Limits...
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.

Show MeSH

Related in: MedlinePlus

Overexpression of cdc5ΔN, but not the FAA polo-box mutant (Song et al. 2000), induces a dominant-negative, connected cell morphology. (A) 1,783 (MATa EG123) cells overexpressing either control EGFP (KLY1080), EGFP-cdc5ΔN (KLY1082), or EGFP-cdc5ΔN/FAA (KLY1081) were cultured under the induction conditions for 12 h. Cells were fixed and examined using confocal microscopy. A similar chained cell phenotype was also observed in a W303-1A and S288C genetic background (data not shown). DIC, differential interference contrast; EGFP, EGFP-fusion proteins expressed; control, strain KLY1080; cdc5ΔN, strain KLY1082; cdc5ΔN/FAA, strain KLY1081. Bar: 5 μm. (B) The FAA mutations in the polo-box do not influence the level of cdc5ΔN expression. An equal amount (30 μg) of cell lysate prepared from various strains shown in A was loaded onto each lane. Control, strain 1080; cdc5ΔN, strain 1082; cdc5ΔN/FAA, strain 1081; EGFP-cdc5ΔN, EGFP-fused cdc5ΔN proteins expressed; EGFP, control EGFP lacking Cdc5. Cdc28 protein serves as a loading control for each lane. (C) Introduction of wild-type CDC5 in a low copy centromeric plasmid, but not the cdc5/FAA or the kinase-inactive cdc5/NA (Hardy and Pautz 1996), remedy the chained cell morphology induced by overexpression of cdc5ΔN. Strain KLY1082 transformed with various CDC5 constructs were cultured in YEP-glucose to exponential phase before morphological examination. Vector, YCplac111 vector; CDC5, YCplac111-CDC5; cdc5/FAA, YCplac111-cdc5/W517F/V518A/L530A; cdc5/NA, YCplac111-cdc5/N209A. (D) Quantitation of the reversion of connected cell phenotype by various CDC5 constructs. The same samples shown in C were also counted. Both the kinase activity and an intact polo-box appear to be required for reversing the chained cell morphology.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC2195991&req=5

Figure 2: Overexpression of cdc5ΔN, but not the FAA polo-box mutant (Song et al. 2000), induces a dominant-negative, connected cell morphology. (A) 1,783 (MATa EG123) cells overexpressing either control EGFP (KLY1080), EGFP-cdc5ΔN (KLY1082), or EGFP-cdc5ΔN/FAA (KLY1081) were cultured under the induction conditions for 12 h. Cells were fixed and examined using confocal microscopy. A similar chained cell phenotype was also observed in a W303-1A and S288C genetic background (data not shown). DIC, differential interference contrast; EGFP, EGFP-fusion proteins expressed; control, strain KLY1080; cdc5ΔN, strain KLY1082; cdc5ΔN/FAA, strain KLY1081. Bar: 5 μm. (B) The FAA mutations in the polo-box do not influence the level of cdc5ΔN expression. An equal amount (30 μg) of cell lysate prepared from various strains shown in A was loaded onto each lane. Control, strain 1080; cdc5ΔN, strain 1082; cdc5ΔN/FAA, strain 1081; EGFP-cdc5ΔN, EGFP-fused cdc5ΔN proteins expressed; EGFP, control EGFP lacking Cdc5. Cdc28 protein serves as a loading control for each lane. (C) Introduction of wild-type CDC5 in a low copy centromeric plasmid, but not the cdc5/FAA or the kinase-inactive cdc5/NA (Hardy and Pautz 1996), remedy the chained cell morphology induced by overexpression of cdc5ΔN. Strain KLY1082 transformed with various CDC5 constructs were cultured in YEP-glucose to exponential phase before morphological examination. Vector, YCplac111 vector; CDC5, YCplac111-CDC5; cdc5/FAA, YCplac111-cdc5/W517F/V518A/L530A; cdc5/NA, YCplac111-cdc5/N209A. (D) Quantitation of the reversion of connected cell phenotype by various CDC5 constructs. The same samples shown in C were also counted. Both the kinase activity and an intact polo-box appear to be required for reversing the chained cell morphology.

