Figure 3.

SPB localization of Cdk1 depends on Cdc15 kinase activity. (A and B) CDC15 and cdc15-as1 cells were synchronized with α-factor and released at 30°C into YPAD medium with the PP1 analogue 8 to inhibit activity of cdc15-as1 kinase (D’Aquino et al., 2005). Anaphase cells with a 4–10-µm spindle were examined for the Cdk1-GFP localization. n = 38 for CDC15, n = 43 for cdc15-as1. The arrows in A point toward the Cdk1-GFP signal at mSPBs. (C) Localization of Cdc15-GFP and cdc15-as1-GFP in anaphase. Two representative cells are shown for each strain grown in YPAD at 30°C in the presence of PP1 analogue 8. The first Cdc15-GFP and cdc15-as1-GFP pictures were taken under identical conditions. The second pictures in this row are linear enhancements of the first pictures. Bar, 5 µm. (D) Protein levels of Cdc15-GFP and cdc15-as1-GFP detected with anti-GFP antibody. Anti-Tub2 was used as loading control. (E) The Dbf2–Mob1 kinase complex is not required for SPB association of Cdk1 in anaphase. Synchronized wild-type, mob1-67, and dbf2-2 cells were grown in YPAD at 37°C after the release of the α-factor block. Anaphase cells were examined for SPB localization of Cdk1-GFP. (F) Quantification of E. n > 75 anaphase cells per strain. Bars, 5 µm.