Fields, London WC2A 3LY, UK,

    Johanna Roostalu, Nicholas I. Cade & Thomas Surrey

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  1. Johanna Roostalu
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Contributions

J.R. and T.S. designed the study; J.R. generated the reagents and performed the experiments; J.R. and N.I.C. analysed the data; J.R. and T.S. wrote the manuscript.

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Correspondence to Johanna Roostalu.

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Integrated supplementary information

Supplementary Figure 3 TPX2 reduces catastrophes at the microtubule minus ends.

Modified box-and-whiskers plot showing the microtubule minus end catastrophe frequencies in the absence (control) and presence of 5 nM full-length mGFP-TPX2 and 250 nM mGFPTPX2mini, as indicated. Number of measured microtubule minus ends (total): control—n = 93, mGFP-TPX2—n = 63, mGFP-TPX2mini—n = 54. Number of catastrophes (total): control—n = 608, mGFP-TPX2—n = 168, mGFP-TPX2mini—n = 153. Microtubule growth time (total): control—395,972 s, mGFP-TPX2—276,060 s, mGFP-TPX2mini—235,408 s. For the modified box-and-whiskers plot the boxes range from 25th to 75th percentile, the whiskers span from 10th to 90th percentile, the horizontal line marks the mean value. Data were pooled from two datasets. Errors are SEM. ∗p ≤ 0.05; (only displayed for comparisons with control); determined for the comparison of mean values analysing raw data (Tukey’s test in conjunction with One Way ANOVA).

Supplementary Figure 4 Purified recombinant proteins used in this study.

Uncropped Coomassie Blue-stained SDS-PAGE gels of a biotinylated human TPX2, TPX2ΔN, and TPX2mini and similarly tagged monomeric Drosophila kinesin-1 rigor mutant and human chTOG. Note that all biotinylated TPX2 constructs and the kinesin-1 mutant have a BAPmTagBFP fused to their N-termini. The chTOG contains an mTagBFP-BAP tag at its Cterminus. The double bands visible for the constructs are not due to the protein degradation but a likely a consequence of different folding and/or maturation states of mTagBFP running with different molecular weights (as observed for mCherry, see for example, ref. 65). Massspectrometry analysis revealed very similar peptide coverage for faster and slower migrating forms of each protein displaying these double bands (data not shown). (b) Human SNAPTPX2 and SNAP-TPX2mini, (c) Human chTOG, chTOG-mGFP, and human EB1, (d) Human importin α and importin β. 1 μg of protein is loaded in all cases. Note that parts of Supplementary Fig. 2c are also depicted on Fig. 1b.

Supplementary Figure 5 Single molecule characterisation of TPX2 binding to growing microtubule ends and to GMPCPP microtubules.

(a) Example plots showing the time course of the measured fluorescence intensity, the calculated transition probability and the binarised probability of mGFP-TPX2mini at a growing microtubule end. (b) Single molecule dwell time and waiting time distributions of 5 nM mGFP-TPX2mini at growing microtubule ends (conditions as in Fig. 4e), with mono-exponential fits (magenta). (c) Average spatial distribution of SNAP-TPX2mini single molecule fluorescence intensities for two different time windows after start of microtubule growth; this agrees with similar measurements performed using microtubule end tracking and comet analysis (Fig. 4b). Averages of 139,000 (<4 min) and 160,000 (>4 min) frames were used to generate the curves. (d) Kymographs showing 50 pM mGFP-TPX2mini (green in merge) binding to GMPCPPstabilised Atto565-labelled microtubules (blue in merge) either in the absence or presence of additional 181 nM Aleax647-labelled SNAP-TPX2mini (magenta in merge), always in the absence of free tubulin. (e) The dissociation rate constant koff, and association rate ron for the conditions shown in d demonstrate that also in the presence of excess TPX2, turnover remains dynamic (7,327 and 6,029 binding events, respectively). (f) Kymographs showing 10 pM of full-length mGFP-TPX2 (green in merge) binding to GMPCPP-stabilised Atto565-labelled microtubules (blue in merge) either in the absence (left) or presence (right) of additional 11 nM Alexa647-labelled SNAP-TPX2 (magenta in merge), both in the absence of free tubulin. Scale bars as indicated.

Supplementary Figure 6 Surface-immobilised TPX2 induces microtubule ‘stub’ formation in TPX2 and tubulin concentration dependent manner.

