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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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Genetic interactions between a dominant-negative cdc5ΔN mutant and cyk2Δ/myo1ΔΔ. (A) Cyk2 and Myo1 rings are largely normal in the connected cells. Often, the presence of enlarged Myo1 rings (arrows) and contraction size rings (barbed arrows) were also present. Bar: 5 μm. (B) Actin polarizes normally, but fails to relocalize to bud-necks. Strain KLY1083 cultured under the induction conditions for 12 h were fixed and stained with rhodamine-phalloidin and DAPI to visualize actin and nuclei, respectively. Bar: 5 μm. (C) Overexpression of EGFP-cdc5ΔN in cyk2Δ or myo1Δ mutants, but not in cdc10Δ or swe1Δ mutants, results in a synthetic cytokinetic defect. Strain KLY1083 carrying an additional cdc10Δ (KLY1589 and KLY1590) cyk2Δ (KLY1591), myo1Δ (KLY1593), or swe1Δ (KLY1439) mutation was generated. These strains were streaked onto YEP-glucose and YEP-galactose to examine synthetic growth defect with overexpression of EGFP-cdc5ΔN. Since both cdc10Δ and cyk2Δ mutants possess an apparent temperature sensitivity for growth, plates were incubated at 23°C for 4 d before photography. Two independently generated cdc10Δ mutants (KLY1589 and KLY1590) were used to confirm the absence of a synthetic defect with overexpression of EGFP-cdc5ΔN. (D) Introduction of swe1Δ or overexpression of GAL-SIC1 do not influence the chained-cell phenotype induced by overexpression of cdc5ΔN. Typical cell morphologies (DIC images) were shown after culturing strain KLY1083, strain KLY1439, and SKY1779 under the induction conditions for 10 h. The chained-cell phenotypes from these strains were undistinguishable. cdc5ΔN, strain KLY1083 cells; cdc5ΔN swe1Δ, strain KLY1439 cells; cdc5ΔN GAL-SIC1, strain SKY1779 cells. Bar: 5 μm. (E) Introduction of swe1Δ abolishes the cell-cycle delay induced by overexpression of cdc5ΔN. To carry out cell-cycle analyses, cells expressing control EGFP-cdc5ΔN/FAA (KLY1229), EGFP-cdc5ΔN (KLY1083), or EGFP-cdc5ΔN, swe1Δ (KLY1439) were arrested with 5 μg of α-factor in YEP-raffinose for 3 h, washed, and transferred into YEP-galactose medium. Samples were taken at the indicated time points and subjected to Western analyses with an anti–Clb2 antibody. Deletion of swe1 abolishes a septin-checkpoint–dependent cell-cycle delay, but did not influence the chained-cell phenotype induced by overexpression of cdc5ΔN (see text for details). Arrows indicate the time point with maximum Clb2 levels, whereas arrowheads indicate the time point with the lowest level of Clb2. The level of Clb2 and Cdc28 proteins in the indicated lanes were determined using ImageQuant. The numbers are the relative levels (folds) of Clb2 in comparison to the lowest level (arrowheads). *Cross-reacting protein with the anti–Clb2 antibody. cdc5ΔN/FAA, strain KLY1229; cdc5ΔN, strain KLY1083; cdc5ΔN swe1Δ, strain KLY1439.
