Phosphatidylinositol 4,5-bisphosphate is required for invasive growth in Saccharomyces cerevisiae

Polarized growth is critical for a range of cellular processes and responses to different environments. Phosphatidylinositol phosphates (PIPs) are important for actin cytoskeleton organization, vesicle trafficking (exo- and endocytosis) and transcription – all of which are required for polarized cell growth (Di Paolo and De Camilli, 2006; Strahl and Thorner, 2007; Vicinanza et al., 2008; Kwiatkowska, 2010; Saarikangas et al., 2010; Shewan et al., 2011). The phosphatidylinositol PtdIns(4,5)P₂ is a major PIP species, generated by phosphorylation of PtdIns(4)P at position 5 by type I phosphatidylinositol-4-phosphate 5-kinases (PI4P5K). In the budding yeast Saccharomyces cerevisiae, PtdIns(4,5)P₂ is generated by the conserved PI4P5K Mss4p which localizes to cortical patches PIK patches at the plasma membrane (Desrivières et al., 1998; Homma et al., 1998; Audhya and Emr, 2002; Ling et al., 2012), where the majority of this lipid is found. This sole yeast PI4P5K – a member of the type I subfamily – is essential for cell viability and actin cytoskeleton organization (Desrivières et al., 1998; Homma et al., 1998). In response to different signals, transient site-specific changes in PtdIns(4,5)P₂ levels result in membrane recruitment and activation of signaling proteins which contain particular domains that bind this phospholipid (Di Paolo and De Camilli, 2006; Lemmon, 2008; Vicinanza et al., 2008). For example, pleckstrin homology (PH), basic rich (BR) and AP180 N-terminal homology (ANTH) domains, which specifically bind PtdIns(4,5)P₂, are found in a range of proteins including those implicated in actin organization, exocytosis, endocytosis, membrane trafficking and cell division (e.g. septins) (Audhya and Emr, 2002; Sun et al., 2005; He et al., 2007; Takahashi and Pryciak, 2007; Orlando et al., 2008; Zhang et al., 2008; Bertin et al., 2010). Mss4p is critical for plasma membrane targeting of components of the yeast pheromone response (i.e. Ste5p) and the yeast cell wall integrity MAP-kinase (MAPK) pathways (i.e. Rom2p) and hence PtdIns(4,5)P₂ is important for responses to cell wall perturbations and pheromone (Audhya and Emr, 2002; Garrenton et al., 2006; Garrenton et al., 2010).

Tor kinases play an important role in nutrient sensing (Barbet et al., 1996; Rohde et al., 2008) and pseudohyphal growth, particularly in response to nitrogen deprivation (Cutler et al., 2001; Orlova et al., 2006). Furthermore, genetic and physical interactions between either Mss4p or PtdIns(4,5)P₂-binding proteins and Tor-kinase-containing complexes (TORCs) have been observed (Audhya et al., 2004; Fadri et al., 2005; Berchtold et al., 2012). For example, Slm1p and Slm2p, both PH domain containing PtdIns(4,5)P₂-binding proteins, interact with TORCs (Audhya et al., 2004; Fadri et al., 2005; Berchtold et al., 2012). Mss4p mediated PtdIns(4,5)P₂ production, Slm1p/Slm2p and Tor kinases are also important for sphingolipid signaling (Audhya et al., 2004; Kobayashi et al., 2005; Bultynck et al., 2006; Daquinag et al., 2007; Roelants et al., 2011; Berchtold et al., 2012; Liu et al., 2012). Starved yeast cells respond to the addition of glucose or ergosterol, the major sterol of S. cerevisiae, by rapid changes in PIPs (Dahl and Dahl, 1985; Kaibuchi et al., 1986; Frascotti et al., 1990). With respect to glucose, these changes are characterized by rapid increases in the levels of PtdIns(4,5)P₂ metabolites including phosphatidic acid, PI and PIP (Kaibuchi et al., 1986; Kato et al., 1989; Frascotti et al., 1990) and also PtdIns(4,5)P₂ levels. Ras2p, which is required for filamentous growth, has been implicated in these PIP increases (Kaibuchi et al., 1986; Kato et al., 1989; Frascotti et al., 1990) which require phospholipase C1 (Plc1p) (Coccetti et al., 1998).

PtdIns(4,5)P₂ is likely to play a role in responses to cell wall perturbation (Audhya and Emr, 2002; Morales-Johansson et al., 2004) and is important for responses to mating pheromone (Garrenton et al., 2006; Garrenton et al., 2010). A unique dedicated MAP kinase pathway is critical for each of these processes, i.e. the cell wall integrity and the mating pheromone-dependent MAPK cascades (Chen and Thorner, 2007). In response to limiting nutrients S. cerevisiae undergo filamentous growth, which is regulated by both MAPK and Ras2/cAMP-dependent protein kinase A (PKA) pathways (Brückner and Mösch, 2012; Cullen and Sprague, 2012). In haploid and diploid cells this nutrient-regulated growth state is frequently referred to as invasive growth and pseudohyphal growth, respectively. These signaling pathways regulate filamentous growth by inducing the transcription of a range of genes, one of the most important being the adhesion flocculin FLO11. Filamentous growth is characterized by three visible changes: cell elongation, increased cell–cell and cell–substrate adhesion due to cell surface changes and alteration in budding pattern to a unipolar-distal budding pattern (Mösch and Fink, 1997; Madhani, 2000).

Given the roles of PtdIns(4,5)P₂ in response to varied stresses including nutrient deprivation and cell wall integrity we examined whether Mss4p and hence this lipid were required for S. cerevisiae filamentous growth. We have carried out a genetic screen for mss4 mutants specifically defective in haploid invasive growth and show that Mss4p is required for this process, in particular cell elongation, induction of the Flo11p flocculin and adhesion to a solid substrate. Our results indicate that PtdIns(4,5)P₂ is critical for haploid invasive growth and suggest that a polarized distribution of this lipid is important for polarized growth in response to nutrient deprivation.

