Kinetochore components are required for central spindle assembly

Abstract

A critical structure poised to coordinate chromosome segregation with division plane specification is the central spindle that forms between separating chromosomes after anaphase onset. The central spindle acts as a signalling centre that concentrates proteins essential for division plane specification and contractile ring constriction. 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-1(PRC1) and the motor ZEN-4(MKLP-1) are required for proper central spindle structure during its elongation. 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-1/2(CENP-F) and CLS-2(CLASP); and is negatively regulated by kinetochore-associated protein phosphatase 1 activity. This in turn promotes central spindle localization of CLS-2(CLASP) 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.

Figure 1. SPD-1^PRC1 and ZEN-4^MKLP1 are required for central spindle stabilization and bundling

(a) Central spindle assembly in the indicated strains. Timings are relative to anaphase onset. (b) Kymographic assay for central spindle mechanical integrity analysis. (c) Functional assay for central spindle density analysis. (d) Schematics of SPD-1^PRC1 and ZEN-4^MKLP1 binding to overlapping microtubule plus-ends. (e) Kymographs for the indicated conditions. (f) 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. (g) Percentage of one-cell embryos that displayed a breaking apart central spindle in the indicated conditions. (h) Central spindle assembly and phenotypes for the indicated conditions. The white arrows indicate the presence of remaining microtubules connected to each set of separating sister chromatids and that initially formed at the central spindle before it broke apart. (i) Quantification of central spindle intensity after spd-1^PRC1(RNAi) (GFP∷β-Tub, p<0.0001; GFP∷AIR-2, p<0.0001) and zen-4^MKLP1(RNAi) (GFP∷β-Tub, p<0.0001; GFP∷AIR-2, p<0.0001). The mean is shown for n=31, 11 and 21 embryos for control, spd-1(RNAi) and zen-4(RNAi), respectively. Data was aggregated over 3 independent experiments. One-way ANOVA was used to determine significance. (j) Central spindle assembly and phenotypes for the indicated conditions. (k) Quantification of central spindle intensity after gpr-1/2+spd-1^PRC1(RNAi) (GFP∷β-Tub, p=0.0750; GFP∷AIR-2, p=0.0103) and gpr-1/2+zen-4^MKLP1(RNAi) (GFP∷β-Tub, p=0.1578; GFP∷AIR-2, p=0.1047). The mean is shown for n=31, 20 and 16 embryos for gpr-1/2(RNAi), gpr-1/2+spd-1(RNAi) and gpr-1/2+zen-4(RNAi), respectively. Data was aggregated over 3 independent experiments. One-way ANOVA was used to determine significance. Error bars

Figure 2. A subset of kinetochore proteins are involved in central spindle assembly

(a) Schematics of kinetochore sub-complexes analyzed. (b) Kymographs for the indicated conditions. (c) 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. (d) Percentage of one-cell embryos that displayed a breaking apart central spindle in the indicated conditions. (e) Central spindle assembly and phenotypes for the indicated conditions. (f) Quantification of central spindle intensity after ndc-80(RNAi) (GFP∷β-Tub, p<0.0001; GFP∷AIR-2, p=0,7065), zwl-1 ^ZWILCH(RNAi) (GFP∷β-Tub, p<0.0001; GFP∷AIR-2, p=0.0417) and bub-1(RNAi) (GFP∷β-Tub, p<0.0001; GFP∷AIR-2, p<0.0001). The mean is shown for n=31, 13, 33 and 35 embryos for control, ndc-80(RNAi), zwl-1(RNAi) and bub-1(RNAi), respectively. Data was aggregated over 3 independent experiments. One-way ANOVA was used to determine significance. Error bars represent the SEM. Scale bars, 5 μm.

Figure 3. The BUB-1/HCP-1/2^CENPF/CLS-2^CLASP pathway is essential for initiating central spindle formation

(a) Schematics of components of the BUB-1 kinetochore branch. (b) Fixed control embryos were stained to visualize DNA, α-Tubulin, BUB-1, HCP-1/2^CENP-F, CLS-2^CLASP and KNL-1 in anaphase. (c) Fluorescence intensity for indicated GFP-tagged proteins along the central spindle at anaphase onset (black dots), and 20 or 50 seconds after anaphase onset (respectively dark and light green dots). 0 μm corresponds to the position of chromosomes at anaphase onset. Black arrows indicate the spindle pole localization of CLS-2^CLASP. The sample size (number of embryos analyzed) is provided in the figure and was generated by aggregation over 2 independent experiments. Error bars represent the SEM. (d) Kymographs for the indicated conditions. (e) Chromosome to chromosome distance after anaphase onset for the indicated conditions. 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. (f) Percentage of one-cell embryos that displayed a breaking apart central spindle in the indicated conditions. (g) Central spindle assembly and phenotypes for the indicated conditions. (h) Quantification of central spindle intensity after cls-2^CLASP(RNAi) (GFP∷β-Tub, p=<0.0001; GFP∷AIR-2, p<0.0001) and hcp-1/2^CENP-F(RNAi) (GFP∷β-Tub, p=<0.0001; GFP∷AIR-2, p<0.0001). The mean is shown for n=31, 17 and 13 embryos for control, cls-2(RNAi) and hcp-1/2 (RNAi), respectively. Data was aggregated over 3 independent experiments. One-way ANOVA was used to determine significance. Error bars represent the SEM. (i) Central spindle assembly and phenotypes for the indicated conditions. (j) Quantification of central spindle intensity after gpr-1/2+cls-2^CLASP(RNAi) (GFP∷β-Tub, p<0.0001; GFP∷AIR-2, p<0.0001). The mean is shown for n=31 and 17 embryos for gpr-1/2(RNAi) and gpr-1/2+cls-2(RNAi) respectively. Data was aggregated over 3 independent experiments. One-way ANOVA was used to determine significance. Error bars represent the SEM. Scale bars, 5 μm.

