Ectopic expression of Cdc5 causes spindle assembly in cdc34-1 cells. (a) cdc34-1 cells carrying GAL–CDC5 (US5276) or GAL–cdc5^KD (US5308) were synchronized in G1 by α-factor treatment in YEP+Raff and released into YEP+Raff+Gal medium. The scale bar represents 2 μm. Upper Panel: Spc42–GFP signal and FACS profile of cells at 300 min. Numbers represent percentage of cells with distinctly visible Spc42–GFP dots representing two well-separated SPBs. (b) cdc34-1 MET–CDC5 cells carrying GAL–CIN8–cmyc₃ (US5311) were grown in Raff+Met medium containing α-factor for 2 hrs at 24 °C and then transferred to Raff+Gal+Met medium containing α-factor for 90 min to induce Cin8 expression. Cells were then released at 37 °C into methionine-deficient glucose medium containing cycloheximide. The fate of Cin8 pulse was determined by western blotting.

Phosphorylation of Cdh1 by Cdc28 and Cdc5. (a) Cdc28–Clb2–HA₃ and Cdc5–HA₃ immunoprecipitated from nocodazole-treated wild-type (WT) cells (US1165, US3259) were used in kinase reactions with GST or GST–Cdh1 as substrates. For the two-step kinase reaction (lanes 6 and 7; see Methods), ‘1’ denotes the first kinase used and ‘2’ denotes the second kinase used after removal of the first kinase. To demonstrate substrate specificities, Cdc28–Clb2 and Cdc5 kinase activities were assayed using histone H1 and casein as substrates. (b) Phosphorylation of GST–Cdh1, GST–Cdh1–m4A and GST–Cdh1–mS_ps4A by immunoprecipitated Cdc28–Clb2–HA₃ and Cdc5–HA₃. Designations ‘1’ and ‘2’ in lanes 3, 6 and 9 represent sequential exposure to the two kinases, as described in a. (c) Schematic representation of putative Cdc5 phosphorylation sites in Cdh1. (d) The blot shows sequential phosphorylation of GST–Cdh1, GST–Cdh1–S125A, GST–Cdh1–S259A and GST–Cdh1–S125A S259A by Cdc28–Clb2–HA₃ and Cdc5–HA₃ kinases.

Functional redundancy between Cdc5 and APC^Cdh1-inhibitor Acm1 in SPB separation and involvement of Cdc5 in nuclear export of Cdh1. (a) acm1Δ, cdc5Δ and cdc5Δ acm1Δ cells carrying GAL–CDC5 (US5300, US5301, US5302) with endogenously tagged CIN8–cmyc₃ were synchronized in G1 in YEP+Raff+Gal and then released into YEP+Glu medium at 24 °C. Samples were collected for Spc42–GFP visualization, electron microscopy and western blotting. The scale bar represents 2 μm. Numbers indicate percentage of cells with two Spc42–GFP dots at 80 min for acm1Δ and cdc5Δ cells, and at 160 min for cdc5Δ acm1Δ cells (as doubling time in these cells is twice as long). EM micrograph shows the state of SPBs at 240 min. Scale bar for EM image, 0.2 μm. Insets show a magnified view of Spc45–GFP dots. (b) Stationary phase cells of cdc5Δ acm1Δ and cdc5Δ acm1Δ cdh1Δ strains harbouring GAL–CDC5 (US5302, US5376) were obtained by growth on YEP+Raff+Gal medium at 24 °C for three days and were subsequently inoculated into YEP+Glu at 24 °C. Cells at 240 min are shown. (c) G1-synchronized cdc28-as1 GAL–CDC5 (US5373) and msn5Δ cdc28-as1 GAL–CDC5 (US5374) cells were released into YEP+Raff+Gal containing 1NM-PP1 for 300 min. Samples were collected for Cdh1–GFP visualization and immunofluorescence staining. Scale bars represent 2 μm (a, b, c). Numbers represent percentage of cells with nuclear Cdh1–GFP and spindles at 300 min. Full-length blots for section a are presented in Supplementary Information, Fig. S5–2.

