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Vol. 18, Issue 1, 265-281, January 2007
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Department of Molecular Biology and Biotechnology, Sheffield University, Sheffield S10 2TN, United Kingdom
Submitted May 11, 2006;
Revised October 20, 2006;
Accepted October 26, 2006
Monitoring Editor: Daniel Lew
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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; Kibbler et al., 2003). A striking feature of its biology is its capacity to grow in different morphological forms: yeast, pseudohyphae, and hyphae (Berman and Sudbery, 2002; Sudbery et al., 2004). The morphology of the yeast form closely resembles that of the budding yeast Saccharomyces cerevisiae. Hyphae consist of chains of long cells with unconstricted, parallel-sided walls. Pseudohyphae also consist of chains of elongated cells, but they display constrictions at the mother-daughter cell neck and at septal junctions. In addition to differences in cell shape, hyphae are distinguished from both yeast and pseudohyphae by three further characteristics (Sudbery et al., 2004). First, growth is intensely polarized to the hyphal tip and proceeds continuously throughout the cell cycle, whereas the growth of buds in yeast and pseudohyphae is restricted to the tip only for the first part of the cell cycle (Soll et al., 1985; Crampin et al., 2005). Second, septin rings, which organize the formation of the primary septum, form at the mother-bud neck in yeast and pseudohyphae shortly before bud emergence at the start of the cell cycle (Sudbery, 2001; Warenda and Konopka, 2002). However, hyphal germ tube evagination from an unbudded yeast cell occurs before the start of the cell cycle (Hazan et al., 2002). A band of septin bars forms at the base of the germ tube and as it elongates, a true septin ring forms 5–15 µm away from the mother cell (Sudbery, 2001; Warenda and Konopka, 2002). The appearance of this septin ring is coincident with the start of the cell cycle (Finley and Berman, 2005). Third, nuclear division takes place across the mother/bud neck in yeast and pseudohyphae, but within the germ tube in hyphae (Sudbery, 2001). Understanding the molecular mechanisms responsible for these differences will not only increase understanding of this important pathogen, but it may serve as a useful model for fundamental aspects of cell biology relevant to all eukaryotic cells.
We recently showed that polarized growth in C. albicans hyphae is driven by a different mechanism compared with that in yeast and pseudohyphae (Crampin et al., 2005). Hyphal growth depends on a Spitzenkörper, whereas in yeast and pseudohyphae polarized growth depends on a polarisome. A Spitzenkörper is a vesicle-rich apical body that acts as a vesicle supply center to concentrate the delivery of secretory vesicles to the growing tip (Bartnicki-Garcia et al., 1989; Virag and Harris, 2006). First studied in S. cerevisiae, the polarisome is a multiprotein complex containing Bni1, Spa2, Bud6, and Pea2 forming a surface crescent at the tip of young buds (Evangelista, 1997; Sheu et al., 1998). The polarisome facilitates the nucleation of actin cables by the formin Bni1 (Sagot et al., 2002; Evangelista et al., 2002). Secretory vesicles are transported along these actin cables by the type V myosin, Myo2, complexed to its regulatory light chain, Mlc1 (Bretscher, 2003).
In S. cerevisiae, polarized growth and the formation and organization of the septin rings are ultimately controlled by the Cdc42 GTPase (Johnson and Pringle, 1990; Johnson, 1999). Like all Rho-type GTPases, Cdc42 cycles between GDP- and GTP-bound forms. Cdc24 acts as a guanine nucleotide exchange factor (GEF) to mediate the formation of the GTP-bound Cdc42 (Zheng et al., 1994; Johnson, 1999). Activation of GTPase activity, to return Cdc42 to the GDP-bound form, is mediated by the GTPase-activating proteins (GAPs) Rga1, Rga2, and Bem3 (Zheng et al., 1994; Stevenson et al., 1995; Smith et al., 2002; Caviston et al., 2003). Classically, GTPases such as Cdc42 act as molecular switches, the GTP-bound form being the active state. Thus, Cdc24 would be expected to be a positive regulator and the GAPs to be negative regulators of Cdc42 signaling. As expected, loss of Cdc24 results in a similar phenotype to loss of Cdc42, that is, cells are unable to form buds and mutants arrest as large unbudded cells (Hartwell, 1974; Adams et al., 1990).
