Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

  • Article
  • Published:

Microtubule minus-end regulation at spindle poles by an ASPM–katanin complex

Nature Cell Biology volume 19, pages 480–492 (2017)Cite this article

Subjects

An Erratum to this article was published on 29 June 2017

This article has been updated

Abstract

ASPM (known as Asp in fly and ASPM-1 in worm) is a microcephaly-associated protein family that regulates spindle architecture, but the underlying mechanism is poorly understood. Here, we show that ASPM forms a complex with another protein linked to microcephaly, the microtubule-severing ATPase katanin. ASPM and katanin localize to spindle poles in a mutually dependent manner and regulate spindle flux. X-ray crystallography revealed that the heterodimer formed by the N- and C-terminal domains of the katanin subunits p60 and p80, respectively, binds conserved motifs in ASPM. Reconstitution experiments demonstrated that ASPM autonomously tracks growing microtubule minus ends and inhibits their growth, while katanin decorates and bends both ends of dynamic microtubules and potentiates the minus-end blocking activity of ASPM. ASPM also binds along microtubules, recruits katanin and promotes katanin-mediated severing of dynamic microtubules. We propose that the ASPM–katanin complex controls microtubule disassembly at spindle poles and that misregulation of this process can lead to microcephaly.

This is a preview of subscription content, access via your institution

Access options

Buy this article

  • Purchase on Springer Link
  • Instant access to full article PDF
Buy now

Prices may be subject to local taxes which are calculated during checkout

Figure 1: ASPM and katanin form a complex at spindle poles.
Figure 2: The ASPM–katanin complex regulates spindle orientation and poleward flux.
Figure 3: The interaction between ASPM and katanin requires a conserved repeat sequence of ASPM and the p60N/p80C heterodimer.
Figure 4: Structure of the p60N/p80C/ASPMp complex.
Figure 5: ASPM associates with microtubule minus ends in vitro and in cells.
Figure 6: The katanin p60/p80 complex binds and bends microtubule ends in cells and in vitro.
Figure 7: The ASPM–katanin complex severs microtubules and blocks minus-end growth in vitro.
Figure 8: Dissociation of katanin from ASPM impairs spindle orientation and poleward flux.

Similar content being viewed by others

Change history

  • 22 May 2017

    In the original version of this Article, the name of author A. F. Maarten Altelaar was coded wrongly, resulting in it being incorrect when exported to citation databases. This has now been corrected, though no visible changes will be apparent.

References

  1. Morris-Rosendahl, D. J. & Kaindl, A. M. What next-generation sequencing (NGS) technology has enabled us to learn about primary autosomal recessive microcephaly (MCPH). Mol. Cell Probes 29, 271–281 (2015).

    Article  CAS  PubMed  Google Scholar 

  2. Dammermann, A., Desai, A. & Oegema, K. The minus end in sight. Curr. Biol. 13, R614–R624 (2003).

    Article  CAS  PubMed  Google Scholar 

  3. Rogers, G. C., Rogers, S. L. & Sharp, D. J. Spindle microtubules in flux. J. Cell Sci. 118, 1105–1116 (2005).

    Article  CAS  PubMed  Google Scholar 

  4. Saunders, R. D., Avides, M. C., Howard, T., Gonzalez, C. & Glover, D. M. The Drosophila gene abnormal spindle encodes a novel microtubule-associated protein that associates with the polar regions of the mitotic spindle. J. Cell Biol. 137, 881–890 (1997).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  5. Wakefield, J. G., Bonaccorsi, S. & Gatti, M. The Drosophila protein asp is involved in microtubule organization during spindle formation and cytokinesis. J. Cell Biol. 153, 637–648 (2001).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  6. Fish, J. L., Kosodo, Y., Enard, W., Paabo, S. & Huttner, W. B. Aspm specifically maintains symmetric proliferative divisions of neuroepithelial cells. Proc. Natl Acad. Sci. USA 103, 10438–10443 (2006).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  7. van der Voet, M. et al. NuMA-related LIN-5, ASPM-1, calmodulin and dynein promote meiotic spindle rotation independently of cortical LIN-5/GPR/Gα. Nat. Cell Biol. 11, 269–277 (2009).

