Loss of CDC5 Function in Saccharomyces cerevisiae Leads to Defects in Swe1p Regulation and Bfa1p/Bub2p-Independent Cytokinesis

Corresponding author: Kyung S. Lee, National Cancer Institute, NIH, 9000 Rockville Pike, Bldg. 37, Rm. 3D25, Bethesda, MD 20892., kyunglee{at}pop.nci.nih.gov (E-mail)

Communicating editor: N. A. JENKINS

	ABSTRACT

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

In many organisms, polo kinases appear to play multiple roles during M-phase progression. To provide new insights into the function of budding yeast polo kinase Cdc5p, we generated novel temperature-sensitive cdc5 mutants by mutagenizing the C-terminal domain. Here we show that, at a semipermissive temperature, the cdc5-3 mutant exhibited a synergistic bud elongation and growth defect with loss of HSL1, a component important for normal G₂/M transition. Loss of SWE1, which phosphorylates and inactivates the budding yeast Cdk1 homolog Cdc28p, suppressed the cdc5-3 hsl1 defect, suggesting that Cdc5p functions at a point upstream of Swe1p. In addition, the cdc5-4 and cdc5-7 mutants exhibited chained cell morphologies with shared cytoplasms between the connected cell bodies, indicating a cytokinetic defect. Close examination of these mutants revealed delayed septin assembly at the incipient bud site and loosely organized septin rings at the mother-bud neck. Components in the mitotic exit network (MEN) play important roles in normal cytokinesis. However, loss of BFA1 or BUB2, negative regulators of the MEN, failed to remedy the cytokinetic defect of these mutants, indicating that Cdc5p promotes cytokinesis independently of Bfa1p and Bub2p. Thus, Cdc5p contributes to the activation of the Swe1p-dependent Cdc28p/Clb pathway, normal septin function, and cytokinesis.

IN various organisms, polo kinases have been shown to regulate diverse cellular and biochemical events at different stages of M phase (for reviews see LANE and NIGG 1997 ; GLOVER et al. 1998 ). Data obtained from higher eukaryotic organisms show that, at the G₂/M transition, polo kinase activates cyclin B1-dependent Cdc2 activity through activation of Cdc25C phosphatase (KUMAGAI and DUNPHY 1996 ) and also through direct phosphorylation and targeting of cyclin B1 to the nucleus (TOYOSHIMA-MORIMOTO et al. 2001 ). In budding yeast, it is widely appreciated that mitotic entry is tightly linked with proper septin ring organization at the bud neck. A defect in septin assembly causes a Swe1p-dependent G₂ delay, which results in a filamentous phenotype due to the inability of buds to switch from polarized to isotropic growth (LEW and REED 1993 ; BARRAL et al. 1999 ; EDGINGTON et al. 1999 ). A recent report suggests that the polo homolog, Cdc5p, is a potential negative regulator of Swe1p (BARTHOLOMEW et al. 2001 ), which inhibits Cdc28 (Cdk1 of budding yeast) by phosphorylating a conserved tyrosine at position 19 (BOOHER et al. 1993 ). Hsl1p, whose activity depends on proper septin function (BARRAL et al. 1999 ), acts as a negative regulator of Swe1p (MA et al. 1996 ). Both Hsl1p and its adaptor Hsl7p are required for the bud-neck localization and degradation of Swe1p (MCMILLAN et al. 1999 ; SHULEWITZ et al. 1999 ) and thus play a critical role for the Swe1p-dependent Cdc28p activation pathway at the G₂/M transition. During anaphase in mammalian cells or at mitotic exit in budding yeast, polo kinase activates the anaphase-promoting complex (APC; DESCOMBES and NIGG 1998 ; SHIRAYAMA et al. 1998 ), which in turn ubiquitinates the mitotic cyclins, leading to their degradation and the inactivation of Cdk1p. This step is thought to be a prerequisite for the initiation of cytokinesis.

Cytokinesis is a highly coordinated cellular process achieved by contractile ring formation; subsequent contraction of this ring divides one cell into two cells. Temporal and spatial regulation of the cytokinetic machinery is pivotal to ensuring equal partitioning of genomic and cellular materials into two dividing cells. In budding yeast, the future cytokinesis site is specified early in the cell cycle and cleavage is achieved by an actomyosin-based contractile ring, followed by septum formation to separate two dividing cells (BI et al. 1998 ; LIPPINCOTT and LI 1998B ). Four septin proteins (Cdc3p, Cdc10p, Cdc11p, and Cdc12p; for review see LONGTINE et al. 1996 ) are the major structural components of the filaments located at the bud neck (FRAZIER et al. 1998 ) and form a ring encircling the mother-bud neck. Septin rings appear to play a key role in recruiting much of the cytokinesis machinery to the mother-bud neck. In a septin-dependent manner, Myo1p (myosin II) assembles into a ring at the future budding site early in the cell cycle, whereas F-actin is recruited to the ring just prior to spindle disassembly and contraction (BI et al. 1998 ; LIPPINCOTT and LI 1998A , LIPPINCOTT and LI 1998B ). The septin ring disassembles and relocalizes to the future budding site at the time of contraction (LIPPINCOTT and LI 1998A ). In addition to roles in cytokinesis, septins appear to be critical for diverse cellular functions such as in chitin deposition (DEMARINI et al. 1997 ), bud site selection (CHANT et al. 1995 ), pheromone-induced morphogenesis (GIOT and KONOPKA 1997 ), mother-daughter cell compartmentalization (BARRAL et al. 2000 ; TAKIZAWA et al. 2000 ), and the coordination of mitotic entry with morphogenesis (CARROLL et al. 1998 ; BARRAL et al. 1999 ; EDGINGTON et al. 1999 ; SHULEWITZ et al. 1999 ; LONGTINE et al. 2000 ).

