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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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Direct interactions between cdc5ΔN and septins may lead to aberrant septin ring structures. Strain 1783 expressing a GST-cdc5ΔN under control of GAL1 promoter (KLY1053) were additionally integrated with TUB1-GFP (KLY1069), YFP-CDC3 (KLY1075), CYK2-GFP (KLY1071), or MYO1-GFP (KLY1073). These cells were cultured under the induction conditions for 12 h and subjected to microscopic examination. (A) Overexpression of GST-cdc5ΔN induces an apparent G1 arrest. Internal cell bodies possess disassembled spindles, whereas peripheral cell bodies possess elongated spindles, suggesting that only the cells at the edge continue to divide (see text). Bar: 5 μm. (B) Induction of various aberrant septin structures by overexpression of GST-cdc5ΔN. It appeared that a large fraction of septin double rings (visualized as YFP-Cdc3) were distantly placed from each other (open arrowheads) or that septin rings were disassembled with remnants of the YFP-Cdc3 signal present at the mother-bud neck (arrowheads). In addition, tiny septin ring structures (arrows) or abnormally large rings (barbed arrows) without an apparent bud formation were often present (see text for detail). Bar: 5 μm. (C) Two-hybrid interactions between Cdc5 and septins. Diagram shows structures of various Cdc5 constructs used in these analyses. To enhance the protein stability, a destruction-box–deficient form of Cdc5 (Song et al. 2000) was used in place of the wild-type Cdc5. Grey boxes indicate the kinase domain in the NH2 terminus of Cdc5. Closed boxes in the COOH-terminal domain indicate the polo-box, whereas the gray indicates the polo-box with FAA mutations. The numbers in the table indicate the Miller units of β-galactosidase activity averaged from two independent experiments. Cl, Cla I; RV, Eco RV; Sn, Sna BI; Cdc5, Cdc5 lacking the NH2-terminal residues 6–71 (Song et al. 2000); cdc5ΔC, COOH-terminal domain deletion; cdc5ΔN, NH2-terminal domain deletion; cdc5ΔN/FAA, NH2-terminal domain deletion with FAA mutations in the polo-box; a and b, control plasmids: pEG202-NLS (DNA binding domain fusion vector) and pJG4-5 (activation domain fusion vector). (D) Coimmunoprecipitation of cdc5ΔN with Cdc11. To examine in vivo interactions between cdc5ΔN and septins, cellular lysates (supernatant of 15,000 g; S15) were prepared from strains bearing CDC11-TEV-9myc at the CDC11 locus and expressing either GAL1-EGFP-cdc5ΔN (SKY1732) or GAL1-EGFP-cdc5ΔN/FAA (SKY1734). Cdc11 was immunoprecipitated with an anti–Myc antibody cross linked to sepharose beads. To elute Cdc11 and its associated proteins, immunoprecipitates were digested with TEV protease. The resulting eluates were mixed with Laemmli sample buffer and subjected to SDS-PAGE and Western blot analyses with either anti–GFP antibody or anti–Cdc11 antibody. To determine the efficiency of coprecipitation, a fraction of S15 before immunoprecipitation was also loaded. Due to the TEV cleavage, Cdc11 protein eluted from immunoprecipitates possesses a molecular size smaller than Cdc11-TEV-9Myc. cdc5ΔN, S15 lysates from SKY1732; cdc5ΔN/FAA, S15 lysates from SKY1734, S15, a fraction of S15 lysates; bead, anti–Flag antibody crossed linked to sepharose beads; anti–Myc, anti–Myc antibody crossed linked to sepharose.