    Article  CAS  PubMed  Google Scholar 

  8. Higgins, J. et al. Human ASPM participates in spindle organisation, spindle orientation and cytokinesis. BMC Cell Biol. 11, 85 (2010).

    Article  PubMed  PubMed Central  CAS  Google Scholar 

  9. Ito, A. & Goshima, G. Microcephaly protein Asp focuses the minus ends of spindle microtubules at the pole and within the spindle. J. Cell Biol. 211, 999–1009 (2015).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  10. Schoborg, T., Zajac, A. L., Fagerstrom, C. J., Guillen, R. X. & Rusan, N. M. An Asp–CaM complex is required for centrosome-pole cohesion and centrosome inheritance in neural stem cells. J. Cell Biol. 211, 987–998 (2015).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  11. Ripoll, P., Pimpinelli, S., Valdivia, M. M. & Avila, J. A cell division mutant of Drosophila with a functionally abnormal spindle. Cell 41, 907–912 (1985).

    Article  CAS  PubMed  Google Scholar 

  12. do Carmo Avides, M. & Glover, D. M. Abnormal spindle protein, Asp, and the integrity of mitotic centrosomal microtubule organizing centers. Science 283, 1733–1735 (1999).

    Article  CAS  PubMed  Google Scholar 

  13. Morales-Mulia, S. & Scholey, J. M. Spindle pole organization in Drosophila S2 cells by dynein, abnormal spindle protein (Asp), and KLP10A. Mol. Biol. Cell 16, 3176–3186 (2005).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  14. Bond, J. et al. ASPM is a major determinant of cerebral cortical size. Nat. Genet. 32, 316–320 (2002).

    Article  CAS  PubMed  Google Scholar 

  15. Roll-Mecak, A. & McNally, F. J. Microtubule-severing enzymes. Curr. Opin. Cell Biol. 22, 96–103 (2010).

    Article  CAS  PubMed  Google Scholar 

  16. McNally, F. J. & Vale, R. D. Identification of katanin, an ATPase that severs and disassembles stable microtubules. Cell 75, 419–429 (1993).

    Article  CAS  PubMed  Google Scholar 

  17. Hartman, J. J. et al. Katanin, a microtubule-severing protein, is a novel AAA ATPase that targets to the centrosome using a WD40-containing subunit. Cell 93, 277–287 (1998).

    Article  CAS  PubMed  Google Scholar 

  18. Mishra-Gorur, K. et al. Mutations in KATNB1 cause complex cerebral malformations by disrupting asymmetrically dividing neural progenitors. Neuron 84, 1226–1239 (2014).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  19. Hu, W. F. et al. Katanin p80 regulates human cortical development by limiting centriole and cilia number. Neuron 84, 1240–1257 (2014).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  20. McNally, K. P., Bazirgan, O. A. & McNally, F. J. Two domains of p80 katanin regulate microtubule severing and spindle pole targeting by p60 katanin. J. Cell Sci. 113, 1623–1633 (2000).

    Article  CAS  PubMed  Google Scholar 

  21. Srayko, M., Buster, D. W., Bazirgan, O. A., McNally, F. J. & Mains, P. E. MEI-1/MEI-2 katanin-like microtubule severing activity is required for Caenorhabditis elegans meiosis. Genes Dev. 14, 1072–1084 (2000).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  22. McNally, K., Audhya, A., Oegema, K. & McNally, F. J. Katanin controls mitotic and meiotic spindle length. J. Cell Biol. 175, 881–891 (2006).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  23. McNally, K. P. & McNally, F. J. The spindle assembly function of Caenorhabditis elegans katanin does not require microtubule-severing activity. Mol. Biol. Cell 22, 1550–1560 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  24. McNally, K. et al. Katanin maintains meiotic metaphase chromosome alignment and spindle structure in vivo and has multiple effects on microtubules in vitro. Mol. Biol. Cell 25, 1037–1049 (2014).

