Abstract
A critical structure poised to coordinate chromosome segregation with division plane specification is the central spindle that forms between separating chromosomes after anaphase onset1,2. The central spindle acts as a signalling centre that concentrates proteins essential for division plane specification and contractile ring constriction3. However, the molecular mechanisms that control the initial stages of central spindle assembly remain elusive. Using Caenorhabditis elegans zygotes, we found that the microtubule-bundling protein SPD-1PRC1 and the motor ZEN-4MKLP-1 are required for proper central spindle structure during its elongation4,5,6,7,8,9. In contrast, we found that the kinetochore controls the initiation of central spindle assembly. Specifically, central spindle microtubule assembly is dependent on kinetochore recruitment of the scaffold protein KNL-1, as well as downstream partners BUB-1, HCP-1/2CENP-F and CLS-2CLASP; and is negatively regulated by kinetochore-associated protein phosphatase 1 activity. This in turn promotes central spindle localization of CLS-2CLASP and initial central spindle microtubule assembly through its microtubule polymerase activity. Together, our results reveal an unexpected role for a conserved kinetochore protein network in coupling two critical events of cell division: chromosome segregation and cytokinesis.
This is a preview of subscription content, access via your institution
Access options
Subscribe to this journal
Receive 12 print issues and online access
$209.00 per year
only $17.42 per issue
Rent or buy this article
Prices vary by article type
from$1.95
to$39.95
Prices may be subject to local taxes which are calculated during checkout
Similar content being viewed by others
Change history
08 June 2015
In the version of this Letter originally published, the following sentence was omitted from the Acknowledgements: 'B.L. is supported by a post-doctoral fellowship from FRM (ARF20140129055).' This has been corrected in the online versions of the Letter.
References
Lee, K-Y., Davies, T. & Mishima, M. Cytokinesis microtubule organisers at a glance. J. Cell Sci. 125, 3495–3500 (2012).
Uehara, R. & Goshima, G. Functional central spindle assembly requires de novo microtubule generation in the interchromosomal region during anaphase. J. Cell Biol. 202, 623–636 (2010).
Glotzer, M. The molecular requirements for cytokinesis. Science 307, 1735–1739 (2005).
Raich, W. B., Moran, A. N., Rothman, J. H. & Hardin, J. Cytokinesis and midzone microtubule organization in Caenorhabditis elegans require the kinesin-like protein ZEN-4. Mol. Biol. Cell 9, 2037–2049 (1998).
Verbrugghe, K. J. & White, J. G. SPD-1 is required for the formation of the spindle midzone but is not essential for the completion of cytokinesis in C. elegans embryos. Curr. Biol. 14, 1755–1760 (2004).
Schuyler, S. C., Liu, J. Y. & Pellman, D. The molecular function of Ase1p: evidence for a MAP-dependent midzone-specific spindle matrix. Microtubule-associated proteins. J. Cell Biol. 160, 517–528 (2003).
Jiang, W. et al. PRC1: a human mitotic spindle-associated CDK substrate protein required for cytokinesis. Mol. Cell 2, 877–885 (1998).
Mollinari, C. et al. PRC1 is a microtubule binding and bundling protein essential to maintain the mitotic spindle midzone. J. Cell Biol. 157, 1175–1186 (2002).
Powers, J., Bossinger, O., Rose, D., Strome, S. & Saxton, W. A nematode kinesin required for cleavage furrow advancement. Curr. Biol. 8, 1133–1136 (1998).
Hu, C. K., Ozlu, N., Coughlin, M., Steen, J. J. & Mitchison, T. J. Plk1 negatively regulates PRC1 to prevent premature midzone formation before cytokinesis. Mol. Biol. Cell 23, 2702–2711 (2012).
Subramanian, R. et al. Insights into antiparallel microtubule crosslinking by PRC1, a conserved nonmotor microtubule binding protein. Cell 142, 433–443 (2010).
Bieling, P., Telley, I. A. & Surrey, T. A minimal midzone protein module controls formation and length of antiparallel microtubule overlaps. Cell 142, 420–432 (2010).