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Figure 7: Genetic interactions between a dominant-negative cdc5ΔN mutant and cyk2Δ/myo1ΔΔ. (A) Cyk2 and Myo1 rings are largely normal in the connected cells. Often, the presence of enlarged Myo1 rings (arrows) and contraction size rings (barbed arrows) were also present. Bar: 5 μm. (B) Actin polarizes normally, but fails to relocalize to bud-necks. Strain KLY1083 cultured under the induction conditions for 12 h were fixed and stained with rhodamine-phalloidin and DAPI to visualize actin and nuclei, respectively. Bar: 5 μm. (C) Overexpression of EGFP-cdc5ΔN in cyk2Δ or myo1Δ mutants, but not in cdc10Δ or swe1Δ mutants, results in a synthetic cytokinetic defect. Strain KLY1083 carrying an additional cdc10Δ (KLY1589 and KLY1590) cyk2Δ (KLY1591), myo1Δ (KLY1593), or swe1Δ (KLY1439) mutation was generated. These strains were streaked onto YEP-glucose and YEP-galactose to examine synthetic growth defect with overexpression of EGFP-cdc5ΔN. Since both cdc10Δ and cyk2Δ mutants possess an apparent temperature sensitivity for growth, plates were incubated at 23°C for 4 d before photography. Two independently generated cdc10Δ mutants (KLY1589 and KLY1590) were used to confirm the absence of a synthetic defect with overexpression of EGFP-cdc5ΔN. (D) Introduction of swe1Δ or overexpression of GAL-SIC1 do not influence the chained-cell phenotype induced by overexpression of cdc5ΔN. Typical cell morphologies (DIC images) were shown after culturing strain KLY1083, strain KLY1439, and SKY1779 under the induction conditions for 10 h. The chained-cell phenotypes from these strains were undistinguishable. cdc5ΔN, strain KLY1083 cells; cdc5ΔN swe1Δ, strain KLY1439 cells; cdc5ΔN GAL-SIC1, strain SKY1779 cells. Bar: 5 μm. (E) Introduction of swe1Δ abolishes the cell-cycle delay induced by overexpression of cdc5ΔN. To carry out cell-cycle analyses, cells expressing control EGFP-cdc5ΔN/FAA (KLY1229), EGFP-cdc5ΔN (KLY1083), or EGFP-cdc5ΔN, swe1Δ (KLY1439) were arrested with 5 μg of α-factor in YEP-raffinose for 3 h, washed, and transferred into YEP-galactose medium. Samples were taken at the indicated time points and subjected to Western analyses with an anti–Clb2 antibody. Deletion of swe1 abolishes a septin-checkpoint–dependent cell-cycle delay, but did not influence the chained-cell phenotype induced by overexpression of cdc5ΔN (see text for details). Arrows indicate the time point with maximum Clb2 levels, whereas arrowheads indicate the time point with the lowest level of Clb2. The level of Clb2 and Cdc28 proteins in the indicated lanes were determined using ImageQuant. The numbers are the relative levels (folds) of Clb2 in comparison to the lowest level (arrowheads). *Cross-reacting protein with the anti–Clb2 antibody. cdc5ΔN/FAA, strain KLY1229; cdc5ΔN, strain KLY1083; cdc5ΔN swe1Δ, strain KLY1439.

Mentions: Inhibition of cytokinesis in connected cells could be the result of a failure either in recruitment of cytokinetic materials or in the function of cytokinetic structures. To investigate these possibilities, strain KLY1053 was additionally integrated with CDC3-YFP (KLY1075), CYK2-GFP (KLY1071), or MYO1-GFP (KLY1073) under control of their own promoters. These strains grew normally with apparently normal ring structures when cultured in YEP-glucose (data not shown). Upon induction of GST-cdc5ΔN, cells were then examined to determine whether these components formed normal ring structures. (Fig. 6 B and 7 A). At an early stage of induction, distinct septin ring structures were observed at all the mother-bud necks of connected cells, indicating that continuous budding events have occurred in the absence of cytokinesis at the previous mother-bud necks. Upon inducing for 12 h, double YFP-Cdc3 rings were found to be distantly placed at the mother-bud neck or remnants of YFP-Cdc3 were placed at one side of the mother-bud neck, suggesting a disturbance in maintaining properly organized septin ring structures or a defect in septin ring disassembly and relocalization. In addition, unusually wide or tiny rings of Cdc3 were occasionally observed at the axial position of incipient budding site without formation of a noticeable bud (Fig. 6 B), suggesting that a fraction of cell bodies were capable of relocalizing septins to the future budding sites but failed to assemble or maintain proper septin ring structures. Taken together, in the presence of dominant-negative cdc5ΔN, septin rings appear to be assembled normally at the growing edge of the connected cells, thereby permitting continuous budding events. However, these ring structures are cytokinesis incompetent, resulting in the generation of connected cell morphology. As with septin ring structures persisting at most of the mother-bud necks of connected cells, both Cyk2-GFP and Myo1-GFP ring structures were also remained at these sites (Fig. 7 A). This observation further supports the notion that cytokinetic processes have failed in these cells.


A novel function of Saccharomyces cerevisiae CDC5 in cytokinesis.