Yeast invasive, filamentous growth requires major rearrangements of the actin cytoskeleton together with the activation of two different signal transduction pathways, a MAPK and cAMP-dependent PKA pathway (Brückner and Mösch, 2012; Cullen and Sprague, 2012). We postulated that, similar to the role of PIPs in mammalian cells and the yeast pheromone response, phosphatidylinositol phosphates might be important for polarized growth in response to limiting nutrient conditions. We set out to determine if the PI4P5K Mss4p is required for yeast haploid invasive growth. A randomly mutagenized library of mss4 mutants was screened for alleles specifically defective in invasive growth. We focused on the Mss4p catalytic kinase domain and a portion of this domain (amino acid 467–601) was mutagenized by error prone PCR (Fig. 1A). A mss4Δ strain, which was kept alive with a plasmid copy of MSS4 driven by the GAL1 promoter, was used to isolate mss4 alleles defective for haploid invasive growth. Over 12,000 yeast colonies were screened for their ability to undergo invasive growth on YEPD medium and ten mutants were identified (Fig. 1B) which exhibited various degrees of invasive growth defects. Four mutants, mss4-f9, -f12, -f13 and -f22, whose defects were reproducibly observed upon plasmid rescue and retransformation, were further studied. Sequencing revealed mss4-f9 and -f22 had a mutation, which resulted in the replacement amino acid arginine 590 by a stop codon. In addition mutations, which changed lysine 597 to arginine in mss4-f9 and isoleucine 566 to asparagine, threonine 574 to isoleucine and lysine 597 to asparagine in mss4-f22 were identified. Mutants mss4-f12 and -f13 both had the same unique mutation, which resulted in the amino acid residue serine 573 changed to a proline. Each individual amino acid change was recreated by mutagenesis and the changes S573P (mss4-f12) and R590* (mss4-f9) are responsible for the invasive growth defects (Table 1; Fig. 1C) and will be hereafter referred to as mss4-f12 and mss4-f9, respectively (Fig. 1A). To rule out the possibility that yeast cells were able to read through the mss4-f9 mutation (R590*) and thereby generate full-length kinase, the portion of MSS4 3′ to the newly introduced stop codon was also removed. Both of these recreated mutants were functional for viability as cells grew on 5-FOA-containing medium. Fig. 1C shows that both mss4-f9 and mss4-f12 mutants are defective in invasive growth, with little to no cells of the latter mutant invading the agar surface. The mss4-f9 mutant had an intermediate defect with less invasion compared to wild-type cells yet increased invasion compared to mss4-f12 cells. These mutants were also defective in invasive growth on low ammonia medium (supplementary material Fig. S1); however, due to poor invasive growth in this condition, subsequent analyses were carried out with low glucose medium. We examined the location of the amino acid changes in mss4-f9 and mss4-f12 mutants in an alignment of PIP-kinases from a range of organisms and the arginine 590 amino acid residue altered in mss4-f9 is not conserved in other PIP-kinases (Fig. 1A). The stop codon in the mss4-f9 mutant results in a truncation of the conserved catalytic domain removing regions of this domain that are likely to be involved in substrate binding, catalysis and plasma membrane targeting (Kunz et al., 2000; Arioka et al., 2004). Strikingly, the serine residue 573, which is altered in mss4-f12 is highly conserved in all PIP-kinases examined. This residue is in proximity to other residues implicated in binding to PIP lipids. Together, these results demonstrate that the PI4P5K Mss4p is necessary for yeast invasive growth.

MSS4 is essential for viability and necessary for organization of the actin cytoskeleton (Desrivières et al., 1998; Homma et al., 1998). The mutations in both mss4-f9 and mss4-f12 are localized to the catalytic kinase domain and therefore we examined the budding growth of these mutants in different solid medium (Fig. 1D). These two mutants grew similar to wild-type strains on rich medium (YEP) containing 2% glucose. When grown on medium containing reduced glucose levels (YEP0.2%D) or non-fermentable carbon sources, such as ethanol or glycerol, the mss4-f12 mutant grew less well than wild-type cells while the growth of mss4-f9 cells was less affected. YEP medium containing 0.2% glucose, 2% ethanol, or 2% glycerol induces invasive growth in wild-type cells more efficiently than YEP medium with 2% glucose (Cullen and Sprague, 2000). Microscopic examination of the yeast colonies on YEP0.2%D revealed that both mss4 mutants do not form crenelated colonies in contrast to wild-type cells but rather form colonies with a smooth surface similar to growth on YEP2%D (Fig. 1E) (Halme et al., 2004; Casalone et al., 2005). Similar differences in colony morphology were observed in ethanol and glycerol containing medium (supplementary material Fig. S2). These alterations in colony morphology are correlated with invasive growth (Reynolds and Fink, 2001; Casalone et al., 2005). As MSS4 is important for mating pheromone response (Garrenton et al., 2010), we examined whether the mss4-f12 cells could respond to mating pheromone and mate efficiently. Both MSS4 and mss4-f12 cells formed shmoos in response to mating pheromone (data not shown) and halo assays revealed identical pheromone-dependent growth arrest (supplementary material Fig. S3A). Furthermore, pheromone-dependent gene induction using the FUS1-lacZ reporter revealed little to no difference with the wild-type (supplementary material Fig. S3B). In quantitative matings the mss4-f12 strain exhibited a minor mating defect (mating efficiency 12.0±1.2% versus 23.5±2.4% for MSS4 strain), which was not substantially affected with an enfeebled mating partner (2.2% for mss4-f12 strain versus 7.3% for MSS4 strain). Together, our results indicate that these mss4 mutants are specifically defective in invasive growth.

We next examined the expression levels of Mss4p in wild-type and mutant strains to determine if the invasive growth defect could be due to altered Mss4p levels. Fig. 2A shows an immunoblot of cells expressing either Mss4p mutant, Mss4-f9p, or Mss4-f12p, as the sole PI4P5K using an anti-Mss4p polyclonal serum directed against the Mss4p N-terminus. As expected, Mss4-f9p migrated faster than wild-type Mss4p due to the stop codon at position 590. Furthermore, this mutant was expressed at a lower level compared to Mss4p, whereas Mss4-f12p is expressed at similar or greater levels compared to Mss4p. As the mss4-f12 mutant exhibited a strong invasive growth defect, we examined whether this defect could be due to an altered level of Mss4p. However, two results argued against this explanation. Firstly, the invasive growth defect of each mutant was complemented by a wild-type copy of MSS4 (supplementary material Fig. S4), indicating that these mutants are recessive. Secondly, we overexpressed Mss4p using the Tet promoter and examined if this resulted in an invasive growth defect. In the absence of the repressor doxycycline, Mss4p is highly overexpressed yet of invasive growth was not affected (Fig. 2B). These results indicate that the invasive growth defects of mss4-f9 and mss4-f12 mutants are unlikely to be due to alterations in the level of this PI4P5K.