A growing body of evidence from various organisms suggests that polo kinases also play important roles in regulating cytokinesis (OHKURA et al. 1995 ; MUNDT et al. 1997 ; BAHLER et al. 1998 ; CARMENA et al. 1998 ; LEE et al. 1999 ; SONG and LEE 2001 ; TANAKA et al. 2001 ). In budding yeast, overexpression of CDC5 or of its mammalian functional homolog Plk1 leads to the induction of additional septin ring structures (LEE and ERIKSON 1997 ; LEE et al. 1999 ), whereas overexpression of the C-terminal domain of CDC5 (cdc5N) induces a dominant-negative cytokinesis defect, likely by disturbing septin structures through a direct interaction between Cdc11p/Cdc12p and cdc5pN (SONG and LEE 2001 ). Recent studies have shown that, besides their roles in mitotic exit, components in the mitotic exit network (MEN) such as Cdc5p, Tem1p, Cdc15p, Mob1p, and Cdc14p are required for actin ring formation at the mother-bud neck (JIMENEZ et al. 1998 ; FRENZ et al. 2000 ; LEE et al. 2001A ). The Tem1p GTPase appears to contribute to actomyosin and septin dynamics during cytokinesis (LIPPINCOTT et al. 2001 ). In addition, Bub2p, a putative GTPase activating protein (GAP), has been shown to be important in restraining actin ring formation and cytokinesis (LEE et al. 2001B ). Bfa1p and Bub2p are closely related to Schizosaccharomyces pombe Byr4p and Cdc16p, respectively, which constitute a two-component GAP for the Spg1p GTPase (FURGE et al. 1998 ), suggesting that a Bfa1p/Bub2p heteromeric complex negatively regulates Tem1p. Taken together, in addition to their role at mitotic exit, the MEN contributes to cytokinesis and Bfa1p/Bub2p negatively regulates the MEN.

Studies have shown that the C-terminal noncatalytic domain of polo kinases appears to play a critical role in their subcellular localization (LEE et al. 1998 ; SONG et al. 2000 ). Overexpression of a kinase-inactive form of CDC5 under control of the GAL1 promoter suppresses a GAL1-SWE1-induced cell growth defect (BARTHOLOMEW et al. 2001 ), whereas overexpression of cdc5N inhibits cytokinesis (SONG and LEE 2001 ). However, it is unknown whether the C-terminal domain of Cdc5p plays an important role in a Swe1p-dependent pathway or in cytokinesis under physiological conditions. To explore these possibilities, we generated novel temperature-sensitive cdc5 mutants by randomly mutagenizing the C-terminal domain of Cdc5p. Our data demonstrate that Cdc5p is required for the Swe1p-dependent Cdc28p/Clb activation pathway, normal septin function, and cytokinesis. Multiple mitotic defects observed with C-terminal domain mutants suggest that the C-terminal region of Cdc5p may be a multi-functional domain able to interact with various cellular proteins at different points of M phase.

	MATERIALS AND METHODS

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

Strains, growth conditions, and cell counts:
Yeast strains used in this study are shown in Table 1. Cells were cultured in YEP (1% yeast extract, 2% Bacto-peptone) supplemented with 2% glucose. Synthetic minimal medium (SHERMAN et al. 1986 ) supplemented with the appropriate nutrients was employed to select for plasmid maintenance. Yeast transformation was carried out by the lithium acetate method (ITO et al. 1983 ). All the cells were counted after separating cell aggregates by sonicating with sonicator model W-225R (Heat systems-Ultrasonics, Plainview, NY) at 40% duty with no. 4 output for 4 sec.

PCR mutagenesis of the C-terminal domain of Cdc5:
The C-terminal domain of Cdc5p was mutagenized by polymerase chain reaction. To facilitate analyses of various mutants, CDC5 was C-terminally tagged with three copies of hemagglutinin (HA) epitope tag to generate YCplac22-CDC5-HA3. A HpaI/PacI fragment, which includes amino acid residues 366–705, but does not include the HA epitope, was mutagenized as described previously (MUHLRAD et al. 1992 ). These PCR products were digested with HpaI and PacI. The fragments obtained were inserted into YCplac22-CDC5/W517F/V518A/L530A (SONG et al. 2000 ) digested with corresponding enzymes. The resulting cdc5 mutant library was transformed into the cdc5 + YCplac33-CDC5 strain to examine the growth phenotype after shuffling the URA3-based CDC5 plasmid on 5-fluoroorotic acid (5-FOA) plates. All potential temperature-sensitive alleles of CDC5 were sequenced to determine the mutation sites in the C-terminal domain.

Strain construction:
Temperature-sensitive alleles of CDC5, which do not support cell viability at 37°, were integrated at the TRP1 locus of a W303-1A-derived cdc5 strain (KLY2372) that is kept viable by the presence of a URA3-based YCplac33-CDC5. After shuffling the YCplac33-CDC5 plasmid on 5-FOA plates, these mutants were subjected to further analyses. To facilitate analyses of these mutants, TUB1-GFP or CDC10-YFP (SONG et al. 2000 ) was integrated and expressed under the native promoter. To alleviate the mitotic exit defect of cdc5 mutant strains, a dominant allele of CDC14 (CDC14^TAB6-1; SHOU et al. 2001 ) was integrated at the HIS3 locus. Both a bfa1::his5⁺ and a bub2::his5⁺ were generated by the one-step gene disruption method described previously (LONGTINE et al. 1998 ).

