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. 2023 Feb 17;12:e82579. doi: 10.7554/eLife.82579

Figure 4. BHC module integrity is essential for spindle assembly and accurate chromosome segregation.

(A) Schematic of BHC-module integrity mutants. (B) Stills from live imaging of meiosis I in indicated conditions. Microtubules (GFP::TBA-2α-tubulin or GFP::TBB-2β-tubulin) in green, chromosomes (mCherry::HIS-11H2B) in magenta. Time in seconds relative to anaphase I onset. Graphs indicate quantifications as referred to in color key. (C) Plots of spindle density (corrected GFP intensity, left) and spindle area (right) 45 s before anaphase I onset. Kruskal-Wallis multiple comparisons, alpha = 0.05, *p<0.05, ***p<0.001, ****p<0.0001, n.s. not significant. Error bars, Mean with standard deviation. (D) Schematic of the CLS-2FL::GFP::HCP-11154-1386 protein fusion. The HCP-111154-1386 fragment contains the KTD. (E) Localization of CLS-2FL::GFP::HCP-11154-1386 (green) in metaphase I, and magnification of a single meiosis I chromosome. DNA (mCherry::HIS-11H2B) in magenta (n=13). (F) Embryonic lethality and (G) meiotic defects rescue assays of indicated depletions by CLS-2FL::GFP::HCP-11154-1386. Scale bars, full spindle 5 µm, single chromosome details 1 µm.

Figure 4—source data 1. Panel B source data.
Quantifications of the meiotic defects observed in indicated conditions.
Figure 4—source data 2. Panel C source data.
Quantifications of spindle density and spindle size during meiosis I in indicated hcp-2(ijm6) mutants upon depletion of endogenous hcp-1. Raw quantifications and corrected intensities of GFP::TBA-2α-tubulin signal (GFP Integrated Intensity/Background Noise). Details of statistical analyses are provided.
Figure 4—source data 3. Panel F source data.
Embryonic viability assay of hcp-2(ijm6) mutant, cls-2FL::gfp::hcp-111541154-1386 transgenic worms upon depletion of hcp-1 and/or cls-2.
Figure 4—source data 4. Panel G source data.
Quantifications of the meiotic defects in hcp-2(ijm6) mutant, cls-2FL::gfp::hcp-111541154-1386 transgenic worms upon depletion of hcp-1 and/or cls-2.

Figure 4.

Figure 4—figure supplement 1. Artificial kinetochore and ring localization of CLS-2 is not sufficient to rescue loss of BHC module integrity.

Figure 4—figure supplement 1.

(A) Stills from live imaging of BUB-1∆KD::GFP transgenic oocytes. Microtubules (GFP::TBB-2β-tubulin) in green, chromosomes (mCherry::HIS-11H2B) in magenta. Right panels indicate quantifications of meiotic defects shown in the color key. Time in seconds relative to anaphase I onset. (B) Localization of indicated GFP::HCP-1 fusion proteins (green) during the first zygotic mitosis (n≥9). Centrosomes (GFP::TBG-1ɣ-tubulin) and indicated GFP::HCP-1 deletion mutants in green, chromosomes (mCherry::HIS-11H2B) in magenta. (C) Schematic of the CLS-2∆CTD::GFP::HCP-11154-1386 protein fusion. (D) Localization of CLS-2∆CTD::GFP::HCP-11154-1386 in metaphase I, relative to KNL-1 marked kinetochores (KNL-1::mCherry, magenta), in hcp-2∆ mutants depleted of endogenous hcp-1 and cls-2 (n=11). (E) Stills from live imaging of meiosis I (left) and color-coded meiotic defects scored (right) in CLS-2∆CTD::GFP::HCP-11154-1386, hcp-2∆ worms, in indicated conditions. Time in seconds relative to anaphase I onset. Scale bars, 5 µm.
Figure 4—figure supplement 1—source data 1. Panel A source data.
Quantifications of the meiotic defects in bub-1∆KD::mCherry transgenic worms.
Figure 4—figure supplement 1—source data 2. Panel E source data.
Quantifications of the meiotic defects observed in hcp-2(ijm6) mutant, cls-2∆CTD::gfp::hcp-111541154-1386 transgenic worms upon depletion of cls-2 or both hcp-1 and cls-2.