Rac1 and Cdc42 Have Different Roles in Candida albicans Development

ABSTRACT We investigated the role of the highly conserved G protein Rac1 in the opportunistic pathogen Candida albicans. We identified and disrupted RAC1 and show here that, in contrast to CDC42, it is not necessary for viability or serum-induced hyphal growth but is essential for filamentous growth when cells are embedded in a matrix. Rac1 is localized to the plasma membrane, yet its distribution is more homogenous than that of Cdc42, with no enrichment at the tips of either buds or hyphae. In addition, fluorescence recovery after photobleaching results indicate that Rac1 and Cdc42 have different dynamics at the membrane. Furthermore, overexpression of Rac1 does not complement Cdc42 function, and conversely, overexpression of Cdc42 does not complement Rac1 function. Thus, Rac1 and Cdc42, although highly similar to one another, have different roles in C. albicans development.

protein is not necessary for viability or actin cytoskeleton organization. Rac1 is essential for filamentous growth when cells are embedded in an agar matrix yet is not required for seruminduced hyphal growth, in contrast to Cdc42. We also show that Rac1 is localized to the plasma membrane with different dynamics from those of Cdc42. Furthermore, overexpression of Rac1 does not complement Cdc42 functions. Altogether, our results indicate that although they are highly similar to one another, Rac1 and Cdc42 have fundamentally different roles in C. albicans development.

MATERIALS AND METHODS
Strain construction. The strains and primers used for this study are listed in Tables 1 and 2, respectively. The rac1⌬/RAC1 mutant (PY156) was obtained by transforming an auxotrophic his Ϫ ura Ϫ arg Ϫ BPW17 strain (27) with a rac1⌬-1::URA3 PCR product generated with the RAC1.P1 and RAC1.P2 primers and pGemUra3. This deletion removed the majority of the RAC1 open reading frame (ORF), leaving 44 bp 3Ј of the ATG and 30 bp 5Ј of the stop codon. A rac1⌬-2::HIS1 cassette was generated by first cloning RAC1 using the RAC1.P3 and -P4 primers into pCR2.1-TOPO (Invitrogen) and then inserting a BamHI-XbaI-blunted HIS1 gene fragment from pGemHis1. This rac1⌬-2::HIS1 cassette was transformed into PY156, and URA ϩ HIS ϩ prototrophs were selected. This second deletion also removed the majority of the RAC1 ORF, leaving 28 bp 3Ј of the ATG and 32 bp 5Ј of the stop codon. pExpArg constructs were digested with StuI and targeted to the RP10 locus in the appropriate strains.
Growth conditions. Cells were grown at 30°C in yeast extract-peptone-dextrose (YEPD) medium unless indicated otherwise. Serum induction was carried out as described previously (3), using overnight cultures grown in YEPD that were back diluted in YEPD and grown for approximately 6 h prior to the addition of 50% serum. Embedded medium conditions were as described previously (6), using YEP and sucrose.
Microscopy. Colonies and cells were imaged as described previously (3). The actin cytoskeleton was visualized as described previously (19), except that Alexa 488-phalloidin (Molecular Probes) was used and images were taken as described elsewhere (2). Fluorescence recovery after photobleaching (FRAP) analysis was performed on a Zeiss LSM510 Meta inverted confocal microscope using a Plan-Apo 63ϫ (numerical aperture, 1.4) objective. Images were captured every 0.4 second at 1 or 2% maximum laser intensity. Bleaching was performed at 90% laser intensity using 10 ϫ 0.6-ms photobleaching scans on a bud or hyphal tip circular area of 1.1 m 2 . Data analysis was done with Excel (Microsoft), and the average intensity of the bleached area was normalized to photobleaching during image acquisition, using the average intensity of an adjacent cell. Regression analysis to determine the FRAP t 1/2 was done using a one-phase exponential association function in SigmaPlot 9, as follows: where k is the rate constant and t 1/2 is 0.69/k. General techniques. Western blot and quantitative reverse transcription-PCR (qRT-PCR) analyses were carried out as described previously (3,4). Cdc42-Rac interactive binding domain (CRIB) pull-down experiments were performed as described previously (24), using the CRIB domain of S. cerevisiae Ste20 (amino acids 304 to 346) cloned into the pGEX 6P-2 plasmid (a gift from D. McCusker). Exponentially growing cells were lysed in GPLB (20 mM Tris-Cl, pH 7.4, 150 mM NaCl, 5 mM MgCl 2 , 5 mM glycerol phosphate, 1 mM dithiothreitol, Boehringer Mannheim protease inhibitor cocktail, 0.5% NP-40) by glass bead agitation in a Ribolyser. The supernatant collected after centrifugation at 5,000 ϫ g was incubated with CRIB-glutathione S-transferase-glutathione-agarose for 1 h at 4°C. After three successive washes with GPLB, proteins were eluted with Laemmli buffer and separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE).