Kinase assays and Western analyses:
Cell lysates were prepared in TED buffer [40 mM Tris-Cl (pH 7.5), 0.25 mM EDTA, 1 mM dithiothreitol, 1 mM 4-(2-aminoethyl)-benzenesulfonyl fluoride (Pefabloc; Boehringer Mannheim, Indianapolis), 10 µg/ml pepstatin A, 10 µg/ml leupeptin] with an equal volume of glass beads (Sigma, St. Louis). To measure the Cdc5p-HA3-associated kinase activity, the obtained lysates were spun at 15,000 x g for 10 min, and the resulting supernatants were subjected to immune complex kinase assays using an anti-HA antibody. Western analyses were carried out with either anti-HA antibody or anti-Cdc28 antibody as described previously (SONG et al. 2000 ). Proteins that interact with antibodies were detected by the enhanced chemiluminescence Western detection system (Amersham Biosciences, Piscataway, NJ).

Cell staining and immunofluorescence microscopy:
To visualize plasma membranes, cells were stained with DiI (Molecular Probes, Eugene, OR) as described previously (LIPPINCOTT and LI 1998A ). To determine whether septa are formed between the cell bodies, calcofluor staining was carried out as previously described (PRINGLE 1991 ; LIPPINCOTT and LI 1998A ) with a fluorescent brightener 28 (Sigma), and then serial sections were obtained using a confocal microscope with a 100-nm interval.

Indirect immunofluorescence was performed as described previously (LEE et al. 1998 ). Actin was localized using rhodamine-conjugated phalloidin (Molecular Probes). DNA was visualized with 4',6-diamidino-2-phenylindole (DAPI). Confocal fluorescent images were collected with a Leica TCS spectrophotometer confocal microscope.

	RESULTS

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

Generation of novel cdc5 C-terminal domain mutants:
Several studies with cdc5-1 and msd2-1 mutants have revealed that Cdc5p plays an important role in activating the APC, thereby inactivating Cdc28p/Clb activity (CHARLES et al. 1998 ; SHIRAYAMA et al. 1998 ). In addition, overexpression studies (BARTHOLOMEW et al. 2001 ; SONG and LEE 2001 ) have suggested that Cdc5p may also contribute to a Swe1p-dependent pathway and to cytokinesis. However, whether Cdc5p contributes to these events under physiological conditions has not been clear. In an attempt to generate novel cdc5 mutants, defective at a step other than mitotic exit, the C-terminal domain of Cdc5p was mutagenized by PCR and conditional alleles were isolated. Among these, three novel mutants (cdc5-3, cdc5-4, and cdc5-7; Fig 1A) that exhibited a temperature-sensitive cell growth defect (Fig 1B) were chosen for further characterization. Both Cdc5p wild-type and mutant proteins were epitope tagged with three copies of HA to enable immunoprecipitation and detection by immunoblot. The previously characterized cdc5-1 mutant was used as a comparison. All three mutants grew well at 23°, but they all exhibited a more severe temperature-sensitive growth defect than did the cdc5-1 mutant at 37° (Fig 1B). To test whether these new cdc5 alleles were dominant or recessive, each of these mutants was mated with a haploid wild-type strain (KLY1548). The resulting diploids did not exhibit any detectable cell growth or morphological defects at 37° (data not shown), indicating that all three newly generated cdc5 alleles are recessive. To investigate whether these three mutants have a cell cycle defect similar to that of cdc5-1, flow cytometry analyses were carried out. Cells were arrested with -factor at 23° for 3 hr and then released into fresh medium prewarmed at 37°. Under these conditions, wild-type CDC5 cells go through the cell cycle normally (Fig 1C). As with the cdc5-1 mutant, however, all three newly generated cdc5 mutants were arrested at a point after achieving a 2N DNA content (Fig 1C). Staining of the mutants with DAPI and an antimicrotubule antibody revealed that, as with cdc5-1, 80–90% of these mutant cells were arrested with divided nuclei and elongated spindles upon shifting the temperature to 37° for 3.5 hr (data not shown). These observations suggest that the primary defect of these three mutants is likely to be in exiting mitosis.

View larger version (38K): In this window In a new window Download PPT slide   Figure 1. (A) Protein sequence alignment between wild-type Cdc5p and the C-terminal domain mutants. Wild-type amino acid residues and their corresponding mutant residues are shown in the single-letter amino acid code. Numbers in wild-type Cdc5p (CDC5) indicate positions of each amino acid residue. , the deletion of R581 residue. (B) Temperature-sensitive growth defect of novel cdc5 mutants. Cultures were grown on YEP + 2% glucose at the indicated temperature for 3 days. CDC5, KLY2470; cdc5-1, KLY2458; cdc5-3, KLY2460; cdc5-4, KLY2462; cdc5-7, KLY2464. (C) Flow cytometry analyses of wild-type and cdc5 mutants. Cells were arrested with -factor for 3 hr at 23°, washed, and transferred into prewarmed YEP + 2% glucose medium at 37°. Samples were taken at the indicated time points. All the cdc5 mutants arrest with 2N DNA content, whereas the wild-type strain cycles normally. CDC5, KLY2470; cdc5-1, KLY2458; cdc5-3, KLY2460; cdc5-4, KLY2462; cdc5-7, KLY2464.

To examine the protein expression levels and associated kinase activities of these mutants, Western analyses and immune complex kinase assays were carried out using exponentially growing cells. At the permissive temperature, the expression level of the cdc5-1 mutant protein was at a level similar to that of wild-type CDC5, whereas the expression levels of the cdc5-3, cdc5-4, and cdc5-7 mutants were somewhat reduced. At 37°, the steady-state expression levels of the cdc5-3, cdc5-4, and cdc5-7 mutants were severalfold lower than that of wild-type CDC5 (Fig 2A). Largely consistent with this reduction in protein expression level, the overall kinase activities associated with the cdc5-3p, cdc5-4p, or cdc5-7p immunoprecipitates from equal amounts of each S15 fraction were significantly lower than that of the wild-type Cdc5p at 37° (Fig 2B).

