mih1Δ cells have an increased cell size and undergo delayed entry into mitosis. (A) Cells of the indicated genotypes were grown to log phase in YPD media at room temperature. Cell size was measured using a Z2 Coulter counter. (B) Wild-type and mih1Δ cells were synchronized with α-factor and released into fresh YPD media and samples were taken at 10-min intervals. Cells were fixed and stained with an anti-tubulin antibody and the percentage of cells with short spindles was determined. Over 200 cells were analyzed for each time point. The experiment was repeated at least three times with matched controls performed in parallel on the same day, and the same overall trend was observed. Error bars are not displayed because the timing of release from α-factor arrest varies from day to day. (C) Wild-type and mih1Δ cells were synchronized with α-factor and released into fresh YPD media and samples were taken at 10-min intervals. Cells were fixed and the percentage of cells with small buds was determined. Over 200 cells were analyzed for each time point. The experiment was repeated at least three times with matched controls performed in parallel on the same day, and the same overall trend was observed. Error bars are not displayed because the timing of release from α-factor arrest varies from day to day. (D) Wild-type and mih1Δ cells carrying CLB5-3XHA cells were synchronized with α-factor and released into fresh YPD media and samples were taken at 10-min intervals. Western blotting was used to monitor the behavior of Clb5-3XHA. (E) Wild-type cells were synchronized with α-factor and released into fresh YPD media and samples were taken at 10-min intervals. Western blotting was used to monitor the behavior of Swe1, Clb2, and Cdk1 tyrosine 19 phosphorylation. The exposures of the Western blots were normalized using background bands to allow direct comparison of the signals from each strain. The Swe1 protein migrates as a disperse group of phosphorylated forms above a 97 kD size standard, whereas Clb2 and Cdk1 migrate approximately at 50 and 35 kD, respectively.

PP2A^Cdc55 is required for dephosphorylation of Mih1. (A) PPH22 pph3Δ pph21Δ and pph22-12 pph3Δ pph21Δ cells were grown to log phase in YPD media at room temperature. After taking a sample (0-min time point), the cells were shifted to the restrictive temperature and samples were collected after 1 h. The phosphorylation state of Mih1 was followed by Western blotting. (B) Western blot analysis of Mih1 phosphorylation in log-phase populations of wild-type, cdc55Δ, sit4Δ, and rts1Δ cells. As a control, mih1Δ cells were included in the analysis. The phosphorylation state of Mih1 was monitored by Western blotting. (C) Wild-type and cdc55Δ cells were synchronized with α-factor and released into fresh YPD media and samples were taken at 10-min intervals. Western blotting was used to monitor the behavior of Mih1. (D) Immunoaffinity-purified phosphorylated 3XHA-Mih1 was treated with immunoaffinity purified Cdc55-3XHA. For controls, purified 3XHA-Mih1 was also incubated with λ phosphatase or a control elution from a nonspecific immunoaffinity column. The phosphorylation state of Mih1 was monitored by Western blotting. (E) Cells of the indicated genotypes were grown to log phase at room temperature. After taking a 0-min time point, the cells were shifted to the restrictive temperature. Samples were collected at 1, 2, and 3 h after shifting to 34°C. Western blotting was used to monitor the behavior of Mih1. The apparent upward shift in mobility of Mih1 in glc7-12 cells at 3 h was not reproducible and may have been caused by nutrient limitation after 3 h in culture. (F) Wild-type and cdc55Δ cells were synchronized with α-factor and released into fresh YPD media and samples were taken at 10-min intervals. Western blotting was used to monitor the behavior of Clb2 and Cdk1 tyrosine 19 phosphorylation. The exposures of the Western blots were normalized using background bands to allow direct comparison of the signals from each strain.

