Figure 1. BUB-1 inhibits chromosome biorientation.

(A) Assay for kinetochore-microtubule attachment formation and chromosome congression. GFP::γ-Tub is used to measure the pole to pole distance, and KNL-1::mCherry is used to measure the chromosome span in the spindle pole axis. (B) Kymographs generated from embryos expressing GFP::γ-Tub and KNL-1::mCherry, for the different indicated conditions. Horizontal scale bar, 5 μm; Vertical scale bar, 20 s. (C) Chromosome span and pole to pole distance as functions of time after NEBD for the indicated conditions. Top right corner: Arrowhead, spindle pole bump following BUB-1 depletion. (D) Schematics of BUB-1 at kinetochores recruiting its downstream partners HCP-1/2^CENP-F and CLS-2^CLASP. (E) Top: Representative images from time-lapse movies showing BUB-1 dependent localisations of GFP::HCP-1^CENP-F, GFP::HCP-2^CENP-F and CLS-2^CLASP::GFP on chromosomes (H2B::mCherry), at metaphase. Bottom: Quantification of the GFP signal on chromosomes at metaphase. Mann Whitney tests were used to determine significance (GFP::HCP-1 p < 0.0001, GFP::HCP-2 p = 0.0003, CLS-2::GFP p < 0.0001). Scale bar, 5 μm. (F) Immunofluorescent staining of kinetochores (KNL-1) and microtubules (DM1α) in Δhcp-2 zygotes at metaphase in the indicated conditions. Scale bar, 5 μm. Magnifications of the kinetochore region (highlighted by a dashed rectangle) are shown on the right of each panel. Arrows point to bent merotelic kinetochores in the BUB-1-depleted zygote. Arrowheads show a mono-oriented chromosome in the HCP-1^CENP-F-depleted zygote. Scale bar, 1 μm. (G) Representative images of kinetochores (KNL-1::mCherry, green) and spindle poles (GFP::γ-Tub, magenta), at different times from the onset of chromosome segregation, for the indicated conditions. White arrows point towards sister kinetochores. White asterisks indicate the presence of kinetochore stretches. Scale bar, 5 μm. (H) Quantification of the percentage of embryos with chromosomes engaged in amphitelic, merotelic and mono-oriented attachments, in the indicated conditions. Error bars represent the SEM.

Figure 1—figure supplement 1. HCP-1/2^CENP-F and CLS-2^CLASP downstream of BUB-1 prevent premature chromosome segregation.

(A) Representative images from time-lapse movies showing BUB-1 dependent localizations of GFP::HCP-1^CENP-F, GFP::HCP-2^CENP-F and CLS-2^CLASP::GFP on chromosomes (H2B::mCherry), at different times relative to NEBD and Anaphase onset. (B) Quantifications of the integrated GFP::HCP-1^CENP-F, GFP::HCP-2^CENP-F, and CLS-2^CLASP::GFP signals measured on chromosomes as a function of time from NEBD, for the indicated conditions. (C) Schematic of the separase-sensor allowing detection of anaphase onset by the loss of GFP signal from centromeres. (D) Representative images from time-lapse movies of embryos expressing H2B::mCherry and the GFP-tagged anaphase sensor, in the indicated conditions. (E) Quantifications of the integrated GFP signal measured on chromosomes as a function of time from NEBD, for the indicated conditions. Arrows indicate the time of chromosome segregation onset for the different conditions. Error bars represent the SEM. Scale bars, 5 μm.

Figure 1—figure supplement 2. Characterization of the Δhcp-1 and Δhcp-2 alleles.

(A) Western blot against HCP-2^CENP-F, in the indicated strains, using polyclonal antibodies targeting the HCP-2^CENP-F N-Terminal or C-Terminal region. α-Tubulin is used as a loading control. (B) Kymographs generated from embryos expressing GFP::γ-Tub and KNL-1::mCherry for the indicated conditions. (C) Chromosome span and pole to pole distance as functions of time after NEBD for the indicated conditions. (D) Western blot against HCP-1^CENP-F, in the indicated strains, using polyclonal antibodies targeting HCP-1^CENP-F C-Terminal region. α-Tubulin is used as a loading control. (E) Kymographs generated from embryos expressing H2B::mCherry, for the different indicated conditions. (F) Representative fixed zygotes in metaphase, stained for DNA, α-Tubulin, BUB-1, and HCP-1/2^CENP-F for the indicated conditions. Error bars represent the SEM. Horizontal scale bars, 5 μm; Vertical scale bars, 20 s.