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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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Cdc28/Clb2 activity cycles during the early stages of connected cells. (A) Overexpression of EGFP-cdc5ΔN, but not the corresponding FAA mutant, accumulates cells with a DNA content >2C (G2/M). To carry out flow cytometric analyses, cells expressing control EGFP (KLY1080), EGFP-cdc5ΔN (KLY1083), or EGFP-cdc5ΔN/FAA (KLY1229) were arrested with α-factor in YEP-raffinose for 3 h, washed, and transferred into YEP-galactose medium to an OD600 of 0.05. Samples were taken at the indicated time points, fixed, and subjected to flow cytometry analyses. Strain KLY1083 begins to accumulate cells with a DNA content >2C (120 min) as the first cycle completes, whereas both strains KLY1080 and KLY1299 complete the cell cycle normally. Accumulation of cells with higher DNA contents appears to be the result of induction of connected cells (see text for details). Control, strain KLY1080; cdc5ΔN, strain KLY1083; cdc5ΔN/FAA, strain KLY1229; 1C, 1C DNA content; 2C, 2C DNA content. (B) Cdc28/Clb2 activity appears to fluctuate normally in strain KLY1083 overexpressing EGFP-cdc5ΔN. To examine the cell cycle progression, cellular lysates prepared from the same cells harvested in A were subjected to Western blot analyses to examine changes in Clb2 and EGFP-cdc5ΔN levels upon release from a α-factor arrest. The level of Cdc28 was determined as a loading control for each lane. The same lysates were also used to carry out anti–Clb2 immune complex kinase assays to measure the Clb2-associated histone H1 kinase activities. The cycling Cdc28/Clb2 activities in KLY1083 strain indicate that the apparent G1 arrest (see Fig. 6a and Fig. d) observed with connected cells was not in effect in an early stage of chained cells. Samples beyond 240 min after release were not taken because of a lack of cell cycle synchrony. *Cross-reacting protein in the anti–Clb2 blot. Control, strain KLY1080; cdc5ΔN, strain KLY1083; cdc5ΔN/FAA, strain KLY1229; EGFP-cdc5ΔN, EGFP-fused cdc5ΔN proteins; EGFP, control EGFP lacking Cdc5; H1, histone H1 kinase activity.
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Figure 5: Cdc28/Clb2 activity cycles during the early stages of connected cells. (A) Overexpression of EGFP-cdc5ΔN, but not the corresponding FAA mutant, accumulates cells with a DNA content >2C (G2/M). To carry out flow cytometric analyses, cells expressing control EGFP (KLY1080), EGFP-cdc5ΔN (KLY1083), or EGFP-cdc5ΔN/FAA (KLY1229) were arrested with α-factor in YEP-raffinose for 3 h, washed, and transferred into YEP-galactose medium to an OD600 of 0.05. Samples were taken at the indicated time points, fixed, and subjected to flow cytometry analyses. Strain KLY1083 begins to accumulate cells with a DNA content >2C (120 min) as the first cycle completes, whereas both strains KLY1080 and KLY1299 complete the cell cycle normally. Accumulation of cells with higher DNA contents appears to be the result of induction of connected cells (see text for details). Control, strain KLY1080; cdc5ΔN, strain KLY1083; cdc5ΔN/FAA, strain KLY1229; 1C, 1C DNA content; 2C, 2C DNA content. (B) Cdc28/Clb2 activity appears to fluctuate normally in strain KLY1083 overexpressing EGFP-cdc5ΔN. To examine the cell cycle progression, cellular lysates prepared from the same cells harvested in A were subjected to Western blot analyses to examine changes in Clb2 and EGFP-cdc5ΔN levels upon release from a α-factor arrest. The level of Cdc28 was determined as a loading control for each lane. The same lysates were also used to carry out anti–Clb2 immune complex kinase assays to measure the Clb2-associated histone H1 kinase activities. The cycling Cdc28/Clb2 activities in KLY1083 strain indicate that the apparent G1 arrest (see Fig. 6a and Fig. d) observed with connected cells was not in effect in an early stage of chained cells. Samples beyond 240 min after release were not taken because of a lack of cell cycle synchrony. *Cross-reacting protein in the anti–Clb2 blot. Control, strain KLY1080; cdc5ΔN, strain KLY1083; cdc5ΔN/FAA, strain KLY1229; EGFP-cdc5ΔN, EGFP-fused cdc5ΔN proteins; EGFP, control EGFP lacking Cdc5; H1, histone H1 kinase activity.

