INTRODUCTION

Top Abstract Introduction Materials and Methods Results Discussion References

The proper control of polarized growth and cellular morphology is crucial for both eukaryotic developmental processes and the specialized function of diverse cell types. For example, the formation of polarized cell structures is important for such diverse processes as the interaction of helper T cells with antigen-presenting B cells (25), nutrient absorption by the microvilli of epithelial cells (37), and flower pollen tube growth (3). The mechanisms by which cells mediate and regulate polarized cell growth are poorly understood.

In the budding yeast Saccharomyces cerevisiae, polarized growth is required for budding during vegetative growth and for projection formation during the mating response. Bud growth occurs in two phases, apical growth and isotropic growth (28). Apical bud growth occurs in G₁, when cell wall deposition is restricted to the tip of the bud. Upon entry into mitosis, buds switch to isotropic growth, in which growth still occurs in the bud but is no longer restricted to the tip and instead occurs throughout the entire bud surface (28). The balance between apical and isotropic bud growth determines the shape of the resulting cell. The identification of components important for these different growth phases and how they are regulated is therefore important for understanding how cell shape is controlled in yeast.

Yeast polarized cell growth involves the coordinated function of polarity-regulating proteins, organization of cytoskeletal components, and regulation of signal transduction cascades (11, 30). Numerous components important for polarized cell growth in yeast are known; however, the number that have been shown to function specifically during apical growth is limited. Thus far, only a few proteins, the polarity components Spa2p, Pea2p, Bud6p, and Bni1p and the Pak1 homolog Ste20p, have been shown to be specifically required for apical growth (44). Many yeast proteins important for polarized growth have functional homologs in other organisms, suggesting that the molecular mechanisms underlying these processes are highly conserved among eukaryotes.

One important aspect of polarized growth is cell wall synthesis and remodeling (7). Yeast cells maintain a rigid cell wall that protects them from osmotic and mechanical stress. This wall must be partially degraded in a localized fashion for polarized cell growth to occur. Localized degradation of cell wall components must also occur following cytokinesis to allow separation of mother and daughter cells. It is likely that cell wall remodeling is tightly controlled both spatially and temporally by the cell polarization and cell cycle regulatory machinery; failure to do so would presumably result in cell lysis. How cells accomplish this control is not well understood.

To identify genes involved in apical growth, we have employed a transposon-based mutagenesis system (42) to screen for mutations that alter the elongated bud morphology of cells arrested during the apical growth phase. Here, the characterization of one gene identified in this screen, CBK1, is reported. This gene is predicted to encode a highly conserved protein kinase; we demonstrate that CBK1 is important for apical growth, mating projection formation, cell separation, and the transcriptional regulation of many cell wall-related genes.

	MATERIALS AND METHODS

Top Abstract Introduction Materials and Methods Results Discussion References

Yeast strains and media. Yeast strains used in this study (Table 1) were congenic with S288c and W303. Standard genetic methods and growth media were used as described previously (18). Deletions of the entire protein coding regions of CBK1, ACE2, and HYM1 were produced by the PCR method described by Baudin et al. (2). Deletions were confirmed by PCR. Strains containing 3xHA-tagged CBK1 alleles were generated using plasmids from an ordered collection of transposon-mutagenized genomic clones. The details of this method have been described previously (43) and can be accessed online at http://ygac.med.yale.edu/. Correct tagging was confirmed by PCR and immunoblot analysis.

Morphological analysis and fluorescence microscopy. Exponentially growing cells were treated as indicated and fixed for 1 h with 3.7% formaldehyde. Fixed cells were washed and resuspended in phosphate-buffered saline (PBS) plus 1.2 M sorbitol. For cell shape analysis, the length (long axis relative to the birth pole) and width (maximum distance perpendicular to the length) of budded cells were measured from images recorded using a Sensys charge-coupled device camera and Imagepoint Lab Spectrum software (Signal Analytics Corporation). To quantify the severity of cell separation defects, exponentially growing cells were fixed and the number of cells in random clusters was recorded and averaged. For quantitative analysis of mating projection formation defects, early log phase cells were treated with -factor (Sigma, St. Louis, Mo.) at 5 µg/ml for 2 h, fixed, and analyzed. Mating projections that had a length (distance from base to tip of projection) greater than half the diameter of the cell were scored as normal. We stained F-actin with rhodamine-phalloidin (Molecular Probes, Eugene, Oreg.) using previously described methods (31). To visualize localization of cell wall chitin, cells were treated with calcofluor at a final concentration of 2 µg/ml. Stained cells were analyzed by fluorescence microscopy using a Leitz Aristoplan microscope. Images were captured using a Sensys charge-coupled device camera and Imagepoint Lab Spectrum software (Signal Analytics Corporation); subsequent image processing was done using Adobe PhotoShop software (Adobe Systems, Sunnyvale, Calif.).

