Figure 3.
Dissecting the molecular linkage between Spc105 and dynein and its functional contribution to accurate chromosome segregation. (A) Representative images of BubR1 (magenta) in cells expressing WT Spc105 or the Δ267–383-Spc105 deletion mutant (green). (B) Quantification of BubR1 intensities relative to EGFP signal in WT and Δ267–383-expressing cells (WT; n = 140 kinetochores from 25 cells and 2 replicates, Δ267–383; n = 135 kinetochores from 24 cells and 2 replicates). (C) Representative images of Rod (magenta) in cells expressing WT Spc105 or the deletion mutant (green). (D) Quantification of Rod intensity relative to EGFP signal in WT and Δ267–383-expressing cells (WT; n = 142 kinetochores from 25 cells and 2 replicates, Δ267–383; n = 140 kinetochores from 25 cells and 2 replicates). (E) Representative confocal Z-planes of control and BubR1 dsRNA-treated fixed cells stained for Spc105 (green) and Rod (magenta). (F) Quantification of Rod:Spc105 intensity ratios in control and BubR1-depleted cells (control; n = 105 kinetochores from 21 cells and 2 replicates, BubR1 RNAi; n = 102 kinetochores from 20 cells and 2 replicates). (G) Schematic of DmBubR1 highlighting the KEN box (orange), TPR motif (cyan), GLEBS (green), and KARD (pink) domains, and homology region (HR) to HsBub1 (magenta). The putative binding partner for each motif/domain is indicated in parentheses and the region (aa 734–872) deleted in the BubR1 mutant is shown. (H) Representative confocal Z-planes of metaphase cells transiently transfected with BubR1-EGFP or BubR1-Δ734–872-EGFP stained for EGFP (green) and Rod (magenta). (I) Quantification of Rod:EGFP intensity ratios in control (BubR1-EGFP) and BubR1-Δ734-872-EGFP-expressing cells (control; n = 60 kinetochores from 12 cells, BubR1-Δ734–872; n = 55 kinetochores from 11 cells). (J) Examples of the chromosome segregation categories quantified in K and L (Δ267-383 is green, DNA is magenta). (K) Percentage of anaphase/telophase cells with at least one lagging chromosome in WT- and Δ267-383-expressing cells (n = 150 cells per condition from three replicates). (L) Percentage of WT- and Δ267–383-expressing cells with either bridge or merotelic lagging chromosomes. (M) Representative examples from confocal time-lapses of Δ267–383-expressing cells with persistent merotelic attachments in anaphase. Kymographs of the boxed regions are shown. Reported P values were generated by a randomization method (PlotsOfDifferences) in B, D, F, and I or two-tailed t tests in K and L: * P value <0.05, ** P value <0.001. Boxed regions are shown in the insets. Plots report mean ± SEM. Scale bars: 10 μm (E, J, and M); 5 μm (A, C, and H), 1 μm (insets); 5 μm (horizontal) and 1 min (vertical) in kymographs. Displayed times are min:s.

Dissecting the molecular linkage between Spc105 and dynein and its functional contribution to accurate chromosome segregation. (A) Representative images of BubR1 (magenta) in cells expressing WT Spc105 or the Δ267–383-Spc105 deletion mutant (green). (B) Quantification of BubR1 intensities relative to EGFP signal in WT and Δ267–383-expressing cells (WT; n = 140 kinetochores from 25 cells and 2 replicates, Δ267–383; n = 135 kinetochores from 24 cells and 2 replicates). (C) Representative images of Rod (magenta) in cells expressing WT Spc105 or the deletion mutant (green). (D) Quantification of Rod intensity relative to EGFP signal in WT and Δ267–383-expressing cells (WT; n = 142 kinetochores from 25 cells and 2 replicates, Δ267–383; n = 140 kinetochores from 25 cells and 2 replicates). (E) Representative confocal Z-planes of control and BubR1 dsRNA-treated fixed cells stained for Spc105 (green) and Rod (magenta). (F) Quantification of Rod:Spc105 intensity ratios in control and BubR1-depleted cells (control; n = 105 kinetochores from 21 cells and 2 replicates, BubR1 RNAi; n = 102 kinetochores from 20 cells and 2 replicates). (G) Schematic of DmBubR1 highlighting the KEN box (orange), TPR motif (cyan), GLEBS (green), and KARD (pink) domains, and homology region (HR) to HsBub1 (magenta). The putative binding partner for each motif/domain is indicated in parentheses and the region (aa 734–872) deleted in the BubR1 mutant is shown. (H) Representative confocal Z-planes of metaphase cells transiently transfected with BubR1-EGFP or BubR1-Δ734–872-EGFP stained for EGFP (green) and Rod (magenta). (I) Quantification of Rod:EGFP intensity ratios in control (BubR1-EGFP) and BubR1-Δ734-872-EGFP-expressing cells (control; n = 60 kinetochores from 12 cells, BubR1-Δ734–872; n = 55 kinetochores from 11 cells). (J) Examples of the chromosome segregation categories quantified in K and L (Δ267-383 is green, DNA is magenta). (K) Percentage of anaphase/telophase cells with at least one lagging chromosome in WT- and Δ267-383-expressing cells (n = 150 cells per condition from three replicates). (L) Percentage of WT- and Δ267–383-expressing cells with either bridge or merotelic lagging chromosomes. (M) Representative examples from confocal time-lapses of Δ267–383-expressing cells with persistent merotelic attachments in anaphase. Kymographs of the boxed regions are shown. Reported P values were generated by a randomization method (PlotsOfDifferences) in B, D, F, and I or two-tailed t tests in K and L: * P value <0.05, ** P value <0.001. Boxed regions are shown in the insets. Plots report mean ± SEM. Scale bars: 10 μm (E, J, and M); 5 μm (A, C, and H), 1 μm (insets); 5 μm (horizontal) and 1 min (vertical) in kymographs. Displayed times are min:s.

This Feature Is Available To Subscribers Only

or Create an Account

Close Modal
Close Modal