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Dynamic kinetochore size regulation promotes microtubule capture and chromosome biorientation in mitosis

Nature Cell Biology volume 20, pages 800–810 (2018)Cite this article

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Abstract

Faithful chromosome segregation depends on the ability of sister kinetochores to attach to spindle microtubules. The outer layer of kinetochores transiently expands in early mitosis to form a fibrous corona, and compacts following microtubule capture. Here we show that the dynein adaptor Spindly and the RZZ (ROD–Zwilch–ZW10) complex drive kinetochore expansion in a dynein-independent manner. C-terminal farnesylation and MPS1 kinase activity cause conformational changes of Spindly that promote oligomerization of RZZ-Spindly complexes into a filamentous meshwork in cells and in vitro. Concurrent with kinetochore expansion, Spindly potentiates kinetochore compaction by recruiting dynein via three conserved short linear motifs. Expanded kinetochores unable to compact engage in extensive, long-lived lateral microtubule interactions that persist to metaphase, and result in merotelic attachments and chromosome segregation errors in anaphase. Thus, dynamic kinetochore size regulation in mitosis is coordinated by a single, Spindly-based mechanism that promotes initial microtubule capture and subsequent correct maturation of attachments.

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Fig. 1: Spindly recruits dynein to compact kinetochores after microtubule attachment.
Fig. 2: Kinetochores expand by forming a structurally stable kinetochore sub-module.
Fig. 3: Spindly and RZZ are essential for kinetochore expansion.
Fig. 4: Spindly stimulates RZZ-Spindly polymerization in vitro and in vivo.
Fig. 5: A structural conformation of Spindly prevents RZZS oligomerization.
Fig. 6: Release of Spindly autoinhibition promotes its interaction with RZZ.
Fig. 7: MPS1 promotes RZZS meshwork formation and kinetochore expansion.
Fig. 8: The expanded kinetochore module interacts with microtubule lattices and prevents biorientation.

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Acknowledgements

We thank all lab members for suggestions and discussions. We are grateful to A. Murachelli for help with EM data figure preparation; to E. von Castelmur, T. Heidebrecht and Y. Hiruma for help with Spindly structure experiments; to J. Vaughan for help with ExM; to R. Gassmann for sharing unpublished results and Spindly constructs; to I. Cheeseman, S. Lens and R. Medema for reagents; and to A. de Graaf of the Hubrecht Imaging Center. The Horizon 2020 iNEXT project (653706) provided financial support and access to EM infrastructures. This work is part of the Oncode Institute which is partly financed by the Dutch Cancer Society. This work was further supported by the Netherlands Organisation for Scientific Research (NWO) (gravitation program CancerGenomiCs.nl; VICI grant (865.12.004 to G.J.P.L.K.)), the Dutch Cancer Society (KWF/HUBR-11080 to G.J.P.L.K.), and the ERC (675737 to A.M.). V.G. is supported by the Proteins@Work initiative of the Netherlands Proteomics Centre.

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Authors and Affiliations

  1. Oncode Institute, Hubrecht Institute–KNAW and University Medical Center Utrecht, Utrecht, The Netherlands

    Carlos Sacristan, Eelco Tromer & Geert JPL Kops

  2. Department of Biochemistry, The Netherlands Cancer Institute, Amsterdam, The Netherlands

    Misbha Ud Din Ahmad, Alexander Fish & Anastassis Perrakis

  3. Department of Mechanistic Cell Biology, Max Planck Institute of Molecular Physiology, Dortmund, Germany

    Jenny Keller & Andrea Musacchio

  4. Section Cell Biology, Center for Molecular Medicine, University Medical Center Utrecht, Utrecht University, Utrecht, The Netherlands

    Job Fermie, Vincent Groenewold & Judith Klumperman

  5. Biocomputing Unit, National Center for Biotechnology (CSIC), Darwin 3, Campus Universidad Autónoma, Madrid, Spain

    Roberto Melero & José María Carazo

  6. Centre for Medical Biotechnology, Faculty of Biology, University Duisburg-Essen, Universitätsstraße, Essen, Germany

    Andrea Musacchio

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  1. Carlos Sacristan
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Contributions

C.S. and G.J.P.L.K. conceived the project. C.S., G.J.P.L.K., M.U.D.A., A.P., J.K. and A.M designed experiments and interpreted data. C.S. performed the cell biology experiments. M.U.D.A. and J.K. performed the in vitro experiments with the help of A.F. J.F. and J.K. performed and analysed the electron microscopy experiments. V.G. performed the cross-linking experiments. E.T. performed the comparative sequence analysis. R.M. and J.M.C performed the electron microscopy of Spindly. C.S. and G.J.P.L.K. wrote the manuscript with the help of A.P. and A.M. and the input of the rest of authors.