C. albicans
Rac1 identification. C. albicans ORF19.6237 was identified by a BLAST search (1) using the human Rac1 sequence. A protein sequence alignment of this ORF with those of Rac1 proteins from different organisms, such as Y. lipolytica, Ustilago maydis, Caenorhabditis elegans, Drosophila melanogaster, and human, is illustrated in Fig. 1. ORF19.6237 is highly identical to the Rac1 proteins in all these species, with an overall sequence identity, shown in gray, of approximately 60%. ORF19.6237 has the conserved amino acid sequence TKXD at positions 116 to 119 (shifted one amino acid due to an insertion of aspartic acid at position 108) which is found in all Rac proteins, in contrast to the conserved sequence TQXD found in all Cdc42 proteins (7), including C. albicans Cdc42. Furthermore, ORF19.6237 has additional Rac-specific residues, including a tryptophan at position 56 which is critical for Rac signaling specificity (8), in contrast with the phenylalanine which is found at this position in Cdc42 proteins. These invariant Rac-specific amino acid residues indicate that the C. albicans ORF19.6237 protein is part of the Rac family of GTPases, and henceforth it will be referred to as Rac1. The most striking difference in C. albicans Rac1 is a stretch of 43 amino acids unique to this protein. In C. albicans, as in other organisms, Rac1 and Cdc42 are also highly similar to one another (57% overall sequence identity), suggesting that these two G proteins may have similar functions.

Rac1 is not required for C. albicans viability.
To determine the importance of Rac1 in C. albicans development, we successively disrupted the two copies of RAC1. RAC1 gene disruption was verified by PCR ( Fig. 2A) and by qRT-PCR (Fig.  2B). RAC1 mRNA was undetectable in rac1⌬/rac1⌬ mutant cells. In wild-type cells, RAC1 mRNA levels were threefold lower than those of CDC42. Dilutions of rac1⌬/rac1⌬ mutant cells spotted on YEPD medium (Fig. 2C) showed that growth of this RAC1 deletion strain was similar to that of the wild-type cells, and furthermore, doubling times in liquid medium were indistinguishable between rac1⌬/rac1⌬ mutant and wild-type cells. These results indicate that Rac1, in contrast to Cdc42 (3,17,21), is not required for C. albicans viability. We next examined the actin cytoskeleton in the rac1⌬/rac1⌬ mutant strain, and Fig. 2D shows no significant differences in actin patch and cable organization between this deletion mutant and the wild type. These results indicate that Rac1 is not required for actin organization in C. albicans.
C. albicans Rac1 is a bona fide G protein.
We constructed a fusion of Rac1 with green fluorescent protein (GFP), which migrates with an apparent M w larger than that of the corresponding GFP-Cdc42 fusion (Fig. 3A), in agreement with the difference in ORF sizes. G proteins cycle between an inactive GDP-bound state and an active GTP-bound state. We mea- sured the amount of active Rac1 by using a CRIB pull-down assay (24); the CRIB domain only binds to Rac1-GTP or Cdc42-GTP. Figure 3B illustrates the results of such a pulldown experiment for strains expressing GFP-Rac1 (WT) and its dominant active (G12V) or dominant negative (T17N) version. The level of Rac1 in the GTP-bound form determined from three independent experiments was 0.4% Ϯ 0.1% of the total Rac1 input. The level of Cdc42-GTP determined similarly was 0.5% Ϯ 0.2% of the total Cdc42 input (data not shown).
These results indicate that Rac1 binds GTP to the same extent as Cdc42 in C. albicans. Rac1 is critical for filamentation in embedded media. Different environments can trigger C. albicans filamentous growth. To investigate the role of Rac1 in such responses, we examined the growth of the RAC1 deletion mutant in response to different stimuli. Cdc42 is required for C. albicans invasive