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Figure 6: Direct interactions between cdc5ΔN and septins may lead to aberrant septin ring structures. Strain 1783 expressing a GST-cdc5ΔN under control of GAL1 promoter (KLY1053) were additionally integrated with TUB1-GFP (KLY1069), YFP-CDC3 (KLY1075), CYK2-GFP (KLY1071), or MYO1-GFP (KLY1073). These cells were cultured under the induction conditions for 12 h and subjected to microscopic examination. (A) Overexpression of GST-cdc5ΔN induces an apparent G1 arrest. Internal cell bodies possess disassembled spindles, whereas peripheral cell bodies possess elongated spindles, suggesting that only the cells at the edge continue to divide (see text). Bar: 5 μm. (B) Induction of various aberrant septin structures by overexpression of GST-cdc5ΔN. It appeared that a large fraction of septin double rings (visualized as YFP-Cdc3) were distantly placed from each other (open arrowheads) or that septin rings were disassembled with remnants of the YFP-Cdc3 signal present at the mother-bud neck (arrowheads). In addition, tiny septin ring structures (arrows) or abnormally large rings (barbed arrows) without an apparent bud formation were often present (see text for detail). Bar: 5 μm. (C) Two-hybrid interactions between Cdc5 and septins. Diagram shows structures of various Cdc5 constructs used in these analyses. To enhance the protein stability, a destruction-box–deficient form of Cdc5 (Song et al. 2000) was used in place of the wild-type Cdc5. Grey boxes indicate the kinase domain in the NH2 terminus of Cdc5. Closed boxes in the COOH-terminal domain indicate the polo-box, whereas the gray indicates the polo-box with FAA mutations. The numbers in the table indicate the Miller units of β-galactosidase activity averaged from two independent experiments. Cl, Cla I; RV, Eco RV; Sn, Sna BI; Cdc5, Cdc5 lacking the NH2-terminal residues 6–71 (Song et al. 2000); cdc5ΔC, COOH-terminal domain deletion; cdc5ΔN, NH2-terminal domain deletion; cdc5ΔN/FAA, NH2-terminal domain deletion with FAA mutations in the polo-box; a and b, control plasmids: pEG202-NLS (DNA binding domain fusion vector) and pJG4-5 (activation domain fusion vector). (D) Coimmunoprecipitation of cdc5ΔN with Cdc11. To examine in vivo interactions between cdc5ΔN and septins, cellular lysates (supernatant of 15,000 g; S15) were prepared from strains bearing CDC11-TEV-9myc at the CDC11 locus and expressing either GAL1-EGFP-cdc5ΔN (SKY1732) or GAL1-EGFP-cdc5ΔN/FAA (SKY1734). Cdc11 was immunoprecipitated with an anti–Myc antibody cross linked to sepharose beads. To elute Cdc11 and its associated proteins, immunoprecipitates were digested with TEV protease. The resulting eluates were mixed with Laemmli sample buffer and subjected to SDS-PAGE and Western blot analyses with either anti–GFP antibody or anti–Cdc11 antibody. To determine the efficiency of coprecipitation, a fraction of S15 before immunoprecipitation was also loaded. Due to the TEV cleavage, Cdc11 protein eluted from immunoprecipitates possesses a molecular size smaller than Cdc11-TEV-9Myc. cdc5ΔN, S15 lysates from SKY1732; cdc5ΔN/FAA, S15 lysates from SKY1734, S15, a fraction of S15 lysates; bead, anti–Flag antibody crossed linked to sepharose beads; anti–Myc, anti–Myc antibody crossed linked to sepharose.

Mentions: To construct plasmids for two-hybrid analyses, CDC3, CDC10, CDC11, and CDC12 were PCR-amplified using genomic clones (gifts of John Chant, Harvard University, Cambridge, MA) as templates. The EcoRI-XhoI (artificially introduced restriction enzyme sites at the 5′ and 3′ ends of each open reading frame) fragments were ligated into pJG4-5 plasmid digested with corresponding enzymes. This results in in-frame fusion of full-length septins to activation domain. To generate a LexA DNA binding domain–fused CDC5 construct, a 2-kb XbaI fragment from pSK1006 was inserted into EcoRI-digested, end-filled pEG202-NLS (Origene Technologies Inc.). Various forms of CDC5 mutants were constructed by PCR amplification or enzymatic deletion (see Table and Fig. 6 C). To construct LEU2-based GAL-SIC1 plasmid, pRDB608 (a gift of Raymond Deshaies, California Institute of Technology, Pasadena, CA) was digested with NaeI and AatII to swap a URA3 fragment for a LEU2 fragment of pRS305. The resulting pSK1996 was digested with BstXI to achieve a targeted integration of GAL-SIC1 at the LEU2 locus.


A novel function of Saccharomyces cerevisiae CDC5 in cytokinesis.