View larger version (73K): In this window In a new window Download PPT slide   Figure 2. (A) Expression levels of wild-type and cdc5 mutants. Both Cdc5p wild-type and mutant proteins were epitope tagged with three copies of HA to enable immunoprecipitation and detection by immunoblot (see MATERIALS AND METHODS). Total cellular protein (100 µg of each) was analyzed to determine expression levels of Cdc5p-HA3 with an anti-HA antibody (top) or to determine the level of Cdc28p as an internal loading control (bottom). Lysates were prepared either from cultures grown at 23° (left) or from cultures shifted to 37° for 3.5 hr (right). (B) Kinase activities of wild-type and mutant forms of Cdc5p. Kinase activities were determined by carrying out in vitro immune complex kinase assays with supernatants obtained from 15,000 x g centrifugation for 15 min (S15). Twenty-eight milligrams of S15 fractions from each mutant were subjected to immunoprecipitation with 5 µg of anti-HA antibody. Levels of immunoprecipitated Cdc5p-HA3 protein were determined by anti-HA Western blotting (top). Kinase activities of various forms of Cdc5p-HA3 were measured using casein as in vitro substrate (bottom). no tag, KLY2372 without HA3 tag at the CDC5 locus; cdc5-1, KLY2458; cdc5-3, KLY2460; cdc5-4, KLY2462; cdc5-7, KLY2464; CDC5, KLY2470.

Bypass of mitotic arrest reveals additional defects in cdc5 mutants:
Previous studies with cdc5 mutants (cdc5-1 and msd2-1) revealed that Cdc5p plays a role in mitotic exit (CHARLES et al. 1998 ; SHIRAYAMA et al. 1998 ). CDC14^TAB6-1 was isolated in a genetic screen for tab (telophase arrest bypassed) mutants that bypass the requirement for CDC15 and TEM1 function in mitotic exit (SHOU et al. 2001 ). The cdc5-1 mutant bearing the CDC14^TAB6-1 allele grew normally without any apparent defect at 37°, confirming the previous finding that the cdc5-1 is defective in mitotic exit (Fig 3A). To examine whether the cdc5-3, cdc5-4, or cdc5-7 defects could also be alleviated by the CDC14^TAB6-1 allele, CDC14^TAB6-1 was integrated at the HIS3 locus. The resulting cdc5-3 CDC14^TAB6-1, cdc5-4 CDC14^TAB6-1, and cdc5-7 CDC14^TAB6-1 strains grew relatively well at 37° (Fig 3A). However, all three mutants exhibited a chained cell morphology to varying degrees and an elongated bud phenotype (see below). Since the cdc5-1 CDC14^TAB6-1 mutant grows normally under these conditions, the slow growth rate of the cdc5-3 CDC14^TAB6-1, cdc5-4 CDC14^TAB6-1, and cdc5-7 CDC14^TAB6-1 mutants suggests that they possess additional uncharacterized defects in addition to the mitotic exit defect.

View larger version (66K): In this window In a new window Download PPT slide   Figure 3. (A) Alleviation of the growth defect of cdc5 mutants by a dominant allele of CDC14 (CDC14TAB6-1). Both wild-type and various cdc5 mutants were integrated with a dominant CDC14TAB6-1 allele at the HIS3 locus and their growth phenotype was examined after culturing the cells on YEP + 2% glucose at 37° for 3 days. (Left) CDC5, KLY2470; cdc5-1, KLY2458; cdc5-3, KLY2460; cdc5-4, KLY2462; cdc5-7, KLY2464. (Right) CDC5 CDC14TAB6-1, KLY2970; cdc5-1 CDC14TAB6-1, KLY2946; cdc5-3 CDC14TAB6-1, KLY2950; cdc5-4 CDC14TAB6-1, KLY2954; cdc5-7 CDC14TAB6-1, KLY2958. (B) Alleviation of elongated bud phenotype by swe1. To examine bud elongation in the swe1 background, swe1 was introduced into the cdc5 mutants harboring an integrated copy of the CDC14TAB6-1 allele. Strains were cultured at 37° for 3.5 hr prior to fixation. The results are the average and standard deviation derived from two independent experiments. More than 300 cells of each sample were counted after sonication. CDC5 CDC14TAB6-1, KLY2970; cdc5-3 CDC14TAB6-1, KLY2950; cdc5-4 CDC14TAB6-1, KLY2954; cdc5-7 CDC14TAB6-1, KLY2958; cdc5-3 CDC14TAB6-1 swe1, KLY3076; cdc5-4 CDC14TAB6-1 swe1, KLY3122; cdc5-7 CDC14TAB6-1 swe1, KLY3155.

Cdc5p functions in a Swe1p-dependent Cdc28p/Clb2p activation pathway:
To further characterize any additional defects in the cdc5-3 CDC14^TAB6-1, cdc5-4 CDC14^TAB6-1, and cdc5-7 CDC14^TAB6-1 strains, cells were prepared after culturing them at 37° for 3.5 hr. In contrast to CDC5 CDC14^TAB6-1, all three mutants exhibited a significant fraction of cells with large buds (Fig 3B), suggesting that they possess a defect, or defects, at a late stage of the cell cycle. Close examination of cell morphologies after sonication revealed that, unlike the CDC5 CDC14^TAB6-1 and cdc5-1 CDC14^TAB6-1 mutants, all three additional cdc5 mutants exhibited a chained cell morphology of three to five cell bodies in 17–20% of the population. In addition to this phenotype, the cdc5-3 CDC14^TAB6-1 strain exhibited an elongated bud morphology in 13% of the population, whereas other mutants possessed elongated buds in <5% of the total population (Fig 3B). Bud elongation defects occur when yeast cells fail to switch from apical growth to isotropic growth at the time of mitotic onset (BARRAL et al. 1999 ; EDGINGTON et al. 1999 ), a transition that requires a function of the Cdc28p/Clb complex (LEW and REED 1993 ). Since an elongated bud morphology associated with mutations in the Cdc28p/Clb activation pathway can be prevented by the loss of SWE1 function (BARRAL et al. 1999 ; EDGINGTON et al. 1999 ; MCMILLAN et al. 1999 ; SHULEWITZ et al. 1999 ; LONGTINE et al. 2000 ), a swe1 mutation was introduced into the three cdc5 mutants. As expected, introduction of a swe1 into the cdc5-3 CDC14^TAB6-1 mutant abrogated the elongated bud phenotype; <1% of these cells exhibited an elongated bud morphology after culturing at 37° for 3.5 hr (Fig 3B). In addition, a swe1 partially alleviated the growth defect of the cdc5-3 CDC14^TAB6-1 mutant (data not shown). Under the same conditions, introduction of a swe1 into the CDC5 CDC14^TAB6-1 or cdc5-1 CDC14^TAB6-1 strains did not appear to alter the cell morphologies (data not shown).

