Figure 5.
Localization of dynein targeting factors and accurate chromosome segregation require sticky IDRs and protein binding motifs in Spc105. (A) Representative images of BubR1 (magenta) in cells expressing WT Spc105 or the HsKI chimera (green). (B) Quantification of BubR1 intensity relative to EGFP signal in WT and HsKI-expressing cells (WT; n = 150 kinetochores from 30 cells and three replicates, HsKI; n = 145 kinetochores from 29 cells and three replicates). (C) Representative images of Rod (magenta) in cells expressing WT Spc105 or the HsKI chimera (green). (D) Quantification of Rod intensity relative to EGFP signal in WT and HsKI-expressing cells (WT; n = 150 kinetochores from 30 cells and three replicates, HsKI; n = 150 kinetochores from 30 cells and three replicates). (E) Representative images of BubR1 (magenta) in cells expressing WT Spc105 or the FUS-HsKI chimera (green). (F) Quantification of BubR1 intensity relative to EGFP signal in WT and FUS-HsKI-expressing cells (WT; n = 150 kinetochores from 30 cells and three replicates, FUS-HsKI; n = 150 kinetochores from 30 cells and three replicates). (G) Representative images of Rod (magenta) in cells expressing WT Spc105 or the FUS-HsKI chimera (green). (H) Quantification of Rod intensity relative to EGFP signal in WT and FUS-HsKI-expressing cells (WT; n = 155 kinetochores from 31 cells and three replicates, FUS-HsKI; n = 160 kinetochores from 32 cells and three replicates). (I) Percentage of anaphase cells with at least one lagging chromosome in WT-, deletion mutant-, and chimera-expressing cells (n = 300 cells per condition from three replicates). (J) Model for how Spc105 and BubR1 contribute to the recruitment of a core population of RZZ at bioriented kinetochores that reduces the Ndc80 complex’s affinity for kMTs. A single Spc105 molecule is shown for clarity, but we propose that it is assembled into higher-order multimers via sticky IDRs (pink circle). Dynein relieves the weakening of kMT attachment affinity by the core pool of RZZ by stripping the complex in fly cells. Boxed regions are shown in the insets. All plots report mean ± SEM. Reported P values were generated by a randomization method (PlotsOfDifferences) in B, D, F, and H and two-tailed t tests in I. **P < 0.001. Different letters above each column in I indicate significant differences (P < 0.05) determined by two-tailed t tests for all pairwise combinations. Shared letters indicate that the difference is not significant (P > 0.05). Scale bars: 5 μm (A, C, E, and G), 1 μm (all insets).

Localization of dynein targeting factors and accurate chromosome segregation require sticky IDRs and protein binding motifs in Spc105. (A) Representative images of BubR1 (magenta) in cells expressing WT Spc105 or the HsKI chimera (green). (B) Quantification of BubR1 intensity relative to EGFP signal in WT and HsKI-expressing cells (WT; n = 150 kinetochores from 30 cells and three replicates, HsKI; n = 145 kinetochores from 29 cells and three replicates). (C) Representative images of Rod (magenta) in cells expressing WT Spc105 or the HsKI chimera (green). (D) Quantification of Rod intensity relative to EGFP signal in WT and HsKI-expressing cells (WT; n = 150 kinetochores from 30 cells and three replicates, HsKI; n = 150 kinetochores from 30 cells and three replicates). (E) Representative images of BubR1 (magenta) in cells expressing WT Spc105 or the FUS-HsKI chimera (green). (F) Quantification of BubR1 intensity relative to EGFP signal in WT and FUS-HsKI-expressing cells (WT; n = 150 kinetochores from 30 cells and three replicates, FUS-HsKI; n = 150 kinetochores from 30 cells and three replicates). (G) Representative images of Rod (magenta) in cells expressing WT Spc105 or the FUS-HsKI chimera (green). (H) Quantification of Rod intensity relative to EGFP signal in WT and FUS-HsKI-expressing cells (WT; n = 155 kinetochores from 31 cells and three replicates, FUS-HsKI; n = 160 kinetochores from 32 cells and three replicates). (I) Percentage of anaphase cells with at least one lagging chromosome in WT-, deletion mutant-, and chimera-expressing cells (n = 300 cells per condition from three replicates). (J) Model for how Spc105 and BubR1 contribute to the recruitment of a core population of RZZ at bioriented kinetochores that reduces the Ndc80 complex’s affinity for kMTs. A single Spc105 molecule is shown for clarity, but we propose that it is assembled into higher-order multimers via sticky IDRs (pink circle). Dynein relieves the weakening of kMT attachment affinity by the core pool of RZZ by stripping the complex in fly cells. Boxed regions are shown in the insets. All plots report mean ± SEM. Reported P values were generated by a randomization method (PlotsOfDifferences) in B, D, F, and H and two-tailed t tests in I. **P < 0.001. Different letters above each column in I indicate significant differences (P < 0.05) determined by two-tailed t tests for all pairwise combinations. Shared letters indicate that the difference is not significant (P > 0.05). Scale bars: 5 μm (A, C, E, and G), 1 μm (all insets).

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