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

Direct interactions between cdc5ΔN and septins may lead to aberrant septin ring structures. Strain 1783 expressing a GST-cdc5ΔN under control of GAL1 promoter (KLY1053) were additionally integrated with TUB1-GFP (KLY1069), YFP-CDC3 (KLY1075), CYK2-GFP (KLY1071), or MYO1-GFP (KLY1073). These cells were cultured under the induction conditions for 12 h and subjected to microscopic examination. (A) Overexpression of GST-cdc5ΔN induces an apparent G1 arrest. Internal cell bodies possess disassembled spindles, whereas peripheral cell bodies possess elongated spindles, suggesting that only the cells at the edge continue to divide (see text). Bar: 5 μm. (B) Induction of various aberrant septin structures by overexpression of GST-cdc5ΔN. It appeared that a large fraction of septin double rings (visualized as YFP-Cdc3) were distantly placed from each other (open arrowheads) or that septin rings were disassembled with remnants of the YFP-Cdc3 signal present at the mother-bud neck (arrowheads). In addition, tiny septin ring structures (arrows) or abnormally large rings (barbed arrows) without an apparent bud formation were often present (see text for detail). Bar: 5 μm. (C) Two-hybrid interactions between Cdc5 and septins. Diagram shows structures of various Cdc5 constructs used in these analyses. To enhance the protein stability, a destruction-box–deficient form of Cdc5 (Song et al. 2000) was used in place of the wild-type Cdc5. Grey boxes indicate the kinase domain in the NH2 terminus of Cdc5. Closed boxes in the COOH-terminal domain indicate the polo-box, whereas the gray indicates the polo-box with FAA mutations. The numbers in the table indicate the Miller units of β-galactosidase activity averaged from two independent experiments. Cl, Cla I; RV, Eco RV; Sn, Sna BI; Cdc5, Cdc5 lacking the NH2-terminal residues 6–71 (Song et al. 2000); cdc5ΔC, COOH-terminal domain deletion; cdc5ΔN, NH2-terminal domain deletion; cdc5ΔN/FAA, NH2-terminal domain deletion with FAA mutations in the polo-box; a and b, control plasmids: pEG202-NLS (DNA binding domain fusion vector) and pJG4-5 (activation domain fusion vector). (D) Coimmunoprecipitation of cdc5ΔN with Cdc11. To examine in vivo interactions between cdc5ΔN and septins, cellular lysates (supernatant of 15,000 g; S15) were prepared from strains bearing CDC11-TEV-9myc at the CDC11 locus and expressing either GAL1-EGFP-cdc5ΔN (SKY1732) or GAL1-EGFP-cdc5ΔN/FAA (SKY1734). Cdc11 was immunoprecipitated with an anti–Myc antibody cross linked to sepharose beads. To elute Cdc11 and its associated proteins, immunoprecipitates were digested with TEV protease. The resulting eluates were mixed with Laemmli sample buffer and subjected to SDS-PAGE and Western blot analyses with either anti–GFP antibody or anti–Cdc11 antibody. To determine the efficiency of coprecipitation, a fraction of S15 before immunoprecipitation was also loaded. Due to the TEV cleavage, Cdc11 protein eluted from immunoprecipitates possesses a molecular size smaller than Cdc11-TEV-9Myc. cdc5ΔN, S15 lysates from SKY1732; cdc5ΔN/FAA, S15 lysates from SKY1734, S15, a fraction of S15 lysates; bead, anti–Flag antibody crossed linked to sepharose beads; anti–Myc, anti–Myc antibody crossed linked to sepharose.