    Article  PubMed  PubMed Central  Google Scholar 

  25. Loughlin, R., Wilbur, J. D., McNally, F. J., Nedelec, F. J. & Heald, R. Katanin contributes to interspecies spindle length scaling in Xenopus. Cell 147, 1397–1407 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  26. Bailey, M. E., Sackett, D. L. & Ross, J. L. Katanin severing and binding microtubules are inhibited by tubulin carboxy tails. Biophys. J. 109, 2546–2561 (2015).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  27. Cheung, K. et al. Proteomic analysis of the mammalian katanin family of microtubule-severing enzymes defines KATNBL1 as a regulator of mammalian katanin microtubule-severing. Mol. Cell Proteomics 15, 1658–1669 (2016).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  28. Elting, M. W., Hueschen, C. L., Udy, D. B. & Dumont, S. Force on spindle microtubule minus ends moves chromosomes. J. Cell Biol. 206, 245–256 (2014).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  29. Sikirzhytski, V. et al. Direct kinetochore-spindle pole connections are not required for chromosome segregation. J. Cell Biol. 206, 231–243 (2014).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  30. Gai, M. et al. ASPM and CITK regulate spindle orientation by affecting the dynamics of astral microtubules. EMBO Rep. 17, 1396–1409 (2016).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  31. Zhang, D., Rogers, G. C., Buster, D. W. & Sharp, D. J. Three microtubule severing enzymes contribute to the ‘Pacman-flux’ machinery that moves chromosomes. J. Cell Biol. 177, 231–242 (2007).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  32. Ganem, N. J., Upton, K. & Compton, D. A. Efficient mitosis in human cells lacking poleward microtubule flux. Curr. Biol. 15, 1827–1832 (2005).

    Article  CAS  PubMed  Google Scholar 

  33. Iwaya, N. et al. A common substrate recognition mode conserved between katanin p60 and VPS4 governs microtubule severing and membrane skeleton reorganization. J. Biol. Chem. 285, 16822–16829 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  34. Jiang, K. et al. Microtubule minus-end stabilization by polymerization-driven CAMSAP deposition. Dev. Cell 28, 295–309 (2014).

    Article  CAS  PubMed  Google Scholar 

  35. Brouhard, G. J. & Rice, L. M. The contribution of αβ-tubulin curvature to microtubule dynamics. J. Cell Biol. 207, 323–334 (2014).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  36. Janosi, I. M., Chretien, D. & Flyvbjerg, H. Modeling elastic properties of microtubule tips and walls. Eur. Biophys. J. 27, 501–513 (1998).

    Article  CAS  PubMed  Google Scholar 

  37. Wang, H., Brust-Mascher, I., Civelekoglu-Scholey, G. & Scholey, J. M. Patronin mediates a switch from kinesin-13-dependent poleward flux to anaphase B spindle elongation. J. Cell Biol. 203, 35–46 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  38. Rogers, G. C. et al. Two mitotic kinesins cooperate to drive sister chromatid separation during anaphase. Nature 427, 364–370 (2004).

    Article  CAS  PubMed  Google Scholar 

  39. Gaetz, J. & Kapoor, T. M. Dynein/dynactin regulate metaphase spindle length by targeting depolymerizing activities to spindle poles. J. Cell Biol. 166, 465–471 (2004).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  40. Desai, A., Verma, S., Mitchison, T. J. & Walczak, C. E. Kin I kinesins are microtubule-destabilizing enzymes. Cell 96, 69–78 (1999).

    Article  CAS  PubMed  Google Scholar 

  41. Montenegro Gouveia, S. et al. In vitro reconstitution of the functional interplay between MCAK and EB3 at microtubule plus ends. Curr. Biol. 20, 1717–1722 (2010).

    Article  CAS  PubMed  Google Scholar 

  42. Tanenbaum, M. E. et al. A complex of Kif18b and MCAK promotes microtubule depolymerization and is negatively regulated by Aurora kinases. Curr. Biol. 21, 1356–1365 (2011).

    Article  CAS  PubMed  Google Scholar 

  43. Zhang, D. et al. Drosophila katanin is a microtubule depolymerase that regulates cortical-microtubule plus-end interactions and cell migration. Nat. Cell Biol. 13, 361–370 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  44. Taverna, E., Gotz, M. & Huttner, W. B. The cell biology of neurogenesis: toward an understanding of the development and evolution of the neocortex. Annu. Rev. Cell Dev. Biol. 30, 465–502 (2014).