Figure 4. PP1-modulated KNL-1-dependent recruitment of BUB-1/HCP-1/2^CENP-F/CLS-2^CLASP controls central spindle assembly

(a) Schematics of mutations engineered in KNL-1. (b) Fluorescence intensity of GFP-tagged CLS-2 along the central spindle at anaphase onset (black dots), and 20 or 50 seconds after anaphase onset (respectively dark and light green dots) in indicated KNL-1 mutants. All experiments were performed in absence of endogenous KNL-1. 0 μm corresponds to the position of chromosomes at anaphase onset. Black arrows indicate the spindle pole localization of CLS-2^CLASP. The sample size (number of embryos analyzed) is provided in the figure and was generated by aggregation over 2 independent experiments. Error bars represent the SEM. (c) Quantification of average CLS-2^CLASP fluorescence intensity centred on chromosome position at anaphase onset. Each value is normalized against the WT average intensity at 0 s. Error bars represent the SEM. (d) Kymographs for the indicated conditions. (e) 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. (f) Percentage of one-cell embryos that displayed a breaking apart central spindle in the indicated conditions. (g) Central spindle assembly and phenotypes for the indicated conditions. (h) Quantification of central spindle intensity in KNL-1 A85-505 (GFP∷β-Tub, p<0.0001; GFP∷AIR-2, p=0.0002) and KNL-1 RRASA (GFP∷β-Tub, p<0.0001; GFP∷AIR-2, p=0.0003). The mean is shown for n=20, 22 and 21 embryos for KNL-1 WT, KNL-1 Δ85-505 and KNL-1 RRASA respectively. Data was aggregated over 3 independent experiments. One-way ANOVA was used to determine significance. (i) Central spindle assembly and phenotypes for the indicated conditions. (j) 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. Scale bars, 5 μm.

Figure 5. CLS-2^CLASP activity is required for central spindle assembly and a two-stage model of central spindle organization

(a) Schematics of CLS-2^CLASP domain organization and engineered mutations. (b) Microtubule polymerization timecourses with indicated concentrations of CLS-2^CLASP∷6xHIS. Data shown represent one out of 3 independent experiments. (c) Central spindle assembly and phenotypes for the indicated conditions. (d) Quantification of central spindle intensity in CLS-2^CLASP WT and CLS-2^CLASP 3A (p<0.0001). The mean is shown for n=10 embryos in each condition. Data was aggregated over 3 independent experiments. A Student t-test was used to determine significance. (e) Percentage of one-cell embryos that displayed a breaking apart central spindle in the indicated conditions. (f) 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. (g) GFP-tagged EBP-1^EB1 localization at the central spindle in the indicated conditions. (h) Quantification of EBP-1^EB1 intensity at the central spindle intensity in KNL-1 WT, KNL-1 Δ85-505 (p<0.0001), KNL-1 RRASA (p<0.0001) and cls-2^CLASP(RNAi) (p<0.0001). The mean is shown for n=13, 13, 10 and 10 embryos for KNL-1 WT, KNL-1 Δ85-505, KNL-1 RRASA and cls-2(RNAi) respectively. Data was aggregated over 3 independent experiments. One-way ANOVA was used to determine significance. (i) Quantification of average (CLS-2^CLASP and SPD-1^PRC1, between 10 and 80 sec after anaphase onset) or total (β-tubulin and EBP-1^EB1, between 8 and 80 sec after anaphase onset) fluorescence intensity at the central spindle over time. The mean is shown for n=13, 12, 13 and 11 embryos for GFP∷β-Tub, CLS-2∷GFP, SPD-1∷GFP and EBP-1∷GFP respectively. Data was aggregated over 2 independent experiments. Error bars represent the SEM. (j) A two-stage model of central spindle organization. BUB-1, HCP-1/2^CENP-F and CLS-2^CLASP are concentrated at the kinetochore during metaphase in a KNL-1-dependent manner opposed by PP1 activity. This concentration allows their timely translocation in the central spindle region at anaphase onset. Central spindle localized CLS-2^CLASP promotes microtubule formation via its microtubule assembly promoting activity. As CLS-2^CLASP leaves the central spindle region, SPD-1^PRC1 and ZEN-4^MKLP1 accumulate and, through their microtubule cross-linking activity, stabilize the central spindle during its elongation. Scale bars, 5 μm.