Since the Hsl1p-Hsl7p pathway plays a critical role in triggering a Swe1p-dependent mitotic delay in response to a septin organization defect, we examined whether Cdc5p genetically interacts with this pathway. The cdc5-3 CDC14^TAB6-1 hsl1 triple mutant grew slowly at 23°, but failed to grow at 34° (Fig 4A). Upon shifting the cultures to 37° for 3.5 hr, the triple mutant exhibited an enhanced elongated bud morphology when compared to either the cdc5-3 CDC14^TAB6-1 double or the hsl1 single mutant (Fig 4B). Under the same conditions, the cdc5-3 CDC14^TAB6-1 hsl1 swe1 mutant grew well with a cellular morphology similar to that of a wild-type strain (Fig 4A and Fig B). Together, these data suggest that Cdc5p functions at a point upstream of Swe1p, most likely in a pathway distinct from that of Hsl1p and Hsl7p.

View larger version (105K): In this window In a new window Download PPT slide   Figure 4. (A) Cdc5p functions in the Swe1p-dependent pathway. Introduction of a swe1 suppressed the synthetic lethality between cdc5-3 CDC14TAB6-1 and hsl1. Strains were cultured on YEP + 2% glucose at the indicated temperatures for 3 days. 1, isogenic wild-type KLY1546; 2, KLY3080 (cdc5-3 CDC14TAB6-1); 3, KLY2868 (hsl1); 4, KLY3170 (cdc5-3 CDC14TAB6-1 hsl1); 5, KLY3173 (cdc5-3 CDC14TAB6-1 hsl1 swe1). (B) Alleviation of enhanced bud elongation defect of the cdc5-3 CDC14TAB6-1 hsl1 mutant by swe1. Strains cultured at 23° overnight were shifted to 37° for 3.5 hr and then fixed. Strains used are the same as in A. Bar, 5 µm.

Loss of CDC5 function results in a cytokinetic defect:
A defect in cytokinesis may occur as a result of a delay at the G₂/M transition. Thus, we examined the effect of loss of SWE1 on the cytokinesis defect of various cdc5 mutants. Introduction of a swe1 into the cdc5-4 CDC14^TAB6-1 or cdc5-7 CDC14^TAB6-1 mutants did not influence the severity of the chained cell morphology, suggesting that this phenotype is not the result of a SWE1-dependent cell cycle delay. However, introduction of a swe1 into the cdc5-3 CDC14^TAB6-1 mutant decreased, but did not abolish, this phenotype (Fig 3B). To directly investigate cytokinetic defects in these mutants, strains bearing the CDC14^TAB6-1 allele were grown exponentially at 37° for 5 hr and subjected to DiI staining to reveal the cytoplasmic membrane structures of the connected cells. Under these conditions, 53% of connected cells (>80 internal mother-bud necks were counted for each mutant, and the peripheral mother-bud necks were excluded from counting) generated from these three mutants (cdc5-3 CDC14^TAB6-1, cdc5-4 CDC14^TAB6-1, or cdc5-7 CDC14^TAB6-1) possessed shared cytoplasm (Fig 5A), suggesting that loss of CDC5 function resulted in a cytokinetic defect. To visualize chitin deposition and septum formation in the cdc5 mutants, cells were stained with calcofluor and then subjected to serial optical sectioning using a confocal microscope. Most of the mother-bud necks of connected cells possessed discontinuous calcofluor signals in focal planes bisecting the cell bodies longitudinally (Fig 5B). These observations, taken together with those obtained by DiI staining, suggest that cytokinesis is either inhibited or delayed in most of the bud necks between connected cell bodies of these cdc5 mutants.

View larger version (76K): In this window In a new window Download PPT slide   Figure 5. (A) Cytokinetic defect in cdc5-7 CDC14TAB6-1 mutant. Strain KLY2958 was cultured in YEP + 2% glucose liquid medium at 23° and then shifted to 37° for an additional 5 hr before fixation with 3.7% formaldehyde. In more than one-half of the connected cell bodies (n = 113) examined, impaired membrane closure was evident by the presence of continuous DiI staining between the cell bodies (arrows). Bar, 5 µm; DIC, differential interference contrast; DiI, DiI staining. (B) Inhibition of septum formation at the internal mother-bud necks. The same samples as in A were stained with calcofluor to reveal septum structure. Cells were then subjected to confocal microscopy with a series of 100-nm sections to examine septum formation between the connected cell bodies. Discontinuous calcofluor signals (arrows) were evident in large fractions (70%, n = 51) of mother-bud necks of the connected cell bodies, indicating a failure of septum formation. Bar, 5 µm.