    Article  CAS  PubMed  Google Scholar 

  45. Ran, F. A. et al. Genome engineering using the CRISPR-Cas9 system. Nat. Protoc. 8, 2281–2308 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  46. Hockemeyer, D. et al. Efficient targeting of expressed and silent genes in human ESCs and iPSCs using zinc-finger nucleases. Nat. Biotechnol. 27, 851–857 (2009).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  47. Scholz, J., Besir, H., Strasser, C. & Suppmann, S. A new method to customize protein expression vectors for fast, efficient and background free parallel cloning. BMC Biotechnol. 13, 12 (2013).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  48. Schuck, P. Size-distribution analysis of macromolecules by sedimentation velocity ultracentrifugation and lamm equation modeling. Biophys. J. 78, 1606–1619 (2000).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  49. Kabsch, W. Xds. Acta Crystallogr. D Biol. Crystallogr. 66, 125–132 (2010).

    CAS  PubMed  PubMed Central  Google Scholar 

  50. Adams, P. D. PHENIX: a comprehensive Python-based system for macromolecular structure solution. Acta Crystallogr. D Biol. Crystallogr. 66, 213–221 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  51. Emsley, P., Lohkamp, B., Scott, W. G. & Cowtan, K. Features and development of Coot. Acta Crystallogr. D Biol. Crystallogr. 66, 486–501 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  52. Orchard, S. et al. The MIntAct project–IntAct as a common curation platform for 11 molecular interaction databases. Nucleic Acids Res. 42, D358–D363 (2014).

    Article  CAS  PubMed  Google Scholar 

Download references

Acknowledgements

We thank A. Prota for the help in refining the crystal structures, M. Boxem, C. Janke and M. Mikhaylova for the gift of materials, and the beamline scientists at beamlines X06DA of the Swiss Light Source (Paul Scherrer Institut, Villigen, Switzerland) for technical assistance with the X-ray data collection. This work was supported by the European Research Council Synergy grant 609822 and Netherlands Organization for Scientific Research (NWO) CW ECHO grant (711.011.005) to A.A., the EMBO long-term and Marie Curie IEF fellowships to L.R., grants from the Swiss National Science Foundation (31003A_166608 to M.O.S. and 31003A_163449 to R.A.K.), a NWO VIDI grant (723.012.102) to A.F.M.A. and as part of the National Roadmap Large-scale Research Facilities of the Netherlands (project number 184.032.201) to A.F.M.A. and A.J.R.H. The structural data reported in this paper are available in PDB (PDB code 5LB7).

Author information

Author notes

  1. Kai Jiang and Lenka Rezabkova: These authors contributed equally to this work.

Authors and Affiliations

  1. Department of Biology, Cell Biology, Faculty of Science, Utrecht University, Padualaan 8, 3584 CH Utrecht, The Netherlands

    Kai Jiang, Shasha Hua, Qingyang Liu & Anna Akhmanova

  2. Division of Biology and Chemistry, Laboratory of Biomolecular Research, Paul Scherrer Institut, 5232 Villigen PSI, Switzerland

    Lenka Rezabkova, Guido Capitani, Richard A. Kammerer & Michel O. Steinmetz

  3. Biomolecular Mass Spectrometry and Proteomics, Bijvoet Center for Biomolecular Research, Utrecht Institute for Pharmaceutical Sciences and The Netherlands Proteomics Centre, Utrecht University, Padualaan 8, 3584 CH Utrecht, The Netherlands

    A. F. Maarten Altelaar & Albert J. R. Heck

Authors
  1. Kai Jiang
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Lenka Rezabkova
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Shasha Hua
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Qingyang Liu
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Guido Capitani
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. A. F. Maarten Altelaar
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Albert J. R. Heck
    View author publications

    You can also search for this author in PubMed Google Scholar

  8. Richard A. Kammerer
    View author publications

    You can also search for this author in PubMed Google Scholar

  9. Michel O. Steinmetz
    View author publications

    You can also search for this author in PubMed Google Scholar

  10. Anna Akhmanova
    View author publications

    You can also search for this author in PubMed Google Scholar

Contributions

K.J., L.R., S.H., R.A.K., M.O.S. and A.A. designed experiments, analysed data and wrote the paper. A.A. coordinated the project. K.J. and S.H. performed cellular and in vitro reconstitution experiments. L.R. performed biophysical experiments. L.R. and G.C. performed crystallography experiments. Q.L., A.F.M.A. and A.J.R.H. performed and analysed mass spectrometry experiments.

