Genetic Analysis Reveals That FLO11 Upregulation and Cell Polarization Independently Regulate Invasive Growth in Saccharomyces cerevisiae

Corresponding author: Stephen J. Kron, University of Chicago, Center for Molecular Oncology, 924 E. 57th St., Rm. R322, Chicago, IL 60637., skron{at}midway.uchicago.edu (E-mail)

Communicating editor: J. RINE

	ABSTRACT

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

Under inducing conditions, haploid Saccharomyces cerevisiae perform a dimorphic transition from yeast-form growth on the agar surface to invasive growth, where chains of cells dig into the solid growth medium. Previous work on signaling cascades that promote agar invasion has demonstrated upregulation of FLO11, a cell-surface flocculin involved in cell-cell adhesion. We find that increasing FLO11 transcription is sufficient to induce both invasive and filamentous growth. A genetic screen for repressors of FLO11 isolated mutant strains that dig into agar (dia) and identified mutations in 35 different genes: ELM1, HSL1, HSL7, BUD3, BUD4, BUD10, AXL1, SIR2, SIR4, BEM2, PGI1, GND1, YDJ1, ARO7, GRR1, CDC53, HSC82, ZUO1, ADH1, CSE2, GCR1, IRA1, MSN5, SRB8, SSN3, SSN8, BPL1, GTR1, MED1, SKN7, TAF25, DIA1, DIA2, DIA3, and DIA4. Indeed, agar invasion in 20 dia mutants requires upregulation of the endogenous FLO11 promoter. However, 13 mutants promote agar invasion even with FLO11 clamped at a constitutive low-expression level. These FLO11 promoter-independent dia mutants establish distinct invasive growth pathways due to polarized bud site selection and/or cell elongation. Epistasis with the STE MAP kinase cascade and cytokinesis/budding checkpoint shows these pathways are targets of DIA genes that repress agar invasion by FLO11 promoter-dependent and -independent mechanisms, respectively.

MANY simple fungal organisms are capable of switching between yeast-form growth, where ovoid cells separate subsequent to mitosis, to filamentous growth, in which cells form chains that remain physically attached via a persistent cytoplasmic or cell wall connection. Significantly, hyphal filament formation may be a key virulence factor in pathogenic fungi such as Candida albicans and Ustilago maydis, contributing to local spread and tissue invasion. Filamentous growth in the budding yeast Saccharomyces cerevisiae is pseudohyphal, characterized by chains of polarized, elongated cells that display a prolonged budded period, mitotic delay, apical polar-budding pattern, and increased agar invasion (KRON and GOW 1995 ). The switch between yeast-form and filamentous growth is a highly regulated process responsive to nutrient availability and other environmental stimuli (GIMENO et al. 1992 ), suggesting that budding yeast filamentation may be a foraging response to nutrient deprivation (BANUETT 1998 ; GALE et al. 1998 ; MITCHELL 1998 ).

In S. cerevisiae, physiologic regulation of pseudohyphal differentiation and resulting agar invasion depends upon activation of the Ras2 small GTP-binding protein (GIMENO et al. 1992 ). Ras2 activates both the STE mitogen-activated protein (MAP) kinase and cAMP-dependent protein kinase pathways to promote filamentous differentiation (GIMENO et al. 1992 ; MOSCH and FINK 1997 ; MADHANI and FINK 1998 ; AHN et al. 1999 ; MOSCH et al. 1999 ). Even though the signaling pathways are fairly well characterized (reviewed in BANUETT 1998 ; MADHANI and FINK 1998 ), their receptors remain to be fully described. One candidate, the ammonium permease Mep2, can stimulate Gpa2 to increase cAMP concentrations in the absence of NH⁺₄ (LORENZ and HEITMAN 1998 ). Gpr1 may also function as a carbon and/or nitrogen starvation sensor during pseudohyphal growth (LORENZ et al. 2000B ; TAMAKI et al. 2000 ). Key outputs of filamentous signaling are likely to be regulated via transcriptional control and may include targets in multiple signal transduction and morphogenetic pathways.

In yeast, cell cycle progression is tightly coupled to bud morphogenesis and cell polarity (reviewed by KRON and GOW 1995 ; LEW and REED 1995 ). Mutations in cell cycle regulators such as the Elm1, Hsl1, or Hsl7 kinases, the Grr1 F-box protein, the mitotic cyclin Clb2, the Cdc28 cyclin-dependent kinase and the Fkh1,2 mitotic transcription factors lead to prolonged budded period, increased cell polarization, constitutive pseudohyphal growth, and increased agar invasion (BLACKETER et al. 1993 ; AHN et al. 1999 ; EDGINGTON et al. 1999 ; LOEB et al. 1999 ; HOLLENHORST et al. 2000 ; ZHU et al. 2000 ). Mutating Swe1, a kinase that antagonizes Cdc28's mitotic activity, attenuates filamentous growth and blocks the effects of elm1, hsl1, and hsl7 but does not suppress response to filamentous signaling (KRON et al. 1994 ; AHN et al. 1999 ; EDGINGTON et al. 1999 ). Presumably, other cell cycle regulators are bona fide targets of the cAMP-dependent kinase and/or STE MAP kinase signaling pathways, but clear links have yet to be established. Apical polar bud site selection in filamentous cells may also be directly regulated by filamentous signaling or may be downstream of the mitotic delay.

Unlike our detailed knowledge of signaling and cell cycle pathways, we understand very little regarding the downstream biophysical mechanisms that permit these processes. During invasion, the individual cells in the colony must displace or degrade the matrix, suggesting the importance of changes in cell-cell and cell-substrate interactions. Indeed, stimulation of the STE MAP kinase (MAPK) pathway increases expression of FLO11, a cell-surface flocculin, and PGU1, an enzyme that degrades extracellular pectin (MADHANI et al. 1999 ). FLO11 expression is also coupled to STA1, 2, and 3 glycoamylase gene expression, suggesting a link between starch degradation and invasive growth (LAMBRECHTS et al. 1996 ). FLO11 expression is required for haploid invasive growth as well as diploid pseudohyphal differentiation (LAMBRECHTS et al. 1996 ; LO and DRANGINIS 1998 )—flo11 mutant cells do not form chains or invade agar but instead form nonadherent piles on the agar surface. Mep2, cAMP-dependent protein kinase, the MAPK cascade, and other pathways coordinately control FLO11 expression via a highly complex promoter (GAGIANO et al. 1999A , GAGIANO et al. 1999B ; GALITSKI et al. 1999 ; PAN and HEITMAN 1999 ; RUPP et al. 1999 ).

FLO11 possesses the largest promoter region of any yeast open reading frame (ORF), containing at least four upstream activating sites and nine upstream repression sites (RUPP et al. 1999 ). Several mechanisms of invasive growth repression have already been described. Mutation of the SFL1 transcriptional repressor derepresses FLO11 expression and invasive growth (ROBERTSON and FINK 1998 ). Dig1 and Dig2 cooperate with Kss1 to repress Ste12 and inhibit haploid invasive growth (COOK et al. 1996 ; TEDFORD et al. 1997 ; BARDWELL et al. 1998 ). The STA10 repressor functions via FLO8, a transcriptional activator (GAGIANO et al. 1999B ). In addition, mutations in IRA1, a GTPase activating protein gene, lead to hyperactivation of Ras2 and increased FLO11 transcription (RUPP et al. 1999 ).

To identify additional FLO11 transcriptional repression mechanisms, we screened for gene disruptions that induce haploid invasive growth and identified mutations in 35 genes. Validating the screen, the majority of these mutations upregulate FLO11 expression from its endogenous promoter and depend upon this increased expression for their effects. Significantly, we also identified mutations that enhance invasion even when FLO11 transcription is clamped by a low-level constitutive promoter. This class of mutations has its primary effects on cell polarity rather than cell adhesion. We find that enhancing one or more of the physical processes of cell-cell adhesion, budding polarity, elongation, or other unidentified mechanisms can increase invasion in haploids and diploids.

	MATERIALS AND METHODS

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

Plasmids:
To generate plasmid cassettes for promoter replacement with the adh⁺ promoter from Schizosaccharomyces pombe (pSP2) or the CDC28 promoter from S. cerevisiae (pSP10), the Sp adh⁺ promoter was amplified from plasmid spADH-CLB2 (AMON 1997 ) using primers SalSpADHp and Bgl SpADHp and the CDC28 promoter was amplified from base pairs -350 to -1 relative to the start site of the CDC28 ORF from genomic DNA with primers SalCDC28p and BglCDC28p (see Table 2). The PCR products were digested with SalI and BglII and ligated into pFA6 kanMX6 GALp (LONGTINE et al. 1998 ) digested with SalI and BglII to excise the GAL promoter.

Yeast strains, media, and genetic methods:
The yeast strains used in this study are listed in Table 1. Strains were derived in the 1278b genetic background (GRENSON et al. 1966 ; LIU et al. 1993 ) using standard genetic methods. Standard yeast culture media and filamentous growth media were prepared as previously described (KRON et al. 1994 ; AHN et al. 1999 ). Yeast media were obtained from United States Biochemical (Cleveland) and other reagents from Fisher Scientific (Pittsburgh) and Sigma (St. Louis). G418 (Life Technologies) was added to YPD agar at 0.2 mg/ml. Synthetic low ammonium medium (SLAD) was prepared with 50 µM ammonium sulfate. Uracil was added to SLAD medium to a concentration of 0.2 mM or histidine to a concentration of 0.3 mM to make SLAD +Ura or SLAD +Ura +His. Haploid matings, diploid sporulations, and tetrad dissections were performed as described (SHERMAN et al. 1986 ). Yeast were transformed using lithium acetate transformation (GIETZ et al. 1992 ) or electroporation (SIMON 1993 ).

PCR disruption (LONGTINE et al. 1998 ) was used to replace SWE1, BUD8, RAS2, and KSS1 in both MATa and MAT 1278b cells with the kanMX G418 resistance marker. SWE1 was deleted from positions +3 to +2372, relative to the start site, BUD8 from positions +1 to +1851, RAS2 from positions -76 to +983, and KSS1 from positions +1 to +1107. Similarly, the defined promoter from -2800 to -1, 5' to the FLO11 open reading frame (RUPP et al. 1999 ), was replaced with the promoter of Sp adh⁺ or CDC28 by amplification of pSP2 or pSP10 with primers FLO11p-1 and FLO11p-2 (Table 2), transformation, and G418 selection. SIR4 and HML deletions were constructed using plasmids pRS42sir4::HIS3+ (KIMMERLY and RINE 1987 ) and pJR826 (J. RINE, personal communication). To construct pseudodiploids, haploid MATa strain SKY760 was transformed with plasmid B2185 [CEN MAT URA3] (MOSCH and FINK 1997 ).

Isolation of mutants that dig into agar:
Insertional mutagenesis of a wild-type 1278b haploid was performed by homologous recombination with a Tn3::lacZ::LEU2 transposon-mutagenized yeast genomic DNA library (BURNS et al. 1994 ). DNA aliquots from 14 pools of the Tn3::lacZ::LEU2 library were combined, digested with NotI, and used to transform yeast strain SKY760 (MATa ura3-52 his3::hisG leu2::hisG). Twenty independent transformations yielded 5000 and 15,000 colonies per SC -Leu plate, for a total of 180,000 LEU⁺ transformants. After 3 days of growth at 22°, the transformation plates were directly screened for digs into agar (dia) mutants. Noninvasive cells were washed away under gently running distilled water and the plates were rubbed with a gloved finger for 1 min. Using transmitted light and a Zeiss Stemi 2000-C (Thornwood, NY) stereomicroscope, plates were then visually scanned for sites of agar invasion. A total of 388 colonies were recovered from the agar plates with a sharp-tipped toothpick and inoculated onto SC -Leu plates. Putative dia mutants and controls were streaked onto YPD and SC -Leu plates, grown for 3 days at 22°, and washed under running water as before. Isolates more invasive than SKY760 wild-type haploids on both YPD and SC -Leu medium were retained. The 194 remaining dia mutants were mated back to the wild-type SKY2606 (MAT ura3-52 leu2::hisG) and diploids were selected on SC -His -Leu medium. Five isolates were sterile and the genomic locus of the lacZ insertion was directly determined in these strains. A total of 189 presumed dia/DIA heterozygotes were analyzed by sporulation and tetrad dissection. Segregants from at least 4 four-spore tetrads from each cross were analyzed for growth on SC -Leu and for YPD agar invasion. In 95 of the 189 crosses, the LEU2 marker and hyperinvasive phenotype cosegregated in a 2:2 pattern, confirming a MATa ura3-52 his3::hisG leu2::hisG dia::LEU2 genotype. Homozygous diploids of each dia mutant were created by crossing MATa and MAT segregants obtained from the backcross to SKY2606.

Identification of dia insertions:
The insertion site of the Tn3::lacZ::LEU2 insertion was determined by vectorette PCR (Botstein laboratory, http://genome-www.stanford.edu/group/botlab/protocols/vectorette.html). Genomic DNA was isolated from MATa segregants of each dia mutant digested with RsaI or AluI and ligated to annealed anchor bubble primers. Sequences adjoining the insertion site were amplified using primers Insertamp1 and Insertamp2 (Table 2). PCR products were gel-purified and sequenced using Insertamp1 and an ABI cycle sequencing kit (Perkin-Elmer, Norwalk, CT). DNA homology searches were performed using the Saccharomyces Genome Database BLAST (http://genome-www2.stanford.edu/cgi-bin/SGD/nph-blast2sgd) and National Center for Biotechnology Information BLAST (http://www.ncbi.nlm.nih.gov/BLAST/) services. In 29 cases at least one of the insertions occurred within the ORF and the disruption is positively identified. In 2 cases (TAF25 and GND1) insertions were within 100 bp of the translational start site. In the remaining 4 cases (DIA1, PGI1, CDC53, and SKN7), the insertions were at least 150 bp from any annotated ORF and were named based on the most proximal annotated ORF 3' to the insertion.

Photomicrography:
Microcolonies were imaged through the agar and plastic petri dish using a Zeiss Axiovert 25 with bright-field illumination and a 32x LD Achroplan or 10x CP-Achromat objective. A Photometrics Sensys 1600 charge-couple device camera and IPLab Spectrum image-acquisition software (Signal Analytics, Vienna, VA) were used to capture images. Images were converted to gray scale and filtered to remove noise and enhance contrast in Photoshop (Adobe, Mountain View, CA).

Quantitative invasive growth assay:
Cells to be tested for quantitative invasive growth were streaked onto thin (0.5 cm) YPD plates to obtain colonies derived from single cells. Colonies were grown at 30° for 3 days. A 1-cm-diameter circle was punched out of the agar at the outer edge of the plate where colonies had grown from single cells. The agar plug was rinsed with water and gently scraped with a plastic spatula to remove the cells not penetrating the surface of the agar. Nonadherent cells were collected, sonicated, and counted on a hemacytometer to obtain the number of cells that did not invade the agar. The rinsed piece of agar was placed into a 15-ml polypropylene tube with 5 ml of water and microwaved for 5 sec until the agar was completely melted. The tube was centrifuged at 2000 x g for 5 min and the supernatant was carefully removed by Pasteur pipette. The cell pellet was washed once with 5 ml water and recentrifuged, and 50 µl of 1 mg/ml Zymolyase (Seikagaku Corp., Tokyo) was added to resuspend the pellet. The cells were incubated for 15 min at 30°, brought to 1 ml final volume in water, and counted by hemacytometer to determine the number of cells that invaded the agar. The fraction of cells that invaded the agar was calculated as the number of cells that invaded the agar divided by the sum of the number of cells that did not invade the agar and the number that did invade the agar. Invasion was measured in three independent experiments for each dia strain and normalized to the invasion of SKY760 (MATa ura3-52 his3::hisG leu2::hisG DIA).

Cell flocculation assay:
A flocculation assay was adapted from BONY et al. 1998 . Yeast strains grown to saturation in 10 ml liquid YPD medium overnight at 30° were deflocculated by two washes in 50 mM sodium citrate, 5 mM EDTA, pH 3.0 buffer followed by sonication for 10 min. Cells were resuspended to a concentration of 10⁸ cells/ml in 5 ml of sodium citrate buffer and calcium chloride was added to a final concentration of 20 mM to induce flocculation. Culture tubes were inverted 50 times/min for 10 min and then left standing vertically. After 10 min, 0.2 ml of the cell suspension was removed from just below the meniscus and added to 1 ml of 0.25 M EDTA, pH 8.0. The flocculation level is expressed as the difference in optical density at 600 nm between the deflocculated cell suspension in 0.25 M EDTA and the sample after 10 min of settling. Flocculation for each dia strain was normalized to that of strain SKY760 (MATa ura3-52 his3::hisG leu2::hisG DIA). Flocculation was measured in three independent experiments for each strain.

Cell elongation measurements:
Yeast strains were incubated overnight in liquid YPD medium at 30°, transferred to fresh YPD, and grown to an OD₆₀₀ of 0.6. Cells were photographed in Nomarski contrast using a Zeiss Axioskop with a 100x oil-immersion objective, a Photometrics Sensys 1600 CCD camera, and IPLab image-acquisition software. NIH Image software was used to manually trace 100 cells and obtain major and minor axis lengths. The reported elongation is the ratio of major to minor axis lengths.

Northern blot analysis:
Yeast strains to be analyzed were incubated in liquid YPD medium overnight at 30°, diluted 100-fold into fresh YPD, and incubated to an OD₆₀₀ of 0.8. Cells were washed in ice water and total RNA was harvested by phenol:chloroform extraction followed by ethanol precipitation. For each sample, 20 µg of total RNA was separated by electrophoresis on a formaldehyde gel and transferred by capillary action to a 0.2-µm-pore-size nylon membrane. DNA probes (1000-bp regions at the 5' end of the FLO11 and ACT1 open reading frames) were amplified and radiolabeled by PCR and then gel-purified. Hybridizations and washes were performed according to SAMBROOK et al. 1989 . FLO11 expression was normalized to ACT1 expression. For each dia strain, this ratio was then normalized to the FLO11/ACT1 expression ratio of strain SKY760 (MATa ura3-52 his3::hisG leu2::hisG DIA). At least three independent measurements of FLO11/ACT1 expression levels were measured for each strain.

Bud scar staining:
Haploid budding pattern was determined by calcofluor staining as described by PRINGLE et al. 1989 . Cells were grown to an OD₆₀₀ of 0.6 in YPD at 30°. Aliquots of 10⁷ cells were fixed at room temperature for 1 hr in 3.7% formaldehyde, rinsed twice in water, resuspended in 200 µl of 1 µg/ml calcofluor white (Fluorescent Brightener 28, Sigma) in water, incubated in the dark at 22° for 30 min, washed five times with 1 ml water, and resuspended to a final volume of 25 µl. Stained cells were observed by epifluorescence microscopy using 365-nm excitation and blue emission filters and photographed using a Zeiss Axioskop, a 100x oil-immersion objective, Photometrics Sensys 1600 CCD camera, and IPLab image-acquisition software. Cells with between two and five obvious bud scars were divided into two classes, axial or polar, based on the predominant bud scar distribution. Over 100 cells were analyzed in each of three separate experiments for each strain.

	RESULTS

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

Expression of FLO11 correlates with agar invasion:
To test whether FLO11 expression directly regulates haploid invasive growth, we constructed strains that express different levels of FLO11 by replacing the endogenous FLO11 promoter with the promoters from the S. pombe adh⁺ gene or the S. cerevisiae CDC28 gene. In addition to altering basal FLO11 expression, these replacement promoters prevent FLO11 from being induced or repressed such as by the cAMP-dependent kinase or STE MAPK pathways. As shown in Fig 1, SpADHp-FLO11 expression in YPD liquid is 0.4 times wild-type expression while CDC28p-FLO11 expression is 4.2 times greater than that of wild-type FLO11. Wild-type haploids invade YPD agar to a minimal extent after 3 days with occasional single cells penetrating the matrix surface, but SpADHp-FLO11 cells do not invade at all. CDC28p-FLO11 cells invade the agar significantly more than wild-type cells with microcolonies of cells growing multiple cell layers below the agar surface. We conclude that increasing FLO11 expression alone is sufficient to induce invasive growth in haploid cells.

View larger version (39K): In this window In a new window Download PPT slide   Figure 1. Extent of cell invasion of rich media correlates with FLO11 expression and the endogenous FLO11 promoter is required for filamentation in response to low nitrogen. (a) Northern blot analysis of FLO11 and ACT1 expression in haploid strains reveals that the SpADHp-FLO11 is expressed at 0.4 times wild-type FLO11 expression while CDC28p-FLO11 is expressed 4.2 times wild-type FLO11 expression. Haploid strains with FLO11 under its endogenous promoter (b), the SpADH promoter (c), or the CDC28 promoter (d) were streaked on YPD medium to obtain single cells. YPD plates were incubated at 21° for 96 hr. Cells that did not penetrate the agar were removed by rubbing the plate with a finger under running water. Homozygous diploid strains with FLO11 under its own promoter (e), the SpADH promoter (f), or the CDC28 promoter (g) were streaked on SLAD medium to obtain single cells. SLAD plates were incubated at 21° for 24 hr. Images of representative colonies are shown. Bar (b), 100 µm for b–d. Bar (e), 50 µm for e–g.

Replacement of the endogenous FLO11 promoter also affects cell filamentation on low ammonia media (Fig 1). SpADHp-FLO11/SpADHp-FLO11 diploids do not elongate appreciably or form filaments. CDC28p-FLO11/CDC28p-FLO11 diploids elongate slightly and form pseudohyphae but do not form filaments as well as FLO11p-FLO11/FLO11p-FLO11 diploids. These results indicate that quantitative levels of FLO11 expression can affect both cell and colony morphology in response to a low-nitrogen signal and that FLO11 may possess functions in addition to mediating cell-cell adhesion.

Isolation of mutations that enhance invasive growth:
On the basis of our results from modulating FLO11 expression and its effects on both haploid invasive growth and filamentous differentiation, we hypothesized that regulation of FLO11 expression from its endogenous promoter may be mediated in part via repression. Thus, we performed a genetic screen to identify potential negative regulators of FLO11 expression by enhanced agar invasion in the mutants. To isolate dia mutants, we mutagenized a 1278b-derived MATa haploid strain (SKY760) by integrating a transposon-mutagenized genomic library containing random Tn3::lacZ::LEU2 insertions (BURNS et al. 1994 ). After 3 days of growth at 30°, 180,000 transformants were directly screened for hyperinvasive growth on the SC-Leu transformation plates. A total of 388 colonies that physically penetrated the surface of the agar were recovered onto SC-Leu medium and then streaked onto SC-Leu and YPD medium. Of the 388 picks, 189 retested as significantly more invasive than wild-type haploids on both synthetic and YPD media. Each of the 189 putative dia mutants was analyzed by a cross to a DIA strain (SKY2606, MAT ura3-52 leu2::hisG) to determine if a single mutation linked to the LEU2 marker was responsible for the hyperinvasive growth. Five dia strains were sterile and these were directly analyzed for insertion locus. In three, the insertions disrupt the SIR4 gene and two fall in SIR2. SIR2 and SIR4 are required for transcriptional silencing of the silent mating type -locus (IVY et al. 1986 ).

Each of the remaining 184 strains was analyzed by tetrad dissection. The LEU2 marker segregated 2:2 and was genetically linked to the hyperinvasive phenotype in 94 of these strains. The insertion sites were determined in these as well. Altogether, the 99 dia mutants comprised 60 different insertions in or adjacent to 35 different genes (Table 3). Of these, 31 were previously characterized in other genetic studies while 4 are novel. Of these 4, DIA1 (YMR316w) does not possess significant homology to any characterized ORFs; DIA2 (YOR080w) encodes an F-box protein; DIA3 (YDL024c) is homologous to acid phosphatases such as PHO5 and PHO11; DIA4 (YHR011w) is homologous to SES1, a seryl tRNA synthase.

We measured the magnitude of the increase in invasion of each dia strain relative to a wild-type haploid strain (Table 3). For wild-type haploids, 0.02 ± 0.01% of cells invade after 4 days of incubation at 30°. In this screen we were able to detect insertional mutants that were as little as 25 times more invasive than wild type (e.g., MED1, HSL1, HSL7, MSN5, DIA2, and DIA4). At the upper end, we detected an insertion in SKN7, which is about 200 times as invasive as wild-type haploids. Images of wild-type and several dia mutant strain haploid colonies that have penetrated the surface of the agar are shown in Fig 2.

View larger version (95K): In this window In a new window Download PPT slide   Figure 2. Haploid dia mutant colonies invade agar. dia strains were streaked on YPD medium to obtain single cells and grown for 96 hr at 21°. Cells that did not penetrate the agar were removed by rubbing the plate with a finger under running water. Representative colonies were imaged. Bar, 100 µm.

Most dia mutations increase FLO11 expression:
Ectopic induction of FLO11 can enhance haploid invasive growth (Fig 1). To determine which of the dia mutations may enhance invasion by upregulating FLO11, we measured FLO11 transcription by Northern blot analysis (Fig 3 and Table 4). In each dia strain, the expression of FLO11 was normalized to the expression of ACT1 and this ratio was normalized to the FLO11:ACT1 expression ratio of wild-type cells. Of the 35 dia mutants, 25 display elevated FLO11 expression (>1.5-fold wild type) while 10 do not significantly upregulate FLO11. These 10 dia mutations likely enhance invasive growth through a mechanism other than induction of FLO11 expression.

View larger version (92K): In this window In a new window Download PPT slide   Figure 3. Upregulation of FLO11 transcription is required for hyperinvasive phenotype of some dia strains. (a) Northern analysis of FLO11 and ACT1 expression in haploid strains reveals that many dia strains upregulate FLO11 (lanes 3, 5, 6, 7, 12, 13, and 14) while others do not (lanes 4, 8, 9, 10, and 11). (b–m) Haploid dia strains were streaked on YPD medium to obtain single cells. YPD plates were incubated at 21° for 96 hr. Cells that did not penetrate the agar were removed by rubbing the plate with a finger under running water. Images of representative colonies are shown.

An increase in FLO11 expression in dia mutants may be a determinant of their hyperinvasive phenotype or a secondary effect and not required for agar invasion. To address whether upregulation of FLO11 is necessary for haploid invasion in each dia strain, we crossed SpADHp-FLO11 into each dia mutant background. While the S. pombe adh⁺ promoter yields a reduced level of FLO11 expression (Fig 3A), importantly, this mutation likely prevents the cell from stimulating or repressing FLO11 transcription through any of its normal physiologic regulatory mechanisms. Indeed, by Northern analysis, the SpADHp-FLO11 construct results in the same, low level of FLO11 expression in each dia mutant as in the wild-type DIA haploid strain (data not shown).

As in a wild-type DIA strain, SpADHp-FLO11 completely abolishes haploid invasion in 22 of the dia mutants (Table 4 and Fig 3). In many of these mutants, it is likely that derepression of FLO11 expression is both necessary and sufficient for their hyperinvasive phenotype. However, within this group, gtr1-100, med1-100, and taf25-100 do not upregulate FLO11 from the endogenous promoter. Such strains appear to require more FLO11 than that supplied by the SpADH promoter and/or may induce invasion through a FLO11-independent mechanism.

In 12 dia strains, although invasion is attenuated when expression of FLO11 is clamped by SpADH-FLO11, it is not abrogated. Further, elm1-100 can invade agar as well with SpADH-FLO11 as the native FLO11 promoter. These 13 dia mutants presumably invade by a pathway that does not require enhanced FLO11 expression. Not surprisingly, 7 of these dia strains, ax1-100, bud10-100, bud3-100, bud4-100, sir2-100, sir4-100, and bem2-100, are among those that do not increase FLO11 transcription from the native promoter. However, 6 of these dia strains, elm1-100, dia2-100, bpl1-100, aro7-100, pgi1-100, and gnd1-100, are among those that were found to upregulate FLO11. Save for elm1-100, this FLO11 induction likely promotes invasion, but a FLO11-independent mechanism must contribute. Thus, while regulation of FLO11 transcription provides an important mechanism of invasive growth control, FLO11 upregulation is not essential for invasive growth. One or more FLO11-independent mechanisms are likely to be sufficient for invasive growth.

Secondary phenotypic characterization of dia mutants:
We examined the dia mutants further to determine if cellular processes other than agar invasion were affected and to investigate potential mechanisms for FLO11-independent agar invasion. We hypothesized that elongated cell shape and polarized bud site selection, in addition to increased cell-cell adhesion, may be independent determinants of agar invasion in the dia mutants. Thus, we measured the ratio of major to minor axes in individual cells of each dia strain and the fraction of cells that exhibit polar bud site selection as opposed to the expected axial budding. On the basis of these results (Table 5), we separated the dia mutations into four classes. Class 1 mutations (3 genes) exhibit both significant polar bud site selection and cell elongation as compared to wild-type DIA haploids. Class 2 mutations (12 genes) perform polarized budding but remain round while class 3 mutants (2 genes) are elongated but display predominantly axial budding. Class 4 mutants (18 genes), like wild-type DIA cells, bud in an axial pattern and remain round. Examples of bud scar staining that illustrate budding pattern and individual cell morphology for representative members of each class are shown in Fig 4.

View larger version (126K): In this window In a new window Download PPT slide   Figure 4. Some dia mutations affect cell morphology and bud site selection. Haploid dia strains were grown to log growth phase in YPD at 30°, fixed in formaldehyde, and stained with calcofluor white to visualize bud scars. Images of representative cells are shown. Class 1 mutations (b and c) elongate cells and cause predominantly polar bud site selection. Class 2 mutations (d–f) cause polar budding but not cell elongation. Class 3 (g) mutations elongate cells but maintain axial budding. Class 4 mutations (h and i) do not affect bud site selection or cell morphology. Bar, 5 µm.

Not surprisingly, among the class 4 genes, where mutants display budding and cell shape similar to that of a DIA wild-type strain, most increase FLO11 expression and require the native promoter for their phenotype. We found that bud site selection had a significant effect on invasion independent from derepression of the FLO11 promoter. The majority of class 1 and 2 dia strains, all of which exhibit an increase in polar budding, are able to invade agar without upregulating FLO11. In turn, nearly all class 3 and 4 dia mutant strains, those that do not display an increase in polar budding, no longer invade agar at the low levels of FLO11 expression permitted by the SpADH promoter. In detail, most polar-budding but round, class 2 haploids—bud3-100, bud4-100, bud10-100, axl1-100, bem2-100, sir2-100, and sir4-100—tend neither to upregulate FLO11 expression nor to require the native promoter for their invasive phenotype (Table 4 and Table 5) while pgi1-100 and gnd1-100 mutations enhance both polar budding as well as FLO11 expression. By contrast to polar budding, cell elongation alone, as in class 3 mutants, is insufficient to cause agar invasion as evidenced by the lack of invasion in the SpADHp-FLO11 grr1-100 and SpADHp-FLO11 cdc53-100 strains. Nonetheless, cell elongation may augment agar invasion given a critical level of FLO11 expression. taf25-100, gtr1-100, and med1-100 are the only three dia mutants that increase haploid invasion without increasing FLO11 expression or cell polarity.

In addition to measuring changes in cell polarity, we also assayed each dia mutant for flocculation (Table 5) to determine if the mutations affect cell-cell adhesion. FLO11 has been implicated in both invasive growth and calcium-dependent flocculation (LO and DRANGINIS 1996 ). Enhanced flocculation tends to correlate with polarized budding, but there are several notable exceptions. In fact, several of the class 2 mutations that do not upregulate FLO11 expression (bud3-100, bud4-100, bud10-100, and axl1-100) still display increased flocculence. Among the 12 class 2 mutants, only 4 (sir2-100, sir4-100, gnd1-100, and dia2-100) do not show at least a fourfold increase in flocculation. Of the 3 class 1 mutants, elm1-100 is 20 times as flocculent as wild-type cells while hsl1-100 and hsl7-100 show no increase in flocculence. grr1-100 is hyperflocculent while cdc53-100 is not. On the other hand, 11 of the 18 class 4 mutants that are neither polar budding nor elongated are no more flocculent than wild-type haploids. Thus, some of the pathways that repress agar invasion may also repress flocculation, but flocculation is not necessary for agar invasion.

Surprisingly, SpADHp-FLO11 does not affect flocculation of any of the dia mutant strains (data not shown), suggesting that upregulation of another adhesin or other factors may be responsible for increased flocculation in these strains. In turn, the decreased agar invasion in SpADHp-FLO11 dia strains suggests that increased cell-cell adhesion may not be sufficient to explain FLO11-induced agar invasion.

Haploid invasive growth is distinct from diploid invasive growth and pseudohyphal differentiation:
Haploid invasive growth pathways have been linked to diploid pseudohyphal growth through their common regulation via the cAMP-dependent kinase and STE MAPK pathways. We constructed homozygous diploids of each dia mutation and measured agar invasion of each dia/dia strain on rich YPD medium as well as cell elongation in response to low nitrogen on SLAD medium to determine whether enhanced haploid invasive growth correlates with altered diploid invasion and/or pseudohyphal differentiation. Table 5 shows complete results of these assays while Fig 5 provides representative images of colonies that penetrated the surface of YPD medium or colonies growing on SLAD medium.

View larger version (128K): In this window In a new window Download PPT slide   Figure 5. Some dia/dia mutations enhance diploid agar invasion and/or pseudohyphal differentiation. Diploid dia/dia strains were streaked on YPD medium (a–f) or SLAD +Ura medium (g–l) to obtain single cells. YPD plates were incubated at 21° for 96 hr. Then cells that did not penetrate the agar were removed by rubbing the plate with a finger under running water. SLAD+Ura plates were incubated at 21° for 36 hr. Representative colonies were imaged. Bar (a), 100 µm for a–f. Bar (g), 50 µm for g–l.

Wild-type diploid cells are significantly more invasive than wild-type haploid cells, possibly resulting from a switch to polarized bud site selection in diploids. Invasive growth of dia/dia mutant strains is greater than that of wild-type diploids for 17 of the mutants. Invasion is decreased relative to the wild-type diploid in 4 of the dia/dia mutant strains (ydj1-100/ydj1-100, taf25-100/taf25-100, gtr1-100/gtr1-100, and bpl1-100/bpl1-100). In the remaining 14 dia/dia strains, invasion is not significantly different between dia/dia mutants and wild-type diploids. Every elongated haploid strain (classes 1 and 3) is also hyperinvasive as a diploid yet only 3 class 2 mutants (polar budding, round morphology) are hyperinvasive as diploids.

In addition to failing to induce diploid invasive growth, class 2 dia/dia mutations typically do not affect pseudohyphal growth. Only pgi1-100/pgi1-100 and gnd1-100/gnd1-100 strains are hyperfilamentous. However, each dia mutation that induces cell elongation in haploids (class 1 and 3 strains) also enhances diploid dia/dia filamentation on SLAD medium. Among the class 4 dia/dia mutants, diploid invasive growth does not appear to be linked to low-nitrogen stimulated filamentation. In some strains, such as ira1-100/ira1-100, filamentation can be induced without hyperinvasion but in other strains, such as ssn3-100/ssn3-100, invasiveness increases while filamentation remains unchanged. In fact, reciprocal regulation of invasion and pseudohyphal differentiation can occur as in a gcr1-100/gcr1-100 strain (Fig 5).

Polar bud site selection enhances agar invasion:
Diploids require Bud8 for bipolar budding (ZAHNER et al. 1996 ) and filamentous growth (MOSCH and FINK 1997 ). Mutants bud proximal to their birth scars, in a pattern similar to normal haploid axial bud site selection. We assayed the effects of a bud8 deletion on the invasiveness of each dia mutant. Deletion of BUD8 switches all of the dia mutant strains except elm1-100 and bem2-100 to predominantly proximal budding. elm1-100 bud site selection remains polar in a bud8 while bem2-100 budding is random in both BUD8 and bud8 backgrounds (data not shown). Each dia mutant strain that buds in a polar manner, except elm1-100 and bem2-100, requires BUD8 expression for invasion (Fig 6 and Table 6). Several of these strains (bud3-100, bud4-100, bud10-100, axl1-100, sro4-100, hsl1-100, hsl7-100, sir2-100, and sir4-100) do not overexpress FLO11 whereas others (ydj1-100, pgi1-100, gnd1-100, aro7-100, and dia2-100) do. Therefore, polarity is an important factor in allowing each of these strains to invade, regardless of the level of FLO11 expression. BUD8 deletion does not affect invasion in axial-budding strains (Table 6) or cell elongation in any of the strains tested (data not shown).

View larger version (63K): In this window In a new window Download PPT slide   Figure 6. Class 2 dia mutations require polar bud site selection for cell invasion. Haploid dia strains were streaked on YPD to obtain single cells. The plates were incubated at 21° for 96 hr. Cells that did not penetrate the agar were removed by rubbing the plate with a finger under running water. Representative colonies were imaged. Bar, 100 µm.

To further test our hypothesis that polar budding is sufficient to cause agar invasion given a basal level of FLO11 expression, we measured agar invasion in diploid (MATa/MAT) and pseudodiploid (MATa [MAT CEN URA3]) strains (Fig 7). Diploid cells bud at the distal pole 95 ± 4% of the time while pseudodiploids form 93 ± 5% of new buds at the distal pole. Diploids invade agar more avidly than haploids, while pseudodiploids are more invasive than either haploids or diploids. This increased invasiveness requires BUD8 since MATa bud8::kan^r [MAT CEN URA3] and MATa/MAT bud8::kan^r/bud8::kan^r strains do not invade, consistent with the notion that polar budding causes invasiveness.

View larger version (103K): In this window In a new window Download PPT slide   Figure 7. Polar bud site selection is responsible for agar invasion in pseudodiploids and diploids. Haploid (a–c), diploid (d and e), and pseudodiploid carrying plasmid B2185 [MAT CEN URA3] (g and h) strains were streaked on YPD to obtain single cells. The plates were incubated at 21° for 96 hr. Cells that did not penetrate the agar were removed by rubbing the plate with a finger under running water. Representative colonies were imaged. Bar, 100 µm.

Sir2 and Sir4 repress the silent mating locus. Therefore, the sir2-100 and sir4-100 strains express both mating type a and -specific genes and should behave as pseudodiploids. These two dia mutants perform polar budding and require BUD8 for invasion. To test whether the expression of the silent mating locus is responsible for their phenotypes, we deleted the HML silent locus in a MATa sir4::HIS3 strain. This sir4 hml strain mates as MATa, no longer invades agar, and resumes axial budding in 98 ± 1% of new buds, suggesting that expression of both mating type loci promotes agar invasion by inducing polar budding.

Most dia strains do not require RAS2 activity for hyperinvasion:
To test whether any of the dia mutations activate RAS2 to cause hyperinvasive growth, we crossed each dia strain to a ras2::kan^r strain to obtain dia ras2 segregants. Then we compared agar invasion by dia RAS2 colonies and dia ras2 colonies (Table 6 and Fig 8). Surprisingly, only three mutants, ira1-100, bpl1-100, and ssn3-100, require Ras2 activity for hyperinvasion. Ira1 is a GTPase-activating protein for Ras2 (TANAKA et al. 1989 ) and may inhibit agar invasion by inactivating Ras2. Bpl1 catalyzes biotinylation of proteins and its relation to Ras2 is unclear (CRONAN and WALLACE 1995 ). Ssn3 is a cyclin-dependent kinase homolog and a subunit of the RNA polymerase II mediator complex involved in Mig1-independent glucose repression along with Ssn8 and Srb8 (BALCIUNAS and RONNE 1995 ). Nonetheless, ssn8-100 and srb8-100 do not require Ras2 for agar invasion. The remainder of the dia strains activate invasive growth either downstream of RAS2 or via a parallel pathway.

View larger version (80K): In this window In a new window Download PPT slide   Figure 8. Hyperinvasion of dia strains, except ira1-100, bpl1-100, and ssn3-100, does not require Ras2 activity. Haploid dia strains were streaked on YPD to obtain single cells. The plates were incubated at 21° for 96 hr. Cells that did not penetrate the agar were removed by rubbing the plate with a finger under running water. Representative colonies were imaged. ira1-100, bpl1-100, and ssn3-100 invasion is abrogated by RAS2 deletion but srb8-100 invades via a RAS2-independent mechanism. Bar, 100 µm.

DIA gene interactions with Kss1 MAPK invasive growth signaling:
Kss1 is a MAP kinase that can both stimulate and inhibit the filamentous and hyperinvasive growth pathway (MADHANI et al. 1997 ; BARDWELL et al. 1998 ). Unphosphorylated Kss1 binds and represses Ste12, a transcriptional activator involved in pheromone signaling and filamentous response. Kss1 phosphorylation by Ste7 results in phosphorylation and activation of Ste12 and decreases the affinity of Kss1 for Ste12 and Dig1, relieving repression. By crossing each dia mutant strain to a kss1 strain, we determined whether the stimulatory and/or inhibitory effects of Kss1 are required for their hyperinvasive growth. Pathway analysis with kss1 is potentially superior to ste12 or other mutations that abrogate signal transduction as kss1 confers a low constitutive level of signaling, reducing the likelihood of false epistasis (BARDWELL et al. 1998 ).

We compared invasive growth of dia KSS1 strains with dia kss1 strains (Table 6 and Fig 9). KSS1 deletion reduces invasion in six dia strains: ydj1-100, zuo1-100, hsc82-100, pgi1-100, gnd1-100, and gtr1-100. Ydj1, Zuo1, and Hsc82 are involved in protein folding and cell stress response (LOUVION et al. 1998 ; LU and CYR 1998 ; YAN et al. 1998 ). Pgi1 catalyzes the isomerization of glucose-6-phosphate to fructose-6-phosphate while Gnd1 catalyzes the reduction of 6-phosphogluconate to ribulose-5-phosphate (LOBO and MAITRA 1982 ; DICKINSON 1991 ). Gtr1 is a GTP-binding protein required for phosphate transport (BUN-YA et al. 1992 ). These proteins may provide links between metabolic regulation and invasive growth. Paradoxically, gtr1-100 does not increase FLO11 expression, a known target of the MAPK invasive growth signaling pathway, suggesting KSS1 may affect invasive growth through FLO11-independent mechanisms in addition to inducing FLO11 transcription. However, it is unclear whether each of these genes requires the stimulatory or inhibitory activity of Kss1.