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Figure 6: Direct interactions between cdc5ΔN and septins may lead to aberrant septin ring structures. Strain 1783 expressing a GST-cdc5ΔN under control of GAL1 promoter (KLY1053) were additionally integrated with TUB1-GFP (KLY1069), YFP-CDC3 (KLY1075), CYK2-GFP (KLY1071), or MYO1-GFP (KLY1073). These cells were cultured under the induction conditions for 12 h and subjected to microscopic examination. (A) Overexpression of GST-cdc5ΔN induces an apparent G1 arrest. Internal cell bodies possess disassembled spindles, whereas peripheral cell bodies possess elongated spindles, suggesting that only the cells at the edge continue to divide (see text). Bar: 5 μm. (B) Induction of various aberrant septin structures by overexpression of GST-cdc5ΔN. It appeared that a large fraction of septin double rings (visualized as YFP-Cdc3) were distantly placed from each other (open arrowheads) or that septin rings were disassembled with remnants of the YFP-Cdc3 signal present at the mother-bud neck (arrowheads). In addition, tiny septin ring structures (arrows) or abnormally large rings (barbed arrows) without an apparent bud formation were often present (see text for detail). Bar: 5 μm. (C) Two-hybrid interactions between Cdc5 and septins. Diagram shows structures of various Cdc5 constructs used in these analyses. To enhance the protein stability, a destruction-box–deficient form of Cdc5 (Song et al. 2000) was used in place of the wild-type Cdc5. Grey boxes indicate the kinase domain in the NH2 terminus of Cdc5. Closed boxes in the COOH-terminal domain indicate the polo-box, whereas the gray indicates the polo-box with FAA mutations. The numbers in the table indicate the Miller units of β-galactosidase activity averaged from two independent experiments. Cl, Cla I; RV, Eco RV; Sn, Sna BI; Cdc5, Cdc5 lacking the NH2-terminal residues 6–71 (Song et al. 2000); cdc5ΔC, COOH-terminal domain deletion; cdc5ΔN, NH2-terminal domain deletion; cdc5ΔN/FAA, NH2-terminal domain deletion with FAA mutations in the polo-box; a and b, control plasmids: pEG202-NLS (DNA binding domain fusion vector) and pJG4-5 (activation domain fusion vector). (D) Coimmunoprecipitation of cdc5ΔN with Cdc11. To examine in vivo interactions between cdc5ΔN and septins, cellular lysates (supernatant of 15,000 g; S15) were prepared from strains bearing CDC11-TEV-9myc at the CDC11 locus and expressing either GAL1-EGFP-cdc5ΔN (SKY1732) or GAL1-EGFP-cdc5ΔN/FAA (SKY1734). Cdc11 was immunoprecipitated with an anti–Myc antibody cross linked to sepharose beads. To elute Cdc11 and its associated proteins, immunoprecipitates were digested with TEV protease. The resulting eluates were mixed with Laemmli sample buffer and subjected to SDS-PAGE and Western blot analyses with either anti–GFP antibody or anti–Cdc11 antibody. To determine the efficiency of coprecipitation, a fraction of S15 before immunoprecipitation was also loaded. Due to the TEV cleavage, Cdc11 protein eluted from immunoprecipitates possesses a molecular size smaller than Cdc11-TEV-9Myc. cdc5ΔN, S15 lysates from SKY1732; cdc5ΔN/FAA, S15 lysates from SKY1734, S15, a fraction of S15 lysates; bead, anti–Flag antibody crossed linked to sepharose beads; anti–Myc, anti–Myc antibody crossed linked to sepharose.
Mentions: To construct plasmids for two-hybrid analyses, CDC3, CDC10, CDC11, and CDC12 were PCR-amplified using genomic clones (gifts of John Chant, Harvard University, Cambridge, MA) as templates. The EcoRI-XhoI (artificially introduced restriction enzyme sites at the 5′ and 3′ ends of each open reading frame) fragments were ligated into pJG4-5 plasmid digested with corresponding enzymes. This results in in-frame fusion of full-length septins to activation domain. To generate a LexA DNA binding domain–fused CDC5 construct, a 2-kb XbaI fragment from pSK1006 was inserted into EcoRI-digested, end-filled pEG202-NLS (Origene Technologies Inc.). Various forms of CDC5 mutants were constructed by PCR amplification or enzymatic deletion (see Table and Fig. 6 C). To construct LEU2-based GAL-SIC1 plasmid, pRDB608 (a gift of Raymond Deshaies, California Institute of Technology, Pasadena, CA) was digested with NaeI and AatII to swap a URA3 fragment for a LEU2 fragment of pRS305. The resulting pSK1996 was digested with BstXI to achieve a targeted integration of GAL-SIC1 at the LEU2 locus.

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