Corresponding authors

Correspondence to Kai Jiang, Richard A. Kammerer, Michel O. Steinmetz or Anna Akhmanova.

Ethics declarations

Competing interests

The authors declare no competing financial interests.

Integrated supplementary information

Supplementary Figure 1 Characterization of ASPM knock-in cell lines.

(a) Genotyping of GFP and strep-GFP-ASPM knock-in cells used in this study. (b) Immunostaining for α-tubulin and DNA (DAPI) in GFP-ASPM knock-in HeLa cells during mitosis. White arrows indicate the localization of ASPM to the microtubule minus ends in the central spindle in telophase. Scale bar, 5 μm.

Supplementary Figure 2 ASPM and katanin regulate spindle architecture.

(a) western blotting with the indicated antibodies in control, ASPM knockout and p80 knockout cells (left, HeLa; right, U2OS PA-GFP-α-tubulin). Note that the knockout of p80 caused the concomitant loss of p60, in agreement with the fact that the two subunits form a tight complex. (b) Sequencing results of ASPM knockout cell lines used in this study. 1 nt insertion will result in p.N71KfsX31 or p.E72RfsX30. (c–d) Immunostaining of γ-tubulin, DNA (DAPI), and NuMA (c) or p150Glued (d) in control, ASPM knockout, and p80 knockout HeLa cells. Scale bar, 5 μm. In HeLa cells, the intensity of NuMA and p150Glued at the spindle pole remained largely unchanged in both ASPM and p80 knockout cells. (e) Quantification of spindle pole intensities of NuMA, and p150Glued as shown in panels c, d. For NuMA intensity, n = 164 spindle poles, control; n = 172, ASPM knockout; n = 176, p80 knockout. For p150Glued intensity, n = 160, control; n = 166, ASPM knockout; n = 170, p80 knockout. (f,g) Immunostaining for α-tubulin, CEP135 and DNA in control, ASPM knockout and p80 knockout HeLa cells (f, bipolar spindles with two centrosomes; g, bipolar and multipolar spindles with >2 centrosomes in p80 knockout cells). Scale bar, 5 μm. (h) Quantification of the distance between two centrosomes in control, ASPM knockout and p80 knockout HeLa cells as shown in f. n = 67 cells, control; n = 61, ASPM knockout, n = 90, p80 knockout. (i) Quantification of centrosome numbers as shown in f,g. 470 cells, control; 469, ASPM knockout; 572, p80 knockout (2 experiments). (j) Quantification of the average distance between two spindle poles in photoactivation experiments in U2OS cells as shown in Fig. 2e. n = 26 cells, control; n = 29, ASPM knockout; n = 26, p80 knockout. The standard deviations of the distance between the two poles during imaging were small (∼0.3 μm), which means that the spindle length kept constant during imaging. Compared to control, spindle length in ASPM and p80 knockout HeLa (h) and U2OS (j) cells shows 6 ∼ 10% reduction. Data represent mean ± s.d.∗, P < 0.05, ∗∗∗, P < 0.001, Mann-Whitney U test. Unprocessed original scans of blots are shown in Supplementary Fig. 8. Source data for panels e,h,i and j can be found in Supplementary Table 5.

Supplementary Figure 3 The interaction between ASPM and katanin requires a conserved repeat sequence of ASPM and the p60N/p80C heterodimer.

(a) Alignment of katanin-binding linear motifs of ASPM from several vertebrate species. Note the complete conservation of the phenylalanine residue corresponding to F352 in the third repeat of mouse ASPM (red arrowhead below the alignment). (b) Streptavidin pull down assays with extracts of HEK293T cells expressing Biotinylation tagged (Bio)-GFP-tagged wild-type (WT) p80 or its indicated mutants together with GFP-tagged WT p60 or the indicated mutants. None of the analyzed mutations perturbed the p60–p80 interaction. See also Supplementary Fig. 8e. (c) Electron density maps of ASPMp in the p60N/p80C/ASPMp complex structure. Only residues S351, F352 and L353 of the ASPMp peptide (LSPDSFLND, residues 347–355 of mouse ASPM) are visible. The SigmaA-weighted 2mFo-DFc (left) and mFo-DFc (right) omit maps (green mash) are contoured at +1.0σ and +3.0σ, respectively.