In addition to the changes in cell–cell adhesion that result in crenelated colonies during filamentous growth, cells elongate and undergo a reorganization of cell polarity. Mss4p is critical for cell morphology and actin cytoskeleton organization (Desrivières et al., 1998; Homma et al., 1998). First we examined the cell morphology of MSS4, mss4-f9 and mss4-f12 mutants. Fig. 3A shows that in rich medium with excess glucose, these mutants exhibit similar morphologies with the mss4-f12 mutant cells slightly larger than wild-type and mss4-f9 cells (average cell long axis MSS4 5.7±0.8 µm, n = 296; mss4-f9 5.7±0.8 µm, n = 318; mss4-f12 6.0±0.8 µm, n = 207). We also examined if these mss4 mutants had an altered bud site selection pattern and found that they budded axially, similar to wild-type cells (wild-type 88%, mss4-f9 85% and mss4-f12 86% axial budding; n = 150–200 cells). Upon glucose depletion, the wild-type formed elongated clumps of cells, whereas the mss4-f12 mutant was substantially defective in cell elongation and appeared not to form cell clumps (Fig. 3B). The mss4-f9 mutant exhibited an intermediate phenotype. As unipolar-distal budding is necessary for invasive growth (Mösch and Fink, 1997; Madhani, 2000) we examined the bud site selection of these mss4 mutants in glucose depletion conditions. Both mutants budded in a unipolar pattern with 88% and 90% unipolar budding for mss4-f9 and mss4-f12 cells, respectively, compared to 85% for wild-type cells (n = 200 cells).

Actin has multiple functions in filamentous growth and reorganization of the actin cytoskeleton is critical during invasive growth (Cali et al., 1998). Hence we examined the distribution of actin cytoskeleton. Fig. 3C shows that actin patches are highly polarized and found predominantly in the buds of mss4-f9 and mss4-f12 cells similar to wild-type controls. In addition, the actin cable distribution appeared to be unaffected in these mss4 mutants, suggesting that the invasive growth defect is not due to a general defect in actin cytoskeleton organization nor in budding pattern. As Mss4p/PtdIns(4,5)P₂ are important for polarized growth (Yakir-Tamang and Gerst, 2009a; Yakir-Tamang and Gerst, 2009b), we investigated the distribution of cell polarity proteins in the mss4-f12 mutant. The Cdc42p effector Gic2p, the exocyst subunit Sec3p and septins are all involved in cell polarity and bind PtdIns(4,5)P₂ (Takahashi and Pryciak, 2007; Orlando et al., 2008; Zhang et al., 2008; Bertin et al., 2010). A reporter for active Cdc42p (Gic2_PBDRFP), the polarisome component Spa2p, Sec3p and the septin subunit Cdc3p localize similarly in MSS4, mss4-f12 and mss4-f9 cells (supplementary material Fig. S5; data not shown). There were somewhat lower Spa2GFP signals in mss4-f12 cells; however, as the signal was polarized we attribute this difference to expression levels.

The cell wall integrity pathway contributes to invasive growth (Birkaya et al., 2009). Hence we examined whether the mss4 mutant with the stronger invasive growth defect was sensitive to the cell wall perturbants Congo red (CR) and Calcofluor white (CFW). Fig. 3D shows that mss4-f12 cells are more sensitive to CR and CFW, although growth of this mutant was still observed. Together our results indicate that while there are no major defects in cell polarity in the mss4 mutants, cell wall integrity is affected.

The cell adhesion flocculin, Flo11p is critical for haploid invasive growth (Lambrechts et al., 1996; Lo and Dranginis, 1998; Rupp et al., 1999; Guo et al., 2000). Upon glucose depletion, this flocculin, which is downstream of both MAP kinase and cAMP PKA pathways, is upregulated (Rupp et al., 1999; Cullen and Sprague, 2000; Kuchin et al., 2002). We used the FLO11 promoter fused to the LacZ to investigate whether the mss4 mutants were able to induce Flo11p expression after glucose depletion. The levels of Flo11–LacZ were determined after 7 hr of growth in medium lacking glucose and Fig. 4A shows that in these conditions wild-type cells induce Flo11p by ∼5-fold. The mss4-f9 mutant induced Flo11p to a lower level, ∼60% of control cells, whereas the mss4-f12 mutant exhibited a further reduction in Flo11p upregulation (∼30%). In amino acid starvation conditions, Flo11p is also required for adhesive growth to solid agar. Hence we examined whether the mss4 mutants could adhere to a solid surface upon His starvation (Roberts and Fink, 1994; Braus et al., 2003; Guldal and Broach, 2006). Fig. 4B shows that in comparison to wild-type cells, mss4-f9 cells were somewhat reduced in adhesion, whereas the mss4-f12 mutant was substantially reduced in adhesion to this solid surface. These results suggest that Mss4p is required for Flo11p upregulation and ultimately adhesion.

We next examined the distribution of active Cdc42p, Spa2p, Sec3p and Cdc3p in wild-type and mss4-f12 cells grown in low glucose (0.2%). In these conditions cells begin to elongate after 6–8 hr and there was still sufficient signals of the reporters. These cell polarity proteins localize similarly in wild-type, mss4-f12 and mss4-f9 cells (supplementary material Fig. S6; data not shown), although a reduction in the signals of Spa2–GFP and Cdc3–GFP was observed in the mss4-f12 mutant. These results indicate that, in glucose depletion conditions, cell polarity is not substantially perturbed in the mss4-f12 mutant.

As both of the identified mss4 mutants have alterations in the catalytic core domain we examined whether these mutants had reduced PtdIns(4,5)P₂ levels in vivo. Phosphatidylinositol phosphates were labeled in vivo with [³³P]P_i, lipids were then extracted and analyzed by TLC. Both mutants exhibited ∼40% increase in PtdIns(4)P levels and a 30–40% decrease in PtdIns(4,5)P₂ levels (Fig. 5A). [³H]inositol labeling was also carried out to determine if complex sphingolipid synthesis was perturbed, as previous studies have suggested that phosphatidylinositol phosphate levels are linked to complex sphingolipid levels (Audhya et al., 2004; Kobayashi et al., 2005; Bultynck et al., 2006; Daquinag et al., 2007; Berchtold et al., 2012). Using inositol labeling, similar results were observed with respect to increased PtdIns(4)P levels and reduced PtdIns(4,5)P₂ levels in the mss4 mutants. However analyses of complex sphingolipids by TLC revealed a substantial increase in the levels of mannosyl-di-inositolphosphate-ceramide [M(IP)₂C] (Fig. 5B). Together these results indicate that the mss4 mutants accumulated PtdIns(4)P and M(IP)₂C and had decreased levels of PtdIns(4,5)P₂ in vivo. We then focused our attention on the strongest mutant, mss4-f12.

While the above biochemical analyses revealed that the mss4-f12 mutant had reduced levels of PtdIns(4,5)P₂, the labeling studies did not provide information on the distribution and levels of PtdIns(4,5)P₂ at the plasma membrane. We used the PtdIns(4,5)P₂ reporter that we had optimized for use in C. albicans (Vernay et al., 2012) to visualize plasma membrane PtdIns(4,5)P₂ in S. cerevisiae. This GFP–PH^Plcδ–PH^Plcδ–GFP reporter, driven by the GAL1 promoter to minimize toxicity associated with long-term expression was integrated at the URA3 locus. Confocal z-sections revealed that this reporter localized predominantly to the plasma membrane and was enriched in small/medium buds (Fig. 5C). In contrast, mss4-f12 cells exhibited an increase in cytoplasmic signal as well as an apparent redistribution of signal in the mother cell. By immunoblot, we observed somewhat higher levels of this PtdIns(4,5)P₂ reporter in the mss4-f12 mutant compared to wild-type MSS4 cells (Fig. 5D). We used a tailor-made MATLAB program to analyze the signal concentration over long axis of the cell (Vernay et al., 2012) and Fig. 5E shows that in the wild-type strain there was an average 2-fold increase in PtdIns(4,5)P₂ concentration going from the back of the mother cell to the tip of the bud. In contrast, the mss4-f12 mutant exhibited little polarized PtdIns(4,5)P₂ distribution with only an average ∼25% increase in PtdIns(4,5)P₂ concentration over the cell long axis. From these sum projections, we also observed an increase in the level of the reporter in the mss4-f12 cells. We determined the ratio of plasma membrane to cytoplasmic signal (from the central z-section) and wild-type cells had an average ratio of 1.98±0.57 (n = 65 cells) compared to that of the mutant 1.46±0.48 (n = 77 cells). Quantitation of the total signal (plasma membrane and intracellular) from central z-sections also revealed a slight increase in the reporter level in the mss4-f12 mutant (average relative value for wild-type MSS4 137±37, n = 65 cells; mss4-f12 156±43, n = 77 cells). These results demonstrate that there is a redistribution of plasma membrane PtdIns(4,5)P₂ levels in mss4-f12 cells resulting in reduced PtdIns(4,5)P₂ polarity.

Mss4p is essential for cell viability and cells expressing kinase defective mutants are not viable (Kobayashi et al., 2005; Ling et al., 2012). Cells expressing the mss4-f12 mutant as the sole MSS4 copy were viable, suggesting that this mutant retained catalytic activity. We used GFP fusions to Mss4p and Mss4-f12p to facilitate immuno-purification for in vitro kinase assays. C-terminal GFP fusions were expressed in a mss4Δ strain and the Mss4-f12–GFP strain exhibited a similar invasive growth defect as the mss4-f12 mutants (supplementary material Fig. S4). Mss4–GFP fusions were purified from these strains and Fig. 6A shows that similar amounts of Mss4p and Mss4-f12p were immunoprecipitated. These agarose immobilized PI4P5Ks were then assayed for their ability to phosphorylate PtdIns(4)P using [γ-³²P]ATP. Fig. 6B shows that Mss4-f12p is catalytically active and phosphorylated PtdIns(4)P to a similar extent as Mss4p. We had difficulties reproducibly immunoprecipitating Mss4-f9–GFP, yet were able to detect low kinase activity, suggesting that this truncated Mss4p has some activity. These in vitro kinase activity assays indicate that mutation of the conserved serine residue at position 573 does not dramatically alter the catalytic activity of this kinase, yet nonetheless perturbs PtdIns(4,5)P₂ levels in vivo.

Mss4p localizes to the plasma membrane and this localization is critical for in vivo PI4P5K activity (Homma et al., 1998; Audhya and Emr, 2003; Ling et al., 2012). To examine Mss4-f9p and Mss4-f12p localization, we used the functional GFP fusions (supplementary material Fig. S7) described above which were expressed from their endogenous promoter. We observed that Mss4–GFP localized to the plasma membrane in both rich and glucose depletion medium (Fig. 6C). Strikingly, we observed a reduction of Mss4-f9–GFP and Mss4-f12–GFP at the plasma membrane (Fig. 6C). In contrast to Mss4-f12–GFP, some Mss4-f9–GFP was observed at the plasma membrane. We carried out crude fractionation to determine whether the Mss4-f9 and Mss4-f12 proteins were soluble. Fig. 6D shows that the majority of the wild-type and mutant Mss4 proteins were found in a 10,000 g pellet with little protein found in the soluble 10,000 g supernatant. Localization and fractionation of Mss4-f9p indicate that there is less of this protein. Our results indicate that the Mss4-f12p mutant is not targeted or maintained at the plasma membrane, despite being associated with cellular membranes.

The induction of Flo11p is dependent on two signaling pathways, the Kss1p MAPK pathway and cAMP-dependent PKA pathway (Rupp et al., 1999; Brückner and Mösch, 2012; Cullen and Sprague, 2012). To determine if Mss4p functioned upstream of the cAMP dependent pathway we examined whether the addition of exogenous cAMP was sufficient to restore the invasive growth defect of mss4-f12 cells. Fig. 7A shows that medium containing 5 mM cAMP, was sufficient to restore invasive growth, similar to that previously observed for a gpa2 mutant (Kübler et al., 1997). We next examined whether expression of the activated MAPKKK allele, ste11-4, could also restore invasive growth of the mss4-f12 mutant. Fig. 7B shows that mss4-f12 cells expressing ste11-4 were still defective in agar invasion. Quantitation of the morphology of MSS4 and mss4-f12 cells expressing ste11-4, revealed that these cells were larger, displayed aberrant morphologies (long axis of MSS4 cells 5.7±0.8 µm, n = 137; mss4-f12 cells 6.2±0.8 µm, n = 84; MSS4 ste4-11 cells 8.1±1.7 µm, n = 109; mss4-f12 ste4-11 cells 7.2±1.0 µm, n = 103) and grew less well. Together these results suggest that Mss4p functions upstream of the cAMP-dependent PKA pathway but not the Kss1p MAPK pathway.

