(A–E) KAR1 or kar1∆15 cells co-expressing Spc72-Venus (green) and Spc98-mT2 (magenta) were synchronized in metaphase using PMET3-CDC20, then released into the cell cycle. (A) Representative SIM images showing the distribution of Spc72-Venus (green) and Spc98-mT2 (magenta). Merged images with cell outlines (scale bar, 2 µm) and cropped images of SPBs by channel or merged (scale bar, 200 nm) are shown: (a) unduplicated, (b, f) side-by-side (c–e and g–i), separated SPBs. (B) Cell cycle progression in terms of the spindle pathway. (C–D) The localization of Spc72-Venus and Spc98-mT2 was quantitated at the indicated times in cells with side-by-side SPBs (C; scale bar, 200 nm) or separated SPBs (D; scale bars: white, 200 nm and black, 1 µm) Total number of cells analyzed by time point was 70, 85, 107, 123, 70, 114, 150, 198 and 125 (KAR1); 96, 160, 103, 70, 128, 139, 320, 205 and 224 (kar1∆15). (E) Boxplots for distribution of spindle length or Spc72 asymmetry index (left) and scattered plot for Spc72 asymmetry index as a function of spindle length at the indicated time points were generated by 3-D SIM analysis. ****, p<0.0001 according to Mann Whitney two-tailed test.