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

Genetic interactions between a dominant-negative cdc5ΔN mutant and cyk2Δ/myo1ΔΔ. (A) Cyk2 and Myo1 rings are largely normal in the connected cells. Often, the presence of enlarged Myo1 rings (arrows) and contraction size rings (barbed arrows) were also present. Bar: 5 μm. (B) Actin polarizes normally, but fails to relocalize to bud-necks. Strain KLY1083 cultured under the induction conditions for 12 h were fixed and stained with rhodamine-phalloidin and DAPI to visualize actin and nuclei, respectively. Bar: 5 μm. (C) Overexpression of EGFP-cdc5ΔN in cyk2Δ or myo1Δ mutants, but not in cdc10Δ or swe1Δ mutants, results in a synthetic cytokinetic defect. Strain KLY1083 carrying an additional cdc10Δ (KLY1589 and KLY1590) cyk2Δ (KLY1591), myo1Δ (KLY1593), or swe1Δ (KLY1439) mutation was generated. These strains were streaked onto YEP-glucose and YEP-galactose to examine synthetic growth defect with overexpression of EGFP-cdc5ΔN. Since both cdc10Δ and cyk2Δ mutants possess an apparent temperature sensitivity for growth, plates were incubated at 23°C for 4 d before photography. Two independently generated cdc10Δ mutants (KLY1589 and KLY1590) were used to confirm the absence of a synthetic defect with overexpression of EGFP-cdc5ΔN. (D) Introduction of swe1Δ or overexpression of GAL-SIC1 do not influence the chained-cell phenotype induced by overexpression of cdc5ΔN. Typical cell morphologies (DIC images) were shown after culturing strain KLY1083, strain KLY1439, and SKY1779 under the induction conditions for 10 h. The chained-cell phenotypes from these strains were undistinguishable. cdc5ΔN, strain KLY1083 cells; cdc5ΔN swe1Δ, strain KLY1439 cells; cdc5ΔN GAL-SIC1, strain SKY1779 cells. Bar: 5 μm. (E) Introduction of swe1Δ abolishes the cell-cycle delay induced by overexpression of cdc5ΔN. To carry out cell-cycle analyses, cells expressing control EGFP-cdc5ΔN/FAA (KLY1229), EGFP-cdc5ΔN (KLY1083), or EGFP-cdc5ΔN, swe1Δ (KLY1439) were arrested with 5 μg of α-factor in YEP-raffinose for 3 h, washed, and transferred into YEP-galactose medium. Samples were taken at the indicated time points and subjected to Western analyses with an anti–Clb2 antibody. Deletion of swe1 abolishes a septin-checkpoint–dependent cell-cycle delay, but did not influence the chained-cell phenotype induced by overexpression of cdc5ΔN (see text for details). Arrows indicate the time point with maximum Clb2 levels, whereas arrowheads indicate the time point with the lowest level of Clb2. The level of Clb2 and Cdc28 proteins in the indicated lanes were determined using ImageQuant. The numbers are the relative levels (folds) of Clb2 in comparison to the lowest level (arrowheads). *Cross-reacting protein with the anti–Clb2 antibody. cdc5ΔN/FAA, strain KLY1229; cdc5ΔN, strain KLY1083; cdc5ΔN swe1Δ, strain KLY1439.
