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. 2006 May 11;25(11):2551–2563. doi: 10.1038/sj.emboj.7601136

Figure 3.

Figure 3

Cin8-bundling activity, not its motor activity or phosphorylation, is required for SPB separation. (A) G1-synchronized cdc28-as1 expressing nondegradable versions of Cin8, Kip1 and Ase1, from their respective native promoter-driven constructs on a CEN vector, were released in the presence of 1NM-PP1. Cells at 240 min after release are shown. The Western blot (rightmost panel) shows stability of the nondegradable versions in G1. (B) clb3Δ clb4Δ clb5Δ GAL-CLB5 cells carrying MET3-CIN8-HA3 were synchronized in G1 in Raff+Gal+Methionine medium and released into methionine-deficient glucose medium. (C) G1-synchronised cdc28Y19E carrying SPC42-GFP at the TRP1 locus and expressing from CEN vector GAL-CIN8-cmyc3, GAL-CIN8 (R196K)-cmyc3 (affecting motor activity), GAL-CIN8 (F467A)-cmyc3 (affecting microtubule binding) or GAL-CIN8 (R394A, H396A, E871A)-cmyc3 (affecting bundling) were released into YEP+Raff+Gal at 37°C. Numbers indicate percentage of cells with two SPC42-GFP dots at 240 min. Western blots show the extent of Cin8 expression. (D) (Top panel) Kinetics of spindle formation in cdc28Y19E expressing GAL-CIN8-cmyc3, GAL-KIP1-cmyc3 or GAL-ASE1-cmyc3 on a CEN plasmid or versions carrying mutations at Cdc28 consensus sites mimicking phosphorylated (E) or unphosphorylated forms (A). (Lower panel) Spindle kinetics in cin8-3 kip1Δ and cin8Δ ase1Δ cells expressing phospho-deficient versions at the Cdc28 consensus sites (A).