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

RESULTS

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 (Charleset al. 1998; Shirayamaet al. 1998). In addition, overexpression studies (Bartholomewet 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; Figure 1A) that exhibited a temperature-sensitive cell growth defect (Figure 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° (Figure 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 α-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 (Figure 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 (Figure 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.

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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. Δ, 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 α-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 wildtype CDC5 (Figure 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° (Figure 2B).

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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 × 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 (Charleset al. 1998; Shirayamaet 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 (Shouet 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 (Figure 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° (Figure 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.

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 (Figure 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 (Figure 3B). Bud elongation defects occur when yeast cells fail to switch from apical growth to isotropic growth at the time of mitotic onset (Barralet al. 1999; Edgingtonet 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 (Barralet al. 1999; Edgingtonet al. 1999; McMillanet al. 1999; Shulewitzet al. 1999; Longtineet al. 2000), a swe1Δ mutation was introduced into the three cdc5 mutants. As expected, introduction of a swe1Δ 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 (Figure 3B). In addition, a swe1Δ partially alleviated the growth defect of the cdc5-3 CDC14^TAB6-1 mutant (data not shown). Under the same conditions, introduction of a swe1Δ into the CDC5 CDC14^TAB6-1 or cdc5-1 CDC14^TAB6-1 strains did not appear to alter the cell morphologies (data not shown).

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Figure 3.

—(A) Alleviation of the growth defect of cdc5 mutants by a dominant allele of CDC14 (CDC14^TAB6-1). Both wild-type and various cdc5 mutants were integrated with a dominant CDC14^TAB6-1 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 CDC14^TAB6-1, KLY2970; cdc5-1 CDC14^TAB6-1, KLY2946; cdc5-3 CDC14^TAB6-1, KLY2950; cdc5-4 CDC14^TAB6-1, KLY2954; cdc5-7 CDC14^TAB6-1, KLY2958. (B) Alleviation of elongated bud phenotype by swe1Δ. To examine bud elongation in the swe1Δ background, swe1Δ was introduced into the cdc5 mutants harboring an integrated copy of the CDC14^TAB6-1 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 CDC14^TAB6-1, KLY2970; cdc5-3 CDC14^TAB6-1, KLY2950; cdc5-4 CDC14^TAB6-1, KLY2954; cdc5-7 CDC14^TAB6-1, KLY2958; cdc5-3 CDC14^TAB6-1 swe1Δ, KLY3076; cdc5-4 CDC14^TAB6-1 swe1Δ, KLY3122; cdc5-7 CDC14^TAB6-1 swe1Δ, KLY3155.

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 hsl1Δ triple mutant grew slowly at 23°, but failed to grow at 34° (Figure 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 hsl1Δ single mutant (Figure 4B). Under the same conditions, the cdc5-3 CDC14^TAB6-1 hsl1Δ swe1Δ mutant grew well with a cellular morphology similar to that of a wild-type strain (Figure 4, A and 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.

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Figure 4.

—(A) Cdc5p functions in the Swe1p-dependent pathway. Introduction of a swe1Δ suppressed the synthetic lethality between cdc5-3 CDC14^TAB6-1 and hsl1Δ. Strains were cultured on YEP + 2% glucose at the indicated temperatures for 3 days. 1, isogenic wild-type KLY1546; 2, KLY3080 (cdc5-3 CDC14^TAB6-1); 3, KLY2868 (hsl1Δ); 4, KLY3170 (cdc5-3 CDC14^TAB6-1 hsl1Δ); 5, KLY3173 (cdc5-3 CDC14^TAB6-1 hsl1Δ swe1Δ). (B) Alleviation of enhanced bud elongation defect of the cdc5-3 CDC14^TAB6-1 hsl1Δ mutant by swe1Δ. 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 swe1Δ 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 swe1Δ into the cdc5-3 CDC14^TAB6-1 mutant decreased, but did not abolish, this phenotype (Figure 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 (Figure 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 (Figure 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.

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Figure 5.

—(A) Cytokinetic defect in cdc5-7 CDC14^TAB6-1 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 α-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 α-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 (Figure 6, A and 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 (Figure 6, A and 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).

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 Figure 6C legend) as evidenced by unusually extended YFP-Cdc10p signals across the mother-bud neck of large-budded cell bodies (Figure 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 α-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 (Huet al. 2001; Leeet al. 2001b), suggesting that Cdc5p controls the MEN by directly regulating Bfa1p function. Since the MEN is required for normal cytokinesis (Jimenezet al. 1998; Frenzet al. 2000; Leeet 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 bfa1Δ or a bub2Δ 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 bfa1Δ or a bub2Δ 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 bfa1Δ and cdc5-7 bub2Δ 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.

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Figure 6.

—Aberrant septin function in a cdc5-7 CDC14^TAB6-1 mutant. (A) The cdc5-7 CDC14^TAB6-1 mutant expressing YFP-CDC10 under its native promoter (KLY3072) was arrested in G₁ with α-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 CDC14^TAB6-1 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 CDC14^TAB6-1 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 α-factor block. (C) The cdc5-7 CDC14^TAB6-1 (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.