Yck1 and 2 are required for Mih1 hyperphosphorylation. (A) yck2-2 yck1Δ and yck2-2 yck1Δ swe1Δ cells were grown to log phase at room temperature in YPD media and shifted to 34°C for 6 h. (B) YCK1 YCK2 and yck2-2 yck1Δ cells were synchronized with α-factor and released into fresh YPD media at 30°C and samples were taken at 10-min intervals. Western blotting was used to monitor the behavior of Mih1, Swe1, Cdk1 tyrosine 19 phosphorylation, and Clb2. The exposures of the Western blots were normalized using background bands to allow direct comparison of the signals from each strain. (C) Isogenic YCK1 YCK2, yck2-2 yck1Δ, and yck2-2 yck1Δ swe1Δ cells were synchronized with α-factor and released into fresh YPD media at 30°C and samples were taken at 10-min intervals. Cells were fixed and stained with an anti-tubulin antibody and the percentage of cells with short spindles was determined. Over 200 cells were analyzed for each time point. The experiment was repeated at least three times with matched controls performed in parallel on the same day, and the same overall trend was observed. Error bars are not displayed because the timing of release from α-factor arrest varies from day to day.

Mih1 undergoes dramatic changes in phosphorylation state during the cell cycle. (A) Wild-type cells were synchronized with α-factor and released into fresh YPD media and samples were taken at 10-min intervals. Western blotting was used to monitor the behavior of Mih1, Swe1, and Clb2. The differently phosphorylated forms of Mih1 migrate between 57 and 97 kD size standards. (B) Phosphorylated 3XHA-Mih1 was immunoaffinity purified and treated with λ phosphatase. The phosphorylation state of Mih1 was monitored by Western blotting.

Mih1 phosphorylation is dependent on the Yck1 and 2 kinases. (A) Western blot analysis of Mih1 phosphorylation in log-phase populations of wild-type, mck1Δ, sky1Δ, ste20Δ, and cla4Δ cells. (B) Purified dephosphorylated 3XHA-Mih1 was incubated with purified TAP-tagged kinases at 30°C for 45 min in the presence of ATP. For each kinase, a control reaction was performed in which no phosphatase was added to ensure that the purified kinase did not introduce background bands to the anti-Mih1 Western blot. The phosphorylation state of Mih1 was monitored by Western blotting. (C) YCK1 YCK2, YCK1 yck2Δ, and yck2-2 yck1Δ cells were grown to log phase in YPD media. After taking a sample, the cells were shifted to 34°C and samples were collected at 1 and 2 h. The phosphorylation state of Mih1 was followed by Western blotting. (D) Purified 3XHA-Mih1 was incubated with the indicated combinations of purified Cdk1-Clb2-3XHA, Yck1-TAP, and dephosphorylated Mih1 at 30°C for 1 h in the presence of ATP. The phosphorylation state of Mih1 was monitored by Western blotting.

Cdk1 regulates Mih1 phosphorylation and dephosphorylation. (A) Log-phase cdk1-as cells were synchronized in G1 with α-factor and released into fresh YPD lacking supplemental adenine. At the indicated times, the cells were split into two aliquots, 1NM-PP1 was added to one aliquot, and samples were collected at 10-min intervals. For the control, an equivalent amount of DMSO was added. The phosphorylation state of Mih1 was monitored by Western blotting. (B) cdk1-as cells were grown to log phase at room temperature and synchronized in G1 with α-factor. The cells were then released into fresh YPD lacking supplemental adenine. At 80 min, the culture was divided in half. 1NM-PP1 was added to one half, and samples were collected at 0, 2.5, 5, and 10 min after the addition of 1NM-PP1. For the control, an equivalent amount of DMSO was added. The phosphorylation state of Mih1 was monitored by Western blotting. (C) Wild-type cells were grown to log phase at room temperature and synchronized in G1 with α-factor. The cells were then released into fresh YPD lacking supplemental adenine. At 80 min, the culture was divided in half. 1NM-PP1 was added to one half, and samples were collected at 0, 2.5, 5, and 10 min after the addition of 1NM-PP1. For the control, an equivalent amount of DMSO was added. The phosphorylation state of Mih1 was monitored by Western blotting.