Figure S1.
Characterization of Spc105-1-400-EGFP oligomerization, Bub3 exclusion from Spc105 clusters, and depletions of DHC, Rod, and BubR1. (A) Representative maximum projection of confocal Z-sections of a mitotic cell expressing Spc105-1-400-EGFP. (B and C) Spinning-disc confocal time-lapse of cluster assembly in a cell with high expression of Spc105-1-400-EGFP (C) Zoomed view of the boxed region (yellow dashed) in B showing a cluster fusion event. (D) Representative confocal Z-planes of control and DHC RNAi cells stained for CID (magenta) and DHC (green). (E) Quantification of DHC ratioed to CID fluorescence (n = 50 kinetochores from 10 cells for each condition). (F) Cell averages (five kinetochores quantified per cell) of DHC to CID ratios for DHC dsRNA-treated cells relative to the mean DHC:CID ratio of control cells as a measure of the DHC RNAi penetrance. (G) Representative confocal Z-planes of control and Rod RNAi cells stained for Spc105 (magenta) and Rod (green). (H) Quantification of Rod ratioed to Spc105 fluorescence (n = 50 kinetochores from 10 cells for each condition). (I) Cell averages (five kinetochores quantified per cell) of Rod to Spc105 ratios for Rod dsRNA-treated cells relative to the mean Rod:Spc105 ratio of control cells as a measure of the Rod RNAi penetrance. (J) Representative confocal Z-plane of a cell coexpressing Bub3-EGFP (green) and oligomerized clusters of Spc105-1-1722-mCherry-Cry2 (magenta). The boxed region is highlighted in the insets. The average fold enrichment for the cell (from five clusters) is indicated in the image. (K) Quantification of the fold enrichment of the signal of Bub3-EGFP in cluster regions relative to the local background signal (n = 40 clusters from eight cells). (L) Representative maximum projections of confocal Z-sections of control and BubR1 dsRNA-treated fixed cells stained for Spc105 (green), BubR1 (red), and tubulin (blue). (M) Cell averages of BubR1:Spc105 ratios for BubR1 dsRNA-treated cells relative to the mean BubR1:Spc105 ratio of control cells as a measure of the BubR1 RNAi penetrance (control; n = 20 cells from two replicates, BubR1 RNAi; n = 21 cells from two replicates). (N) T-COFFEE alignment of human (Hs) Bub1 and D. melanogaster (Dm) BubR1 aa sequences identifying a motif in Drosophila BubR1 with homology to a region in human Bub1 implicated in RZZ recruitment to kinetochores. Boxed regions are highlighted in the insets. Bar plots report mean ± SD. Scale bars, 10 μm; 1 μm (C and insets).

Characterization of Spc105-1-400-EGFP oligomerization, Bub3 exclusion from Spc105 clusters, and depletions of DHC, Rod, and BubR1. (A) Representative maximum projection of confocal Z-sections of a mitotic cell expressing Spc105-1-400-EGFP. (B and C) Spinning-disc confocal time-lapse of cluster assembly in a cell with high expression of Spc105-1-400-EGFP (C) Zoomed view of the boxed region (yellow dashed) in B showing a cluster fusion event. (D) Representative confocal Z-planes of control and DHC RNAi cells stained for CID (magenta) and DHC (green). (E) Quantification of DHC ratioed to CID fluorescence (n = 50 kinetochores from 10 cells for each condition). (F) Cell averages (five kinetochores quantified per cell) of DHC to CID ratios for DHC dsRNA-treated cells relative to the mean DHC:CID ratio of control cells as a measure of the DHC RNAi penetrance. (G) Representative confocal Z-planes of control and Rod RNAi cells stained for Spc105 (magenta) and Rod (green). (H) Quantification of Rod ratioed to Spc105 fluorescence (n = 50 kinetochores from 10 cells for each condition). (I) Cell averages (five kinetochores quantified per cell) of Rod to Spc105 ratios for Rod dsRNA-treated cells relative to the mean Rod:Spc105 ratio of control cells as a measure of the Rod RNAi penetrance. (J) Representative confocal Z-plane of a cell coexpressing Bub3-EGFP (green) and oligomerized clusters of Spc105-1-1722-mCherry-Cry2 (magenta). The boxed region is highlighted in the insets. The average fold enrichment for the cell (from five clusters) is indicated in the image. (K) Quantification of the fold enrichment of the signal of Bub3-EGFP in cluster regions relative to the local background signal (n = 40 clusters from eight cells). (L) Representative maximum projections of confocal Z-sections of control and BubR1 dsRNA-treated fixed cells stained for Spc105 (green), BubR1 (red), and tubulin (blue). (M) Cell averages of BubR1:Spc105 ratios for BubR1 dsRNA-treated cells relative to the mean BubR1:Spc105 ratio of control cells as a measure of the BubR1 RNAi penetrance (control; n = 20 cells from two replicates, BubR1 RNAi; n = 21 cells from two replicates). (N) T-COFFEE alignment of human (Hs) Bub1 and D. melanogaster (Dm) BubR1 aa sequences identifying a motif in Drosophila BubR1 with homology to a region in human Bub1 implicated in RZZ recruitment to kinetochores. Boxed regions are highlighted in the insets. Bar plots report mean ± SD. Scale bars, 10 μm; 1 μm (C and insets).

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