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Correspondence to Geert JPL Kops.

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Supplementary Figure 1 Inhibition of mitotic kinases is not sufficient to compact expanded kinetochores. Related to Figure 1.

(a-b) Scheme of workflow (a) and representative images (b) of immunostains of nocodazole-treated HeLa cells incubated with inhibitors of Aurora B (ZM-447439), PLK1 (BI-2636) and MPS1 (Cpd-5). The experiment was repeated at least three times with similar results.

Supplementary Figure 2 Multiple sequence alignment of dynein adaptors. Related to Figure 1.

Multiple sequence alignment of the CC1, CC2 and Spindly box motifs found in a selection of metazoan orthologs of the human spindly-like dynein-dynactin adaptors SPINDLY, TRAK1/2, HAP1 BICD1/2 and BICDR1/2 (supplementary sequences 2). Metazoan species: Homo sapiens (human), Mus musculus (mouse), Xenopus tropicalis (frog), Danionrerio (zebra fish), Drosophila melanogaster (fruit fly), Caenorhabditis elegans (worm), Nematostella vectensis (starlet sea anemone). Sequences used can be found in Supplementary Tables 4 and 5.

Supplementary Figure 3 Characterization of dynein-binding motifs of Spindly. Related to Figure 1.

(a) Immunoblot showing Spindly knockdown and expression of siRNA-resistant GFP-tagged versions of Spindly in mitotic population. The experiment was repeated twice with similar results. (b) Representative immunofluorescence images of HeLa cells transfected with siRNA to Spindly and treated or not with doxycycline (Dox) to induce the expression of GFP-Spindly variants, and immunostained for the indicated antigens. The experiment was repeated at least three times with similar results. The same images of FL and SpindlyΔN are shown in Figure 1f. (c) Representative images of nocodazole-treated HeLa cells transfected with siRNAs to Spindly and expressing the indicated GFP-Spindly variants, and immunostained for the indicated antigens. Quantification is shown in Figure 1c. (d) Representative images of metaphase HeLa cells transfected with siRNA to Spindly and expressing the indicated GFP-Spindly variants, and immunostained for the indicated antigens. Quantification is shown in Figure 1e. The same images of FL, SpindlyΔSB and SpindlyΔCCS are shown in Figure 1f. FL, Full length Spindly.

Supplementary Figure 4 Characterization of kinetochore expansion. Related to Figure 3.

(a) Cartoon depicting the effect of ZW10 or Spindly depletion on the expandable module of the kinetochore (green), KMN network (blue) and CCAN (red). (b-c) Immunostaining with the indicated antibodies (b) and quantification (c) of kinetochore levels in cells from one of the experiments shown in Figure 3e,f. The graph in (c) shows the mean kinetochore intensity normalized to the values of the control. Control is siGAPDH. See Supplementary Table 3 for source data.

Supplementary Figure 5 Polymerization of the RZZ complex by SpindlyΔN. Related to Figure 4.

(a) Example of a HeLa cell expressing GFP-SpindlyΔN and showing a cytoplasmic filament positive for Zwilch and ROD. The experiment was repeated at least three times with similar results.

Supplementary Figure 6 Spindly protein analysis. Related to Figure 5.

(a) Coomassie staining of SDS-PAGE gels of the constructs used in Figure 5. (b) Volume of correlation plot from SEC-SAXS analysis for Spindly1-440 and SpindlyFL. Guinier extrapolated dataset plotted as q.I(q) (left panel) and integrated area of q (right panel). The calculated Vc and Rg values for Spindly1-440 are 1096 Å2 and 76Å, respectively. For, SpindlyFL, the Vc and Rg values are 1562 Å2 and 88 Å, respectively. The molecular mass69 calculated is 128 KDa and 220 KDa for Spindly1-440 and SpindlyFL, respectively. See Supplementary Table 3 for source data. (c) SEC-MALLS analysis of Spindly1-440 and SpindlyFL. The absolute molar mass in daltons (log scale) is plotted against elution volume. Calculated molar masses corresponding to the gel filtration peaks are shown as light (Spindly1-440) or dark blue (SpindlyFL) curves. For comparison, the expected molar masses for a monomer, dimer and a trimer are also plotted. The analysis shows that Spindly1-440 is dimeric in solution whereas in similar conditions SpindlyFL exists as a trimer. The experiment was repeated twice with similar results. See Supplementary Table 3 for source data. (d) Cross-linking proteomics analysis of SpindlyFL. All the cross-linked residues (boxes in grey) found in two independent experiments are represented in the X and Y axis. All the lysines present in Spindly are depicted in red. The encircled areas (b,c,d) mark several long-range interactions between the N-terminal region (1-250) and the lysines encircled in purple, suggesting intramolecular interactions between the N-terminal domain and the middle region of Spindly. See Supplementary Table 3 for source data.