However, the role of the GAPs is not so straightforward. S. cerevisiae mutants lacking Cdc42 GAPs show defects in both bud morphology and septin ring organization (Gladfelter et al., 2002; Smith et al., 2002; Caviston et al., 2003). Septin rings are sometimes absent from the bud neck and found instead at the bud tip or within an elongated daughter cell. In other cells septins remain at the neck but form a misorganized septin band, consisting of longitudinal bars, which resembles the basal septin band that forms in C. albicans germ tubes (Sudbery, 2001). Mutations have been isolated in the effector domain of Cdc42, which specifically affect septin ring formation. Two of these mutants, cdc42Y32H and cdc42V36TK94E, are defective in GTPase activity and are suppressed by multicopy RGA1 (Gladfelter et al., 2002). Moreover, a temperature-sensitive septin mutant, cdc12-6, is suppressed by multicopy RGA1 or BEM3 (Caviston et al., 2003). These observations suggest that Cdc42-directed GAPs may play a positive role in organizing the septin ring. Two explanations have been advanced for this unexpected conclusion. First, Cdc42 cycling between GTP- and GDP-bound forms may be required for proper septin ring formation (Gladfelter et al., 2002). Second, GAPs may directly participate in the formation of septin rings (Caviston et al., 2003). These hypotheses are not mutually exclusive.
Further regulation of Cdc42 activity may be imposed by the Rho-guanine dissociation inhibitor (Rho-GDI) Rdi1. Rho-GDIs extract their target Rho-GTPase from membranes and maintain them in the cytosol, block the dissociation of GDP necessary for the exchange of GDP for GTP, and may interfere with association of the GTPase with its targets (DerMardirossian and Bokoch, 2005). Rdi1 is the only S. cerevisiae Rho-GDI, and it coimmunoprecipitates with both Cdc42 and Rho1; so, it may regulate both of these GTPases. However, its deletion has no obvious phenotypic effects (Masuda et al., 1994; Koch et al., 1997).
Cdc42 not only controls septin ring formation and organization but also controls the delivery and docking of secretory vesicles to the bud tip, a process that is required for polarized growth to occur (Pruyne et al., 2004). Cdc42 is therefore likely to play a key role in promoting the characteristic aspects of hyphal morphology in C. albicans, as hyphal elongation depends on continuous polarized growth toward the hyphal tip (Soll et al., 1985; Crampin et al., 2005). Several lines of evidence confirm that this is the case. First, in S. cerevisiae cdc42 alleles have been isolated that specifically reduce pseudohyphal growth, and when strains of C. albicans were constructed with the equivalent alleles, the capacity for hyphal growth was greatly reduced (vanden Berg et al., 2004). Second, promoter shutdown experiments show that in C. albicans, Cdc42 and its GEF Cdc24 are required for viability, and their ectopic expression from a nonnative promoter interferes with hyphal induction (Ushinsky et al., 2002; Bassilana et al., 2003). Third, expression of CDC42G12V, encoding a constitutively GTP-bound form was found to be lethal (Ushinsky et al., 2002), as it was in S. cerevisiae (Ziman et al., 1991). C. albicans yeast cells conditionally expressing Cdc42G12V arrested with a multibudded morphology, whereas cells induced to form hyphae were swollen and showed reduced polarity.
These experiments show that proper regulation of Cdc42 is critical for normal hyphal development. In S. cerevisiae, loss of all of the Cdc42 GAP proteins encoded by Rga1/2 and Bem3 would be expected to result in elevated levels of activated Cdc42, yet in contrast to the phenotype of cells expressing CDC42G12V, cells lacking the Cdc42 GAPs show hyperpolarized bud growth and the ectopic septin ring formation — features reminiscent of hyphal formation in C. albicans (Gladfelter et al., 2002; Smith et al., 2002; Caviston et al., 2003). This suggests that the Cdc42 GAPs may play an important role in modulating Cdc42 action with respect to morphological switching. Another protein that may play a role in modulating Cdc42 activity is Rdi1. Here, we investigate the role of Cdc42 GAPs and Rdi1 in C. albicans to test the hypothesis that these proteins provide the additional elements of Cdc42 regulation necessary for the control of the morphological transitions between yeast, pseudohyphae, and hyphae.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Techniques for Microscopy
Wide field epifluorescence microscopy was carried out using a Delta Vision Linux IV microscope (Applied Precision Instruments, Seattle, WA) with an Olympus 100x UPlanApo NA 1.35 lens (Olympus, Tokyo, Japan). Images were deconvolved using the SoftWoRx image analysis software supplied with the microscope. Where parts of images were expanded to show greater detail, the SoftWoRx Interpolated Zoom facility was used to smooth pixilation. Unless otherwise stated images are projections of deconvolved Z stacks. Quantification of fluorescence was carried out using the SoftWoRx Data Inspector function by using images that had been deconvolved but not subjected to any other form of processing. Where intensity between images was compared, the images were captured on the same day with the same exposure time. Differential interference contrast (DIC) images were acquired with a DMLB microscope by using a 100x UPlan Apo NA 1.35 objective (Leica, Wetzlar, Germany) and were captured with a model CCD-1800-v camera (Princeton Instruments, Monmouth Junction, NJ) controlled by Openlab version 2.5 software (Improvision, Warwick, United Kingdom). Cell dimensions were measured in cells fixed with 2.5% formaldehyde by using the dimension-measuring facility in Openlab. Where dimensions of strains were directly compared the cultures were grown in parallel on the same day. Images from both the Leica and Delta Vision microscopes were exported as TIFF files, which were edited for size and contrast in Adobe Photoshop version 7.0 (Adobe Systems, Mountain View, CA).