Carmena, M., Wheelock, M., Funabiki, H. & Earnshaw, W. C. The chromosomal passenger complex (CPC): from easy rider to the godfather of mitosis. Nat. Rev. Mol. Cell Biol. 13, 789–803 (2012).
van der Horst, A. & Lens, S. M. Cell division: control of the chromosomal passenger complex in time and space. Chromosoma 123, 25–42 (2014).
Grill, S. W., Gönczy, P., Stelzer, E. H. & Hyman, A. A. Polarity controls forces governing asymmetric spindle positioning in the Caenorhabditis elegans embryo. Nature 409, 630–633 (2001).
Cheeseman, I. M. The Kinetochore. Cold Spring Harb. Perspect. Biol. 6, a015826 (2014).
Desai, A. et al. KNL-1 directs assembly of the microtubule-binding interface of the kinetochore in C. elegans. Genes Dev. 17, 2421–2435 (2003).
Cheeseman, I. M., MacLeod, I., Yates, J. R. III, Oegema, K. & Desai, A. The CENP-F-like proteins HCP-1 and HCP-2 target CLASP to kinetochores to mediate chromosome segregation. Curr. Biol. 15, 771–777 (2005).
Oegema, K., Desai, A., Rybina, S., Kirkham, M. & Hyman, A. A. Functional analysis of kinetochore assembly in Caenorhabditis elegans. J. Cell Biol. 153, 1209–1226 (2001).
Cheeseman, I. M. et al. A conserved protein network controls assembly of the outer kinetochore and its ability to sustain tension. Genes Dev. 18, 2255–2268 (2004).
Gassmann, R. et al. A new mechanism controlling kinetochore-microtubule interactions revealed by comparison of two dynein-targeting components: SPDL-1 and the Rod/Zwilch/Zw10 complex. Genes Dev. 22, 2385–2399 (2008).
Cheeseman, I. M., Chappie, J. S., Wilson-Kubalek, E. M. & Desai, A. The conserved KMN network constitutes the core microtubule-binding site of the kinetochore. Cell 127, 983–997 (2006).
Essex, A., Dammermann, A., Lewellyn, L., Oegema, K. & Desai, A. Systematic analysis in Caenorhabditis elegans reveals that the spindle checkpoint is composed of two largely independent branches. Mol. Biol. Cell 20, 1252–1267 (2009).
Inoue, Y. H. et al. Mutations in orbit/mast reveal that the central spindle is comprised of two microtubule populations, those that initiate cleavage and those that propagate furrow ingression. J. Cell Biol. 166, 49–60 (2004).
Liu, J. et al. PRC1 cooperates with CLASP1 to organize central spindle plasticity in mitosis. J. Biol. Chem. 284, 23059–23071 (2009).
Krenn, V., Overlack, K., Primorac, I., Van Gerwen, S. & Musacchio, A. KI motifs of human Knl1 enhance assembly of comprehensive spindle checkpoint complexes around MELT repeats. Curr. Biol. 24, 29–39 (2014).
Vleugel, M. et al. Arrayed BUB recruitment modules in the kinetochore scaffold KNL1 promote accurate chromosome segregation. J. Cell Biol. 203, 943–955 (2013).
Zhang, G., Lischetti, T. & Nilsson, J. A minimal number of MELT repeats supports all the functions of KNL1 in chromosome segregation. J. Cell Sci. 127, 871–884 (2014).
Moyle, M. W. et al. A Bub1-Mad1 interaction targets the Mad1-Mad2 complex to unattached kinetochores to initiate the spindle checkpoint. J. Cell Biol. 204, 647–657 (2014).
Espeut, J., Cheerambathur, D. K., Krenning, L., Oegema, K. & Desai, A. Microtubule binding by KNL-1 contributes to spindle checkpoint silencing at the kinetochore. J. Cell Biol. 196, 469–482 (2012).
London, N. & Biggins, S. Mad1 kinetochore recruitment by Mps1-mediated phosphorylation of Bub1 signals the spindle checkpoint. Genes Dev. 28, 140–152 (2014).
Srayko, M., Kaya, A., Stamford, J. & Hyman, A. A. Identification and characterization of factors required for microtubule growth and nucleation in the early C. elegans embryo. Dev. Cell 9, 223–236 (2005).
Al-Bassam, J. & Chang, F. Regulation of microtubule dynamics by TOG-domain proteins XMAP215/Dis1 and CLASP. Trends Cell Biol. 21, 604–614 (2011).