Supplementary Figure 4 The CH1-CH3 fragment of C. elegans ASPM-1 binds microtubule minus-end in cells.

(a,b) The worm ASPM-1 CH1–CH3 fragment associates with minus ends of free microtubules (a) or minus ends freshly generated by photoablation (b) in interphase MRC5 cells. Green lightning bolts indicate the sites of photoablation. Scale bars, 2 μm.

Supplementary Figure 5 The katanin p60/p80 heterodimer decorates and bends microtubule ends.

(a) TIRFM live cell imaging and kymographs of colocalization of GFP-p60/p80C and EB3-TagRFP in MRC5 cells. Scale bars: horizontal, 2 μm; vertical, 10 s. (b) Live cell images of a single microtubule plus end bound to GFP-p60/p80C and EB3-TagRFP in MRC5 cells. White arrow, breaking of a bent microtubule end. Scale bar, 1 μm. (c) Live imaging of MRC5 cells expressing individual katanin subunits together with EB3-TagRFP. Scale bar, 2 μm. (d) TIRFM time lapse images showing the complete severing of dynamic microtubules by p60/SNAP-Alexa647-p80C at 750 nM in a flow-in experiment. Time represented as min:sec. Scale bar, 2 μm. (e–f) Maximum intensity projections of time lapse images of GFP-p60/p80 WT or mutants in HeLa cells. Imaging was performed using TIRFM with 500 ms exposure in a stream mode. 100–200 images representing consecutive frames were used to make projections. Mutations of R615A, K618A, G607A, V608A, D609A and I610A in p80 completely abolish the end-binding activity of p60/p80 in cells. Scale bar, 2 μm. (g) Protein sequence alignment of p60N and p80C from Caenorhabditis elegans, Chlamydomonas reinhardtii, Tetrahymena thermophila, Arabidopsis thaliana, Drosophila melanogaster , Danio rerio, Xenopus tropicalis, Homo sapiens and Mus musculus. The residues that are essential for microtubule end binding are indicated with red dots. Mutating charged residues denoted by black dots to alanine had no effect on end binding in vivo. Red triangles indicate the residues interacting with ASPM, which are conserved in vertebrates (red dashed rectangle). Numbers displayed on top of the sequence alignment are based on mouse katanin sequences.

Supplementary Figure 6 Characterization of wild-type katanin p60/p80C and its mutants, ASPM mutant and the ASPM–katanin complex at different concentrations.

(a) The p60/p80C R615A mutant does not bind microtubule ends at 25 or 75 nM. Time represented as min:sec. Scale bars, 2 μm. (b) The p60/p80C Y574A mutant can decorate, bend and break microtubule ends at 75 nM but not at 25 nM. White arrow, breaking of a bent microtubule end. Time represented as min:sec. Scale bars, 2 μm. (c) Wild-type (WT) p60/p80C and the indicated mutants can efficiently sever GMPCPP-stabilized microtubules at 75 nM but not at 25 nM. See Fig. 7a for quantification. Scale bar, 2 μm. (d–g) The p60/p80C R615A mutant but not the Y574A mutant at 25 nM can be recruited onto microtubule lattice and perform severing in the assay with dynamic microtubules in the presence of 30 nM ASPM. White arrows indicate the sites where severing events occur. Time represented as min:sec. f, n = 64 (WT), 59 (R615A) and 59 (Y574A) microtubules; g, Data are the mean of 2 experiments. Scale bar, 2 μm. (h–j) 30 nM ASPM F302A/F377A mutant does not recruit p60/p80C to microtubule lattice and does not promote severing. i, n = 62 (WT) and 60 (F302A/F377A) microtubules; j, data are the mean of 2 experiments. Severing frequency data for WT in panels g and j were replotted from Fig. 7f (30 nM). (k–m) 5 nM ASPM F302A/F377A mutant does not recruit p60/p80C to dynamic microtubule minus ends and does not induce their blocking. l, n = 19 (WT) and 29 (F302A/F377A) microtubules; m, n = 19 (WT) and 33 (F302A/F377A) microtubules. Scale bars, horizontal, 2 μm; vertical, 1 min. Data represent mean ± s.d. Source data for panels f, g, i, j, l and m can be found in Supplementary Table 5.