Aberrant septin function and actin recruitment defect in the cdc5-4 and the cdc5-7 mutants:
Septins play a critical role in recruiting cytokinetic machinery to the bud neck. They form a ring structure at the future budding site prior to bud emergence. At the time of cytokinesis, this ring disassembles concurrently with spindle disassembly (LIPPINCOTT and LI 1998A ). To investigate whether the septin structures function normally in the cytokinetically defective cdc5-7 CDC14^TAB6-1 mutant, a YFP-fused CDC10, a septin component of neck filaments, was integrated into the genome and expressed under native CDC10 promoter control. When the CDC5 CDC14^TAB6-1 strain was arrested with -factor for 3 hr at 37° and released into prewarmed medium, cells went through the cell cycle without any noticeable septin defects. In contrast, at 120 min after release, 25% of the cdc5-7 CDC14^TAB6-1 mutant cells developed a chained cell phenotype with apparent defects in the morphology and subcellular localization of the septin rings. To closely monitor the septin defect in this mutant, cells with three cell bodies were carefully examined as a function of time upon releasing from -factor block. At 90 min, 82% of cells with three cell bodies did not exhibit septin rings at the second bud neck. At 180 min, 52% of these cells exhibited this defect as buds grew. These observations are suggestive of a delay in septin relocalization to, and assembly at, this site (Fig 6A and Fig B). Consistent with this observation, the percentage of cells with septin rings at both bud necks increased from 18% at 90 min to 43% at 180 min (Fig 6A and Fig B). A similar septin relocalization/assembly defect was observed in the cdc5-4 CDC14^TAB6-1 mutant (data not shown). Provision of a centromeric CDC5 plasmid completely rescued this defect (data not shown).

View larger version (60K): In this window In a new window Download PPT slide   Figure 6. Aberrant septin function in a cdc5-7 CDC14TAB6-1 mutant. (A) The cdc5-7 CDC14TAB6-1 mutant expressing YFP-CDC10 under its native promoter (KLY3072) was arrested in G1 with -factor at 37° for 3 hr. Upon releasing these cells into fresh medium prewarmed at 37°, cells were harvested at the indicated time points and sonicated prior to examining the septin ring morphologies. A large fraction of cells at 150 min (61%, n = 203) did not possess readily visible septin rings at the second mother-bud necks with small buds (arrow in no. 1 cell of Cdc10p + Tub panel). In addition, 30% (n = 205) of cells possessed second septin rings in the absence of disassembly of the first septin ring (no. 2 cell of Cdc10p + Tub panel). More than 98% (n = 150) of the cells with four or more cell bodies did not possess septin rings at the first cytokinetic sites (arrow in no. 3 cell of Cdc10p + Tub panel). Recruited actin rings (arrows in the Actin panel) were rarely visible in the connected cells, suggesting that these cell bodies are still capable of recruiting cytokinetic machineries. Under the same conditions, the CDC5 CDC14TAB6-1 strain (KLY3075) did not exhibit any noticeable septin defect (top). A copy of a TUB-GFP fusion was additionally integrated to visualize the cell cycle stages of individual cell bodies in the connected cells. DIC, differential interference contrast; Cdc10p + Tub, YFP-Cdc10p + GFP-tubulin; Actin, actin-phalloidin staining; DAPI, DNA staining. Superimposed images are shown in overlays. Bar, 5 µm. (B) Quantification of cells with aberrant septin rings in the cdc5-7 CDC14TAB6-1 mutant. Cells defective in septin disassembly or assembly were quantified using the same samples prepared in A. Approximately 200 cells with three cell bodies were counted at each time point. Cells were classified into three groups: septin ring at the first bud neck (no. 1 neck only), at the first and second bud necks (no. 1 and no. 2 neck), and at the second neck (no. 2 neck only). Percentages of cells in each group were determined at each time point and plotted as a function of time after release from -factor block. (C) The cdc5-7 CDC14TAB6-1 (strain KLY3578) cells grown at 23° were shifted to 37° for 4 hr and then harvested to examine the septin ring morphologies. In 23% (n = 1261) of the population, septin rings are loosely organized, as revealed by YFP-Cdc10p signals. Septin rings are considered aberrant when the width (the longitudinal length that spans the two cell bodies) is longer than the height (the length along the mother-bud neck). Weak actin bars (arrows) were still visible in 65% (n = 212) of the cells with aberrant septin rings. Bar, 5 µm.

Since the delayed septin relocalization/assembly might have resulted from a defect in septin structure or stability, we carefully examined septin ring morphologies after culturing the cells at 37° for 4 hr. In asynchronously growing cells, 35% (n = 220) of the cdc5-4 mutant and 54% (n = 232) of the cdc5-7 mutant developed abnormally elongated septin rings (Table 2; see the definition of abnormal septin rings in the Fig 6C legend) as evidenced by unusually extended YFP-Cdc10p signals across the mother-bud neck of large-budded cell bodies (Fig 6C). Immunostaining with an anti-Cdc11 antibody resulted in a similar septin ring morphology as visualized with the YFP-Cdc10p fusion protein (data not shown). Provision of a centromeric CDC5 plasmid completely rescued this defect (data not shown). Under the same conditions, the cdc5-1 mutant, which is apparently defective only in mitotic exit, possessed aberrant septin rings in <2% (n = 300) of the population. In addition, mitotic exit mutants such as tem1-3, cdc15-2, and dbf2-2 did not exhibit this defect (C. J. PARK and K. S. LEE, unpublished data). This observation suggests that induction of an aberrant septin ring is not the result of mitotic exit failure and that cdc5-4 and cdc5-7 are specifically defective in proper septin structure and function.