Supplementary Figure 7 Release of Spindly intramolecular interactions enhances the association of Spindly with RZZ. Related to Figure 6.

(a) Binding of RZZ to the indicated versions of Spindly constructs isolated from insect cells determined by Surface Plasmon Resonance (SPR). The response plotted on the y axis is normalized for the molecular weight of the analyte to yield the stoichiometry of binding. The experiment was repeated three times with similar results. See Supplementary Table 3 for source data. (b) Immunoblot showing GFP-Spindly versions in mitotic cells treated as indicated (Lon, Lonafarnib). The experiment was repeated twice with similar results. (c-f) Representative images (c,d) and quantification (e,f) of relative kinetochore intensities of the indicated GFP-Spindly variants in nocodazole-treated HeLa cells transfected with siRNA to Spindly. The graphs show the mean kinetochore intensity normalized to the values of SpindlyFL. Each dot represents one cell. The sample size in (e) is: FL (n= 54 cells), FL/C602A (n= 55 cells), ΔN (n= 56 cells), ΔN/C602A (n= 56 cells), Δ274 (n= 51 cells) Δ274/C602A (n= 55 cells), Δ287 (n= 55 cells), Δ287/C602A (n= 51 cells) pooled from two independent experiments. See Supplementary Table 3 for source data. The sample size in (f) is: FL (n= 63 cells), FL/C602A (n= 57 cells), ΔCCS (n= 61 cells), ΔCCS/C602A (n= 59 cells) pooled from two independent experiments. See Supplementary Table 3 for source data.

Supplementary Figure 8 Enlarged kinetochores in metaphase have immature attachments. Related to Figure 8.

(a,b) Representative images (a) and quantification of kinetochore levels (b) of Astrin in metaphase cells transfected with siRNA to Spindly and expressing the indicated GFP-Spindly variants. The graph shows the mean kinetochore intensity normalized to SpindlyFL. Each dot represents one cell: FL (n=55 cells), ΔN (n=48 cells) pooled from two independent experiments. Source data can be found in Supplementary Table 3. (c) Representative images of metaphase HeLa cells transfected with siRNA to Spindly, expressing GFP-SpindlyΔN and mCherry-Tubulin, fixed at 37°C or after cold treatment, and stained for indicated antigens. (‘b’, bridging fibre; ‘k’, k-fibres). The asterisk indicates the region of the enlarged kinetochore that normally mediates the lateral interaction with microtubules at 37°C. The experiment was repeated at least three times with similar results. (d) Live-cell imaging of HeLa cells transfected with siRNA to Spindly and expressing GFP-SpindlyΔN and mCherry-Tubulin. (‘Gr’, Growing microtubules; ‘Sh’, Shrinking microtubules; ‘e’ end-on attachment; ‘l’, lateral attachment). Maximum projections of several z-planes are shown. See also Supplementary Movies 5. The experiment was repeated at least three times with similar results. (e) Model of the mechanism of kinetochore expansion and compaction performed by the axis RZZ-Spindly-dynein and MPS1. See discussion for details.

Supplementary information

Supplementary Information

Supplementary Figures 1–8, Supplementary Table and Supplementary Video legends

Reporting Summary

Supplementary Table 1

List of plasmids, primers and RNAi sequences used in this study

Supplementary Table 2

List of antibodies used in this study

Supplementary Table 3

Statistics Source Data

Supplementary Table 4

Related to Figure 1b. All sequences of Spindly used in this study

Supplementary Table 5

Related to Supplementary Figure 2. All sequences used in this study

Supplementary Video 1

Formation of filaments in vitro by RZZS

Supplementary Video 2

Formation of cytoplasmic filaments in cells expressing GFP-SpindlyΔN

Supplementary Video 3

Interaction of expanded kinetochores with microtubules

Supplementary Video 4

Interaction of expanded kinetochores with microtubules

Supplementary Video 5

Interaction of expanded kinetochores with microtubules

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Sacristan, C., Ahmad, M.U.D., Keller, J. et al. Dynamic kinetochore size regulation promotes microtubule capture and chromosome biorientation in mitosis. Nat Cell Biol 20, 800–810 (2018). https://doi.org/10.1038/s41556-018-0130-3

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