Supplementary Figure 7 Purified proteins used for in vitro reconstitution experiments.

(a) Coomassie blue stained gels with strep-GFP tagged ASPM, Asp and ASPM-1 proteins purified from transiently transfected HEK293T cells. The bands corresponding to purified full-length proteins are marked with red asterisks. (b) Coomassie blue stained gels with p60/strep-SNAP-p80C wild-type and mutants, p60N + L/strep-GFP-p80C and p60N + L/strep-SNAP-p80C purified from HEK293T cells and strep-p60/6xHis-GFP-p80C purified from E. coli.

Supplementary Figure 8 Full scans of western blots used to prepare figures, as indicated.

Supplementary information

Supplementary Information

Supplementary Information (PDF 6176 kb)

Supplementary Table 1

Supplementary Information (XLSX 9 kb)

Supplementary Table 2

Supplementary Information (XLSX 14 kb)

Supplementary Table 3

Supplementary Information (XLSX 13 kb)

Supplementary Table 4

Supplementary Information (XLSX 130 kb)

Supplementary Table 5

Supplementary Information (XLSX 106 kb)

Reduced spindle flux in ASPM and katanin knockout cells.

Photoactivation of PA-GFP-α-tubulin in control (left), ASPM knockout (middle) and katanin knockout (right) U2OS metaphase cells. Images were collected using a spinning disk microscope at 2 s interval. Video is sped up 60 times. Time is shown in the format min:sec. (MOV 5999 kb)

Overexpressed katanin p60/p80 complex tracks and bends microtubule ends in cells.

MRC5 cells were co-transfected with GFP-p60, p80 (dark) and EB3-TagRFP. Images were collected using a TIRF microscope in stream mode (2 frames/s). Video is sped up 15 times. Time is shown in the format min:sec. (MOV 1481 kb)

Katanin p60/p80C severs dynamic microtubules at 300 nM in vitro.

Microtubules were first polymerized in the tubulin polymerization reaction mix (20 μM unlabeled tubulin and 0.5 μM X-rhodamine-tubulin in MRB80 buffer supplemented with 50 mM KCl). Subsequently, the tubulin polymerization reaction mix supplemented with 300 nM p60/SNAP-Alexa647-p80C and 1 mM ATP was flowed into the reaction chamber. Images were collected using a TIRF microscope at a 3 s interval. Video is sped up 15 times. Time is shown in the format min:sec. (MOV 3936 kb)

Katanin p60N + L/p80C bends and breaks a microtubule end in vitro.

Microtubules were polymerized in the presence of 1 μM p60N + L/GFP-p80C, 20 μM unlabeled tubulin and 0.5 μM X-rhodamine-tubulin in MRB80 buffer supplemented with 50 mM KCl. Images were collected using a TIRF microscope at 2 s interval. Video is sped up 30 times. Time is shown in the format min:sec. (MOV 181 kb)

30 nM ASPM and 25 nM katanin p60/p80C sever dynamic microtubules in vitro.

Microtubules were first polymerized in the tubulin polymerization reaction mix (20 μM unlabeled tubulin and 0.5 μM X-rhodamine-tubulin in MRB80 buffer supplemented with 50 mM KCl). Subsequently, tubulin polymerization reaction mix supplemented with 30 nM ASPM, 25 nM p60/SNAP-Alexa647-p80C and 1 mM ATP was flowed into the reaction chamber. Images were collected using a TIRF microscope at a 3 s interval. Video is sped up 15 times. Time is shown in the format min:sec. (MOV 3526 kb)

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Jiang, K., Rezabkova, L., Hua, S. et al. Microtubule minus-end regulation at spindle poles by an ASPM–katanin complex. Nat Cell Biol 19, 480–492 (2017). https://doi.org/10.1038/ncb3511

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/ncb3511

This article is cited by

Search

Advanced search

Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing