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  • Letter
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Kinetochore components are required for central spindle assembly

A Corrigendum to this article was published on 30 June 2015

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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.

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Figure 1: SPD-1PRC1 and ZEN-4MKLP1 are required for central spindle stabilization and bundling.
Figure 2: A subset of kinetochore proteins are involved in central spindle assembly.
Figure 3: The BUB-1/HCP-1/2CENP-F/CLS-2CLASP pathway is essential for initiating central spindle formation.
Figure 4: PP1-modulated KNL-1-dependent recruitment of BUB-1/HCP-1/2CENP-F/CLS-2CLASP controls central spindle assembly.
Figure 5: CLS-2CLASP activity is required for central spindle assembly and a two-stage model of central spindle organization.

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  • 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.

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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

Authors

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

Correspondence to Julien Dumont.

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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)

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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

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