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

Figure 1. The N-terminal MELT repeats of KNL-1, but not BUB-3, are required for BHC module kinetochore targeting in oocytes.

(A) Schematic of kinetochore and ring domain protein organization around a bivalent chromosome during metaphase I in a C. elegans oocyte. CPC: Chromosome Passenger Complex. (B) Immunolocalization of BUB-1 (left) and quantification of BUB-1 signal at kinetochores (right) in bub-3(ok3437) mutants (bub3∆, n=58) compared to wild type controls (n=58). Error bars, Mean and standard deviation. Unpaired t-test, alpha = 0.05, p<0.0001. (C) Schematic of KNL-1::mCherry protein fusions. (D–F) Localization of BUB-1::GFP (D), GFP::HCP-1 (E) and CLS-2::GFP (F) in worms carrying full length or MELT-deleted KNL-1::mCherry (KNL-1FL and KNL-1∆85-505 respectively, n≥10). (G) Localization of CLS-2::GFP at ring domains in knl-1-depleted oocytes (left) with corresponding schematic (right). (H) Localization of BUB-1::mCherry in gei-17-depleted oocytes (n=29) compared to controls (n=25). Scale bars 5 µm, 1 µm in insets.

Figure 1—source data 1. Panel B source data.
Quantification of BUB-1 signal at kinetochores in bub-3(ok3437) mutants (bub-3∆) and in control N2. Integrated intensity measurements corrected by integrated background noise (Integrated signal intensity/Average noise) of immunolocalized BUB-1 at kinetochores in fixed oocytes of bub-3(ok3437) mutants (bub-3∆) compared to N2 wild type controls. Descriptive statistics and details of unpaired t-test for comparison of samples are provided.
elife-82579-fig1-data1.xlsx (136.2KB, xlsx)

Figure 1.

Figure 1—figure supplement 1. BUB-3 is not required for BHC module kinetochore targeting.

Figure 1—figure supplement 1.

(A) Left: Western blot of full-protein extracts. BUB-1 (top) and α-tubulin (TUB-α, bottom) in wild type and bub-3(ok3437) mutants (bub-3∆). The number of worms extracted is indicated (n worms). Center: Quantification and linear regression of corrected intensities of BUB-1 and TUB-α in wild type and bub-3∆ extracts. Right: Percentage of BUB-1 and TUB-α signals in bub-3∆ relative to wild type. (B) Immunolocalization of CLS-2 (left) and quantification of CLS-2 signal at kinetochores (right) in bub-3(ok3437) mutants (bub3∆, n=58) compared to wild type controls (n=58). Note the ring localization of CLS-2, which is clearly visible on the left- and right-most chromosomes of the wild-type spindle. (C) Stills of metaphase I oocytes from live imaging of endogenously tagged GFP::HCP-1 (left), and quantification of GFP signal/average noise in the whole spindle, measured at the frame preceding spindle rotation at the cortex (right), in bub-3(ok3437) mutants (bub3∆, n=10) compared to controls (n=15). Error bars, Mean and standard deviation. Unpaired t-test, alpha = 0.05, p<0.0001. Scale bars, 5 µm.
Figure 1—figure supplement 1—source data 1. Panel A source data 1.
Folder containing raw images and uncropped annotated images of Western blot of BUB-1 and tubulin in full-protein extracts of wild type and bub-3(ok3437) (bub-3∆) mutant worms.
Figure 1—figure supplement 1—source data 2. Panel A source data 2.
Signal measurements on western blots, performed in Fiji. Simple linear regression was performed using GraphPad Prism.
Figure 1—figure supplement 1—source data 3. Panel B source data.
Quantification of CLS-2 signal at kinetochores in bub-3(ok3437) mutants (bub-3∆) compared to wild type N2 controls. Signals were measured in Fiji from immunolocalized proteins. Details of statistical analyses are provided.
Figure 1—figure supplement 1—source data 4. Panel C source data.
Quantification of HCP-1 signal at kinetochores in bub-3(ok3437) mutants (bub-3∆) compared to controls. Signals were measured in Fiji from live imaging of GFP::HCP-1. Details of statistical analyses are provided.