Funk, C., Schmeiser, V., Ortiz, J. & Lechner, J. A TOGL domain specifically targets yeast CLASP to kinetochores to stabilize kinetochore microtubules. J. Cell Biol. 205, 555–571 (2014).
Frøkjaer-Jensen, C. et al. Single-copy insertion of transgenes in Caenorhabditis elegans. Nat. Genet. 40, 1375–1383 (2008).
Dumont, J., Oegema, K. & Desai, A. A kinetochore-independent mechanism drives anaphase chromosome separation during acentrosomal meiosis. Nat. Cell Biol. 12, 894–901 (2010).
Schindelin, J. et al. Fiji: an open-source platform for biological-image analysis. Nat. Methods 9, 676–682 (2012).
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).
Acknowledgements
We thank all members of the Tran/Paoletti, Pintard, Doye and Dumont laboratories for support and advice. We are grateful to P. Moussounda and P. Feynerol for providing technical support. We thank C. Janke and N. Tavernier for their help with protein purification. We thank A. Desai and the CGC for worm strains. We thank Y. Kohara for the zen-4MKLP1 cDNA (yk35d10). We are grateful to M. Shirasu-Hiza for critical reading of the manuscript. T.K. is supported by R01-GM074215 (awarded to A. Desai). This work was supported by grants from the ANR (ANR-09-RPDOC-005-01), the FRM (AJE201112) and the Mairie de Paris (Emergence) to J.D., and NIH DP2 OD008773 to J.C.C. B.L. is supported by a post-doctoral fellowship from FRM (ARF20140129055).
Author information
Authors and Affiliations
Contributions
All experiments were conceived by J.D. with input from G.M. and F.E. Experiments were primarily performed and analysed by G.M. and F.E. Biochemistry experiments were performed by B.L. Most of the transgenic strains used here were constructed by M.S. and K.L. T.L. developed the automated central spindle tracking and quantification software. T.K. constructed and provided the OD971 strain. J.C.C. and J.E. constructed and shared several strains used here. G.M., F.E., J.C.C. and J.D. prepared the figures and wrote the manuscript.
Corresponding author
Ethics declarations
Competing interests
The authors declare no competing financial interests.
Integrated supplementary information
Supplementary Figure 1 Spindle and chromosome dynamics.
(a) Chromosome to chromosome distance after anaphase onset for the indicated conditions. The sample size (number of embryos analyzed) is provided in the figure and was generated by aggregation over 3 independent experiments. Error bars represent the SEM. (b) Nuclear envelope breakdown (NEBD) to anaphase onset timing for the indicated conditions. (c) Spindle elongation after anaphase onset (time 0 s) in the indicated conditions. Error bars represent the SEM.
Supplementary Figure 2 CLS-2CLASP and sister chromatid dynamics in indicated conditions.
(a) Localization of GFP-tagged CLS-2CLASP in control or BUB-1 depleted embryos. White dashed lines indicate a 10 pixel thick linescan plotted on Fig. 3c. (b) Chromosome alignment at anaphase onset in indicated RNAi depletions. (c) Sister chromatid alignment and segregation in indicated RNAi depletions. (d) Sister chromatid alignment and segregation in indicated conditions. Scale bars, 5 μm.
Supplementary Figure 3 The KNL-1 (Δ85-505) deletion mutant recruits Mis12, Ndc80 and RZZ complexes at the kinetochore.
Left: Kinetochore localization of GFP-tagged MIS-12 (a), KBP-4Spc24 (b) and CZW-1ZW10 (c) in the indicated KNL-1 background. White dashed lines indicate a 10 pixel thick linescan plotted on the corresponding graphs on the right. Right: Fluorescence intensity of RFP or GFP-tagged KNL-1 (magenta), MIS-12 (a), KBP-4Spc24 (b) and CZW-1ZW10 (c) (green) along the central spindle at metaphase (when chromosomes are first visibly aligned), at anaphase onset, 20 and 50 s after anaphase onset. 0 μm corresponds to the position of chromosomes at anaphase onset. All experiments were performed in the absence of endogenous KNL-1. The sample size for each condition (number of embryos analyzed) is provided in the figure and was generated by aggregation over 2 independent experiments. Error bars represent the SEM. Scale bars, 5 μm.