Mentions: We have previously shown that Cdc5 localizes at spindle poles and cytokinetic neck-filaments (Song et al. 2000). The noncatalytic COOH-terminal domain of Cdc5, which contains the polo-box, is sufficient to localize at both mother-bud neck and spindle poles, whereas introduction of a triple W517F/V518A/L530A mutation (referred as FAA hereafter) in the polo-box abolishes localization (Song et al. 2000). In addition, overexpression of Cdc5, but not the FAA polo-box mutant, can induce ectopic septin ring structures in abnormally elongated buds (Song et al. 2000). These data suggest that the polo-box is critical in the localization and cytokinetic function of Cdc5. Thus, we attempted to inhibit the polo-box function by overexpression of the COOH-terminal domain of Cdc5 (cdc5ΔN). To this end, strain 1783 was integrated with a single copy of GAL1-EGFP, GAL1-EGFP-cdc5ΔN, or GAL1-EGFP-cdc5ΔN/FAA. These cells grow normally without any detectable morphological defect on YEP-glucose (data not shown). However, when grown in YEP-galactose, expression of EGFP-cdc5ΔN (KLY1082) resulted in connected cells. In contrast, cells expressing either control EGFP (KLY1080) or EGFP-cdc5ΔN/FAA (KLY1081) did not yield any detectable morphological changes (Fig. 2 A, top). Microscopic examinations revealed that cells expressing cdc5ΔN yield two fluorescent bands at the mother-bud neck and fluorescent dots in the cytoplasm, whereas the EGFP control and the corresponding FAA mutant yielded only diffuse signals (Fig. 2 A, bottom). The apparent multiple dot signals in strain KLY1082 may be in part attributable to the ability of cdc5ΔN to induce multiple subcellular structures containing Spc42, a component of spindle pole bodies (Song et al. 2000). However, strains KLY1082 and KLY1083 (a strain expressing three copies of EGFP-cdc5ΔN; described below) do not possess any detectable growth defect in the presence of the antimicrotubule drug benomyl (data not shown), suggesting that these strains grow without a significant defect in the spindle checkpoint pathway. Both EGFP-cdc5ΔN and EGFP-cdc5ΔN/FAA were expressed at similar levels, indicating that the observed phenotype associated with EGFP-cdc5ΔN expression was not due to a difference in protein abundance (Fig. 2 B).


A novel function of Saccharomyces cerevisiae CDC5 in cytokinesis.

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

Overexpression of cdc5ΔN, but not the FAA polo-box mutant (Song et al. 2000), induces a dominant-negative, connected cell morphology. (A) 1,783 (MATa EG123) cells overexpressing either control EGFP (KLY1080), EGFP-cdc5ΔN (KLY1082), or EGFP-cdc5ΔN/FAA (KLY1081) were cultured under the induction conditions for 12 h. Cells were fixed and examined using confocal microscopy. A similar chained cell phenotype was also observed in a W303-1A and S288C genetic background (data not shown). DIC, differential interference contrast; EGFP, EGFP-fusion proteins expressed; control, strain KLY1080; cdc5ΔN, strain KLY1082; cdc5ΔN/FAA, strain KLY1081. Bar: 5 μm. (B) The FAA mutations in the polo-box do not influence the level of cdc5ΔN expression. An equal amount (30 μg) of cell lysate prepared from various strains shown in A was loaded onto each lane. Control, strain 1080; cdc5ΔN, strain 1082; cdc5ΔN/FAA, strain 1081; EGFP-cdc5ΔN, EGFP-fused cdc5ΔN proteins expressed; EGFP, control EGFP lacking Cdc5. Cdc28 protein serves as a loading control for each lane. (C) Introduction of wild-type CDC5 in a low copy centromeric plasmid, but not the cdc5/FAA or the kinase-inactive cdc5/NA (Hardy and Pautz 1996), remedy the chained cell morphology induced by overexpression of cdc5ΔN. Strain KLY1082 transformed with various CDC5 constructs were cultured in YEP-glucose to exponential phase before morphological examination. Vector, YCplac111 vector; CDC5, YCplac111-CDC5; cdc5/FAA, YCplac111-cdc5/W517F/V518A/L530A; cdc5/NA, YCplac111-cdc5/N209A. (D) Quantitation of the reversion of connected cell phenotype by various CDC5 constructs. The same samples shown in C were also counted. Both the kinase activity and an intact polo-box appear to be required for reversing the chained cell morphology.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2195991&req=5