View larger version (102K): In this window In a new window Download PPT slide   Figure 9. Some dia strains require Kss1 activity for hyperinvasion of agar. Haploid dia strains were streaked on YPD to obtain single cells. The plates were incubated at 21° for 96 hr. Cells that did not penetrate the agar were removed by rubbing the plate with a finger under running water. Representative colonies were imaged. pgi1-100 and zuo1-100 require Kss1 to enhance agar invasion but adh1-100 does not. Bar, 100 µm.

SWE1 mediates the enhanced invasive growth in hsl1, hsl7, and elm1 mutant strains:
Elm1, Hsl1, and Hsl7 repress cell elongation, filamentous growth, and invasive growth by negatively regulating Swe1, which in turn phosphorylates Cdc28 to inhibit mitosis (EDGINGTON et al. 1999 ). As expected, SWE1 deletion suppresses the cell elongation and hyperinvasion phenotypes of elm1-100, hsl1-100, and hsl7-100 strains (Fig 10).

View larger version (106K): In this window In a new window Download PPT slide   Figure 10. elm1 and hsl7 mutant strains invade in a SWE1-dependent manner. Haploid dia strains were streaked on YPD to obtain single cells. The plates were incubated at 21° for 96 hr. Cells that did not penetrate the agar were removed by rubbing the plate with a finger under running water. Representative colonies were imaged. pgi1-100 and zuo1-100 require Kss1 to enhance agar invasion but adh1-100 does not. Bar, 100 µm.

	DISCUSSION

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

The flocculin gene FLO11 is upregulated by activation of filamentous signaling to mediate cell-cell adhesion during flocculation, invasive growth, and filament formation (LO and DRANGINIS 1998 ). Although a precise role for Flo11 in yeast invasion remains unclear, this flocculin probably contributes to cell-cell and/or cell-matrix adhesion so the multicellular colony can generate sufficient traction force to burrow into the substratum. Previous studies have shown that flo11 deletion mutant cells are completely deficient for agar invasion and filamentous growth (LAMBRECHTS et al. 1996 ; LO and DRANGINIS 1996 ). However, study of the significance of regulated expression of FLO11 is confounded by the complexity of the FLO11 promoter and its responsiveness to multiple stimuli (RUPP et al. 1998; GAGIANO et al. 1999B ). Within the microenvironment of the invasive yeast colony, it is difficult to predict the pattern of expression of FLO11 on a cell-by-cell basis, yet this expression may be critical to the phenotype. By replacing the endogenous FLO11 promoter with the S. pombe adh⁺ and S. cerevisiae CDC28 promoters, we clamped FLO11 expression at a level somewhat below and somewhat above the basal expression of FLO11 in rich liquid media. This constitutive expression had significant effects on both haploid cell invasion as well as diploid cell morphology and filamentation, suggesting that the quantitative level of FLO11 expression and presumably abundance of Flo11 protein on the cell surface are important determinants of both invasive growth and pseudohyphal development. Further, based on the filamentous growth response of diploid cells carrying clamped promoter alleles of FLO11, Flo11 appears to have a role in determining cell shape in addition to its functions in cell adhesion.

Based upon the clear relationship between levels of FLO11 expression and invasive growth, we performed a genetic screen for mutants that dig into agar. We visually screened transposon insertion mutants for clones that display enhanced agar invasion and then measured the FLO11 expression in each of these mutant strains. We identified 35 DIA genes that when mutated confer markedly enhanced invasive growth. The mutations fall in elements of multiple signaling, metabolic, and morphogenesis pathways. Although most DIA genes are novel regulators of invasion and filamentation, among them are several genes previously implicated as negative regulators of agar invasion and/or filamentous growth. Nonetheless, we did not identify several genes whose deletion has previously been reported to promote haploid invasive growth such as CLB2, BCY1, TPK3, SFL1, CTS1, and ACE2 (KING and BUTLER 1998 ; ROBERTSON and FINK 1998 ; AHN et al. 1999 ; PAN and HEITMAN 1999 ). That our screen was not performed to saturation, that we only selected highly invasive mutants, and our use of a transposable element as a mutagen may each have contributed to lack of complete coverage of this phenotype.

Regulation of FLO11 in haploid invasive growth:
A well-described phenotype of flo11 deletion is a nearly complete deficit in diploid filamentous growth. Similarly, FLO11 has been shown necessary for haploid invasion. Our results with S. pombe adh⁺ promoter and CDC28 promoter-driven FLO11 suggest that FLO11 upregulation is also sufficient to drive invasion. As expected, we found that FLO11 induction likely plays a key role in the enhanced invasion displayed by the dia mutants; 26 of the 35 mutations we identified confer significantly increased FLO11 expression. However, to address whether FLO11 upregulation was essential for the enhanced invasion conferred by the dia mutants, we assayed for invasive growth in strains where the endogenous FLO11 promoter was replaced with the S. pombe adh⁺ promoter and thereby clamped at a low, constitutive expression level.

Bypassing the effects of SpADH promoter-driven FLO11 is a relatively high bar to clear. This construct permits only 40% of the FLO11 transcription of haploids grown in YPD with FLO11 under its own promoter. FLO11 expression may fall below a "critical threshold" required for cell invasion. Nonetheless, we did find that many dia strains, including some that upregulate FLO11 expression, could still invade despite this low FLO11 expression level. In particular, strains that bud in a polar manner do not require FLO11 upregulation to be invasive. The only exceptions are hsl1, hsl7, and ydj1, suggesting that increased cell-cell adhesion from higher FLO11 expression is crucial in these strains along with polar bud site selection. In the other polar-budding strains the level of cell-cell adhesion provided by FLO11 may contribute a basal level of cell invasion, which is enhanced by cell elongation or polarized bud site selection.

Agar invasion, flocculation, and filamentation are genetically separable processes:
Among our findings is that haploid agar invasion, flocculation, diploid invasion, and diploid filamentation can be genetically separated. Each process can be activated by the MAP kinase signaling pathway, but pathways unique to each process also exist. For example, mutations that increase polar budding in haploids but do not increase FLO11 expression (e.g., bud3, bud4, bud10, axl1, sir2, and sir4) do not enhance agar invasion or diploid elongation. This class of genes identifies a haploid-specific invasive growth pathway based on polarized bud site selection. In contrast, mutations that enhance cell elongation also increase diploid invasion and filamentation but have no effect on flocculation.

Mutations that do not affect cell elongation or bud site selection have more complex effects on diploid phenotypes. Mutations that derepress FLO11 generally also induce hyperinvasive growth in diploids. Pseudohyphal growth relies on many of the same physical processes as agar invasion including cell adhesion and polarization, so intersection of these pathways is not surprising. For example, ira1/ira1 mutants are extremely filamentous; adh1/adh1 mutations also enhance filamentation. However, the regulatory pathways that affect these processes also diverge. These distinctions can be quite subtle; srb8/srb8 displays increased pseudohyphal growth but ssn3/ssn3 and ssn8/ssn8 do not, even though the three genes appear to function together in the RNA polymerase II complex (BALCIUNAS and RONNE 1995 ). Invasion-specific pathways may diverge from pseudohyphal growth downstream or act independently of Ras2 or Kss1 as ras2 or kss1 mutations are readily bypassed by many dia mutants.

Bud site selection as a determinant of agar invasion:
This screen identified polar bud site selection as a novel mechanism for inducing or enhancing yeast haploid invasive growth that can promote haploid cell invasion without upregulation of FLO11. We identified mutations in 15 different genes that both increase polar budding and induce invasive growth in haploids. As expected, bud8 deletion confers proximal budding to each of these dia strains, except the elm1 and bem2 mutants. The loss of polar budding in the dia bud8 strains is accompanied by suppression of the invasive phenotype, even in strains that induce FLO11 expression (e.g., pgi1, gnd1, ydj1, aro7, and dia2). Interestingly, polar budding in elm1 seems to be independent of normal bud site selection processes, possibly due to septin localization and bud site misassembly at the bud tip (BOUQUIN et al. 2000 ).

Our work is distinct from previous studies (e.g., ROBERTS et al. 1997 ; LO and DRANGINIS 1998 ; GALITSKI et al. 1999 ) that described wild-type haploid cells of the 1278b background as invasive and diploids as noninvasive. Consistent with our findings that polar bud site selection is sufficient to confer a dia phenotype, we find instead that wild-type diploids invade to a degree at least equivalent to many of our dia mutants. In turn, pseudodiploid strains and strains that express both mating type loci also invade significantly more than wild-type haploids. This discrepancy with the literature is likely due to differences in the protocol used to assay invasion and the definition of "invasive growth." We have used a combination of flowing water and manual force to sweep away cells that do not penetrate below the surface of the agar where other assays often rely on the flow of water alone to remove nonadherent cells from the agar plate. We have found that under running water, cell-cell and cell-agar adhesion are sufficient to permit haploid colonies to remain in place unless they are removed by rubbing the plate. Typically, no cells are observed within the agar below a haploid colony. By contrast, the surface cells of a diploid colony are readily washed from the plate by simple rinsing but a core of the diploid colony that has penetrated the agar surface typically remains. We suggest that agar invasion measured by this method is a more reliable and specific measure of capacity for invasive growth as opposed to cell-cell adhesion.

Despite the key role of bud site selection in invasive growth, we find that, like enhanced FLO11 expression, elongated cell morphology is sufficient to induce agar invasion. Mutations in at least two DIA genes, GRR1 and CDC53, confer elongated cell morphology without affecting axial bud site selection. Grr1 interacts with Skp1, a component of the E3 ubiquitin-ligating enzyme complex along with Cdc53 and Cdc4 (PATTON et al. 1998 ). Presumably, mutations in this complex stabilize enhancers of polarized growth. Candidates include G₁ cyclins Cln1 and Cln2, mitotic inhibitors Sic1 and Swe1, and the Cdc42 effector Gic2 (LI and JOHNSTON 1997 ; JAQUENOUD et al. 1998 ; KAISER et al. 1998 ). In addition, Grr1 represses glucose-repressed genes such as HXT glucose transporters (LI and JOHNSTON 1997 ; GANCEDO 1998 ) and possibly other genes that inhibit invasive growth.

Signaling mechanisms that repress invasive growth:
Not all the dia mutants participate directly in limiting FLO11 expression, bud polarity, or cell elongation. Our screen also identified negative regulators of the signaling pathways that control these outputs. The three stress response genes, HSC82, ZUO1, and YDJ1, can be linked to the STE MAP kinase pathway by their dependence on intact KSS1 for their effects. HSC82 encodes the constitutively expressed isoform of the heat-shock protein Hsp90. Hsp90 can induce signaling through Ste11 (LOUVION et al. 1998 ), which in turn activates FLO11 transcription. Ydj1 has a synthetic lethal interaction with Hsp90 (KIMURA et al. 1995 ). The cAMP-dependent protein kinase pathway has been implicated in suppressing certain stress response pathways, leading to invasive growth (STANHILL et al. 1999 ). Mutations in stress response genes may heighten this effect. Also, hyperosmotic shock can induce filamentation and invasion in a Kss1-dependent manner if the high-osmolarity glycerol response pathway is compromised (DAVENPORT et al. 1999 ).

An independent group of mutants includes the RNA Pol II subunits SSN3, SSN8, SRB8, CSE2, and MED1. Mutations in these subunits also release transcriptional repression of glucose-repressible genes (BALCIUNAS and RONNE 1995 ; BALCUINAS et al. 1999), providing a possible link between carbon metabolism and invasive growth. This complex may repress cell invasion in high-glucose conditions but allow invasion during glucose starvation. The glycolytic enzymes PGI1, GND1, and ADH1 also provide a link between carbon regulation and cell invasion. Perhaps an accumulation of metabolic intermediates is responsible for the hyperinvasion. Indeed, certain nonmetabolizable alcohols can induce cell elongation and polarized budding (LORENZ et al. 2000A ). Alcohol-induced filamentation requires an intact STE MAP kinase cascade, as does invasion due to mutations in PGI1 and GND1, suggesting a link between such a metabolite and STE pathway activation rather than glucose repression per se.

Several DIA genes that likely participate in signaling have no clear links to known pathways regulating invasive growth. GTR1, MED1, and TAF25 enhance cell invasion without altering bud site selection, cell morphology, flocculation, or FLO11 expression. As yet unknown factors beyond elongation, polar budding, or cell adhesion may contribute to agar invasion in these mutants. One potential target is enzymatic degradation of the agar, allowing cells to penetrate without significantly increasing the force they generate. The coregulation of the FLO11 adhesin and the PGU1 pectinase (MADHANI et al. 1999 ) as well as the STA1, 2, and 3 glycoamylases (GAGIANO et al. 1999B ) provides a paradigm for coordinated regulation of cell adhesion and matrix degradation, suggesting that agar degrading activities may indeed be targets of invasive growth signaling pathways.

	FOOTNOTES

¹ Present address: Department of Chemical Engineering, University of Wisconsin-Madison, 1415 Engineering Dr., Madison, WI 53706-1691.

	ACKNOWLEDGMENTS

The authors thank A. Amon, A. Dranginis, J. Pringle, J. Rine, M. Snyder, and members of the Kron laboratory for sharing reagents and helpful advice. The authors acknowledge Gerald R. Fink for his seminal contributions to the field and congratulate him on his 60th birthday. This work was supported by National Science Foundation CAREER grant MCB-9875976 and a James S. McDonnell Foundation Scholar Award to S.J.K. S.P.P. is an Amgen Fellow of the Life Sciences Research Foundation.

Manuscript received May 20, 2000; Accepted for publication July 21, 2000.

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