Supplementary Figure 4 GFP-tagged BUB-1, HCP-1CENP-F and CLS-2CLASP recruitment downstream of KNL-1 mutants.
(a, d and g) GFP-tagged BUB-1 (a), HCP-1CENP-F (d) and CLS-2CLASP (g) localization in the indicated conditions. White dashed lines indicate a 10 pixel thick linescan plotted on graph b, e and h. (b, e and h) Fluorescence intensity of GFP-tagged BUB-1 (b), HCP-1CENP-F (e) and CLS-2CLASP (h) (green) and of the various RFP-tagged KNL-1 mutants (magenta) along the central spindle long axis. 0 μm corresponds to the position of chromosomes at anaphase onset. (c, f and i) Quantification of average GFP-tagged BUB-1 (c), HCP-1CENP-F (f) and CLS-2CLASP (i) fluorescence intensity centred on chromosome position at anaphase onset. Each value is normalized against the WT average intensity at 0 s. The sample size for each condition (number of embryos analyzed) is provided in the figure and was generated by aggregation over 2 independent experiments. Error bars represent the SEM. Scale bars, 5 μm.
Supplementary Figure 5 Purification of recombinant CLS-2CLASP and dynamic localization of various proteins at the central spindle in indicated conditions.
(a) Strategy used for recombinant CLS-2CLASP purification from SF9 insect cells (Left). Coomasie-stained gel with 1.5 μg of pure recombinant CLS-2CLASP used in the assay represented on Fig. 5b (Right). (b) GFP-tagged EBP-1EB1, Tubulin, CLS-2CLASP and SPD-1PRC1 localization during anaphase in a one-cell embryo. (c) GFP-tagged CLS-2CLASP recruitment at the central spindle for the indicated conditions. (d) Quantification of GFP-tagged CLS-2CLASP central spindle intensity in control and spd-1PRC1(RNAi) embryos 20 s after anaphase onset (P < 0,0001). The mean is shown for n = 12 and 14 embryos for control and spd-1(RNAi), respectively. Data was aggregated over 2 independent experiments. A Student t-test was used to determine significance. Error bars represent the SEM. Scale bars, 5 μm.
Supplementary information
Supplementary Information
Supplementary Information (PDF 2120 kb)
Supplementary Video 1
Movie montage of the first embryonic division in (Left) GFP::TBB-2β−Tubulinf (yellow) and mCherry::H2B (magenta) or (Right) GFP::AIR-2AuroraB, GFP::γ-Tubulin (green) and mCherry::H2B (magenta) expressing embryos in control and after SPD-1PRC1 or ZEN-4MKLP1 depletion. Images, which are the maximum projection of 4 z-sections, were collected every 10 s and played back at 60x real time (6 images per second) with time 0 corresponding to anaphase onset. (MOV 1869 kb)
Supplementary Video 2
Movie montage of the first embryonic division in (Left) GFP::TBB-2β−Tubulin (yellow) and mCherry::H2B (magenta) or (Right) GFP::AIR-2AuroraB, GFP::γ-Tubulin (green) and mCherry::H2B (magenta) expressing embryos in GPR-1/2 depletion or GPR-1/2 and SPD-1PRC1, ZEN-4MKLP1 or CLS-2CLASP co-depletion. Images, which are the maximum projection of 4 z-sections, were collected every 10 s and played back at 60x real time (6 images per second) with time 0 corresponding to anaphase onset. (MOV 1133 kb)
Supplementary Video 3
Movie montage of the first embryonic division in (Left) GFP::TBB-2β−Tubulin (yellow) and mCherry::H2B (magenta) or (Right) GFP::AIR-2AuroraB, GFP::γ-Tubulin (green) and mCherry::H2B (magenta) expressing embryos in control and after NDC-80, ZWL-1ZWILCH or BUB-1 depletion. Images, which are the maximum projection of 4 z-sections, were collected every 10 s and played back at 60x real time (6 images per second) with time 0 corresponding to anaphase onset. (MOV 890 kb)
Supplementary Video 4
Movie montage of the first embryonic division in β-Tubulin::GFP (yellow) and KNL-1::RFP (magenta) expressing embryos in control and after different CLS-2CLASP depletion conditions (28h or 32h post RNAi injection). Images, which are single z-sections were collected every second and played back at 6x real time (6 images per second) with time 0 corresponding to anaphase onset. (MOV 3524 kb)
