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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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Overexpression of cdc5ΔN results in an inhibition of septum formation between mother-bud necks. Strain KLY1083 was cultured under the induction conditions for 9 h, fixed with formaldehyde, and stained with calcofluor or DiI as described in Materials and Methods. Cells were then subjected to confocal microscopy with a series of 100-nm sections to investigate whether cytoplasms are connected between the chained cells. (A) Calcofluor signals were discontinuous in large fractions (∼70%) of mother-bud necks of the connected cell bodies, indicating a failure of septum formation. The arrow indicates a cell body with bud scars, suggesting that this is likely to be the mother cell responsible for generating other connected cell bodies. Bar: 5 μm. (B) Membrane closure is impaired in ∼50% of the necks examined. However, it was also apparent that heavy patches of DiI stain were present in other bud necks, suggesting that membrane closure was impaired but not completely inhibited by overexpression of cdc5ΔN. Barbed arrows indicate the necks with apparently connected cytoplasm. Bar: 5 μm.
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Figure 4: Overexpression of cdc5ΔN results in an inhibition of septum formation between mother-bud necks. Strain KLY1083 was cultured under the induction conditions for 9 h, fixed with formaldehyde, and stained with calcofluor or DiI as described in Materials and Methods. Cells were then subjected to confocal microscopy with a series of 100-nm sections to investigate whether cytoplasms are connected between the chained cells. (A) Calcofluor signals were discontinuous in large fractions (∼70%) of mother-bud necks of the connected cell bodies, indicating a failure of septum formation. The arrow indicates a cell body with bud scars, suggesting that this is likely to be the mother cell responsible for generating other connected cell bodies. Bar: 5 μm. (B) Membrane closure is impaired in ∼50% of the necks examined. However, it was also apparent that heavy patches of DiI stain were present in other bud necks, suggesting that membrane closure was impaired but not completely inhibited by overexpression of cdc5ΔN. Barbed arrows indicate the necks with apparently connected cytoplasm. Bar: 5 μm.

Mentions: To confirm a cytokinetic defect in strain KLY1083, cells induced for 9 h were stained with calcofluor to examine chitin deposition in cell walls and septum. Strong calcofluor bands were observed between connected cell bodies (Fig. 4 A). To examine whether septum formation is completed in these cells, we performed serial optical sectioning using a confocal microscope. In most of the mother-bud necks examined, calcofluor signals were discontinuous in focal planes bisecting the cell bodies longitudinally, indicating that septa were not completely formed between the connected cell bodies (Fig. 4 A). Interestingly, among the connected cells examined (n = 56), only one cell body, either in center or at one edge of the chains, possessed bud scars (revealed as fluorescent chitin rings), indicating that other cell bodies did not generate daughter cells. Since these cells do not increase in number under the induction conditions (Fig. 3 D), the bud scars on the presumed mother cells are likely to be the result of previous budding events before transferring into induction conditions.


A novel function of Saccharomyces cerevisiae CDC5 in cytokinesis.

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

Overexpression of cdc5ΔN results in an inhibition of septum formation between mother-bud necks. Strain KLY1083 was cultured under the induction conditions for 9 h, fixed with formaldehyde, and stained with calcofluor or DiI as described in Materials and Methods. Cells were then subjected to confocal microscopy with a series of 100-nm sections to investigate whether cytoplasms are connected between the chained cells. (A) Calcofluor signals were discontinuous in large fractions (∼70%) of mother-bud necks of the connected cell bodies, indicating a failure of septum formation. The arrow indicates a cell body with bud scars, suggesting that this is likely to be the mother cell responsible for generating other connected cell bodies. Bar: 5 μm. (B) Membrane closure is impaired in ∼50% of the necks examined. However, it was also apparent that heavy patches of DiI stain were present in other bud necks, suggesting that membrane closure was impaired but not completely inhibited by overexpression of cdc5ΔN. Barbed arrows indicate the necks with apparently connected cytoplasm. Bar: 5 μm.
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Related In: Results  -  Collection

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

Figure 4: Overexpression of cdc5ΔN results in an inhibition of septum formation between mother-bud necks. Strain KLY1083 was cultured under the induction conditions for 9 h, fixed with formaldehyde, and stained with calcofluor or DiI as described in Materials and Methods. Cells were then subjected to confocal microscopy with a series of 100-nm sections to investigate whether cytoplasms are connected between the chained cells. (A) Calcofluor signals were discontinuous in large fractions (∼70%) of mother-bud necks of the connected cell bodies, indicating a failure of septum formation. The arrow indicates a cell body with bud scars, suggesting that this is likely to be the mother cell responsible for generating other connected cell bodies. Bar: 5 μm. (B) Membrane closure is impaired in ∼50% of the necks examined. However, it was also apparent that heavy patches of DiI stain were present in other bud necks, suggesting that membrane closure was impaired but not completely inhibited by overexpression of cdc5ΔN. Barbed arrows indicate the necks with apparently connected cytoplasm. Bar: 5 μm.
Mentions: To confirm a cytokinetic defect in strain KLY1083, cells induced for 9 h were stained with calcofluor to examine chitin deposition in cell walls and septum. Strong calcofluor bands were observed between connected cell bodies (Fig. 4 A). To examine whether septum formation is completed in these cells, we performed serial optical sectioning using a confocal microscope. In most of the mother-bud necks examined, calcofluor signals were discontinuous in focal planes bisecting the cell bodies longitudinally, indicating that septa were not completely formed between the connected cell bodies (Fig. 4 A). Interestingly, among the connected cells examined (n = 56), only one cell body, either in center or at one edge of the chains, possessed bud scars (revealed as fluorescent chitin rings), indicating that other cell bodies did not generate daughter cells. Since these cells do not increase in number under the induction conditions (Fig. 3 D), the bud scars on the presumed mother cells are likely to be the result of previous budding events before transferring into induction conditions.

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