SPB1 indicates the SPB closer to bud where SPB2 is the SPB closer to the mother cell compartment. Yellow or red arrows point the two SPBs in a cell with a normally aligned or misaligned spindle respectively. The numbers 1 and 2 next to the arrows indicate SPB1 and SPB2 respectively. Scale bar: 3 µm. In the Box-Whisker plots showing Bfa1-GFP mean fluorescence intensities, the boxes show the lower and upper quartiles, the whiskers show the minimum and maximal values excluding outliers; circles represent the outliers calculated as values greater or lower than 1.5 times the interquartile range; the line inside the box indicates the median. Asterisks show significant difference according to student’s t-test (p<0.001). Sample sizes for kar9∆ cells were 49 and 23, whereas sample sizes for kar9∆ SPC72-GBP cells were 38 and 14 for each SPB with normal and misaligned spindles respectively. See the accompanying data file for exact p-values (Figure 7—source data 1). (B) SPOC integrity of the indicated cell types. Cells were grown at 23°C and shifted to 30°C for 3 hr to induce the accumulation of cells with misaligned spindles (blue bars). Black bars show the percentage of multi-nucleated phenotypes, which indicates SPOC deficiency N: 100 cells per strain. A representative plot out of three biological replicates is shown. (C) Immunoblot showing Bfa1-GFP mobility shift. Indicated strains arrested in G1 using alpha-factor and released in alpha-factor free, nocodazole containing YPDA medium. Samples were collected after 2.5 hr. Gal1-CDC5 bearing strains were grown and G1-arrested in raffinose and galactose containing medium. Releasing from G1 block in glucose containing medium repressed the Gal1 promoter to maintain Cdc5 depletion. Bfa1-GFP was detected in the total cell extracts of the indicated strains using anti-GFP antibody. Anti-TAT1 antibody was used to detect tubulin as a loading control. Asterisks indicate the hyper-phosphorylated form of Bfa1. A representative blot out of three independent experiments is shown. (D) Model for SPOC activation and Bfa1-SPB remodeling. When the mitotic spindle is correctly aligned, Bfa1 molecules are stably in contact with Nud1 and Spc72, where Cdc5 can phosphorylate and thereby inactivate Bfa1. When the spindle misaligns, Kin4 binds to Spc72 to phosphorylate Bfa1 (Maekawa et al., 2007). Kin4 phosphorylated Bfa1 is recognized by Bmh1 (Caydasi et al., 2014). Bmh1-bound Bfa1 disconnects from Spc72 but remains associated with Nud1, although dynamically. Cdc5 cannot phosphorylate Bfa1 when Bfa1 is disconnected from Spc72.