Western Blots
Western blots were carried out as described previously (Wightman et al., 2004; Crampin et al., 2005). The anti-green fluorescent protein (GFP) monoclonal antibody (mAb) was supplied by Roche Biosciences (Lewes, United Kingdom). Anti tetra-His mAb (QIAGEN, Dorking, Surrey, United Kingdom) was used to recognize Rdi1–6His. Sba1, used as a loading control in Figure 10, is an Hsp90 cochaperone whose total cellular content remains constant under a variety of growth conditions (Millson et al., 2005). Anti-Sba1 polyclonal antiserum was a kind gift from P. Piper (Sheffield University, United Kingdom).
Strain Construction
Strains constructed are listed in Table 1, and the oligonucleotides used are listed in the Supplemental Table 1. Single mutant strains were constructed by sequential deletion of both alleles in the parental strain BWP17 (Wilson et al., 1999). Double and triple mutant strains were constructed using the recyclable URA3 cassette URA3-dpl200 (Wilson et al., 2000). Transformants were screened for transplacement of the targeted gene by using a 5' primer that annealed within the selectable marker of the transplacing cassette and a 3' primer that annealed downstream of the deleted gene. Positive clones were further screened using polymerase chain reaction (PCR) primers that annealed upstream and downstream of the deleted gene. A unique restriction site was used to distinguish the disruption cassette from the wild-type gene if they were predicted to be of the same size. When the URA3 marker was recycled using 5-fluoroorotic acid (FOA), correct excision of the URA3 gene was confirmed by PCR by using a 5' primer that annealed within the portion of the URA3 gene that remained after the recombination event selected by FOA, and a 3' primer that annealed downstream of the targeted gene. Finally, the complete absence of a gene was confirmed using PCR primers that annealed within the deleted region. The absence of a PCR product, when it was present in an appropriate positive control, confirmed the gene had been deleted. The phenotypes reported here were observed in two independent transformants in each case. To confirm that phenotypes were due to the loss of gene function and not to accidental changes that occurred during genetic manipulation, wild-type copies of the genes were reintroduced into the deleted strains. This was done in two ways. In RDI1, the promoter and open reading frame (ORF) were amplified by PCR into CIP10 and reintegrated at the RP10 locus (Brand et al., 2004). Second, in RGA2, BEM3, and RDI1, the coding region was placed under the control of the MET3 promoter by cloning the ORF, amplified by PCR, into pCaEXP, which was then targeted to the RP10 locus (Care et al., 1999). C-terminal fusions to yellow fluorescent protein (YFP) and cyan fluorescent protein (CFP), and N-terminal MET3 promoter-YFP fusions were constructed using linear cassettes generated by PCR as described previously (Gerami-Nejad et al., 2001, 2004).
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). Transplacements were carried out by PCR by using a forward primer that annealed upstream of the point mutation and a reverse primer that annealed downstream of the selectable marker in the pFA plasmid, and which also contained a 5' extension derived from the sequence downstream of the stop codon of the target gene. Transplacements were screened by appropriate PCR assays as described above. The mutated sequence in putative mutants was reamplified by PCR and resequenced to confirm the mutation had been introduced and that the reading frame had been maintained. |
RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The annotation of the C. albicans genome sequence identified single homologues of the S. cerevisiae BEM3, RDI1, and the RGA1/2 gene pair (Braun et al., 2005). We verified these gene notations by carrying out a BLASTP search using the predicted S. cerevisiae protein sequence to search the C. albicans genome for predicted proteins showing similar sequences. CaRga2 (orf19.4593) is 23% identical and 39% similar to ScRga2 over 1138 residues (p = 3E-33). Because the C. albicans orf19.4593 shows a slightly greater similarity to S. cerevisiae RGA2 compared with ScRga1, the C. albicans gene is named RGA2. CaBem3 is 24% identical and 43% similar over 1088 residues to ScBem3 (p = 5E-55). No other C. albicans proteins with GAP domains showed significant homologies to ScRga2 or ScBem3 (E values < –10). To investigate the role of these proteins during morphogenesis, we constructed mutants in which the encoding genes were deleted both singly and in combination. We also constructed strains in which these mutations were combined with deletions of SWE1 to investigate whether the mutant phenotypes were due to the operation of the Swe1-dependent morphogenesis checkpoint (Lew and Reed, 1995). To monitor the effect of the mutations on septin ring, Spitzenkörper formation, and nuclear division we introduced CDC10-YFP, MLC1-YFP, and NOP1-YFP alleles, respectively, into these strains. A full list of the genotypes of the strains constructed is shown in Table 1.
During yeast phase growth, loss of Bem3 had little effect on cell morphology compared with the parent BWP17 strain, and septin rings formed normally (Figure 1, A and B). Loss of Rga2 resulted in a uniform population of elongated yeast cells although septin rings still formed between mother and daughter cells and the bud necks looked normal (Figure 1C). Loss of both Rga2 and Bem3 resulted in a small number of cells having highly polarized buds that lack constrictions at the junction with the mother cell (Figure 1D). In these cells, an ectopic septin ring formed in the elongated bud. In some cells, the bud swelled immediately distal to this ring (Figure 1D, arrow). Loss of Swe1 did not reverse the phenotype resulting from loss of the GAPs, showing that the elongated cell phenotype is not a result of the Swe1-dependent morphogenesis checkpoint (Figure 1E). Thus, Rga2 apparently plays a direct role in controlling bud shape. Finally, loss of Rdi1 did not further exacerbate the elongation resulting from loss of the Cdc42 GAPs (Figure 1F). Cells were still elongated and septin rings formed ectopically in some cells.
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). Hyphae are induced by growth on YEPD at pH 7.0, 37°C, plus 20% serum. To investigate whether cells lacking Cdc42 GAPs acquire hyphal characteristics under conditions when hyphae do not normally form, we investigated the morphology of mutants in pseudohyphal-promoting conditions. Parental, the bem3
/ rga2/ double mutant strain, and bem3/ and the rga2/ single mutant strains were grown to saturation in YEPD medium under conditions that promote yeast form growth, and the yeast cells were then reinoculated into YEPD medium and cultured in conditions favoring pseudohyphal growth. We report here in detail on experiments where cells were grown on YEPD at pH 6.0, 36°C. However, we obtained similar results with cultures grown on YEPD at pH 5.0, 36°C. In such experiments, pseudohyphal cells evaginate buds 40–50 min after reinoculation into fresh medium (Sudbery, 2001). After evagination, samples were withdrawn at intervals and analyzed to record the dimensions of the hyphal germ tube or pseudohyphal bud. Loss of Cdc42 GAPs had a major effect on the morphology of cells grown in conditions that normally induce pseudohyphal growth (Figure 2). Two differences are apparent in the shape of parental BWP17 cells in Figure 2A, and the mutants lacking Cdc42 GAPs shown in Figure 2, C–E. First, the BWP17 cells show constrictions at the mother bud neck (Figure 2A), which are absent in cells lacking both of the Cdc42 GAPs (Figure 2E) but not in cells lacking only one or other of the GAPs (Figure 2, C and D). Second, cells of the double bem3/ rga2/ mutant, and to a lesser extent the rga2/ single mutant, are longer and thinner than the BWP17 cells. Both of these differences are characteristics that distinguish hyphae (Figure 2B) from pseudohyphae (Figure 2A).
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/ mutant, but not the bem3/ mutant, had a greater length and lesser width than the parental BWP17 cells (length-to-width ratios: BWP17 = 2.9 ± 0.4, rga2/ = 3.9 ± 0.4, and bem3/ = 3.0 ± 0.4). The double mutant lacking all Cdc42 GAP activity (bem3/ rga2/) showed a further increase in the length-to-width ratio compared with the rga2/ and bem3/ single mutants (4.6 ± 0.4) after 100 min. Together, these data show that loss of Cdc42 GAP activity results in a change in the morphology of pseudohyphae so that they acquire characteristics of true hyphae, being longer and thinner than wild-type pseudohyphae and lacking a constriction at the mother-bud neck. However, the phenotype of the bem3/ rga2/ double mutant growing in pseudohyphal conditions was still different from that when it was growing in hyphal conditions (Figure 2, E and F). Indeed, the phenotype of bem3/ rga2/ mutant growing in hyphal-inducing conditions was indistinguishable from the BWP17 parental strain (length-to-width ratios: BWP17 = 10.4 ± 0.6 and bem3/ rga2/ = 10.4 ± 0.9). So, the Cdc42 GAPs are not required for hyphal formation in hyphal-inducing conditions, but in their absence, a more hyphal-like shape develops in pseudohyphal conditions, suggesting that Cdc42 GAPs have a role in regulating the processes that lead to the characteristic hyphal morphology.
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/ rga2/ rdi1/ triple mutant and were surprised to observe that under pseudohyphal conditions, the mutant had a less extreme phenotype than the bem3/ rga2/ double mutant (Figure 4H and Table 3). Thus, Rdi1 does not simply act as a negative regulator of Cdc42.
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/ rga2/ strain under the control of the regulatable MET3 promoter (Care et al., 1999). As expected, expression of either RGA2 or BEM3 in the bem3/ rga2/ mutant growing under pseudohyphal-promoting conditions resulted in a reduction in the degree of polarized growth and restored constrictions at the mother/bud neck (Supplemental Figure 1). Thus the characteristic phenotype of the bem3/ rga2/ mutant is solely due to the absence of both Rga2 and Bem3. In a similar way we showed that the effects of the rdi1/ allele on the phenotype of a bem3/ rga2/ mutant was due solely due to loss of Rdi1 (data not shown).
The experiments reported in Figure 2 focus on the role of the Cdc42 GAPs during the establishment of hyphal or pseudohyphal growth during the first few cell cycles after outgrowth from unbudded yeast cells. To investigate the effect over a longer time frame, we investigated the response of the mutants to growth on solid Spider medium, which promotes filamentous growth from colony edges (Liu et al., 1994). (We refer to "filamentous growth" here because the nature of the colony outgrowths on Spider medium has not so far been characterized as hyphae or pseudohyphae.) The bem3/ rga2/ double mutant, and to a lesser extent, the bem3/ and rga2/ single mutants, showed enhanced filamentation compared with the parental strain (Figure 3, A–D). Thus, loss of Cdc42 GAPs not only enhances polarized growth in the short-term but also results in sustained enhancement of filamentous growth on Spider medium. Consistent with its effect on polarized growth in the yeast and pseudohyphal forms, loss of Rdi1 greatly reduced filamentous growth; indeed, the triple mutant lacking both the Cdc42 GAPs and Rdi1 showed less filamentation than the parental BWP17 strain (Figure 3, E and F). Loss of Swe1 abolished filament formation even in the double mutant lacking the Cdc42 GAPs; Figure 3, G and H). This result is surprising because we had previously found that swe1/ mutants formed germ tubes normally in liquid culture when challenged with serum (Wightman et al., 2004), although others have found that the rate of hyphal elongation, and the degree of filamentation on serum agar is reduced in a strain lacking Swe1 (Umeyama et al., 2005). Note that the swe1/ mutation in the strain shown in Figure 3G was generated independently of the swe1/ mutation in the strain shown in Figure 3H.
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), whereas in pseudohyphae Mlc1-YFP localizes to an apical crescent characteristic of a polarisome (Figure 4, A and B) (Crampin et al., 2005). Figure 4, C and D, shows that in most bem3/ rga2/ MLC1/MLC1-YFP cells growing in pseudohyphal-promoting conditions, Mlc1-YFP localizes to a discrete apical spot characteristic of a Spitzenkörper. Another way to visualize the Spitzenkörper is by staining with the amphiphilic membrane dye FM4-64 (Fischer-Parton et al., 2000; Crampin et al., 2005). Figure 4E shows that FM4-64 colocalizes with Mlc1-YFP to a discrete spot in the bem3/ rga2/ MLC1/MLC1-YFP mutant growing in pseudohyphal-promoting conditions. Thus the loss of the Cdc42 GAPs Rga2 and Bem3 results in the formation of a Spitzenkörper in conditions where it would not normally be present. Mlc1-YFP also localized to a Spitzenkörper-like structure in many cells lacking only one or other of the Cdc42 GAPs (Figure 4, F and G).
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). Second, the maximum fluorescence intensity of Mlc1-YFP in the Spitzenkörper is approximately threefold greater compared with that in the polarisome (Crampin et al., 2005). In the bem3/ rga2/ mutant growing under pseudohyphal-inducing conditions, Mlc1-YFP localization to the Spitzenkörper was indeed found to persist as Mlc1-YFP appeared in the cytokinetic ring (Figure 4I). We quantified the morphology and maximum fluorescence intensity of Mlc1-YFP at the tips of pseudohyphal cells of parental and mutant strains at intervals after inoculation of unbudded yeast cells into pseudohyphal growth conditions. For comparison, we also analyzed a parallel culture of BWP17 cells growing as hyphae. Each tip was classified according to whether it displayed a spot, crescent, or showed no fluorescence. We verified these assignments by showing that there was a consistent correlation between the fluorescence intensity and the pattern of localization assigned (see legend to Figure 5). The pattern of Mlc1-YFP localization in parental BWP17 cells growing as hyphae and pseudohyphae is shown in Figure 4, A and B, respectively. As we reported previously (Crampin et al., 2005), in cells growing in the hyphal form, Mlc1-YFP localized to an apical spot that persisted over the whole of the first cell cycle in >80% of the cells (Figure 5A). The remaining 20%, which displayed a crescent that disappeared at 80 min, was probably due to the small proportion of pseudohyphae that are present in such cultures (Sudbery, 2001). In contrast, in the pseudohyphal culture most cells displayed a crescent at early time points, which disappeared at the end of the first cycle (100 min), before reappearing as the second cycle commenced (110–130 min).
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/ rga2/ mutant, the proportion of cells displaying a spot morphology dipped at the end of the first cell cycle (100 min), but instead of apical Mlc1-YFP disappearing completely, the proportion of cells displaying a crescent showed a compensatory increase. Thus, some cell cycle regulation remains in cells lacking Cdc42 GAPs, but unlike pseudohyphae, apical Mlc1-YFP localization persists throughout the cell cycle. Consistent with the Spitzenkörper-like organization of Mlc1-YFP in bem3/ rga2/ mutants, the intensity of fluorescence at the tip was also found to be similar to that of wild-type hyphae and consistently over three- to fourfold greater than that of BWP17 pseudohyphal cells (Figure 5D). Figure 5B shows that a proportion of small pseudohyphal buds displayed an Mlc1-YFP spot rather than a crescent. However, as we observed previously (Crampin et al., 2005), the mean of the peak fluorescence intensity of these spots (194 ± 41) was much less than that of the spots in the BWP17 hyphae (538 ± 39) or the spots in bem3/ rga2/ mutant growing in pseudohyphal-inducing conditions (656 ± 52).
We also quantified the localization of Mlc1-YFP in bem3/ and rga2/ single mutants (Figure 5, E–F). In both cases, a spot was observed in about half of the cells immediately after evagination, but the frequency decreased at later times and the proportion of cells displaying a crescent was consistently higher than the double mutant lacking both Cdc42 GAPs. However, unlike the parental strain, some form of apical localization persisted throughout the cell cycle in most cells. Finally, we quantified the localization of Mlc1-YFP in the bem3/ rga2/ rdi1/ triple mutant (Figure 5G). This showed a similar distribution to the bem3/ rga2/ double mutant. Thus, loss of Rdi1 does not affect the proportion of cells displaying a Spitzenkörper, so the degree of polarized growth must be reduced for some other reason.
The Pattern of Septin Localization Is Altered in the Absence of Cdc42 GAPs
In addition to the shape, a second defining characteristic of hyphal germ tubes is that the septin ring forms within the germ tube where it organizes the formation of the primary septum during cytokinesis. Moreover, a septin band, consisting of longitudinal bars, forms at the base of the germ tube just after evagination, which disappears as the septin ring forms. In contrast, the septin ring forms at the mother bud neck in pseudohyphae (Sudbery, 2001). To investigate whether Rga2 and Bem3 also regulate this aspect of hyphal growth, and have a role in positioning the site of septin ring formation, we examined the pattern of septin ring localization in wild-type and mutant C. albicans cells expressing Cdc10-YFP during growth in pseudohyphal-promoting growth conditions (Figure 6, A–E). As expected, the septin ring formed at the mother-bud neck in all parental BWP17 cells (Figure 6, A and D). However, in cells lacking both Cdc42 GAPs the septin ring formed within the elongated pseudohyphal bud and away from the neck in virtually all cells (Figure 6, B and D). Furthermore, just after evagination of germ tubes, septin bars formed at the base of the bud and a septin cap was transiently visible at the bud tips (Figure 6E). Interestingly, in the bem3/ and rga2/ single mutants the septin ring formed at the mother-bud neck in most cells (Figure 6D). Thus, the Cdc42 GAPs are required for the normal regulation of septin ring localization, but the two Cdc42 GAPs are apparently redundant with respect to this function. The pattern of septin localization in the double mutant lacking both Cdc42 GAPs was not affected by the additional loss of Rdi1 (Figure 6D). Consistent with the ectopic position of septin rings, we also observed that Mlc1-YFP in the contractile cytokinetic ring was located within the germ tube instead of the bud neck location expected in pseudohyphae (Figure 4I).
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). However, it has also been reported that the ectopic septin rings that form in S. cerevisiae cells lacking all Cdc42 GAPs is not Swe1 dependent (Caviston et al., 2003). In agreement with the latter observation, we found that the localization of the septin ring within the pseudohyphal bud of C. albicans cells lacking Cdc42 GAPs was not Swe1 dependent (Figure 6, C and D).
Nuclear Division Occurs within the Germ Tube of Mutants Lacking Cdc42 GAPs
In pseudohyphae mitosis takes place across the mother-bud neck, but in hyphae nuclei migrate out of the mother cell and mitosis takes place within the germ tube, after which one nucleus returns to the mother cell. Thus, the pattern of nuclear division provides another characteristic that distinguishes hyphae from pseudohyphae. We investigated the pattern of mitosis in bem3/ rga2/ cells and parental BWP17 cells, growing in pseudohyphal conditions by expressing Nop1-YFP, a nucleolar protein that can be used to visualize nuclei in living cells (Crampin et al., 2005). Unbudded cells from an overnight culture were reinoculated into conditions promoting pseudohyphal growth and the position of the nuclei determined between 100 and 120 min after inoculation, the period during which the majority of cells underwent the first mitosis. Cells that contained either a single nucleus that was located within the germ tube, or contained two nuclei, both of which were in the germ tube, could be confidently scored as cells where mitosis took place in the germ tube. In cells where the nucleus was positioned across the mother bud neck, the site of mitosis cannot be ascertained with certainty, because these cells could either be cells where mitosis is taking place at this position, or they could be cells where the nucleus would continue to migrate into the germ tube. As expected, the nucleus was positioned across the mother bud neck in parental BWP17 cells, consistent with mitosis occurring at this position (Sudbery, 2001) (Figure 7A). In contrast, mitosis took place within the germ tube in the bem3/ rga2/ mutant cells growing in pseudohyphal conditions (Figure 7B). Quantification at three separate time points showed that 
20% of bem3/ rga2/ cells were undergoing mitosis within the germ tube compared with <1% of parental BWP17 cells (Figure 7C). In contrast, more nuclei were positioned across the mother bud neck in parental BWP17 cells, compared with bem3/ rga2/ cells (Figure 7C). Thus, bem3/ rga2/ cells growing in pseudohyphal conditions show the hyphal pattern of nuclear division.
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). Additionally, the analogous p190GDK1321A mutant of the human Rho-GAP failed to bind to or stimulate the GTPase activity of RhoA (Li et al., 1997). The second class of mutation was the replacement of a conserved arginine that protrudes into the active site of the Rho-GTPase and is thought to stabilize the transition state of the GTPase reaction (Rittinger et al., 1997). In the human Cdc42-GAP, replacement of this arginine by alanine (Cdc42hs-GAPR305A) greatly reduced the catalytic activity of Cdc42-GAP without affecting its affinity for Cdc42hs (Leonard et al., 1998). Similarly, the p190GDR1283L protein lost GAP activity but retained affinity for RhoA (Li et al., 1997). The equivalent mutations in the C. albicans GAP genes are rga2R1015L and bem3R985L. We replaced the critical arginine residue with leucine, because the alanine substitution in Cdc42hs-GAPR305A resulted in a protein with detectable GAP activity.
Strains were constructed in which a GAP gene carrying one of these point mutations was the sole copy of one or other of the GAP genes (e.g., BEM3/BEM3 rga2/rga2R1015L) or in which the mutant gene was the only copy of either GAP gene (e.g., bem3/ rga2/rga2R1015L). A list of the strains constructed is listed in Table 2, which also shows data describing the morphology of the strains growing under pseudohyphal-promoting conditions and the percentage of cells in which the septa formed away from the bud neck. It was necessary to monitor the position of the septa using calcofluor white staining rather than the position of the septin ring, because the process of strain construction resulted in strains with no markers with which to introduce Cdc10-YFP fusions. We have previously shown that the septa forms at the site of the septin ring (Sudbery, 2001). In every case, the point mutation had exactly the same effect as the equivalent deletion allele (Table 2). For example, the rga2/rga2R1015L and rga2/rga2K1061A strains showed a similar degree of enhanced polarized growth as the rga2/ strain; and the bem3/ rga2/rga2R1015L and bem3/ rga2/rga2K1061A strains showed a similar degree of polarized growth to the bem3/ rga2/ strain. Similarly, although the septa predominantly localized to the bud neck in strains that retained a single wild-type copy of either RGA2 or BEM3, it localized within the germ tube/elongated bud in strains in which the only remaining GAP gene carried one of the point mutations (bem3/ rga2/rga2R1015L, bem3/ rga2/rga2K1061A, rga2/ bem3/bem3R985L, and rga2/ bem3/bem3K1025A). Thus, the mutant phenotypes arising from loss of the Cdc42 GAP-encoding genes are due to loss of their GTPase-activating function.
