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A novel function of Saccharomyces cerevisiae CDC5 in cytokinesis.

Song S, Lee KS - J. Cell Biol. (2001)

Bottom Line: Overexpression of the COOH-terminal domain of Cdc5 (cdc5DeltaN), but not the corresponding polo-box mutant, resulted in connected cells.The polo-box-dependent interactions between Cdc5 and septins (Cdc11 and Cdc12) and genetic interactions between the dominant-negative cdc5DeltaN and Cyk2/Hof1 or Myo1 suggest that direct interactions between cdc5DeltaN and septins resulted in inhibition of Cyk2/Hof1- and Myo1-mediated cytokinetic pathways.Thus, we propose that Cdc5 may coordinate mitotic exit with cytokinesis by participating in both anaphase promoting complex activation and a polo-box-dependent cytokinetic pathway.

View Article: PubMed Central - PubMed

Affiliation: Laboratory of Metabolism, Division of Basic Sciences, National Cancer Institute, National Institutes of Health, Bethesda, Maryland 20892, USA.

ABSTRACT
Coordination of mitotic exit with timely initiation of cytokinesis is critical to ensure completion of mitotic events before cell division. The Saccharomyces cerevisiae polo kinase Cdc5 functions in a pathway leading to the degradation of mitotic cyclin Clb2, thereby permitting mitotic exit. Here we provide evidence that Cdc5 also plays a role in regulating cytokinesis and that an intact polo-box, a conserved motif in the noncatalytic COOH-terminal domain of Cdc5, is required for this event. Depletion of Cdc5 function leads to an arrest in cytokinesis. Overexpression of the COOH-terminal domain of Cdc5 (cdc5DeltaN), but not the corresponding polo-box mutant, resulted in connected cells. These cells shared cytoplasms with incomplete septa, and possessed aberrant septin ring structures. Provision of additional copies of endogenous CDC5 remedied this phenotype, suggesting a dominant-negative inhibition of cytokinesis. The polo-box-dependent interactions between Cdc5 and septins (Cdc11 and Cdc12) and genetic interactions between the dominant-negative cdc5DeltaN and Cyk2/Hof1 or Myo1 suggest that direct interactions between cdc5DeltaN and septins resulted in inhibition of Cyk2/Hof1- and Myo1-mediated cytokinetic pathways. Thus, we propose that Cdc5 may coordinate mitotic exit with cytokinesis by participating in both anaphase promoting complex activation and a polo-box-dependent cytokinetic pathway.

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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.
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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.

Mentions: Although it has been shown that polo kinases play important roles in multiple stages of M phase in various eucaryotic organisms, it has not been clear whether budding yeast polo kinase Cdc5 has a role other than in the mitotic exit pathway. To address this issue, we investigated the terminal morphology of a cdc5Δ mutant. Since overexpression of Cdc5 inhibits cell growth (Kitada et al. 1993) and induces abnormal bud elongation (Song et al. 2000), cdc5Δ strains were generated that were kept viable by expressing either its murine functional homologue Plk (KLY1046) or the less-stable cdc5-1–encoded protein (Cheng et al. 1998) (KLY1047) under control of the GAL1 promoter. To reveal arrest phenotypes relating to dynamic spindle structures in mitosis, these strains possess an integrated copy of a GFP-fused TUB1 (Tub1-GFP) under control of its own promoter. Provision of a single copy of endogenous CDC5 into these strains complemented the growth defect associated with the cdc5Δ mutation (data not shown). Both strains grow well on YEP-galactose medium, but are unable to grow on YEP-glucose (Fig. 1 A). When exponentially growing cells were transferred to YEP-glucose medium, Plk was undetectable after 10 h, whereas the cdc5-1 protein disappeared after 3–4 h (Fig. 1 B). The fast removal of cdc5-1 protein, in comparison with Plk, may be due to the presence of two putative destruction boxes in the NH2-terminal sequence of Cdc5 (Shirayama et al. 1998). Consistent with the protein removal kinetics, large-budded cells become abundant ∼6 h after depletion of Plk or 3 h after depletion of cdc5-1. As with cells with separated chromatids, cells with disassembled spindles increased and reached plateau in 8 h for KLY1046 or 4 h for KLY1047 (Fig. 1 B). To determine the percentage of cells arrested at various phases of the cell cycle, both strains were depleted of the Plk or cdc5-1 proteins for 8 or 4 h, respectively, and counted using spindle structures as a cell-cycle marker. We observed that 65% of strain KLY1046 arrested at early anaphase with a short spindle elongated to, or extended just beyond, the mother-bud neck, whereas 25% of the population were arrested at cytokinesis with disassembled spindles (Fig. 1 C). In the case of strain KLY1047, 53% of the cells were arrested in cytokinesis, whereas 3 and 44% of the cell population were arrested at early anaphase and late anaphase/telophase, respectively (Fig. 1 C). The complete absence of unbudded (G1) cells suggests that cells were arrested in terminal phenotypes under these conditions. In contrast, consistent with a late mitosis arrest as reported previously (Kitada et al. 1993; Toczyski et al. 1997), the cdc5-1 mutant arrested homogeneously with elongated spindles (93% of the population) when cultured at 35°C for 3.5 h (Fig. 1 C). Since the contraction of cytokinetic rings occurs at the time of spindle breakdown (Lippincott and Li 1998a), a large population of cdc5Δ cells with disassembled spindles in both strain KLY1046 and strain KLY1047 suggests that Cdc5 activity is required at a step in cytokinesis. In addition, our data suggest that Cdc5 function is required at multiple points of M phase, including cytokinesis.