(a) TIRF microscopy images of flow chamber surfaces pre-incubated with 125 nM of biotinylated mTagBFPtagged proteins visualised by mTagBFP fluorescence. These fields of view correspond to the ones depicting the Atto647N-labelled tubulin channel at the same protein concentrations (125 nM Kin1rigor, 125 nM chTOG, and 125 nM TPX2) on Fig. 5b. Scale bar as indicated. (b) Quantification of surface densities of biotinylated proteins at different concentrations based on mTagBFP-fluorescence. Three different fields of view were imaged for each condition after monitoring the microtubule nucleation using Atto647N-tubulin channel for experiments depicted on Fig. 5b, c. t = 0 when the sample is placed at 30 °C. (c) Quantification of Atto647N-tubulin intensities on different biotinylated protein surfaces (same as Fig. 5c and Supplementary Fig. 4b) at 15 min time point. t = 0 when the sample is placed at 30 °C. (d) Images of time series of TIRF microscopy images of Atto647N-labelled tubulin particles on a glass surfaces pre-incubated with 125 nM biotinylated TPX2 at increasing tubulin concentrations. Scale bar as indicated. (e) Plots of quantified time courses of the mean Atto647N-labelled tubulin intensities measured for the whole field of view at different tubulin concentrations as shown on Supplementary Fig. 4d. (f) Size-exclusion chromatography profiles showing TPX2, tubulin, and combinations of TPX2 and tubulin eluting from Superose 6 Increase column. Protein concentrations and peak elution volumes as indicated.

Supplementary Figure 7 The combined action of TPX2 and EB1 does not stimulate efficient microtubule nucleation and growth in ‘surface’ nucleation assay.

(a) Time series of TIRF microscopy images showing nucleation and growth of Atto647N-labelled microtubules on surfaces with immobilised biotinylated TPX2 (pre-incubated at 125 nM) in the absence (top row) or presence of 100 nM chTOG (middle row), or 100 nM human EB1 (bottom row). Atto647N-labelled tubulin concentration was 12.5 μM. Scale bar as indicated. t = 0 when the sample is placed at 30 °C. (b) Modified box-and-whiskers graph showing the microtubule growth speeds measured for immobilised dynamic microtubules, as in Fig. 3, growing in microtubule nucleation assay buffer in the presence of 7.5 μM tubulin and microtubule binding proteins at the indicated concentrations, as also used in the microtubule nucleation assays in Supplementary Fig. 5a. Number of 25 s microtubule growth intervals observed to calculate the mean growth speeds for each condition: control—n = 590, 100 nM EB1—n = 617, 100 nM TPX2—n = 379, 100 nM chTOG—n = 213. All events are from one dataset each. For the modified box-and-whiskers plot boxes range from 25th to 75th percentile, the whiskers span from 10th to 90th percentile, the horizontal line marks the mean value. ∗∗∗p ≤ 0.001 (only displayed for comparisons with control); determined for the comparison of mean values analysing raw data (Tukey’s test in conjunction with One Way ANOVA).

Supplementary Figure 8 EB1 activity does not synergise with TPX2 or chTOG in stimulating microtubule formation in the ‘solution’ nucleation assay.

(a) Time series of TIRF microscopy images showing Atto647N-labelled microtubules that nucleated in solution in the presence of biotinylated TPX2 for 1 min followed by binding to neutravidin-coated surfaces via biotinylated TPX2 in the absence (first row) or presence (second row) of untagged EB1. (b) Time series of TIRF microscopy images as in a, but now with biotinylated chTOG instead of biotinylated TPX2 in the absence (first row) or presence (second row) of untagged EB1. Atto647N-labelled tubulin concentration was always 12.5 μM. Other protein concentrations and scale bars as indicated.

Supplementary Figure 9 N-terminally truncated TPX2 promotes microtubule nucleation when combined with chTOG in the ‘surface’ nucleation assay.

Time series of TIRF microscopy images showing nucleation and growth of Atto647N-labelled microtubules on surfaces with immobilised biotinylated TPX2ΔN (pre-incubated at 125 nM) in the absence (first row) or presence of 100 nM untagged chTOG, as indicated. Tubulin concentration was 12.5 μM. Scale bar as indicated.

Supplementary Table 1 SEC-MALS analysis of recombinant TPX2 constructs.
Full size table
Supplementary Table 2 Protein expression constructs generated in this study.
Full size table

Supplementary information

Supplementary Information

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Human chTOG is a microtubule polymerase.