Electron microscopy. Exponentially growing cells were harvested by filtration, frozen at high pressure, cryofixed, and embedded in Epon-Araldite resin as described elsewhere (33); freeze substitution was done in 2% osmium tetroxide plus 0.1% uranyl acetate. Serial sections were examined and micrographs were taken using a Philips CM10 electron microscope. Negatives were digitally scanned directly at a 1,600-dot-per-in. resolution using an Epson ES-1200C transparency scanner (Epson, Long Beach, Calif.) to create 8-bit tagged image files, and these images were processed using Adobe Photoshop software (Adobe Systems, Sunnyvale, Calif.).

FITC-ConA analysis of apical growth. To label cell wall mannoproteins, exponentially growing cells were collected, washed once with PBS, and incubated in the dark with fluorescein isothiocyanate (FITC)-concanavalin A (ConA) (Polysciences Inc., Warrington, Pa.) at a final concentration of 100 µg/ml for 10 min at 25°C. The cells were then washed with PBS and returned to growth at 30°C in fresh YPAD medium. After 25 min, cells were fixed, washed with PBS, and observed by fluorescence microscopy. Buds with completely faded staining at the tip were categorized as undergoing apical growth, while buds with uniformly decreased staining throughout the surface were categorized as undergoing isotropic growth. Buds with a gradient of staining that became increasingly faded toward the bud tip were categorized as gradient.

Halo and FUS1-lacZ induction assays. For analysis of pheromone-induced growth arrest, sterile filter disks were saturated with -factor at 5 µg/ml and placed in the middle of freshly plated lawns of exponentially growing MATa bar1 or MATa cbk1 bar1 cells. Plates were photographed after 2 days of growth at 30°C. Analysis of FUS1-lacZ reporter construct induction was carried out using yeast strains (DDY2081 and DDY2082) carrying a centromeric plasmid-borne FUS1-lacZ reporter construct as described elsewhere (51). Results show the averages and standard deviations of three independent trials for each condition.

Analysis of bud site selection. Cells maintained in exponential growth for at least 4 consecutive generations were fixed in 3.7% formaldehyde, washed with PBS, stained with calcofluor at a final concentration of 2 µg/ml, and visualized by fluorescence microscopy. In wild-type diploid cells, bud site selection for the first three budding events was determined by analyzing the position of newly emerging buds in cells with zero, one, or two bud scars (13). Buds emerging from the one-third of the cell opposite the birth scar (a chitin-poor region of daughter cell walls where separation from the mother occurred) were scored as distal. Buds emerging from the one-third of the cell closest to the birth scar were scored as proximal. Buds emerging within the middle third of the cell were scored as medial. The vast majority of cbk1 cells fail to separate promptly following cytokinesis and therefore do not have birth scars. To quantify bud site selection in cbk1 cells, we analyzed small clusters of cells in which a progenitor cell with a birth scar could be clearly identified. The budding history in these clusters can be accurately determined by analyzing the pattern of the chains of cells that emerge from the progenitor cell. The sites chosen for the first three budding events were categorized by the criteria described above. At least 100 cells of each type (first, second, and third buds) were analyzed for each strain.

Gene expression analysis. Northern analysis of CTS1 mRNA levels was conducted by blotting total RNA as described elsewhere (1). ³²P-labelled CTS1, ACT1, and EGT2 probe was produced by random hexamer priming in the presence of [-³²P]dCTP of a gel-purified PCR fragment produced from yeast genomic DNA using primers specific for the appropriate genes. Blots were scanned and quantified using a PhosphorImager and ImageQuant software (Molecular Dynamics, San Jose, Calif.). Whole-genome transcriptional analyses were carried out using genomic DNA microarrays generously provided by Joe DeRisi (University of California, San Francisco), using methods described elsewhere (8); detailed protocols are available at http://zenith.berkeley.edu/~seidel/Protocols/. Briefly, poly(A) mRNA was prepared from asynchronously growing cbk1 mutant (DDY2080) and wild-type (DDY759) cells, as well as from asynchronous cbk1 using a Qiagen Oligotex kit as specified by the manufacturer. Cy3- and Cy5-labeled cDNA probes were prepared and hybridized to DNA arrays. Arrays were scanned using a GSI Luminomics Scannarray 3000 slide scanner, and the resulting images were processed using Scanalyze software (M. Eisen; freely available at http://www.microarrays.org). Spots with a correlation coefficient of less than 0.35 were not included in the expression analysis. The complete set of data is available at http://zenith.berkeley.edu/amad/cgi-bin/index.pl.

Indirect immunolocalization of epitope-tagged Cbk1p. Indirect immunofluorescence was performed as described previously (14). Exponentially growing cells were fixed with 3.7% formaldehyde for 20 min, washed twice with PBS, and spheroplasted at 37°C for various amounts of time with Zymolyase 100T at 5 µg/ml in PBS-1.2 M sorbitol. Cells were then washed twice with PBS-sorbitol and transferred to polylysine-coated slides. Cells were washed once with PBS-0.1% bovine serum albumin (BSA), twice with PBS-0.1% BSA-0.1% NP-40, and once again with PBS-0.1% BSA. Cells were incubated overnight at 4°C with monoclonal anti-hemagglutinin (HA) (16B12; Covance) and rabbit anti-yeast -tubulin (Tub2p) antibodies (gift of F. Solomon, Massachusetts Institute of Technology). After four washes (as previously), cells were incubated with Cy3-conjugated goat anti-mouse antibodies (Jackson ImmunoResearch Laboratories, Inc., West Grove, Pa.) and FITC-conjugated goat anti-rabbit antibodies at room temperature for 1.5 h. Cells were then washed, mounted with a solution containing 4',6'-diamidino-2-phenylindole (DAPI) and analyzed by fluorescence microscopy as described above.

	RESULTS

Top Abstract Introduction Materials and Methods Results Discussion References

Cbk1p belongs to a family of closely related eukaryotic protein kinases. CBK1 was identified in a screen for genes involved in the apical growth phase of bud formation. The details and full results of this screen will be presented elsewhere. When grown at the restrictive temperature, cdc34-2 cells arrest in the apical growth phase and form multiple highly elongated buds (17) (Fig. 1). We introduced an ordered collection of transposon disruption alleles (43) into cdc34-2 cells and screened for mutants with altered bud morphology. We found that cdc34-2 cbk1-mTn cells form multiple buds that are much less elongated than those of cdc34-2 cells (Fig. 1). The average bud length of cdc34-2 cells grown at the restrictive temperature for 7 h is 3.8 ± 1.5 µm (n = 103), while the average bud length of cdc34-2 cbk1-mTn cells is 2.5 ± 1.0 µm (n = 95). This phenotype suggests that the CBK1 gene is required for efficient apical growth.

View larger version (50K): [in this window] [in a new window]   FIG. 1.   Morphology of cdc34-2 and cdc34-2 cbk1 cells grown at the restrictive temperature. Cells were grown to mid-logarithmic phase at 25°C in YPAD, shifted to 37°C for 7 h, and then fixed with formaldehyde and photographed. Bar, 5.0 µm.

View larger version (138K): [in this window] [in a new window]   FIG. 2.   Sequence comparison of Cbk1p-related protein kinases. (A) The predicted amino acid (aa) sequences of Cbk1p from S. cerevisiae, Orb6 from S. pombe (53), Cot-1 from N. crassa (55), human Ndr (34), Drosophila Trc (Ndr) (15), C. elegans SAX-1 (Ndr) (56), N. tabacum kinase X71057 (GenBank accession no. X71057), and Drosophila melanogaster LATS (21, 54) were compared using the program CLUSTALW (48). Kinase domains (19) are shown in black, and the percent amino acid identity of the kinase domains with the Cbk1p kinase domain is shown in white. Regions outside the putative catalytic domain are crosshatched. The insert between subdomains VII and VIII of the catalytic domain (crosshatched region in the middle of the kinase domain) is characteristic of this family of protein kinases. Several members of this kinase family also have glutamine-rich regions in the sequence N terminal of their catalytic domains. (B) Multiple-sequence alignment of the catalytic domain and surrounding sequences of the Cbk1p-related kinases was performed using CLUSTALW. Shading was done using MacBoxShade (M. Baron). Shaded residues are conserved in at least five of the eight sequences. Black shading indicates identity with Cbk1p, while grey shading indicates similarity. Kinase subdomains are labeled above the sequence in roman numerals. Numbered boxed sequences indicate functionally significant regions discussed in the text. Boxed region 1, directly preceding the kinase domain, overlaps the Ca2+/S100B-binding domain of Ndr. Boxed region 2, directly preceding kinase subdomain VIII, indicates a conserved motif that overlaps the activation loop phosphorylation site in human Ndr. Boxed region 3, near the carboxy terminus, corresponds to the hydrophobic motif that is found in many AGC family kinases. Residues corresponding to experimentally determined sites of regulatory phosphorylation in human Ndr are marked with arrows.

Disruption of CBK1 alters cellular morphology and causes cell separation defects. In order to further analyze its cellular function, we constructed strains lacking the entire CBK1 open reading frame in both the S288C and W303 genetic backgrounds. The two strains produced similar results. For brevity, the data presented here are for S288C background strains only, except where otherwise noted. cbk1 cells have no obvious defects in growth rate when incubated on solid medium or in liquid culture, regardless of temperature or genetic background. In addition, cytological analysis indicates that cell cycle progression is not affected in cbk1 cells (data not shown). However, cbk1 cells have altered cellular morphologies (Fig. 3). Wild-type diploid cells are oval shaped (Fig. 3), with an average length/width ratio of 1.4 ± 0.2 (n = 102). In contrast, cbk1 diploid cells are rounder (Fig. 3), with an average length/width ratio of 1.1 ± 0.1 (n = 107). In addition to the changes in cell shape, cbk1 mutants form clumps of cells that are resistant to sonication. The cell separation defect is most severe in diploid cbk1 mutants, with the average clump containing 90 ± 64 (n = 40) cells (Fig. 3). Similar phenotypes are observed in haploid cbk1 cells (data not shown).

View larger version (101K): [in this window] [in a new window]   FIG. 3.   Morphology of wild-type (WT) and cbk1, ace2, and cbk1-mTn3F1 mutant homozygous diploid cells. Cells were grown to early logarithmic phase in YPAD and then fixed with formaldehyde and photographed. Bar, 10 µm.

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View larger version (53K): [in this window] [in a new window]   FIG. 4.   Electron microscopy of high-pressure-frozen-freeze-substituted cbk1 cells. Black arrowheads indicate regions of shared cell wall (CW) material. No cytoplasmic connections were observed in adjacent sections at any of these sites. Bar, 0.5 µm.

Efficiency of apical growth is diminished in cbk1 cells. The decreased hyperpolarized growth observed in cdc34-2 cbk1-mTn cells (Fig. 1) and the round shape of cbk1 cells (Fig. 3) suggest that Cbk1p plays a role in maintaining apical growth. To test this directly, we visually monitored cell surface growth with FITC-ConA, which binds cell wall mannose (50). Exponentially growing wild-type or cbk1 diploid cells were labeled with FITC-ConA for 10 min and then introduced to fresh medium for 25 min. The cells were then fixed and scored for staining pattern. In these experiments, regions where new cell wall growth has occurred have reduced staining intensity. Of the wild-type cells with labeled buds, 36% (n = 132) had a completely unlabeled region at the tip of the bud (Fig. 5), indicating that these cells were in the apical growth phase during the time following FITC-ConA labeling. Nineteen percent exhibited a gradient of staining that decreases toward the bud tip, leaving the tip still partially labeled (Fig. 5), indicating that some apical growth occurred after the time of labeling. Forty-five percent had uniform staining throughout the bud that was of reduced intensity (Fig. 5), indicative of isotropic growth. Thus, in wild-type cells, 55% of buds experienced some apical growth in the 25-min period after they were labeled. In comparison, only 2% (n = 127) of labeled cbk1 buds were completely unlabeled at the tip. However, 42% had a detectable gradient of staining (Fig. 5), indicating that some apical growth had occurred. Fifty-six percent of labeled cbk1 buds had uniformly decreased staining and were thus growing isotropically in the time following labeling (Fig. 5). Therefore, in cbk1 cells, 44% of buds underwent a period of at least partial apical growth after they were labeled. Thus, there was a striking difference in the fractions of wild-type and cbk1 cells with completely unlabeled bud tips (36% in the wild type versus 2% in the cbk1 mutant). This suggests that cbk1 cells are less efficient in restricting growth to a small region at the bud tip. In summary, these results suggest that the duration of the apical growth phase in cbk1 cells is similar to that in the wild type but the ability to concentrate apical growth at the bud tip is decreased during this phase.

View larger version (59K): [in this window] [in a new window]   FIG. 5.   Analysis of apical growth in wild-type (WT) and cbk1 mutant cells by FITC-ConA pulse-labeling. (A) Exponentially growing wild-type and cbk1 mutant diploid cells were pulse-labeled with FITC-ConA for 10 min, washed, and returned to growth in fresh medium. After 25 min, cells were fixed and observed by fluorescence microscopy. FITC-ConA fluorescence images are shown above differential interference contrast images. Staining that is completely faded at the bud tip indicates apical growth (Tip), while uniformly decreased staining throughout the bud indicates isotropic growth (Isotropic). Cells with a gradient of staining that was not completely faded at the bud tip were also observed (Gradient). (B) The pattern of fading in labeled buds was quantitated for wild-type and cbk1 diploid cells. Labeled buds were divided into three categories as described above: labeled buds with a completely faded tip (tip), labeled buds with staining that faded toward the tip but with staining at the tip still visible (gradient), and labeled buds with uniformly faded staining (isotropic). Bar, 5.0 µm.

CBK1 is required for the formation of normal mating projections in response to pheromone. Since polarized growth is critical for mating projection formation, we tested the ability of cbk1 cells to form mating projections in response to pheromone. After a 2-h treatment with -factor mating pheromone, 93% (n = 148) of wild-type cells form normal-sized (at least half a cell diameter in length) mating projections (Fig. 6). In contrast, only 8% (n = 203) of cbk1 cells formed normal mating projections (Fig. 6). Interestingly, a majority (74%) of the cbk1 cells developed one or more small surface bumps (Fig. 6, arrows). While much smaller than normal mating projections, these small protrusions were flanked by regions of increased chitin deposition, as seen in normal mating projections (Fig. 6, insets). In order to rule out the possibility that the mating projection defect observed in cbk1 mutants is due simply to a delayed response to pheromone, we tested the effect of longer treatments with pheromone. After a 6-h incubation with -factor, nearly all wild-type cells formed at least one normal-sized projection (98%, n = 160) and 66% formed two or more normal-sized projections (Fig. 6). In contrast, only 9% (n = 175) of the cbk1 cells formed at least one normal-sized mating projection (Fig. 6). As with the 2-h induction, most (76%) of the cbk1 cells formed small surface bumps and 58% had two or more of these apparently defective mating projections (Fig. 6). These results indicate that Cbk1p is not required for the establishment of a mating projection but is required for maintenance of persistent polarized growth of the structure.

View larger version (91K): [in this window] [in a new window]   FIG. 6.   Pheromone-induced morphology and actin reorganization in wild-type and cbk1 mutant cells. Exponentially growing cells were incubated with -factor (5 µg/ml) for 2 or 6 h, fixed with formaldehyde, and stained with rhodamine-phalloidin to visualize the actin cytoskeleton. White arrows point out small protrusions on the cell surface. Upper left insets show calcofluor staining of chitin. Strains used for the 6-h time point were bar1 mutants. Bar, 5.0 µm.

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View larger version (26K): [in this window] [in a new window]   FIG. 7.   Pheromone-induced growth arrest and transcriptional induction in wild-type and cbk1 mutant cells. (A) Sterile filter disks were saturated with -factor at 5 µg/ml and placed in the middle of freshly plated lawns of wild-type or cbk1 mutant cells. Pictures were taken after 2 days of incubation at 30°C. The strains used were bar1 mutants. (B) Expression of FUS1-lacZ reporter fusion upon exposure to -factor at 5 µg/ml in YPAD medium. Open bars represent wild-type cells; gray bars represent cbk1 mutant cells. -Galactosidase expression levels were measured in Miller units as previously described (55).

Deletion of CBK1 alters the pattern of bud site selection in diploids. Mutants defective in polarized growth often have bud site selection defects (30, 44). We therefore examined the budding pattern of haploid and diploid cbk1 cells by staining with calcofluor, which stains the chitin-rich bud scars (20). Although the separation defect of these cells made analysis difficult, chains of adjacent bud scars can be clearly seen in haploid cbk1 cells. Clusters of heavy calcofluor staining were found in the middle of globular clumps, indicative of a normal haploid axial budding pattern (Fig. 8A).

View larger version (56K): [in this window] [in a new window]   FIG. 8.   Bud site selection in wild-type (WT) and cbk1 mutant cells. (A) Bud scars in haploid and diploid cbk1 cells were stained with calcofluor and photographed. Note the chitin-rich junctions between attached cells. Bar, 5.0 µm. (B) The graphs show the placement of the first three buds in wild-type and cbk1 mutant diploid cells. Bud scars were scored as distal (opposite the birth site), proximal (near the birth site), or medial (see Materials and Methods). At least 100 cells of each type (first, second, and third buds) were analyzed for both strains.

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Deletion of CBK1 affects expression of a range of cell wall-modifying enzymes. Cell separation following cytokinesis requires degradation of the chitin-rich septum between mother and daughter cells. This event is dependent upon expression of a chitinase encoded by the CTS1 gene (26). Strikingly, RNA blot analysis of mRNA abundance indicates that expression of CTS1 is reduced approximately 13-fold in cbk1 cells (Fig. 9). In contrast, expression of the actin and EGT2 (early G₁ transcript) (23) genes was not affected in cbk1 cells. The normal transcription of EGT2, which is temporally coregulated in late M/early G₁ with CTS1 (23), indicates that the effect of CBK1 deletion is not general for late M/early G₁ genes.

View larger version (24K): [in this window] [in a new window]   FIG. 9.   Northern analysis of CTS1 mRNA levels in wild-type and cbk1 mutant cells. (A) Northern blots showing CTS1 (top), ACT1 (middle), and EGT2 (bottom) message levels in wild-type and cbk1 mutant cells. The amount of total RNA loaded in each lane is indicated above the corresponding lane. (B) Relative expression of CTS1, ACT1, and EGT2 mRNAs in wild-type and cbk1 cells, expressed as the ratio of PhosphorImager counts (arbitrary units) of mRNA from wild-type cells over mRNA from cbk1 mutant cells.

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View this table: [in this window] [in a new window]   TABLE 3.   Genes with >2.2-fold higher expression in cbk1 mutant cells

Genetic evidence suggests that Cbk1p functions in different pathways to regulate morphogenesis and cell separation. The expression of CTS1 is regulated by the transcription factor Ace2p (9). Our results indicate that CTS1 expression is greatly reduced in cbk1 mutants; other recent data suggest that Cbk1p may act as an upstream activator of the transcription factor Ace2p to promote the expression of CTS1 (41). To analyze the relationship between CBK1 and ACE2, we constructed ace2 strains and compared their phenotypes to cbk1 mutant phenotypes. The cell separation defect of ace2 cells is comparable in severity to that observed in cbk1 cells, with ace2 diploids forming clumps containing an average of 115 ± 54 cells (n = 37) (Fig. 3). However, unlike cbk1 cells, ace2 cells are not rounder than wild-type cells (Fig. 3); ace2 diploid cells have an average length/width ratio of 1.4 ± 0.2 (n = 101). Additionally, 82% (n = 210) of ace2 cells form normal-sized mating projections after a 2-h treatment with -factor (Fig. 7; also data not shown). These results suggest that Cbk1p acts through downstream components other than Ace2p to promote polarized growth.

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View larger version (53K): [in this window] [in a new window]   FIG. 10.   Localization of Cbk1p by indirect immunofluorescence staining. (A) The chromosomal CBK1 gene was tagged with a triple HA epitope (3xHA) at the indicated positions (see Materials and Methods). Dark arrowheads indicate that the tagged protein retained its function, and the light arrowhead represents the cbk1-mTn3F1 allele, which is partially functional (details are discussed in Results). (B) CBK1::3xHA and untagged haploid strains were stained with anti-HA antibodies, anti-Tub2p antibodies (microtubule staining), and DAPI (DNA staining). Cbk1p::3xHA localizes to both sides of the bud neck late in anaphase (note long spindle length) and remains as a patch on the surface of each recently separated cell. No polarized localization was detected in the untagged strain. For these pictures, the strain with CBK1 tagged at amino acid 259 was used. Bar, 5.0 µm.

Cbk1p localizes to both sides of the bud neck during late anaphase. To determine the intracellular location of Cbk1p, we examined the localization of Cbk1p::3xHA in both functionally tagged strains (amino acids 21 and 259) by indirect immunofluorescence assay using antibodies directed against the HA epitope. The cells were also stained with anti-Tub2p antibodies to help determine the cell cycle stage. In both tagged strains, Cbk1p::3xHA localizes to both sides of the bud neck during late anaphase (Fig. 10B). Some unbudded cells have a single patch of staining. It is likely that most of the patches seen in unbudded cells are sites of recent cell separation, but it is possible that some represent incipient bud sites. Staining is occasionally observed at the tips of very small buds (data not shown) but is never detected at the tips of larger buds. To determine more definitively if Cbk1p localizes to apical growth sites, we examined Cbk1p::3xHA localization in cdc34-2 cells grown at the restrictive temperature. As a control, we simultaneously visualized the polarized-growth component Spa2p using anti-Spa2p serum (14). While Spa2p localizes to the tips of actively growing buds in cdc34-2 cells grown at the restrictive temperature, no staining above the background is observed for Cbk1p::3xHA (data not shown). Immunoblot analysis indicates that Cbk1p is present at wild-type levels in cdc34-2 cells grown at the restrictive temperature (data not shown). Localization of Cbk1p::3xHA is not detected in -factor-treated cells.

hym1 mutants are phenotypically similar to cbk1 mutants, and Hym1p is required for Cbk1p localization. Recently, CBK1 was identified in a screen for genes involved in Sin3p-mediated transcriptional repression (10). The same screen identified another gene, HYM1, which, based on genetic analysis, was proposed to function in a pathway with CBK1 (10). Hym1p is an evolutionarily conserved protein that has homologs in worms, flies, plants, fish, mice, and humans. Interestingly, the Hym1p homolog HymA is important for the morphological development of specialized structures called conidiophores in Aspergillus nidulans (22). To further characterize the function of HYM1 and its relationship to CBK1, we deleted HYM1 and analyzed the resulting phenotypes. As described previously (10), we found that hym1 mutants have cell separation defects (average number of cells per clump, 95 ± 58) that are similar to phenotypes observed in cbk1 mutants (Fig. 11A). In addition, hym1 cells, like cbk1 cells, are rounder than wild-type cells (Fig. 11A). The average length/width ratio of hym1 diploid cells is 1.1 ± 0.1 (n = 103). We tested the ability of haploid hym1 cells to form mating projections in response to pheromone. After a 2-h treatment with -factor, only 6% (n = 199) of hym1 cells form mating projections that are at least half a cell diameter in length (Fig. 11A). As observed with cbk1 cells, -factor-treated hym1 cells form small surface bumps (Fig. 11A). Thus, based on the phenotypes we tested, hym1 mutants are similar to cbk1 mutants.

View larger version (71K): [in this window] [in a new window]   FIG. 11.   hym1 and cbk1 mutants have similar phenotypes, and Hym1p is required for normal Cbk1p localization. (A) Morphology of vegetatively growing and pheromone-treated wild-type (WT) and cbk1, hym1, and cbk1 hym1 mutant cells. Exponentially growing diploid cells were fixed with formaldehyde and photographed. Bar, 10 µm. Exponentially growing haploid cells were treated with -factor at 5 µg/ml for 2 h, fixed, and then photographed. Bar, 5.0 µm. (B) Cbk1p::3xHA was visualized by indirect immunofluorescence assay in wild-type and hym1 mutant cells. No polarized localization of Cbk1p::3xHA is observed in hym1 mutant cells. Bar, 5.0 µm.

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	DISCUSSION

Top Abstract Introduction Materials and Methods Results Discussion References

Cbk1p and Hym1p are important for apical bud growth, mating projection formation, and cell separation. CBK1 was identified in a screen for genes involved in the apical growth phase of bud formation. Analysis of cbk1 mutants indicates that Cbk1p is important for apical bud growth, mating projection formation, and cell separation following cytokinesis. cbk1 cells are rounder than wild-type cells and form abnormally small mating projections in response to pheromone treatment. In addition, direct visualization of the incorporation of new cell wall material using FITC-ConA suggests that cbk1 cells have a decreased ability to concentrate growth at the bud tip during the apical growth phase. These results suggest that Cbk1p is required to maintain polarized growth processes. cbk1 mutants also have a severe defect in postcytokinesis cell separation.

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Cbk1p regulates the expression of a number of genes with cell wall-related functions. Efficient cell separation is dependent on the expression of a chitinase encoded by the gene CTS1 (26); transcription of this gene is dependent on the transcription factor Ace2p (9). We found that CTS1 expression is greatly reduced in cbk1 cells. To determine if deletion of CBK1 affects the expression of other genes, we conducted a genomewide comparison of mRNA transcript levels between wild-type and cbk1 cells. Our results suggest that Cbk1p may exert transcriptional control over a broad range of cell wall modification activities. Expression of the glucanase-encoding gene SCW11, which is also required for efficient mother-daughter separation, as well as the putative glucanase-encoding gene YNR067c, is reduced in cbk1 mutants, while expression of the CHS1 chitin synthase gene and the ECM3, ECM5, and ECM8 cell wall integrity genes is elevated. Strikingly, expression of YER124c, a gene of unknown function, was reduced 12.7-fold in cbk1 cells.

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cbk1

cbk1

Cbk1p and Hym1p appear to function in Ace2p-dependent and -independent pathways that regulate cell morphogenesis and cell separation. Recently, it has been reported that Cbk1p may promote cell separation by acting through Ace2p to promote the expression of CTS1. The cell separation defects of cbk1 cells can be suppressed by dominant gain-of-function ACE2 alleles and partially suppressed by overexpression of CTS1 (41). Additionally, Ace2p interacts with Cbk1p in the two-hybrid system (41). Our results are consistent with this model and suggest that the expression of at least one other gene important for cell separation, SCW11, is also regulated by Cbk1p. Low expression of SCW11 in cbk1 mutants provides a possible explanation for the incomplete suppression of cbk1 cell separation defects by CTS1 overexpression.

ace2

View larger version (17K): [in this window] [in a new window]   FIG. 12.   Model for the regulation of morphogenesis by Cbk1p and Hym1p. Cbk1p functions in two distinct cell morphogenesis pathways: an ACE2-independent pathway that is required for apical growth and mating projection formation and an ACE2-dependent pathway that is required for cell separation. Based on genetic analysis, Hym1p is proposed to act at the same level as Cbk1p. Expression of SCW11 may be Ace2p dependent or require another, unknown, transcription factor. Downstream targets of Cbk1p that promote apical growth and mating projection formation await identification.

Cbk1p and the coordination of gene expression with morphogenesis. Our results and the recent results of Racki et al. (41) strongly suggest that Cbk1p promotes cell separation by stimulating the transcription of genes such as CTS1 and SCW11, which encode enzymes required to digest the chitinous septum between mother and daughter cells. Additionally, our results suggest that Cbk1p regulates the transcription of a number of genes with cell wall-related functions and may thereby control the dynamic balance between cell wall degradation and synthesis during polarized growth.

Direct regulation of cortical activities by Cbk1p. Although our results and the results of others (10) indicate that Cbk1p is involved in the regulation of transcription (a nuclear event), the polarized localization of Cbk1p suggests that it may also function to directly mediate cortical activities. Proper organization of the actin cytoskeleton is essential for polarized growth in yeast (30), and other Cbk1p-related kinases are known to affect actin organization. When N. crassa Cot-1 mutants are shifted to the restrictive temperature, actin polarity is lost and actin patches become uniformly located throughout the hyphae (49). Orb6 has also been shown to be involved in actin organization in fission yeast (53). Recently, it has been suggested that the Drosophila Ndr kinase (Trc) may regulate the actin cytoskeleton (15). Consistent with these results, we have shown that pheromone-treated cbk1 cells are unable to maintain a polarized actin cytoskeleton in growing mating projections. However, vegetatively growing cbk1 cells appear to have normal actin organization (data not shown). Thus, our results suggest that Cbk1p functions to promote pheromone-induced polarization of the actin cytoskeleton. The mechanisms by which Cbk1p and related kinases control organization of the actin cytoskeleton are unknown and await further characterization.