To investigate whether the aberrant septin rings are generated at a specific point of the cell cycle, the cdc5-4 and cdc5-7 mutants were cultured under various conditions and aberrant septin rings were counted. When cells were arrested with -factor at 23° and then released to 37° for 4 hr, 70% of the cdc5-4 mutant and 80% of the cdc5-7 mutant exhibited aberrant septin ring morphologies, which was abrogated in the presence of nocodazole (Table 2). Since the cdc5-4 and cdc5-7 mutants are also defective in mitotic exit, these observations suggest that aberrant septin rings are induced at a point after the nocodazole block, but prior to mitotic exit. Consistent with this notion, both the cdc5-4 and the cdc5-7 mutants bearing the CDC14^TAB6-1 allele (cdc5-4 CDC14^TAB6-1 and cdc5-7 CDC14^TAB6-1, respectively) exhibited significantly decreased aberrant septin structures under the same culture conditions (Table 2).

Since septin ring structure is pivotal for recruiting cytokinetic machinery, the observed defect in septin structure and function may have directly contributed to the cytokinesis defect. Thus, we examined actin localization in cells with fully elongated spindles. Approximately 54% (n = 220) of the cdc5-4 CDC14^TAB6-1 mutant and 46% (n = 216) of the cdc5-7 CDC14^TAB6-1 mutant possessed visible actin rings after 4 hr at 37°, whereas 87% (n = 187) of the CDC5 CDC14^TAB6-1 mutant possessed distinct actin rings under the same conditions (Table 3). Taken together, it is likely that a defect in septin function in the cdc5-4 and cdc5-7 mutants may have resulted in delayed actin recruitment leading to a cytokinetic defect.

Cdc5p contributes to cytokinesis independently of Bfa1p and Bub2p:
It has been recently demonstrated that Cdc5p contributes to the phosphorylation of Bfa1p in vivo (HU et al. 2001 ; LEE et al. 2001B ), suggesting that Cdc5p controls the MEN by directly regulating Bfa1p function. Since the MEN is required for normal cytokinesis (JIMENEZ et al. 1998 ; FRENZ et al. 2000 ; LEE et al. 2001A ), we investigated whether the cytokinetic defect associated with the cdc5-4 and cdc5-7 mutants is due to a failure to negatively regulate the Bfa1p/Bub2p-dependent inhibition of the MEN. As observed with the provision of CDC14^TAB6-1, introduction of a bfa1 or a bub2 suppressed the growth defect of all four cdc5 mutants (cdc5-1, cdc5-3, cdc5-4, and cdc5-7) at 37° (data not shown). However, a bfa1 or a bub2 failed to suppress the chained cell phenotype of the cdc5-3, cdc5-4, and cdc5-7 mutants, when compared with the respective mutants bearing the CDC14^TAB6-1 allele (Table 4). In addition, DiI staining revealed that, among the chained cells, 40% of the cdc5-7 bfa1 and cdc5-7 bub2 mutants possessed connected cytoplasms (data not shown). These observations suggest that Cdc5p contributes to cytokinesis independently of the Bfa1p/Bub2p-dependent regulation of the MEN.

	DISCUSSION

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

Data obtained from various organisms suggest that polo kinases play important roles at multiple points of M-phase progression and that their roles are largely conserved among evolutionarily distant organisms. In budding yeast, ectopic expression of the mammalian polo-like kinase PLK1 complements the mitotic exit defect associated with the cdc5-1 mutation (LEE and ERIKSON 1997 ), indicating that at least polo kinase-dependent APC activation is conserved between budding yeast and mammals. Studies with a dominant-negative cdc5 mutant (SONG and LEE 2001 ) and the work reported here suggest that, like the proposed cytokinetic roles of polo kinases in other eukaryotic organisms (OHKURA et al. 1995 ; MUNDT et al. 1997 ; ADAMS et al. 1998 ; BAHLER et al. 1998 ; CARMENA et al. 1998 ), Cdc5 is required for normal cytokinesis.

Besides its roles in mitotic exit and cytokinesis, Cdc5p has also been implicated in the regulation of Swe1p. BARTHOLOMEW et al. 2001 have reported that overexpression of wild-type CDC5 or kinase-inactive CDC5/N209A under control of the GAL1 promoter leads to Swe1p phosphorylation. In addition, expression of GAL1-CDC5/N209A suppresses Swe1p-dependent elongated bud formation in hsl1 or hsl7 mutants (BARTHOLOMEW et al. 2001 ). These authors have suggested that Cdc5p may function as a negative regulator of Swe1p (BARTHOLOMEW et al. 2001 ). In support of this notion, we have observed that the cdc5-3 CDC14^TAB6-1 mutant exhibits an elongated bud phenotype. The cdc5-3 CDC14^TAB6-1 hsl1 triple mutant exhibited enhanced bud elongation and synthetic lethality at 34°. Introduction of a swe1 into the cdc5-3 CDC14^TAB6-1 hsl1 mutant abrogated this elongated bud phenotype and suppressed the growth defect, indicating that Cdc5p functions in the Swe1p-dependent checkpoint pathway. Defects in septin assembly cause a G₂ delay, resulting in a filamentous phenotype (BARRAL et al. 1999 ; EDGINGTON et al. 1999 ; SHULEWITZ et al. 1999 ; LONGTINE et al. 2000 ). However, only 3% (n = 355) of the cdc5-3 CDC14^TAB6-1 mutant exhibited aberrant septin ring structures, in comparison to 21% (n = 260) of the cdc5-7 CDC14^TAB6-1 mutant under the same conditions. In addition, the cdc5-4 CDC14^TAB6-1 and cdc5-7 CDC14^TAB6-1 mutants, which possess a gross septin defect, exhibited fewer cells with elongated buds than did the cdc5-3 CDC14^TAB6-1 mutant. These observations suggest that the elongated bud morphology in the cdc5-3 CDC14^TAB6-1 mutant may likely be due to a defect in mediating the septin-dependent inactivation of Swe1p rather than to an indirect septin perturbation effect. How Cdc5p relates its activity to other regulatory components of the Swe1p-dependent pathway and regulates the activation of Cdc28p/Clb remains to be investigated further.