Supplementary Video 5
Movie montage of the first embryonic division in (Left) GFP::TBB-2β−Tubulin (yellow) and mCherry::H2B (magenta) or (Right) GFP::AIR-2AuroraB, GFP::γ-Tubulin (green) and mCherry::H2B (magenta) expressing embryos in control and after partial HCP-1/2CENP-F or CLS-2CLASP depletion. Images, which are the maximum projection of 4 z-sections, were collected every 10 s and played back at 60x real time (6 images per second) with time 0 corresponding to anaphase onset. (MOV 2007 kb)
Supplementary Video 6
Movie montage of the first embryonic division in (Left) GFP::TBB-2β−Tubulin (yellow) and mCherry::H2B (magenta) or (Right) GFP::AIR-2AuroraB, GFP::γ-Tubulin (green) and mCherry::H2B (magenta) expressing embryos and either KNL-1 WT::RFP, KNL-1 Δ85-505::RFP or KNL-1 RRASA::RFP in absence of endogenous KNL-1. Images, which are the maximum projection of 4 z-sections were collected every 10 s and played back at 60x real time (6 images per second) with time 0 corresponding to anaphase onset. (MOV 1884 kb)
Supplementary Video 7
Movie montage of the first embryonic division in (Left) GFP::TBB-2β−Tubulin (yellow) and mCherry::H2B (magenta) or (Right) GFP::AIR-2AuroraB, GFP::γ-Tubulin (green) and mCherry::H2B (magenta) expressing embryos and either KNL-1 WT::RFP or KNL-1 RRASA::RFP in absence of endogenous KNL-1 or/and KLP-7MCAK. Images, which are the maximum projection of 4 z-sections were collected every 10 s and played back at 60x real time (6 images per second) with time 0 corresponding to anaphase onset. (MOV 903 kb)
Supplementary Video 8
Movie montage of the first embryonic division in embryos expressing mCherry::TBB-2β−Tubulin (yellow) and GFP::CLS-2CLASP (magenta) WT (left) or 3A (right). Images, which are single z-sections, were collected every 10 s and played back at 60x real time (6 images per second) with time 0 corresponding to anaphase onset. (MOV 951 kb)
Supplementary Video 9
Movie montage of the first embryonic division in embryos expressing GFP::EBP-1EB1 (green) and either KNL-1 WT::RFP, KNL-1 Δ85-505::RFP or KNL-1 RRASA::RFP (magenta) in absence of endogenous KNL-1 or KNL-1 WT::RFP (magenta) in absence of endogenous CLS-2CLASP. Images, which are single z-sections, were collected every 4 s and played back at 24x real time (6 images per second) with time 0 corresponding to anaphase onset. (MOV 3132 kb)
Supplementary Video 10
Movie montage of the first embryonic division in embryos expressing mCherry::H2B (magenta) and either GFP::TBB-2β−Tubulin or CLS-2CLASP ::GFP or SPD-1PRC1 ::GFP (green). Images, which are the maximum projection of 4 z-sections were collected every 10 s and played back at 60x real time (6 images per second) with time 0 corresponding to anaphase onset. (MOV 5951 kb)
Rights and permissions
About this article
Cite this article
Maton, G., Edwards, F., Lacroix, B. et al. Kinetochore components are required for central spindle assembly. Nat Cell Biol 17, 697–705 (2015). https://doi.org/10.1038/ncb3150
Received:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1038/ncb3150
This article is cited by
-
Kinetochore component function in C. elegans oocytes revealed by 4D tracking of holocentric chromosomes
Nature Communications (2023)
-
Kinetochore- and chromosome-driven transition of microtubules into bundles promotes spindle assembly
Nature Communications (2022)
-
CENP-F-dependent DRP1 function regulates APC/C activity during oocyte meiosis I
Nature Communications (2022)
-
Chromosome segregation occurs by microtubule pushing in oocytes
Nature Communications (2017)
-
Erratum: Corrigenda: Kinetochore components are required for central spindle assembly
Nature Cell Biology (2015)