We have isolated a specific mss4 mutant that is defective for invasive growth in low nutrient conditions. The mss4-f12 mutant exhibits cell wall integrity defects, yet distribution of active Cdc42p, the polarisome component Spa2p, the exocyst subunit Sec3p and the septin subunit Cdc3p are largely unaffected. Upon glucose depletion, we show that mss4 mutant cells do not elongate and are substantially defective in FLO11 induction as well as adhesion. This mss4-f12 allele has a highly conserved serine residue in the kinase domain changed to a proline residue. We show that Mss4-f12p is catalytically active in vitro; however, in mss4-f12 cells we observed reduced levels of PtdIns(4,5)P₂ and an accumulation of the complex sphingolipid, M(IP)₂C. Using a reporter to visualize PtdIns(4,5)P₂ in vivo, we demonstrate that mss4-f12 cells have reduced levels of plasma membrane PtdIns(4,5)P₂ as well as a defect in its polarized distribution. In contrast to the distribution of Mss4p, we did not observe the Mss4-f12 protein at the plasma membrane, which nonetheless appeared to be associated with a membrane fraction. Together our results indicate that plasma membrane PtdIns(4,5)P₂ is critical for yeast invasive growth and suggest that this phospholipid functions upstream of the cAMP-dependent PKA signaling pathway required for invasive growth.

From over 12,000 mutagenized copies of mss4, we isolated 10 mutants and confirmed the invasive growth defect in four. Two mutants had an arginine at position 590 replaced by a stop codon and two mutants had a serine residue at position 573 changed to a proline residue. The serine 573 in Mss4p is conserved in all phosphatidylinositol phosphate kinases. Based on the crystal structure of the human type IIβ phosphatidylinositol phosphate kinase, the equivalent residue is part of the kinase catalytic core and is less than 8 Å from residues involved in binding to ATP and phosphatidylinositol phosphate as well as a critically conserved aspartic acid residue that functions as a weak base in kinase catalysis (Rao et al., 1998). However our results indicate that the in vitro kinase activity of Mss4-f12p is similar to that of the wild-type. The positively charged surface of PI4P5Ks together with the substrate-binding region has been proposed to function as a coincidence detector that targets phosphatidylinositol phosphate kinases to the plasma membrane (Fairn et al., 2009). Interestingly, the Mss4-f12p mutant did not localize to the plasma membrane, although serine 573 is not in proximity to residues that have been proposed to be important for membrane binding (Rao et al., 1998; Arioka et al., 2004). We propose that the three positively charged residues that form a patch on the surface of the structure of the human type IIβ phosphatidylinositol phosphate kinase adjacent to the equivalent of the S. cerevisiae Mss4p Ser 573, i.e. Hs Arg 134, Lys 218 and Arg 224 (equivalent to Lys 480, Lys 571 and Arg 577 in S. cerevisiae Mss4p) are involved in plasma membrane binding or stabilization at this membrane.

Our results suggest that sufficient plasma membrane PtdIns(4,5)P₂ is critical for haploid invasive growth in S. cerevisiae. The phosphatidylinositol-specific phospholipase C (Plc1p), which hydrolyzes PtdIns(4,5)P₂ to IP₃ and DAG, is important for pseudohyphal filamentous growth (Ansari et al., 1999). Plc1p binds both the sugar-sensing G-protein-coupled receptor Gpr1p and Gα subunit Gpa2p (Ansari et al., 1999). The pseudohyphal filamentous growth defect, in nitrogen depletion conditions, of the plc1 deletion mutant is rescued either by activating the MAPK or the cAMP-dependent PKA pathway. Plc1p is also required for induction of the transcriptional reporter FG(TyA)::lacZ expression during nitrogen depletion. Perhaps sufficient plasma membrane PtdIns(4,5)P₂ is required during filamentous growth to generate IP₃ and DAG that may be required for signaling. The mss4-f12 mutant grew poorly on non-fermentable carbon sources, which is consistent with the growth defect of the plc1Δ mutant (Flick and Thorner, 1993). However, this mss4 mutant grows at 37°C, in contrast to the plc1Δ mutant (Flick and Thorner, 1993), making unlikely a complete loss of Plc1p activity. Alternatively it is possible that plasma membrane PtdIns(4,5)P₂ is important for targeting Plc1p to this membrane; however, we have been unable to detect Plc1p at the plasma membrane (unpublished observations). Addition of exogenous cAMP, but not expression of an activated MAPKKK allele ste11-4, restored invasive growth in the mss4-f12 mutant, similar to what was observed with a gpa2 mutant (Kübler et al., 1997). These results are consistent with Mss4p functioning upstream of the cAMP-dependent PKA pathway.

Phosphatidylinositol (PI) is a precursor for PIPs and sphingolipids. Growth defects due to inhibition of sphingolipid biosynthesis can be suppressed by Mss4p overexpression, yet levels of complex sphingolipids are unaffected (Kobayashi et al., 2005); however, they are reduced in stt4^ts and mss4^ts strains (Tabuchi et al., 2006). Furthermore, the calcium sensitive phenotype of the csg2 complex sphingolipid biosynthesis mutant was suppressed by a mss4^ts mutation (Dunn et al., 1998). In contrast, inhibition of the plasma membrane PI4-kinase Stt4p by wortmannin resulted in an increase in complex sphingolipids (Brice et al., 2009). Similarly in the mss4-f12 mutant we observed reduced levels of PtdIns(4,5)P₂ and an accumulation of the complex sphingolipid, M(IP)₂C. Furthermore, inhibition of complex sphingolipid biosynthesis results in an increase in plasma membrane PtdIns(4)P and PtdIns(4,5)P₂ (Jesch et al., 2010), suggesting that the increased PI levels observed in the mss4-f12 mutant are likely to contribute to increased complex sphingolipid levels. This common phospholipid is likely to underlie some of the connections between these two lipid biosynthesis pathways (Henry et al., 2012).

Sustained growth in one direction is required for cells to elongate, such as those observed in filamentous yeast and plant pollen tubes. The PI4P5K Mss4p is critical for filamentous growth in Candida albicans (Vernay et al., 2012) and Neurospora crassa (Seiler and Plamann, 2003; Mähs et al., 2012). In the pathogenic fungi C. albicans, Mss4p is critical for invasive filamentous growth and, in particular, the switch between the yeast and filamentous forms (Vernay et al., 2012). In N. crassa, mss4 mutants were identified in a screen for hyphal morphogenesis defects and exhibited general polarity defects (Seiler and Plamann, 2003). Two ts mutants from this screen both have the conserved Tyr at the equivalent of position 744 in S. cerevisiae Mss4p substituted with an Asn, resulting in a complete loss of activity of this Neurospora PI4P5K (Mähs et al., 2012). In tobacco and Arabidopsis, PI4P5Ks are critical for pollen tube growth and polarity (Ischebeck et al., 2008; Sousa et al., 2008; Ischebeck et al., 2010; Ischebeck et al., 2011; Stenzel et al., 2011). Perturbation of this class of lipid kinase results in altered morphologies and reduced pollen tubes elongation (Ischebeck et al., 2008; Sousa et al., 2008; Ischebeck et al., 2010; Ischebeck et al., 2011; Stenzel et al., 2011). Together with our findings that Mss4p is required for invasive growth in S. cerevisiae, suggest that PtdIns(4,5)P₂ plays an important role in sustained unidirectional growth, likely via extensive membrane traffic. Indeed, our studies in C. albicans reveal that a dramatic gradient of PtdIns(4,5)P₂ emanates from the tip of the hyphal filament in wild-type cells.

Our results indicate that the mss4-f12 mutant has reduced levels of plasma membrane PtdIns(4,5)P_2. However, we did not observe Mss4-f12p on the plasma membrane raising the possibility that PtdIns(4,5)P₂ is generated in an intracellular compartment, some of which ends up at the plasma membrane. In addition the polarized distribution of this phospholipid in budding mss4-f12 cells is perturbed, yet we did not observe a substantial alteration in the distribution of four cell polarity proteins. Previous studies using either of the PtdIns(4,5)P₂ reporters, GST–GFP–PH^Plcδ or GFP–PH^Plcδ–PH^Plcδ, did not detect a substantial enrichment of PtdIns(4,5)P₂ in small buds (Stefan et al., 2002; Garrenton et al., 2010); however, an accumulation of this lipid was observed at the bud neck (Garrenton et al., 2010). Using the GFP–PH^Plcδ–PH^Plcδ–GFP reporter we observed enrichment of PtdIns(4,5)P₂ throughout the plasma membrane of small buds with 2–3-fold the concentration of PtdIns(4,5)P₂ in the bud compared to the back of the mother cells. Previous studies have shown using the inositolpolyphosphate 5-phosphatase synaptojanin Sjl2p and the ANTH domain from Sla2p to visualize PtdIns(4,5)P₂ that this phospholipid is localized to endocytosis sites which are concentrated in small buds (Sun et al., 2007; Brach et al., 2011). The loss of PtdIns(4,5)P₂ polarity in the mss4-f12 cells is consistent with the suggestion that membrane traffic via endocytosis and exocytosis is important for invasive growth.

In summary, our results suggest that PtdIns(4,5)P₂ is important for invasive growth in S. cerevisiae and that this phospholipid is a critical regulator for cell elongation in highly polarized cells.

Standard techniques and medium were used for yeast growth and genetic manipulation (Rose et al., 1991). Media was prepared as described (Gimeno et al., 1992; Casalone et al., 2005). Strains, primers and plasmids used are described in supplementary material Tables S1–S3.

The MSS4 ORF was amplified from genomic DNA using Mss4pBamHI/Mss4mNotI. The resulting 2340 bp product was cloned into pRS416 under the control of GAL1-10 promoter (resulting in p416GALpMSS4) and pCM188 (Garí et al., 1997) 3′ of the tetracycline regulatable promoter (resulting in pCM188TetpMSS4). The MSS4 ORF with 704 bp 5′ of the ATG codon was amplified using Mss4pSalI/Mss4mNotI and the resulting 3044 bp product was cloned into pRS414 and pRS415 yielding p414MSS4pMSS4 and p415MSS4pMSS4.

A C-terminal Mss4–GFP fusion was constructed by inserting a unique AatII restriction site in place of the stop codon (using site directed mutagenesis with Mss4pAatIInostop/Mss4mAatIInostop, resulting in p414MSS4pMSS4-AII) and subsequently cloning (Gly-Ala)₅–yeGFP-ADH1_term (PCR amplified from p416GalCdc24HAGFP with AatII and NotI sites at the 5′ and 3′ ends using AatIIpGAyeGFP/ADHtNotI) into p414MSS4pMSS4-AII, yielding p414MSS4pMSS4GFP. p414MSS4pmss4f9GFP was created by inserting a unique AatII replacing the two codons which coded for R590/S591 by site-directed mutagenesis in p414MSS4pMSS4GFP using primers Mss4pAatII1767/Mss4mAatII1767 resulting in p414MSS4pMSS4-590AII-GFP. This MSS4 plasmid, which contained two AatII sites, was digested with AatII, removing the 3′ region after R590 and subsequently religated, resulting in p414MSS4pmss4f9GFP. p414MSS4pmss4f12GFP was created by site-directed mutagenesis using the plasmid p414MSS4pMSS4GFP with Mss4C12pT1717C/Mss4C12pT1717C.

To visualize plasma membrane PtdIns(4,5)P₂ a XhoI/NotI GFP–PH^Plcδ–PH^Plcδ–GFP fragment optimized for expression in C. albicans (Vernay et al., 2012) from pExpARG-pADH1GFP-PH^Plcδ-PH^Plcδ-GFP was cloned into pRS406 with a GAL1-10 promoter inserted at the XhoI site. This resulting plasmid, p406GALpGFP-PH^Plcδ-PH^Plcδ-GFP was linearized with StuI and transformed into wild-type (RAY1990) and mss4-f12 strains (RAY1991). Two independent transformants of each strain (RAY2001/2003 and RAY1993/1999) with this reporter integrated at the URA3 locus were analyzed for invasive growth and GFP expression by immunoblot and fluorescence microscopy.

The mss4 deletion mutants were constructed by PCR-based gene disruption as described using Mss4pKO/Mss4mKO and the pBSLoxPHis5SpLoxP plasmid (Arkowitz and Lowe, 1997; Nern and Arkowitz, 1998) and transformed into the 10560-6B strain carrying p416GALpMSS4. Four unique, silent restriction sites were introduced by site-directed mutagenesis in p414MSS4pMSS4, NheI (bp −3; Mss4pNheI-3/Mss4mNheI-3), BglII (bp 607; Mss4pBglII607/Mss4mBglII607), AatII (bp 1401; Mss4pAatII1401/Mss4mAatII1401), XhoI (bp 1807; Mss4pXhoI1807/Mss4mXhoI180) resulting in p414MSS4pMSS4*.