Mentions: To investigate whether the cell cycle is altered in connected cells, flow cytometry analyses were carried out for the cells expressing control EGFP (KLY1080), EGFP-cdc5ΔN (KLY1083), or EGFP-cdc5ΔN/FAA (KLY1229). Cells arrested with α-factor were transferred into YEP-galactose medium to induce protein expression upon release from a G1 arrest. Strains KLY1080 and KLY1229 appeared to progress through the cell cycle normally, regenerating the 1C DNA-containing (G1) population ∼120 min after release. In contrast, strain KLY1083 expressing EGFP-cdc5ΔN failed to regenerate a distinct 1C (G1) peak, and continued to accumulate a DNA content greater than 2C (G2/M) peak (Fig. 5 A). To further investigate whether these cells go through the cell cycle normally, the levels of Clb2 protein and Cdc28/Clb2-associated kinase activities were examined. Control strain KLY1080 achieved the maximum Cdc28/Clb2 activity (onset of M phase) in 60 and 150 min after release. Strain KLY1229 exhibited a slight delay in the cell cycle in comparison with EGFP control, whereas strain KLY1083 had a 20-min delay. However, in all cases, Clb2 protein levels and Clb2-associated kinase activities appeared to fluctuate normally with similar kinetics (Fig. 5 B), suggesting that overexpression of cdc5ΔN did not efficiently prevent endogenous Cdc5 from activating APC. Upon induction after release, approximately equal amounts of EGFP control, EGFP-cdc5ΔN, and EGFP-cdc5ΔN/FAA proteins were expressed in these cells (Fig. 5 B). These data suggest that overexpression of EGFP-cdc5ΔN appears to selectively inhibit cytokinesis without significantly perturbing the nuclear division cycle during the early stage of connected cells.


A novel function of Saccharomyces cerevisiae CDC5 in cytokinesis.

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

Cdc28/Clb2 activity cycles during the early stages of connected cells. (A) Overexpression of EGFP-cdc5ΔN, but not the corresponding FAA mutant, accumulates cells with a DNA content >2C (G2/M). To carry out flow cytometric analyses, cells expressing control EGFP (KLY1080), EGFP-cdc5ΔN (KLY1083), or EGFP-cdc5ΔN/FAA (KLY1229) were arrested with α-factor in YEP-raffinose for 3 h, washed, and transferred into YEP-galactose medium to an OD600 of 0.05. Samples were taken at the indicated time points, fixed, and subjected to flow cytometry analyses. Strain KLY1083 begins to accumulate cells with a DNA content >2C (120 min) as the first cycle completes, whereas both strains KLY1080 and KLY1299 complete the cell cycle normally. Accumulation of cells with higher DNA contents appears to be the result of induction of connected cells (see text for details). Control, strain KLY1080; cdc5ΔN, strain KLY1083; cdc5ΔN/FAA, strain KLY1229; 1C, 1C DNA content; 2C, 2C DNA content. (B) Cdc28/Clb2 activity appears to fluctuate normally in strain KLY1083 overexpressing EGFP-cdc5ΔN. To examine the cell cycle progression, cellular lysates prepared from the same cells harvested in A were subjected to Western blot analyses to examine changes in Clb2 and EGFP-cdc5ΔN levels upon release from a α-factor arrest. The level of Cdc28 was determined as a loading control for each lane. The same lysates were also used to carry out anti–Clb2 immune complex kinase assays to measure the Clb2-associated histone H1 kinase activities. The cycling Cdc28/Clb2 activities in KLY1083 strain indicate that the apparent G1 arrest (see Fig. 6a and Fig. d) observed with connected cells was not in effect in an early stage of chained cells. Samples beyond 240 min after release were not taken because of a lack of cell cycle synchrony. *Cross-reacting protein in the anti–Clb2 blot. Control, strain KLY1080; cdc5ΔN, strain KLY1083; cdc5ΔN/FAA, strain KLY1229; EGFP-cdc5ΔN, EGFP-fused cdc5ΔN proteins; EGFP, control EGFP lacking Cdc5; H1, histone H1 kinase activity.
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Related In: Results  -  Collection