A novel function of Saccharomyces cerevisiae CDC5 in cytokinesis.

Song S, Lee KS - J. Cell Biol. (2001)

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.
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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.
Mentions: Although it has been shown that polo kinases play important roles in multiple stages of M phase in various eucaryotic organisms, it has not been clear whether budding yeast polo kinase Cdc5 has a role other than in the mitotic exit pathway. To address this issue, we investigated the terminal morphology of a cdc5Δ mutant. Since overexpression of Cdc5 inhibits cell growth (Kitada et al. 1993) and induces abnormal bud elongation (Song et al. 2000), cdc5Δ strains were generated that were kept viable by expressing either its murine functional homologue Plk (KLY1046) or the less-stable cdc5-1–encoded protein (Cheng et al. 1998) (KLY1047) under control of the GAL1 promoter. To reveal arrest phenotypes relating to dynamic spindle structures in mitosis, these strains possess an integrated copy of a GFP-fused TUB1 (Tub1-GFP) under control of its own promoter. Provision of a single copy of endogenous CDC5 into these strains complemented the growth defect associated with the cdc5Δ mutation (data not shown). Both strains grow well on YEP-galactose medium, but are unable to grow on YEP-glucose (Fig. 1 A). When exponentially growing cells were transferred to YEP-glucose medium, Plk was undetectable after 10 h, whereas the cdc5-1 protein disappeared after 3–4 h (Fig. 1 B). The fast removal of cdc5-1 protein, in comparison with Plk, may be due to the presence of two putative destruction boxes in the NH2-terminal sequence of Cdc5 (Shirayama et al. 1998). Consistent with the protein removal kinetics, large-budded cells become abundant ∼6 h after depletion of Plk or 3 h after depletion of cdc5-1. As with cells with separated chromatids, cells with disassembled spindles increased and reached plateau in 8 h for KLY1046 or 4 h for KLY1047 (Fig. 1 B). To determine the percentage of cells arrested at various phases of the cell cycle, both strains were depleted of the Plk or cdc5-1 proteins for 8 or 4 h, respectively, and counted using spindle structures as a cell-cycle marker. We observed that 65% of strain KLY1046 arrested at early anaphase with a short spindle elongated to, or extended just beyond, the mother-bud neck, whereas 25% of the population were arrested at cytokinesis with disassembled spindles (Fig. 1 C). In the case of strain KLY1047, 53% of the cells were arrested in cytokinesis, whereas 3 and 44% of the cell population were arrested at early anaphase and late anaphase/telophase, respectively (Fig. 1 C). The complete absence of unbudded (G1) cells suggests that cells were arrested in terminal phenotypes under these conditions. In contrast, consistent with a late mitosis arrest as reported previously (Kitada et al. 1993; Toczyski et al. 1997), the cdc5-1 mutant arrested homogeneously with elongated spindles (93% of the population) when cultured at 35°C for 3.5 h (Fig. 1 C). Since the contraction of cytokinetic rings occurs at the time of spindle breakdown (Lippincott and Li 1998a), a large population of cdc5Δ cells with disassembled spindles in both strain KLY1046 and strain KLY1047 suggests that Cdc5 activity is required at a step in cytokinesis. In addition, our data suggest that Cdc5 function is required at multiple points of M phase, including cytokinesis.

Bottom Line: Overexpression of the COOH-terminal domain of Cdc5 (cdc5DeltaN), but not the corresponding polo-box mutant, resulted in connected cells.The polo-box-dependent interactions between Cdc5 and septins (Cdc11 and Cdc12) and genetic interactions between the dominant-negative cdc5DeltaN and Cyk2/Hof1 or Myo1 suggest that direct interactions between cdc5DeltaN and septins resulted in inhibition of Cyk2/Hof1- and Myo1-mediated cytokinetic pathways.Thus, we propose that Cdc5 may coordinate mitotic exit with cytokinesis by participating in both anaphase promoting complex activation and a polo-box-dependent cytokinetic pathway.

View Article: PubMed Central - PubMed

Affiliation: Laboratory of Metabolism, Division of Basic Sciences, National Cancer Institute, National Institutes of Health, Bethesda, Maryland 20892, USA.

ABSTRACT
Coordination of mitotic exit with timely initiation of cytokinesis is critical to ensure completion of mitotic events before cell division. The Saccharomyces cerevisiae polo kinase Cdc5 functions in a pathway leading to the degradation of mitotic cyclin Clb2, thereby permitting mitotic exit. Here we provide evidence that Cdc5 also plays a role in regulating cytokinesis and that an intact polo-box, a conserved motif in the noncatalytic COOH-terminal domain of Cdc5, is required for this event. Depletion of Cdc5 function leads to an arrest in cytokinesis. Overexpression of the COOH-terminal domain of Cdc5 (cdc5DeltaN), but not the corresponding polo-box mutant, resulted in connected cells. These cells shared cytoplasms with incomplete septa, and possessed aberrant septin ring structures. Provision of additional copies of endogenous CDC5 remedied this phenotype, suggesting a dominant-negative inhibition of cytokinesis. The polo-box-dependent interactions between Cdc5 and septins (Cdc11 and Cdc12) and genetic interactions between the dominant-negative cdc5DeltaN and Cyk2/Hof1 or Myo1 suggest that direct interactions between cdc5DeltaN and septins resulted in inhibition of Cyk2/Hof1- and Myo1-mediated cytokinetic pathways. Thus, we propose that Cdc5 may coordinate mitotic exit with cytokinesis by participating in both anaphase promoting complex activation and a polo-box-dependent cytokinetic pathway.

Show MeSH
Related in: MedlinePlus