Figure 2: Overexpression of cdc5ΔN, but not the FAA polo-box mutant (Song et al. 2000), induces a dominant-negative, connected cell morphology. (A) 1,783 (MATa EG123) cells overexpressing either control EGFP (KLY1080), EGFP-cdc5ΔN (KLY1082), or EGFP-cdc5ΔN/FAA (KLY1081) were cultured under the induction conditions for 12 h. Cells were fixed and examined using confocal microscopy. A similar chained cell phenotype was also observed in a W303-1A and S288C genetic background (data not shown). DIC, differential interference contrast; EGFP, EGFP-fusion proteins expressed; control, strain KLY1080; cdc5ΔN, strain KLY1082; cdc5ΔN/FAA, strain KLY1081. Bar: 5 μm. (B) The FAA mutations in the polo-box do not influence the level of cdc5ΔN expression. An equal amount (30 μg) of cell lysate prepared from various strains shown in A was loaded onto each lane. Control, strain 1080; cdc5ΔN, strain 1082; cdc5ΔN/FAA, strain 1081; EGFP-cdc5ΔN, EGFP-fused cdc5ΔN proteins expressed; EGFP, control EGFP lacking Cdc5. Cdc28 protein serves as a loading control for each lane. (C) Introduction of wild-type CDC5 in a low copy centromeric plasmid, but not the cdc5/FAA or the kinase-inactive cdc5/NA (Hardy and Pautz 1996), remedy the chained cell morphology induced by overexpression of cdc5ΔN. Strain KLY1082 transformed with various CDC5 constructs were cultured in YEP-glucose to exponential phase before morphological examination. Vector, YCplac111 vector; CDC5, YCplac111-CDC5; cdc5/FAA, YCplac111-cdc5/W517F/V518A/L530A; cdc5/NA, YCplac111-cdc5/N209A. (D) Quantitation of the reversion of connected cell phenotype by various CDC5 constructs. The same samples shown in C were also counted. Both the kinase activity and an intact polo-box appear to be required for reversing the chained cell morphology.
Mentions: We have previously shown that Cdc5 localizes at spindle poles and cytokinetic neck-filaments (Song et al. 2000). The noncatalytic COOH-terminal domain of Cdc5, which contains the polo-box, is sufficient to localize at both mother-bud neck and spindle poles, whereas introduction of a triple W517F/V518A/L530A mutation (referred as FAA hereafter) in the polo-box abolishes localization (Song et al. 2000). In addition, overexpression of Cdc5, but not the FAA polo-box mutant, can induce ectopic septin ring structures in abnormally elongated buds (Song et al. 2000). These data suggest that the polo-box is critical in the localization and cytokinetic function of Cdc5. Thus, we attempted to inhibit the polo-box function by overexpression of the COOH-terminal domain of Cdc5 (cdc5ΔN). To this end, strain 1783 was integrated with a single copy of GAL1-EGFP, GAL1-EGFP-cdc5ΔN, or GAL1-EGFP-cdc5ΔN/FAA. These cells grow normally without any detectable morphological defect on YEP-glucose (data not shown). However, when grown in YEP-galactose, expression of EGFP-cdc5ΔN (KLY1082) resulted in connected cells. In contrast, cells expressing either control EGFP (KLY1080) or EGFP-cdc5ΔN/FAA (KLY1081) did not yield any detectable morphological changes (Fig. 2 A, top). Microscopic examinations revealed that cells expressing cdc5ΔN yield two fluorescent bands at the mother-bud neck and fluorescent dots in the cytoplasm, whereas the EGFP control and the corresponding FAA mutant yielded only diffuse signals (Fig. 2 A, bottom). The apparent multiple dot signals in strain KLY1082 may be in part attributable to the ability of cdc5ΔN to induce multiple subcellular structures containing Spc42, a component of spindle pole bodies (Song et al. 2000). However, strains KLY1082 and KLY1083 (a strain expressing three copies of EGFP-cdc5ΔN; described below) do not possess any detectable growth defect in the presence of the antimicrotubule drug benomyl (data not shown), suggesting that these strains grow without a significant defect in the spindle checkpoint pathway. Both EGFP-cdc5ΔN and EGFP-cdc5ΔN/FAA were expressed at similar levels, indicating that the observed phenotype associated with EGFP-cdc5ΔN expression was not due to a difference in protein abundance (Fig. 2 B).

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