Anaphase spindle localization of phosphorylation mutants of Cin8–3GFP. (A) a, Cin8; b, Cin8-2A, c, Cin8-3A, d, Cin8-3D. Time interval, 1 minute. (B) a, Cin8; b, Cin8-3A; time interval, 20 seconds. Scale bar: 1 μm. Arrows indicate spindle poles; arrowheads indicate the midzone.

Cin8 is differentially phosphorylated during anaphase. Band-retardation assay in whole extracts of cells producing 6Myc–Cin8. Extracts were fractionated on SDS-PAGE and examined by Western blot. Arrowheads indicate the slow-migrating band of Cin8. (A) Cells expressing cdc15-2, cdc14-1 or cdc5-7 and containing 6Myc–Cin8 were grown at 26°C (permissive) and 37°C (restrictive). (B) Wild-type cells were either cycling or arrested at metaphase (by nocodazole), S-phase (by hydroxyurea) and G1 (by α-factor). (C) WT cells or cdc15-2 cells were grown at 37°C for 4 hours. The extracts were either treated with CIP phosphatase (pp) (+) or not (−). (D) cdc15-2 cells expressing either WT Cin8, Cin8-5A (S277, T285, S493, S736, S1010), Cin8-3A (S277, T285, S493) or Cin8-2A (S736, S1010) (indicated on top) were grown at 26°C or 37°C (indicated at the bottom). (E) In vitro phosphorylation of bacterially expressed Cin8-590 by Cdk1 using equal concentrations of the Cdk1 complexes with cyclin Clb2, Clb3 or Clb5 (indicated on top). Coomassie Brilliant Blue (CBB) staining and ³²P autoradiograms of SDS-PAGE fractionation of phosphorylation reaction mixtures are shown (indicated on the left). Proteins corresponding to the various bands are indicated on the right, based on their predicted size. The relative specificity pattern obtained using equal concentrations of the kinase complexes with a reference substrate Histone H1 is shown in the bottom panel (H1). (F) Effect of CDC55 deletion on band-retardation assay of Cin8. Genotypes of cells and variants of Cin8–13Myc are indicated at the top; the various modes of arrest are indicated at the bottom. (G) Effect of Cdc14 overexpression on the slow-migrating band of Cin8–13Myc. 3HA–Cdc14 was produced from a galactose-inducible promoter. Cells were arrested either in S-phase or at late anaphase (indicated on top). In anaphase-arrested cells, Cdc14 was induced by addition of galactose for the times indicated above. The western blots of Cin8–13Myc (top) and 3HA–Cdc14 (bottom) are shown. (H) 6Myc–Cin8 was expressed in WT or spo12Δ cells (indicated on top). Cells were arrested in S-phase and released to fresh medium for 70 minutes (bottom).

Anaphase spindle elongation and morphology in cells that express phosphorylation mutants of Cin8. (A) Representative spindle-elongation kinetics in cells producing WT Cin8 (left), Cin8-3A (middle) and Cin8-3D (right). V, rate of spindle elongation (μm/minute). The phases of spindle elongation are indicated as follows: diamonds, fast phase; squares, slow phase; circles, post-anaphase. (B) Long anaphase spindle morphologies in cells expressing tubulin–GFP and Cin8 (top), Cin8-3A (middle) or Cin8-3D (bottom). Scale bar: 1 μm. Arrowheads indicate small overlapping region of the iMTs.