4 nM human chTOG-mGFP (right, green) binds to the growing end of Atto647N-labelled microtubule (magenta) and increases its growth speed (right), compared to the control microtubule in the absence of chTOG-mGFP (left). Tubulin concentration was 7.5 μM. Time is in minutes. Scale bar is 3 μm. This movie relates to Fig. 1d. (AVI 1688 kb)

Localisation of mGFP-TPX2 on dynamic microtubules.

5 nM mGFP-TPX2 (left, green in merge) binds all along the lattice of Atto647N-labelled growing microtubules (magenta in merge). 0.25 nM mGFP-TPX2 (right, green in merge) binds to the growing ends of Atto647N-labelled microtubules (magenta in merge) and GMPCPP stabilised microtubule ‘seed’ regions. The lower panels show the mGFP-TPX2 channel only. Tubulin concentration was 7.5 μM. Time is in minutes. Scale bars are 3 μm. This movie relates to Fig. 2c–f. (AVI 2934 kb)

Surface-immobilised TPX2 arrests nucleation intermediates.

Microtubule nucleation and growth of Atto647N-labelled microtubules on a surface with immobilised biotinylated Kin1rigor control (pre-incubated at 125 nM, left), with biotinylated chTOG (pre-incubated at 125 nM, middle), or with biotinylated TPX2 (pre-incubated at 125 nM, right), as indicated. Atto647N-labelled tubulin concentration was always 12.5 μM. Time is in minutes. t = 0 when the sample is placed on the microscope at 30 °C. Scale bars are 6 μm. This movie relates to Fig. 5b. (AVI 5658 kb)

The combined action of TPX2 and chTOG synergistically stimulates efficient microtubule nucleation and growth.

Microtubule nucleation and growth of Atto647N-labelled microtubules on a surface with immobilised biotinylated TPX2 (pre-incubated at 125 nM, first and second movie from left) or, for controls, with biotinylated Kin1rigor (pre-incubated at 125 nM, third and fourth movie from left) in either the absence (1st and 3rd movie from left) or presence of 100 nM chTOG (second and fourth movie from left), as indicated. Atto647N-labelled tubulin concentration was always 12.5 μM. Time is in minutes. t = 0 when the sample is placed on the microscope at 30 °C. Scale bars are 6 μm. This movie relates to Fig. 5d. (AVI 9938 kb)

In solution TPX2 nucleates microtubules more efficiently than chTOG.

Nucleation and growth of Atto647N-labelled microtubules that were nucleated in solution in the presence of 25 nM biotinylated chTOG (left), 25 nM biotinylated TPX2 (middle), and 25 nM biotinylated TPX2 and 100 nM untagged chTOG (right) at 30 °C for 1 min, followed by binding to neutravidin-coated surfaces via the biotinylated protein. Atto647N-labelled tubulin concentration was always 12.5 μM. Time is in minutes. t = 0 when the sample is placed at 30 °C. Scale bars are 6 μm. This movie relates to Fig. 6b. (AVI 7160 kb)

N-terminal region of TPX2 is not required for synergistic TPX2/chTOG-dependent efficient microtubule nucleation and growth.

Nucleation and growth of Atto647N-labelled microtubules on a surface with immobilised biotinylated TPX2ΔN (pre-incubated at 125 nM) in the absence (left) or presence (right) of 100 nM chTOG, as indicated. Atto647N-labelled tubulin concentration was 12.5 μM. Time is in minutes. t = 0 when the sample is placed on the microscope at 30 °C. Scale bars are 6 μm. This movie relates to Fig. 7b and Supplementary Fig. 7. (AVI 6823 kb)

Regulation of TPX2 and chTOG-stimulated microtubule nucleation by importins.

Microtubule nucleation and growth on a surface with immobilised biotinylated rigor kinesin (pre-incubated at 125 nM) always in the presence of 100 nM chTOG and 12.5 μM Atto647N-labelled tubulin (magenta), without (first movie from left) and with additional 500 nM importin α/β complex (second movie from left), 100 nM mGFPTPX2 (green, third movie from left), or both 500 nM importin α/β and 100 nM mGFP-TPX2 (green, 4th movie from left), as indicated. Time is in minutes. t = 0 when the sample is placed on the microscope at 30 °C. Scale bars are 6 μm. This movie relates to Fig. 8a. (AVI 6584 kb)

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Roostalu, J., Cade, N. & Surrey, T. Complementary activities of TPX2 and chTOG constitute an efficient importin-regulated microtubule nucleation module. Nat Cell Biol 17, 1422–1434 (2015). https://doi.org/10.1038/ncb3241

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