Figure 6. More Bfa1 localizes to daughter spindle pole body (dSPB) in the absence of BUD14.

(A) Bfa1-GFP signal intensity quantifications at the SPBs of BFA1-GFP mCherry-TUB1 or BFA1-GFP mCherry-TUB1 bud14∆ cells with normal aligned anaphase spindles were plotted on the right. Black and gray lines in the dotplots are the mean value and standard deviation, respectively. Representative still images of cells are shown on the left. (B) Bfa1-GFP signal intensities at the dSPB were plotted in BUD14 and bud14∆ cells bearing empty plasmid (blue and red), as well as bud14∆ cells bearing a ADH-BUD14-containing plasmid (green). (C) Bfa1-GFP signal intensities at the dSPB were plotted in bud14∆ cells bearing empty plasmid (red), ADH1-BUD14-containing plasmid (green) and ADH1-bud14-F379A-containing plasmid (yellow) (D) Analysis of Bfa1-GFP asymmetry at the SPBs of kar9∆, kar9∆ kin4∆, and kar9∆ bud14∆ cells with correctly aligned and misaligned anaphase spindles. Box and Whisker plot shows the ratio of Bfa1-GFP signal intensities at the SPB1 and SPB2, where SPB1 always corresponds to SPB with the greater Bfa1-GFP signal. The box represents first and third quartile while the whiskers show 10–90 percentile of the data. The horizontal line in the box indicates the median of the data. Only comparisons of normal and misaligned spindles are shown in the figure. Representative still images of cells are shown on the right. n: sample size. Scale bar: 2 µm. One-way ANOVA with uncorrected Fisher’s LSD was applied for all statistical analyses. **p<0.01, ***p<0.001, ****p<0.0001. All pairwise comparisons are shown in the corresponding source data files.

Figure 6—figure supplement 1. Bfa1, Bub2, and Tem1 localization in bud14∆ cells during anaphase and metaphase.

(A, B) Bub2-GFP and Tem1-GFP representative images (A) and signal intensity quantifications at the spindle pole bodies (SPBs) (B) of BFA1-GFP mCherry-TUB1 or BFA1-GFP mCherry-TUB1 bud14∆ cells with normal aligned anaphase spindles. Black and gray lines in the dotplots are the mean value and standard deviation, respectively. Representative still images of cells are shown in (A). (C) Bfa1-GFP, Bub2-GFP, and Tem1-GFP signal intensities at the SPBs of cells with metaphase spindles (spindle length = 1.5–2 µm). Graph is plotted as described in (B). (D) Analysis of Bfa1-GFP signal intensities at the SPBs of kar9∆, kar9∆ kin4∆, and kar9∆ bud14∆ cells with correctly aligned and misaligned anaphase spindles. The graph was plotted as in (B). Pairwise comparisons were performed using one-way ANOVA with uncorrected Fisher’s LSD. ***p<0.001, ****p<0.0001, n: sample size. Scale bar: 2 µm.

Figure 6—figure supplement 2. Analysis of Mob1 spindle pole body (SPB) localization.

(A) Percentage of metaphase cells (spindle length = 1.5–2 µm) with Mob1-GFP signal at the SPBs of the indicated cells. Graphs are average of three independent experiments. A minimum of 100 cells were counted from each strain in each experiment. Error bars show standard deviation. (B) Representative still images showing Mob1-GFP localization in metaphase cells. (C, D) Dotplot of Mob1-GFP signal intensity at the SPBs of indicated strains carrying MOB1-GFP mCherry-TUB1 in metaphase (C) and anaphase (D). Black lines represent the mean value, and gray lines show standard deviation. (E) Representative still images of anaphase cells. Scale bar: 2 µm. One-way ANOVA with uncorrected Fisher’s LSD was used for pairwise comparisons. ****p<0.0001.

Figure 6—figure supplement 3. Bfa1-GFP localization during spindle misalignment.

Selected frames from time-lapse series of BFA1-GFP SPC42-eqFP mCherry-TUB1-bearing kar9∆ (A), kar9∆ bud14∆ (B), and kar9∆ kin4∆ (C) gene deletions. Cell boundaries are outlined with a dashed line. Blue arrows indicate the spindle pole bodies (SPBs). Time points from the beginning of the time lapse are indicated on the frames. Scale bar: 2 µm.