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. 2001 Feb 5;152(3):451–470. doi: 10.1083/jcb.152.3.451

Figure 1.

Figure 1

Figure 1

Figure 1

Figure 1

Cells depleted of Plk or cdc5-1 protein arrest at multiple points of M phase. (A) Growth of cdc5Δ mutant conditionally rescued by expressing either GAL1-HA-EGFP-PLK (KLY1046) or GAL1-HA-EGFP-cdc5-1 (KLY1047). Strains were streaked onto either YEP-galactose or YEP-glucose, and incubated for 3 d at 30°C. As a comparison, an isogenic wild-type strain, 1783, was also streaked. Wild-type, 1783 strain; GAL1-PLK, strain KLY1046;GAL1-cdc5-1, strain KLY1047. (B) Depletion of Plk or cdc5-1 protein revealed a large fraction of large-budded cells with disassembled spindles. Strains KLY1046 and KLY1047 growing exponentially in YEP-galactose medium were transferred into YEP-glucose to deplete Plk and cdc5-1 proteins. Upon transfer, samples were taken to analyze the levels of HA-EGFP-Plk and HA-EGFP-cdc5-1 proteins using an anti–HA antibody. Due to an apparent cell lysis phenotype after a prolonged incubation at the restricted temperature, cells were not taken beyond the last indicated time point. At the indicated time points, cells were harvested to determine chromosomal structures with DAPI staining. The same samples were used to examine spindle structures by GFP-tubulin fluorescent signals. Strain KLY1047, but not KLY1046, has accumulated a significant number of cells with elongated spindles (see text). (C) Terminal phenotypes of cdc5Δ or cdc5-1 cells. To determine terminal arresting phenotypes associated with depletion of Plk or cdc5-1 protein, strain KLY1046 was depleted of Plk for 10 h, whereas KLY1047 cells were depleted of cdc5-1 protein for 4 h. Cells arrested at different phases of the cell cycle were scored based on the spindle morphologies. Numbers shown are the average of two independent experiments. (D) Terminal morphology of the cdc5-1 mutant expressing TUB1-GFP (KLY1253), YFP-CDC3 (KLY 1260), CYK2-GFP (KLY1256), or MYO1-GFP (KLY1258). The cdc5-1 cells growing exponentially at 23°C were shifted to 35°C and cultured for an additional 3.5 h. Cells were fixed with 3.7% formaldehyde and harvested to examine the terminal arrest phenotype. The cdc5-1 mutant arrests as large-budded cells with two Cyk2-GFP rings and elongated spindles. Arrows in Tub1 indicate weakly visible elongated spindles, whereas the barbed arrows in Myo1 indicate Myo1-GFP localized at the future budding site. Bar: 5 μm.