The cdc5-4 CDC14^TAB6-1 mutant and the cdc5-7 CDC14^TAB6-1 mutant displayed delayed septin localization to the incipient bud site in chained cells. These mutants also exhibited aberrant septin ring morphologies, as evidenced by the presence of loosely organized YFP-Cdc10p signals at the bud necks. However, these mutants did not appear to possess a significant defect in the G₂/M transition, as judged by lack of an elongated bud morphology at the restrictive temperature. These observations suggest that the apparent septin defect in these mutants is not sufficient to trigger a G₂ delay, although it may be sufficient to contribute to the cytokinetic failure. Close examination of septin structures in the cdc5-4 and cdc5-7 mutants revealed that a high percentage of aberrant septin rings is induced between early mitosis and mitotic exit, a period in which septin disassembly and relocalization do not occur. In contrast, aberrant septin structures were not observed in other mutants in the MEN such as tem1-3, cdc15-2, or dbf2-2 (C. J. PARK and K. S. LEE, unpublished data). These observations suggest that a Cdc5p-dependent mitotic activity is likely to be important for proper septin function and therefore for normal cytokinesis. In addition, a relatively low penetrant defect associated with the cdc5-4 or cdc5-7 mutation suggests that alternative pathway(s) may exist to compensate the cytokinetic defect associated with these mutations. In support of this argument, the cdc5-3 mutant with largely normal septin ring structures (3% of aberrant septin rings in cdc5-3 CDC14^TAB6-1 as opposed to 21% in cdc5-7 CDC14^TAB6-1 as judged by fluorescent YFP-Cdc10p signals at the neck) still exhibits a significant cytokinetic defect even in the swe1 background (Fig 3B). Although it may be difficult to assess the functionality of septins by neck-localized YFP-Cdc10p signals, this observation suggests the possibility of a septin-organization-independent cytokinesis failure in this mutant. A deeper understanding of how Cdc5p contributes to normal cytokinesis and septin function may require identification of additional Cdc5p interacting proteins and physiological substrates important for this event.

Recent reports have shown that mutations in components of the MEN such as Cdc5p, Tem1p, Cdc15p, Dbf2p, and Cdc14p result in a defect in actin ring formation (FRENZ et al. 2000 ; LEE et al. 2001A ). In addition, LIPPINCOTT et. al. (2001) have reported that the tem1^- mutant exhibits a defect in the actin ring and septin ring dynamics, when its mitotic exit defect is alleviated. Cdc5p has been shown to phosphorylate and negatively regulate Bfa1p (HU et al. 2001 ), which may form a two-component GAP with Bub2p for the Tem1p GTPase. In an attempt to examine whether the cytokinetic defect of cdc5-4 and cdc5-7 could be the result of a failure to regulate Bfa1p/Bub2p properly, we examined whether introduction of a bfa1 or a bub2 alleviates the cytokinetic defect associated with loss of CDC5 function. We found that introduction of a bfa1 or a bub2 into the cdc5 mutants resulted in a degree of cytokinetic defect similar to that resulting from the introduction of CDC14^TAB6-1. These observations indicate that the cytokinetic defect associated with the loss of CDC5 function is independent of Bfa1p/Bub2p function or activity (Fig 7). However, whether or not Cdc5p contributes to cytokinesis by activating the MEN downstream of Tem1p is not yet clear. Recently, LEE et al. 2001A have shown that Cdc5p is required for Dbf2p kinase activity even in the absence of Bub2p, suggesting that Cdc5p may also contribute to Dbf2p activity through a Bfa1p/Bub2p-independent pathway. Whether Cdc5p contributes to the regulation of cytokinesis by directly regulating Dbf2p activity or by a yet-unidentified pathway requires further investigation (Fig 7).

View larger version (18K): In this window In a new window Download PPT slide   Figure 7. Model proposing the Bfa1p/Bub2p-independent cytokinetic role of Cdc5p. Cdc5p regulates Bfa1p/Bub2p-dependent MEN activity, which is also shown to be important for septin ring dynamics (LIPPINCOTT et al. 2001 ) and actin ring formation (FRENZ et al. 2000 ; LEE et al. 2001A ). The data reported here show that the cdc5-7 mutant exhibits a cytokinetic defect even in the absence of Bfa1p or Bub2p, indicating the presence of a Bfa1p/Bub2p-independent cytokinetic pathway.

Data obtained from various organisms show that polo kinases play multiple roles during M-phase progression. In mammalian cells, Plk localizes at centrosomes in G₂ and early mitosis and at the midbody in late mitosis and cytokinesis. The C-terminal domain of Plk is sufficient to localize at these sites (Y. S. SEONG and K. S. LEE, unpublished data). In budding yeast, Cdc5p localizes at the spindle pole bodies. Later in the cell cycle, Cdc5p localizes to bud neck in both the polo-box- and the septin-dependent manner (SONG and LEE 2001 ; C. J. PARK and K. S. LEE, unpublished data). The dynamic subcellular localization of polo kinases presages their diverse functions in various organisms. The data presented here suggest that the C-terminal domain of Cdc5p is required for interacting with various physiological binding partners important for G₂/M transition, mitotic exit, and cytokinesis. Although the morphological features and timing of certain events during M-phase progression are strikingly different between budding yeast and mammalian cells, spatial regulation of polo kinases may be critical for coordinating multiple mitotic events in all eukaryotes.

	ACKNOWLEDGMENTS

We are grateful to Dan Ilkovitch for technical support. We thank Jeremy Thorner (University of California-Berkeley) for providing the hsl1 strain, Susan Garfield for helping with confocal microscopy, and Jim McNally and Tatiana Kapova for processing confocal images obtained at the LRBGE Fluorescence Imaging Facility in NCI/DBS.

Manuscript received May 31, 2002; Accepted for publication October 9, 2002.

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