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Figure 5: Cdc28/Clb2 activity cycles during the early stages of connected cells. (A) Overexpression of EGFP-cdc5ΔN, but not the corresponding FAA mutant, accumulates cells with a DNA content >2C (G2/M). To carry out flow cytometric analyses, cells expressing control EGFP (KLY1080), EGFP-cdc5ΔN (KLY1083), or EGFP-cdc5ΔN/FAA (KLY1229) were arrested with α-factor in YEP-raffinose for 3 h, washed, and transferred into YEP-galactose medium to an OD600 of 0.05. Samples were taken at the indicated time points, fixed, and subjected to flow cytometry analyses. Strain KLY1083 begins to accumulate cells with a DNA content >2C (120 min) as the first cycle completes, whereas both strains KLY1080 and KLY1299 complete the cell cycle normally. Accumulation of cells with higher DNA contents appears to be the result of induction of connected cells (see text for details). Control, strain KLY1080; cdc5ΔN, strain KLY1083; cdc5ΔN/FAA, strain KLY1229; 1C, 1C DNA content; 2C, 2C DNA content. (B) Cdc28/Clb2 activity appears to fluctuate normally in strain KLY1083 overexpressing EGFP-cdc5ΔN. To examine the cell cycle progression, cellular lysates prepared from the same cells harvested in A were subjected to Western blot analyses to examine changes in Clb2 and EGFP-cdc5ΔN levels upon release from a α-factor arrest. The level of Cdc28 was determined as a loading control for each lane. The same lysates were also used to carry out anti–Clb2 immune complex kinase assays to measure the Clb2-associated histone H1 kinase activities. The cycling Cdc28/Clb2 activities in KLY1083 strain indicate that the apparent G1 arrest (see Fig. 6a and Fig. d) observed with connected cells was not in effect in an early stage of chained cells. Samples beyond 240 min after release were not taken because of a lack of cell cycle synchrony. *Cross-reacting protein in the anti–Clb2 blot. Control, strain KLY1080; cdc5ΔN, strain KLY1083; cdc5ΔN/FAA, strain KLY1229; EGFP-cdc5ΔN, EGFP-fused cdc5ΔN proteins; EGFP, control EGFP lacking Cdc5; H1, histone H1 kinase activity.
Mentions: To investigate whether the cell cycle is altered in connected cells, flow cytometry analyses were carried out for the cells expressing control EGFP (KLY1080), EGFP-cdc5ΔN (KLY1083), or EGFP-cdc5ΔN/FAA (KLY1229). Cells arrested with α-factor were transferred into YEP-galactose medium to induce protein expression upon release from a G1 arrest. Strains KLY1080 and KLY1229 appeared to progress through the cell cycle normally, regenerating the 1C DNA-containing (G1) population ∼120 min after release. In contrast, strain KLY1083 expressing EGFP-cdc5ΔN failed to regenerate a distinct 1C (G1) peak, and continued to accumulate a DNA content greater than 2C (G2/M) peak (Fig. 5 A). To further investigate whether these cells go through the cell cycle normally, the levels of Clb2 protein and Cdc28/Clb2-associated kinase activities were examined. Control strain KLY1080 achieved the maximum Cdc28/Clb2 activity (onset of M phase) in 60 and 150 min after release. Strain KLY1229 exhibited a slight delay in the cell cycle in comparison with EGFP control, whereas strain KLY1083 had a 20-min delay. However, in all cases, Clb2 protein levels and Clb2-associated kinase activities appeared to fluctuate normally with similar kinetics (Fig. 5 B), suggesting that overexpression of cdc5ΔN did not efficiently prevent endogenous Cdc5 from activating APC. Upon induction after release, approximately equal amounts of EGFP control, EGFP-cdc5ΔN, and EGFP-cdc5ΔN/FAA proteins were expressed in these cells (Fig. 5 B). These data suggest that overexpression of EGFP-cdc5ΔN appears to selectively inhibit cytokinesis without significantly perturbing the nuclear division cycle during the early stage of connected 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.

Show MeSH
Related in: MedlinePlus