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Figure 7: Genetic interactions between a dominant-negative cdc5ΔN mutant and cyk2Δ/myo1ΔΔ. (A) Cyk2 and Myo1 rings are largely normal in the connected cells. Often, the presence of enlarged Myo1 rings (arrows) and contraction size rings (barbed arrows) were also present. Bar: 5 μm. (B) Actin polarizes normally, but fails to relocalize to bud-necks. Strain KLY1083 cultured under the induction conditions for 12 h were fixed and stained with rhodamine-phalloidin and DAPI to visualize actin and nuclei, respectively. Bar: 5 μm. (C) Overexpression of EGFP-cdc5ΔN in cyk2Δ or myo1Δ mutants, but not in cdc10Δ or swe1Δ mutants, results in a synthetic cytokinetic defect. Strain KLY1083 carrying an additional cdc10Δ (KLY1589 and KLY1590) cyk2Δ (KLY1591), myo1Δ (KLY1593), or swe1Δ (KLY1439) mutation was generated. These strains were streaked onto YEP-glucose and YEP-galactose to examine synthetic growth defect with overexpression of EGFP-cdc5ΔN. Since both cdc10Δ and cyk2Δ mutants possess an apparent temperature sensitivity for growth, plates were incubated at 23°C for 4 d before photography. Two independently generated cdc10Δ mutants (KLY1589 and KLY1590) were used to confirm the absence of a synthetic defect with overexpression of EGFP-cdc5ΔN. (D) Introduction of swe1Δ or overexpression of GAL-SIC1 do not influence the chained-cell phenotype induced by overexpression of cdc5ΔN. Typical cell morphologies (DIC images) were shown after culturing strain KLY1083, strain KLY1439, and SKY1779 under the induction conditions for 10 h. The chained-cell phenotypes from these strains were undistinguishable. cdc5ΔN, strain KLY1083 cells; cdc5ΔN swe1Δ, strain KLY1439 cells; cdc5ΔN GAL-SIC1, strain SKY1779 cells. Bar: 5 μm. (E) Introduction of swe1Δ abolishes the cell-cycle delay induced by overexpression of cdc5ΔN. To carry out cell-cycle analyses, cells expressing control EGFP-cdc5ΔN/FAA (KLY1229), EGFP-cdc5ΔN (KLY1083), or EGFP-cdc5ΔN, swe1Δ (KLY1439) were arrested with 5 μg of α-factor in YEP-raffinose for 3 h, washed, and transferred into YEP-galactose medium. Samples were taken at the indicated time points and subjected to Western analyses with an anti–Clb2 antibody. Deletion of swe1 abolishes a septin-checkpoint–dependent cell-cycle delay, but did not influence the chained-cell phenotype induced by overexpression of cdc5ΔN (see text for details). Arrows indicate the time point with maximum Clb2 levels, whereas arrowheads indicate the time point with the lowest level of Clb2. The level of Clb2 and Cdc28 proteins in the indicated lanes were determined using ImageQuant. The numbers are the relative levels (folds) of Clb2 in comparison to the lowest level (arrowheads). *Cross-reacting protein with the anti–Clb2 antibody. cdc5ΔN/FAA, strain KLY1229; cdc5ΔN, strain KLY1083; cdc5ΔN swe1Δ, strain KLY1439.
Mentions: Inhibition of cytokinesis in connected cells could be the result of a failure either in recruitment of cytokinetic materials or in the function of cytokinetic structures. To investigate these possibilities, strain KLY1053 was additionally integrated with CDC3-YFP (KLY1075), CYK2-GFP (KLY1071), or MYO1-GFP (KLY1073) under control of their own promoters. These strains grew normally with apparently normal ring structures when cultured in YEP-glucose (data not shown). Upon induction of GST-cdc5ΔN, cells were then examined to determine whether these components formed normal ring structures. (Fig. 6 B and 7 A). At an early stage of induction, distinct septin ring structures were observed at all the mother-bud necks of connected cells, indicating that continuous budding events have occurred in the absence of cytokinesis at the previous mother-bud necks. Upon inducing for 12 h, double YFP-Cdc3 rings were found to be distantly placed at the mother-bud neck or remnants of YFP-Cdc3 were placed at one side of the mother-bud neck, suggesting a disturbance in maintaining properly organized septin ring structures or a defect in septin ring disassembly and relocalization. In addition, unusually wide or tiny rings of Cdc3 were occasionally observed at the axial position of incipient budding site without formation of a noticeable bud (Fig. 6 B), suggesting that a fraction of cell bodies were capable of relocalizing septins to the future budding sites but failed to assemble or maintain proper septin ring structures. Taken together, in the presence of dominant-negative cdc5ΔN, septin rings appear to be assembled normally at the growing edge of the connected cells, thereby permitting continuous budding events. However, these ring structures are cytokinesis incompetent, resulting in the generation of connected cell morphology. As with septin ring structures persisting at most of the mother-bud necks of connected cells, both Cyk2-GFP and Myo1-GFP ring structures were also remained at these sites (Fig. 7 A). This observation further supports the notion that cytokinetic processes have failed in these cells.

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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Related in: MedlinePlus