To generate random mss4 mutants, the region between the unique AatII and XhoI sites (bp 1401–1807) was mutagenized by error prone PCR using p414MSS4pMSS4* as a template and an unbalanced dNTP mixture (5.8 mM dATP, 5 dCTP, 10.5 mM dGTP and 72.5 mM dTTP) (Barale et al., 2006). The AatII/XhoI fragment was then cloned into p414MSS4pMSS4*. Subsequently this library was transformed into RAY1885 and transformants were selected on SC-Trp glucose plates, which repressed MSS4 expression. Colonies were then cultured overnight in YEPD in 96 well plates and subsequently spotted onto solid YEPD and YEP, incubated for 5 to 10 days. Invasive growth was assessed by washing the plates with a gloved hand, as described (Roberts and Fink, 1994; Guldal and Broach, 2006), and was compared to a control strain. Colonies were screened for invasive growth defects. In mutants that exhibited reduced invasive growth the p416GALpMSS4 plasmid was removed by 5-FOA counter-selection. The invasive growth phenotypes of the mutants were subsequently confirmed on YEPD, YEP and SLAD medium. The mss4-f9 and mss4-f12 mutant alleles were recreated by site-directed mutagenesis using the plasmid p414MSS4pMSS4 and primers mss4C9pA1768T/mss4C9mA1768T and Mss4C12pT1717C/Mss4C12mT1717C, resulting in p414MSS4pmss4f9 and p414MSS4pmss4f12, respectively. The region 3′ of the stop codon in the mss4-f9 mutant was removed by insertion of two AatII sites (one replacing codons encoding for R590/S591 and a second 5′ of the stop codon) by site-directed mutagenesis, digestion with AatII to remove the 3′ region after R590 and subsequent religation resulting in p414MSS4pmss4f9Δ. Individual mss4 point mutants were made by site-directed mutagenesis. All PCR-amplified products and mutants were confirmed by sequencing.

RAY1885 was transformed with p414MSS4pMSS4, p414MSS4pMSS4GFP, p414MSS4pmss4f9, p414MSS4pmss4f9Δ, p414MSS4pmss4f12, p414MSS4pmss4f9GFP and p414MSS4pmss4f12GFP plasmids, the rescuing plasmids were removed with 5-FOA and the invasiveness defects were examined on YEPD, YEP and SLAD medium. The invasive growth defect of these strains was complemented by the p415MSS4pMSS4 plasmid.

Diploid strains (RAY1945 and RAY1949) were generated using Gal inducible HO endonuclease to switch the mating type of a 10560-6B mss4-Δ1::HIS5Sp strain carrying p415MSS4pMSS4. After verification of a MATa isolate by response to pheromone and PCR (Huxley et al., 1990) followed by loss of the pGal-HO plasmid, diploids were generated by mating with RAY1940 and subsequent loss of p414MSS4pMSS4. This diploid was transformed with p414MSS4pMSS4 or p414MSS4pmss4f12 resulting in RAY1945 and RAY1949, respectively. These diploids were sporulated, spores enriched and MATa isolates identified (RAY2012-2015).

The MSS4 ORF was PCR amplified using p416GALMSS4 as a template with Mss4pSalI/Mss4mNotI. The 1922 bp PCR product was digested with SpeI/NotI and the 421 bp fragment cloned into the pGEX-6P plasmid, resulting in pGEX-6PMSS4. The GST fusion protein was expressed in E. coli BL21 cells grown at 37°C and induced with 0.1 mM isopropyl 1-thio-β-D-galactopyranoside for 3 hr. Cells were harvested, resuspended in PBS, lysed by sonication (5×30 sec) and centrifuged 15 min at 10,000 rpm at 4°C. The supernatant was then added to GSH–Sepharose 4B resin (Amersham Biosciences), which was subsequently washed with PBS containing 1 mM PMSF and protease inhibitor mixture (Roche Applied Science). GST–Mss4p was eluted with 50 mM Tris-HCl, pH 8, 10 mM GSH, purity confirmed by SDS-PAGE and rabbits were immunized. For immunoblots, anti-Mss4p (polyclonal; 1∶1000), anti-GFP (polyclonal; 1∶1000) (Nern and Arkowitz, 2000b) and anti-GAPDH (monoclonal; 1∶10,000) were used. Following incubation with peroxidase-conjugated secondary antibodies (Bio-Rad), bands were visualized by enhanced chemiluminescence (luminol-coumaric acid) on an imaging system (Las3000; Fujifilm).

Exponentially growing cell cultures were diluted to 0.1 or 0.5 OD₆₀₀/ml and spotted on YEPD, YEP or SLAD (Gimeno et al., 1992) solid medium either in the presence or absence cAMP (5 mM) for invasive growth assays or spotted on SC-Trp solid medium either in the presence or absence of 10 mM 3-amino-triazole (3AT). After 5 days incubation, cells on the surface were washed off as described (Roberts and Fink, 1994; Guldal and Broach, 2006) for invasive growth and for adhesive growth assays, surface cells were washed off with a gentle stream of H₂O as described (Braus et al., 2003; Guldal and Broach, 2006). Growth in the presence of Calcofluor white and Congo red was carried out as described (Ram and Klis, 2006). Induction of a Fus1LacZ reporter was assayed as described (Nern and Arkowitz, 1998; Nern and Arkowitz, 1999). Quantitative matings were carried out as described (Nern and Arkowitz, 1998; Nern and Arkowitz, 1999) with the wild-type (RAY876) and enfeebled (RAY1563) tester strains.

Strains carrying the YEp355 FLO11-LacZ plasmid (B3782; Rupp et al., 1999) were grown overnight in SC-His-Trp-Ura medium containing glucose. Exponentially growing cells (10 OD₆₀₀) were transferred to medium (20 ml) with or without glucose. After 7 hr incubation, NaF and NaN₃ (10 mM final concentration each) were added to cultures. LacZ activity determinations were carried out with culture extracts as described (Rose and Botstein, 1983) and activity was normalized to the total protein (Bradford, 1976).