Ectopic expression of Cdc5 causes hyperphosphorylation of Cdh1 and SPB separation. (a) cdc28-as1 cells carrying GAL–CDC5 (US5255) or GAL–cdc5^KD (kinase-dead; US5360) were synchronized by α-factor treatment in YEP+Raff and released into YEP+Raff+Gal containing 1NM-PP1 at 24 °C for 300 min. Numbers indicate percentage of cells with two Spc42–GFP dots at 300 min. The scale bar represents 2 μm. (b) Top panel: G1-synchronized cdc28-as1 cells carrying GAL–cmyc₃–CDC5 (US5233) were released into YEP+Raff+Gal containing 1NM-PP1 for 6 h and cells were collected at the indicated times. Western blots show Cin8–HA₃ and Kip1–cmyc₃ levels, and immunoprecipitated HA₃–Cdh1 (with and without alkaline phosphatase (CIP)). cdc28-as1 cells carrying GAL–cmyc₃–cdc5^KD (US5371) were treated as described for the top panel. Bottom panel: cmyc₃–Cdc5 kinase was immunoprecipitated from cdc28-as1 cells carrying either GAL–cmyc₃–CDC5 or GAL–myc₃–cdc5^KD and was used in an in vitro assay to determine kinase activity using casein as a substrate. (c) cdc28-as1 MET–CDC5 cells carrying GAL–CIN8–cmyc₃ (US5310) were grown in Raff+Met medium containing α-factor for 2 h at 24 °C and then transferred to Raff+Gal+Met medium containing α-factor for 90 min to induce Cin8 expression. Cells were then released at 24 °C into methionine-deficient glucose medium containing 1NM-PP1 and cycloheximide (1 mg ml⁻¹) to inhibit protein synthesis). The fate of Cin8 pulse was determined by western blotting. (d) G1-synchronized cdc28-as1, cdc28-as1 expressing non-degradable version of Cin8 (cin8^nd) from endogenous promoter, cdc28-as1 GAL–CDC5 and cdc28-as1 GAL–cdc5^KD (US5312, US5314, US5315, US5341) were released into medium containing 1NM-PP1 and the proteasome inhibitor MG132. All except cin8^nd-carrying strains harboured endogenously-tagged CIN8–HA₃. Ubiquitin conjugates of immunoprecipitated Cin8 were separated on a 6% SDS gel and detected by anti-ubiquitin antibodies. The blot on the right shows a shorter exposure. (e) G1-synchronized cdc28-as1 GAL–CDC5 cells carrying endogenously-tagged cmyc₃–CDC27 and HA₃–CDH1 (US5355) in raffinose medium were released into either glucose or galactose medium containing 1NM-PP1. Immunoprecipitates were analysed by western blotting. Full-length blots for b, c and e are presented in Supplementary Information, Fig. S5–1.