Expression of a Constitutively GTP-bound Cdc42 Allele Does Not Result in the Same Phenotype as Loss of GAP Function
Deletion of the Cdc42 GAPs is predicted to result in an increased level of Cdc42-GTP, which would be expected to produce a similar phenotype to expression of the constitutively GTP bound Cdc42G12V. Previously, it has been that shown that expression of Cdc42G12V from the PCK1 promoter in C. albicans resulted in the formation of aberrant multibudded cells under yeast-inducing conditions, and cells with large, aberrant, branched structures under hyphal-inducing conditions (Ushinsky et al., 2002). However, the effect of expression of Cdc42G12V under pseudohyphal-inducing conditions was not investigated. To investigate whether expression of Cdc42G12V also causes the formation of hyphal characteristics under pseudohyphal conditions, cells of strain CaSU64 (CDC42/PPCK1-CDC42G12V) (Ushinsky et al., 2002) were grown in YPD medium to saturation overnight in conditions that promote yeast-form growth. The resulting unbudded yeast cells were then reinoculated into culture conditions that normally induce pseudohyphal growth and activation of the PCK1 promoter (2% casamino acids, 35°C, pH 5.0). The appearance of such cells, along with a parental control, is shown in Figure 8, A and B. Cells expressing Cdc42G12V were swollen, and the mother bud necks were abnormally large. Interestingly, some of the buds showed tip splitting and branching. Thus, in contrast to loss of Cdc42 GAPs, a predicted increase in the level of Cdc42-GTP by expression of Cdc42G12V does not result in a hyphal like morphology.
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Bem3 Localizes to the Tips of Hyphae and Young Buds; Rga2 Localizes to the Sites of Cytokinesis in Yeast and Pseudohyphae
We next investigated the localization of Rga2, Bem3, and Rdi1 by constructing fusions to YFP. The phenotype of bem3/ rga2 /RGA2-YFP and rga2/ bem3/BEM3-YFP were the same as the bem3/ and rga2/ strains, respectively, showing that the fusions were functional (data not shown). We observed that Bem3-YFP localized to a bright patch at the tips of hyphae (Figure 9A). However, the morphology was different from the discrete Mlc1-YFP spot that characterizes the Spitzenkörper; rather Bem3-YFP fluorescence formed a more diffuse pattern, which in some hyphal tips seemed to consist of a circle surrounding a nonfluorescing center (Figure 9, B and C). So, Bem3 may not be a Spitzenkörper component. Bem3-YFP also localized to a crescent at the tips of young yeast buds (Figure 9, D and E,), which disappeared as the bud increased in size (Figure 9F). Bem3-YFP also localized to the tips of pseudohyphal buds (Figure 9G). We did not observe any signal from Bem3-YFP at the sites of septation in any of the three growth forms. In striking contrast to Bem3-YFP, Rga2-YFP was not present at hyphal tips even in overexposed images (Figure 9H). However, Rga2-YFP was observed at sites of septation in pseudohyphae and yeast (Figure 9, I and J). A faint signal was observed at the tips of young pseudohyphal buds, which was faint or nonexistent in larger buds (Figure 9J). No signal was apparent at the tips of young yeast buds (Figure 9I). Thus, Bem3 is predominantly associated with the tips of hyphae and buds, whereas Rga2 is predominantly associated with sites of septation. Rdi1-YFP localized throughout the cytoplasm of both the mother cell and germ tube (Supplemental Figure 2).
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/ rga2/ mutant suggest that Rga2 and Bem3 may play a physiological role in regulating the formation of hyphal characteristics. However, there is no obvious change in the transcript levels of Rga2 and Bem3 during hyphal induction (Nantel et al., 2002). To investigate the possibility that they are regulated by posttranslation modifications such as phosphorylation, we carried out Western blots by using an anti-GFP mAb and lysates from strains expressing Rga2-YFP or Bem3-YFP. Rga2-YFP showed a band shift specifically in hyphal extracts, which disappeared upon treatment with calf intestinal phosphatase (Figure 9K). Thus, Rga2 is phosphorylated in a hyphal-specific manner, which may provide a mechanism for its down-regulation in hyphae. A band shift of Bem3-YFP was not detected (data not shown).
Apical Localization of Cdc42 Is Enhanced in Cells Lacking the Cdc42 GAPs
In S. cerevisiae, polarized growth is initiated by the recruitment of Cdc42, initially in a actin-independent manner (Ayscough et al., 1997). However, there is accumulating evidence that Cdc42 may participate in a positive feedback loop so that localization of Cdc42 can be a self-reinforcing process (Zajac et al., 2005; Roumanie et al., 2005). If this is the case in C. albicans, then cells with increased Cdc42 activity, because of the loss of the Cdc42 GAPs, will show increased amounts of Cdc42 at sites of polarized growth. To test this hypothesis, we expressed YFP-CDC42 from the MET3 promoter (MET3-YFP-CDC42) in parental BWP17 and bem3/ rga2/ cells growing in derepressing pseudohyphal conditions (SD medium, pH 6.0, 36°C) and took samples at intervals for imaging
GFP) (Roche Biosciences) or an anti-tetra His mAb (QIAGEN). Scale bars = 5 µm.