Mss4Δ strains (RAY1940 and RAY1942) carrying p414MSS4pMSS4GFP and p414MSS4pmss4f12GFP were grown exponentially in SC-Trp medium and cells (50 OD₆₀₀) were harvested by centrifugation. All subsequent steps were carried out at 4°C. Cells were lysed by agitation with glass beads in buffer A (50 mM Tris-HCl pH 7.4, 150 mM NaCl, 1 mM PMSF, 40 µg/ml each of leupeptin, chymostatin, pepstatin A, aprotinin, and antipain) containing 0.1% Triton X-100. Immunoprecipitation was carried out as described (Nern and Arkowitz, 1999) using 10,000 g clarified cell extracts and anti-GFP IgG-Sepharose (from protein A Sepharose [Pharmacia] incubated with anti-GFP sera and then cross-linked with dimethylpimelimidate). Anti-GFP IgG-Sepharose (20 µl) was incubated for 1 hr with clarified cell extracts (1 mg protein) followed by four washes with buffer A containing 0.1% Triton X-100. The amount of immunoprecipitated Mss4p was quantified by SDS-PAGE followed by immunoblot and detection with anti-Mss4p sera. The remaining resin was used for kinase assays as described (Desrivières et al., 1998; Homma et al., 1998). Kinase buffer (40 µl of 50 mM Tris-HCl pH 7.5, 1 mM EGTA, 10 mM MgCl₂, 50 µM ATP) was added to resins and assays were initiated by addition of 5 µCi [γ-³²P]ATP and 80 µM of PtdIns(4)P. After 1 hr at 30°C reactions were stopped by addition of CHCl₃/MeOH (187 µl 1∶2 v/v). The lipids were extracted by addition of 2.4 M HCl (45 µl) and CHCl₃ (190 µl). The lower phase was removed and washed with 184 µl of 1 M HCl/MeOH/CHCl₃ (47∶48∶3 v/v/v) and then dried. Lipids were analyzed by TLC (Reggiori et al., 1997) on silica gel 60 plates (Merck) impregnated with 1.2% potassium oxalate and dried at 100°C for 1 hr. Samples were separated with a CHCl₃/MeOH/(CH₃)₂CO/CH₃COOH/H₂O (42∶30∶12∶12∶12 v/v/v/v/v) solvent system. Autoradiography was carried out and radioactivity was quantified using a phosphoimager (FujiBAS 1000; Fujifilm).

In vivo labeling was performed essentially as described (Desrivières et al., 1998). Exponentially growing cultures (20 OD₆₀₀) (RAY1986 or RAY1991) grown in SC-Trp-His were resuspended in fresh medium (10 OD₆₀₀/ml) and incubated with shaking for 30 min. [³H]Inositol (20 µCi) was added to each culture prior to incubation for an additional 40 min, followed by addition of 6 ml of pre-warmed medium and then another 80 min incubation. NaF and NaN₃ (10 mM final concentration each) were then added to each culture. Lipid extraction and analysis was carried out as described (Guillas et al., 2001). Immediately after NaF/NaN₃ addition, cells were harvested by centrifugation for 5 min at 3000 rpm, resuspended in ice cold water, and centrifuged for 5 min at 10,000 rpm. Cells were then suspended in 400 µl of ice cold CHCl₃/MeOH (1∶1 v/v) and disrupted by vortexing with glass beads for 5×1 min. Following centrifugation for 5 min at 10,000 rpm, the supernatant was recovered. Pellets extracted twice more with 400 µl CHCl₃/MeOH/H₂O (10∶10∶3 v/v/v). The organic phases were pooled and dried using a speed vac. Lipids were subsequently desalted by partitioning between n-butanol and water followed by a back extraction of the butanol phase with water (Krakow et al., 1986). The desalted lipids were then analyzed using 0.2 mm silica gel TLC plates (CHCl₃/MeOH/H₂O; 55∶45∶10 v/v/v solvent system). Radioactivity was detected and quantitated by fluorography (Benghezal et al., 1995). For phosphate labeling, [³³P]H₂PO₄⁻ was used at 1 µCi/OD₆₀₀ cells and lipid extraction carried out similarly. Lipids were analyzed by TLC (CHCl₃/MeOH/25% NH₄OH/H₂O; 90∶70∶4∶16 v/v/v/v) (Munnik et al., 1996).

Differential interference contrast images were acquired with a Leica DMR wide-field microscope (NA 1.32×63 Plan-Apo objective) and colony morphology images were acquired with a Leica MZ6 dissection scope (×10 magnification) (Bassilana et al., 2005). Images were captured with a Princeton Instruments Micromax charge coupled device camera or an Andor Technology Neo sCMOS camera using IPLab (Scanalytics Inc.) v3.5 software or Solis (Andor Technology) v4 software, respectively. The length of the major cell axis was determined by fitting an ellipsoid to the mother cell using ImageJ. Actin was visualized using Alexa-Fluor-488–phalloidin (Pringle et al., 1989) on a DeltaVision deconvolution microscopy (Olympus IX-70) system (Applied Precision) with a NA 1.4 ×60 objective. Images (30×0.15 µm z-sections) were deconvolved using softWoRx software and maximum projections generated. All GFP and RFP images (except PtdIns(4,5)P₂ analyses) were acquired on an Andor Technology Revolution-XD spinning-disk laser confocal microscope (UplanSApo 1.4 NA 100× objective) controlled by v2 iQ2 software with 488-nm or 561-nm laser excitation and 140×140×400 nm voxel size (Vernay et al., 2012). Sum projections were generated by ImageJ. Bars are 5 µm.

For PtdIns(4,5)P₂ distribution, wild-type and mss4-f12 cells were grown overnight at in SC-ura medium containing Fructose (4%) and Galactose (4%), back-diluted the following day into same medium containing Galactose and grown for an additional 6 hr. Cells were washed with PBS prior to analyses on a Zeiss LSM 510 META confocal (Axiovert 200M microscope, Plan-Apo 1.4 NA 63× objective and 488-nm laser excitation) (Bassilana et al., 2005). Voxel size was 140×140×700 nm and the pinhole was set to 1 airy unit.

The semi-automatic MATLAB program, BudPolarity (BP) was used as described (Vernay et al., 2012). Three-dimensional images were converted into 2D images by sum projection. Cell morphology was defined by an entered intensity threshold and the major axis of each cell was determined by an ellipsoid fit. Fluorescence intensity is integrated along the major axis (normalized) to follow concentration. Another Matlab program (HyphalPolarity) was used to determine average intensities of the plasma membrane and cytoplasm (all signal interior to the plasma membrane excluding the vacuole) from the central z-section for each cell (Vernay et al., 2012).

We thank H.-U. Mösch, G. Fink, E. Bi and D. Stone for strains and plasmids. S. Schaub and S. Bogliolo for assistance and M. Bassilana for comments and discussion.