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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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Time-dependent induction of connected cells by overexpression of cdc5ΔN. (A) Strain KLY1083 expressing three copies of GAL1-EGFP-cdc5ΔN homogeneously induced a chained cell phenotype. Typical cell morphologies (DIC images) were shown after culturing these cells under the induction conditions for 7 h (left), 12 h (middle), or 24 h (right). These cells continue to increase cell body numbers without an apparent cell division. Bar: 5 μm. (B) Strain KLY1229 expressing three copies of the corresponding FAA triple mutant failed to induce this phenotype even after 24 h induction. Bar: 5 μm. (C) Quantification of connected cells as a function of time. Percentage of cells in chains was determined by dividing the sum of cells in chains by the total number of cells. cdc5ΔN, strain KLY1083; cdc5ΔN/FAA, strain KLY1229. (D) Strain KLY1083 grows without increasing cell numbers under the induction conditions. Cells expressing control EGFP (KLY1080), EGFP-cdc5ΔN (KLY1083), or EGFP-cdc5ΔN/FAA (KLY1229) were taken at the indicated time points upon transferring cultures into YEP-galactose. Cell number was determined by plating serial dilutions on YEP-glucose and counting the colony numbers. The number of cells at time 0 was 1.2 × 106 cells/ml with an OD600 of 0.05. The resulting cell number and OD600 at each time point were divided by those at time 0 to give relative cell number and OD600. (E) The FAA mutations do not influence the stability of EGFP-cdc5ΔN. EGFP-cdc5ΔN was detected by an anti–GFP antibody, whereas Cdc28 (loading control) was recognized by an anti–Cdc28 antibody. 1X, one copy of cdc5ΔN; 3X, three copies of cdc5ΔN.
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Figure 3: Time-dependent induction of connected cells by overexpression of cdc5ΔN. (A) Strain KLY1083 expressing three copies of GAL1-EGFP-cdc5ΔN homogeneously induced a chained cell phenotype. Typical cell morphologies (DIC images) were shown after culturing these cells under the induction conditions for 7 h (left), 12 h (middle), or 24 h (right). These cells continue to increase cell body numbers without an apparent cell division. Bar: 5 μm. (B) Strain KLY1229 expressing three copies of the corresponding FAA triple mutant failed to induce this phenotype even after 24 h induction. Bar: 5 μm. (C) Quantification of connected cells as a function of time. Percentage of cells in chains was determined by dividing the sum of cells in chains by the total number of cells. cdc5ΔN, strain KLY1083; cdc5ΔN/FAA, strain KLY1229. (D) Strain KLY1083 grows without increasing cell numbers under the induction conditions. Cells expressing control EGFP (KLY1080), EGFP-cdc5ΔN (KLY1083), or EGFP-cdc5ΔN/FAA (KLY1229) were taken at the indicated time points upon transferring cultures into YEP-galactose. Cell number was determined by plating serial dilutions on YEP-glucose and counting the colony numbers. The number of cells at time 0 was 1.2 × 106 cells/ml with an OD600 of 0.05. The resulting cell number and OD600 at each time point were divided by those at time 0 to give relative cell number and OD600. (E) The FAA mutations do not influence the stability of EGFP-cdc5ΔN. EGFP-cdc5ΔN was detected by an anti–GFP antibody, whereas Cdc28 (loading control) was recognized by an anti–Cdc28 antibody. 1X, one copy of cdc5ΔN; 3X, three copies of cdc5ΔN.

Mentions: To examine the phenotype associated with the overexpression of EGFP-cdc5ΔN, strain KLY1083 was cultured under induction conditions in YEP-galactose. Control strains expressing either EGFP alone (KLY1080) or an equal dosage of EGFP-cdc5ΔN/FAA (KLY1229) were grown under the same conditions. When induced, KLY 1083 cells yielded a connected cell phenotype in a time-dependent manner, whereas cells expressing the corresponding FAA mutant appear to divide normally with wild-type morphology (Fig. 3A and Fig. B). Upon inducing for 9 h, 98% of the population exhibited this phenotype, and 30% of them possess >10 connected cell bodies. In contrast, cells expressing the FAA mutant did not show this morphology (Fig. 3 C). In addition, the cell number of strain KLY1083 (when counting the connected cells as one cell) did not increase after shifting to the induction conditions (Fig. 3 D), indicating that cells remained unseparated. However, these cells continued to grow, although slowly, as indicated by an increase in optical density of the cell culture. Under the same conditions, cells expressing either control EGFP (KLY1080) or EGFP-cdc5ΔN/FAA (KLY1229) exhibited a normal increase in both cell number and optical density (Fig. 3 D). Western analyses revealed that both EGFP-cdc5ΔN and EGFP-cdc5ΔN/FAA were expressed at similar levels, and that they were slightly more abundant than that of a single copy EGFP-cdc5ΔN integrant (Fig. 3 E).


A novel function of Saccharomyces cerevisiae CDC5 in cytokinesis.

