(A–D, I, L) Wild type (A40512) and cdc15-as1 (A40524) cells containing an mCherry-TUB1 and KAR9-GFP fusion were treated with 10 μM 1-Na-PP1 for the indicated time period at 25°C on agar pads. (E–H, J, M) Wild type (A40530) and temperature sensitive cdc15-2 (A40529) cells containing the mCherry-TUB1 and KAR9-GFP fusions were heat shocked for the indicated period of time at 37°C on agar pads. (A, E) Cells were imaged and metaphase cells were identified by spindle morphology. Astral microtubule associated Kar9 intensity was determined for both SPBs in metaphase. The two SPBs in each metaphase cell were placed into one of two categories: SPB with high levels of Kar9 bound to emanating microtubules (strong) or the pole with low levels of Kar9 bound to emanating microtubules (weak). Mean and stdev are shown (n > 90 cells). (B, F) Quantifications from (A) and (E) were used to create a weak SPB/strong SPB localization ratio in (B) and (F), respectively. Mean, stdev, and results of t-test are shown. (C, G) Qualitative classification of Kar9 localization for cells quantified in (A) and (E). (D, H) Sample images of Kar9 localization categories quantified in (C) and (G). (I, J) Cells were imaged every 10 s for 100 s. Metaphase cells were identified by spindle morphology and Kar9 localization was quantified using the classifiers described in (H); n > 45 cells. (K–M) Cells were imaged every 5 m for 4 hr. G1 cells were followed until anaphase and Kar9 intensity was measured on astral microtubules 10 min prior to anaphase onset. Mother and daughter bound SPBs were identified during anaphase. Kar9 intensity and Kar9 symmetry index [(dSPB-mSPB)/(dSPB +mSPB)] was determined. Mean, stdev, and results of t-test are shown (n > 60 cells). (N, O) Wild type (A40559), cdc15-as1 (A40556), or kar9Δ (A40562) cells containing SPC42-mCherry and GFP-TUB1 were treated with 10 μM 1-Na-PP1 for 45 min at 25°C on agar pads. Cells were then imaged every 15 m for 3 hr. SPB inheritance was determined by the brightness of the Spc42-mCherry signal entering the bud (N); Mean and stdev of three experiments are shown, t-test. Sample images of Spc42-mCherry signal are shown in (O).