Phosphorylation of Cdh1 by Cdc5 requires priming by Cdc28. (a) Schematic representation of the four Cdk phosphorylation sites constituting the PBB sites in Cdh1. Residues potentially phosphorylated by Cdc28 are underlined and designated 1, 2, 3 and 4. Ser residues (indicated in red) immediately preceding Cdc28 phosphorylation site are highly conserved in PBB sites. (b) G1-synchronized wild-type cells endogenously tagged with CIN8–cmyc₃ carrying either GALL–CDH1, GALL–cdh1–m11A or GALL–cdh1–m4A (US4622, US4623, US4624) in YEP+Raff were released into YEP+Raff+Gal for 300 min and collected for Spc42–GFP visualization, immunoblotting and electron microscopy. The scale bar represents 2 μm (immunofluorescence) and 0.3 μm (electron microscopy). (c) Wild-type cells harbouring GAL–cmyc₃–CDC5 cells and carrying either GALL–HA₃–CDH1, GALL–HA₃–cdh1–m11A, GALL–HA₃–CDH1–m4A or GALL–HA₃–cdh1–mS_ps4A (US4625, US4626, US4627, US4628) were first synchronized in G1 by α-factor treatment in raffinose medium and then released into either glucose or galactose medium for 180 min. In parallel, G1-synchronized cdc28-as1 cells carrying GALL–HA₃–CDH1 and GAL–cmyc₃–CDC5 (US5353) were released into either glucose or galactose medium with or without 1NM-PP1 for 240 min. Immunoprecipitates were analysed by western blotting. (d) G1-synchronized cdc28-as1 GAL–cmyc₃–CDC5 cells with or without GALL–HA₃–cdh1–m4A (US5354, US5354) in YEP+Raff were released into YEP+Raff+Gal medium. Numbers indicate percentage of cells with two well-separated Spc42–GFP dots at 240 min. The scale bar represents 2 μm. Full-length blots for b and c are shown in Supplementary Information, Fig. S5–1.

Absence of Cdc5 delays assembly of short spindle. (a) Stationary-phase cells of cdc28–1N, cdc28–1N cdc5Δ GAL–CDC5 and cdc28–1N cdc5Δ GAL–CDC5 cdh1Δ strains (US4362, US5259, US5364) were obtained by growth on YEP+Raff+Gal medium at 24 °C for 3 days and were subsequently transferred to YEP+Glu medium at 37 °C. Spc42–GFP signals and FACS in cells at 300 min are shown. (b) cdc28–1N carrying either GALL–CDH1 or GALL–cdh1–S125A S259A (US5369, US5372) were synchronized in G1 by α-factor treatment in Raffinose medium and then released into Raff+Gal medium at 37 °C. Samples were collected to analyse the pattern of Spc42–GFP fluorescence and for western blotting. Insets in a and b show a magnified view of Spc42–GFP dots. (c) Spindle length distribution in wild-type and cdc5Δ cells expressing Spc42–GFP (US3786, US5282) was determined by live-cell imaging (3 examples are shown from a total of 16 cells imaged documented in Supplementary Information, Fig. S3). The distance between the Spc42–GFP dots is plotted versus time. The vertical dotted lines represent the time at which the spindle first attains a stable length of 1.5 μm before a phase of rapid increase sets in. The bottom panels show the distribution of time taken to reach a spindle length of 1.5 μm in various cells. (d) Wild-type and cdc5Δ GAL–CDC5 cells carrying HA₃–Cdh1 and Cin8–cmyc₃ (US5322, US4970) were synchronized in G1 by α-factor treatment in YEP+Raff+Gal medium and then released into YEP+Glu medium. Western blots and tubulin immunofluorescence staining of samples collected at various times are shown. Scale bars represent 2 μm (a, b, d). The spindle length distribution is shown in Supplementary Information, Fig. S4a. Full-length blots for b and d are documented in Supplementary Information, Fig. S5–2.

Cdc5 is unstable in 1NM-PP1 treated cdc28-as1 cells. (a) cdc28-as1 cells (US3723) were synchronized in YEP+Raff medium with α-factor treatment and galactose was added to induce Cdc5 for 30 min. Subsequently, cells were released into cycloheximide-containing YEP+Glu medium, with or without 1NM-PP1, and the fate of Cdc5 pulse was determined by western blotting. The abundance of Cdc5 was quantified using a densitometer. (b) A scheme for the regulation of SPB separation involving Cdc28, Cdc5, Cdh1, Acm1 and microtubule binding proteins Cin8 and Kip1. Low level accumulation of Cin8 and Kip1 is sufficient to sever the inter-SPB bridge. Further separation of SPBs for the assembly of a characteristic short spindle requires greater accumulation of these kinesin motors, mediated by Acm1 and Cdc5 polo kinase. Full-length blots are presented in Supplementary Information, Fig. S5–2.