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

Time-dependent induction of connected cells by overexpression of cdc5ΔN. (A) Strain KLY1083 expressing three copies of GAL1-EGFP-cdc5ΔN homogeneously induced a chained cell phenotype. Typical cell morphologies (DIC images) were shown after culturing these cells under the induction conditions for 7 h (left), 12 h (middle), or 24 h (right). These cells continue to increase cell body numbers without an apparent cell division. Bar: 5 μm. (B) Strain KLY1229 expressing three copies of the corresponding FAA triple mutant failed to induce this phenotype even after 24 h induction. Bar: 5 μm. (C) Quantification of connected cells as a function of time. Percentage of cells in chains was determined by dividing the sum of cells in chains by the total number of cells. cdc5ΔN, strain KLY1083; cdc5ΔN/FAA, strain KLY1229. (D) Strain KLY1083 grows without increasing cell numbers under the induction conditions. Cells expressing control EGFP (KLY1080), EGFP-cdc5ΔN (KLY1083), or EGFP-cdc5ΔN/FAA (KLY1229) were taken at the indicated time points upon transferring cultures into YEP-galactose. Cell number was determined by plating serial dilutions on YEP-glucose and counting the colony numbers. The number of cells at time 0 was 1.2 × 106 cells/ml with an OD600 of 0.05. The resulting cell number and OD600 at each time point were divided by those at time 0 to give relative cell number and OD600. (E) The FAA mutations do not influence the stability of EGFP-cdc5ΔN. EGFP-cdc5ΔN was detected by an anti–GFP antibody, whereas Cdc28 (loading control) was recognized by an anti–Cdc28 antibody. 1X, one copy of cdc5ΔN; 3X, three copies of cdc5ΔN.
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Related In: Results  -  Collection

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Figure 3: Time-dependent induction of connected cells by overexpression of cdc5ΔN. (A) Strain KLY1083 expressing three copies of GAL1-EGFP-cdc5ΔN homogeneously induced a chained cell phenotype. Typical cell morphologies (DIC images) were shown after culturing these cells under the induction conditions for 7 h (left), 12 h (middle), or 24 h (right). These cells continue to increase cell body numbers without an apparent cell division. Bar: 5 μm. (B) Strain KLY1229 expressing three copies of the corresponding FAA triple mutant failed to induce this phenotype even after 24 h induction. Bar: 5 μm. (C) Quantification of connected cells as a function of time. Percentage of cells in chains was determined by dividing the sum of cells in chains by the total number of cells. cdc5ΔN, strain KLY1083; cdc5ΔN/FAA, strain KLY1229. (D) Strain KLY1083 grows without increasing cell numbers under the induction conditions. Cells expressing control EGFP (KLY1080), EGFP-cdc5ΔN (KLY1083), or EGFP-cdc5ΔN/FAA (KLY1229) were taken at the indicated time points upon transferring cultures into YEP-galactose. Cell number was determined by plating serial dilutions on YEP-glucose and counting the colony numbers. The number of cells at time 0 was 1.2 × 106 cells/ml with an OD600 of 0.05. The resulting cell number and OD600 at each time point were divided by those at time 0 to give relative cell number and OD600. (E) The FAA mutations do not influence the stability of EGFP-cdc5ΔN. EGFP-cdc5ΔN was detected by an anti–GFP antibody, whereas Cdc28 (loading control) was recognized by an anti–Cdc28 antibody. 1X, one copy of cdc5ΔN; 3X, three copies of cdc5ΔN.
Mentions: To examine the phenotype associated with the overexpression of EGFP-cdc5ΔN, strain KLY1083 was cultured under induction conditions in YEP-galactose. Control strains expressing either EGFP alone (KLY1080) or an equal dosage of EGFP-cdc5ΔN/FAA (KLY1229) were grown under the same conditions. When induced, KLY 1083 cells yielded a connected cell phenotype in a time-dependent manner, whereas cells expressing the corresponding FAA mutant appear to divide normally with wild-type morphology (Fig. 3A and Fig. B). Upon inducing for 9 h, 98% of the population exhibited this phenotype, and 30% of them possess >10 connected cell bodies. In contrast, cells expressing the FAA mutant did not show this morphology (Fig. 3 C). In addition, the cell number of strain KLY1083 (when counting the connected cells as one cell) did not increase after shifting to the induction conditions (Fig. 3 D), indicating that cells remained unseparated. However, these cells continued to grow, although slowly, as indicated by an increase in optical density of the cell culture. Under the same conditions, cells expressing either control EGFP (KLY1080) or EGFP-cdc5ΔN/FAA (KLY1229) exhibited a normal increase in both cell number and optical density (Fig. 3 D). Western analyses revealed that both EGFP-cdc5ΔN and EGFP-cdc5ΔN/FAA were expressed at similar levels, and that they were slightly more abundant than that of a single copy EGFP-cdc5ΔN integrant (Fig. 3 E).

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