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BASSILANA AND ARKOWITZ EUKARYOT. CELL hyphal growth in response to serum (3,23). In contrast, Rac1 is not necessary for such responses, since the RAC1 deletion mutant invaded fetal calf serum (FCS)-containing agar to a similar extent as a wild-type strain (Fig. 4A, upper panel) and formed hyphae (95%) after 3 h of incubation at 37°C in liquid medium (Fig. 4A, lower panel), similar to wild-type cells. Furthermore, rac1⌬/rac1⌬ mutant cells responded to two other inducers of filamentous growth, i.e., Spider medium and Nacetylglucosamine, similar to wild-type cells (data not shown). Mutants such as the efg1⌬/efg1⌬ mutant, which is defective in serum-induced responses (16), can nevertheless respond by filamentous growth when embedded in solid media (9). Similarly, we observed that cdc42⌬/PMETCDC42 mutants, which are unable to form hyphae in YEPD-FCS medium (3), could respond to YEP-sucrose embedded medium by filamentous growth (Fig. 4B, upper panel), although the filaments were not as long as those in the wild-type strain. Strikingly, we did not observe filamentation of rac1⌬/rac1⌬ mutant colonies in such an embedded medium (Fig. 4B, lower panel). This defect was indeed due to the RAC1 deletion, as it was complemented by the reintroduction of RAC1. Wild-type cells, cdc42 mutant cells, and rac1⌬/rac1⌬ mutant cells with reintroduced RAC1 all formed Ͼ90% filamentous colonies, whereas rac1⌬/rac1⌬ mutant cells formed Ͻ5% filamentous colonies (Fig. 4C). Furthermore, rac1⌬/rac1⌬ mutant cells which overexpressed GFP-Rac1 (via the ADH promoter) also formed Ͼ90% filamentous colonies, indicating that GFP-Rac1 is functional. Together, these results indicate that Rac1 is required for filamentation under embedded conditions. Rac1 and Cdc42 have different dynamics at the membrane. In C. albicans, Cdc42 localizes to the plasma membrane, where it is clustered at the tips of growing buds or hyphae (11) (Fig.  5A). Similarly, Rac1 is localized to the plasma membrane during both vegetative and serum-induced hyphal growth (Fig.  5A), yet its membrane localization is more homogenous than that of Cdc42, with little to no enrichment at the tips of either buds, hyphae, or septa. In order to examine the dynamics of these two G proteins, we carried out FRAP experiments using functional GFP fusions (Fig. 4C and 6A and B) in strains in which these fusions were the sole copies of the respective G protein. The fluorescence at the tips of either buds or hyphae of cells expressing GFP-Rac1 and GFP-Cdc42 was photobleached (Fig. 5A), and the recovery of fluorescence over time was monitored (Fig. 5B). The average FRAP t 1/2 values were 5.1 Ϯ 1.5 s (n ϭ 17) and 3.4 Ϯ 1.7 s (n ϭ 12) for Cdc42 in buds and hyphae, respectively. A similar value of ϳ5 s has been observed for Cdc42 in budding S. cerevisiae cells (25). Strikingly, the FRAP t 1/2 of Rac1 was approximately three to four times faster than that of Cdc42, with average FRAP t 1/2 values of 1.4 Ϯ 0.3 s (n ϭ 13) for buds and 1.3 Ϯ 0.4 s (n ϭ 14) for hyphae. Furthermore, the FRAP t 1/2 values for Rac1 were similar irrespective of the location of photobleaching (cell body or hyphae). These results indicate that compared to Cdc42, membrane-associated Rac1 is highly dynamic, and they suggest that these two G proteins have different interactions at the membrane.
Rac1 overexpression does not complement Cdc42 function. Rac1 and Cdc42 have both complementary and overlapping functions in different organisms. To investigate if these two G proteins have overlapping functions in C. albicans, we initially examined the effect of Rac1 overexpression in cdc42⌬/ PMETCDC42 mutant cells with respect to cell viability and serum-induced hyphal growth. As illustrated in Fig. 6A, GFP-Rac1 overexpression did not restore growth to cdc42⌬/ PMETCDC42 mutant cells on a medium which represses CDC42 expression, indicating that Rac1 overexpression cannot complement Cdc42 function for viability. In contrast, overexpression of GFP-Cdc42 enabled cdc42⌬/PMETCDC42 mutants to grow on a repressive medium, indicating that this GFP fusion is functional for viability. When grown in YEPD medium, which contains a low methionine level (semipermissive conditions) (3), cdc42⌬/PMETCDC42 mutant cells appeared larger than wild-type cells (Fig. 6B). The overexpression of Cdc42 or GFP-Cdc42 in the cdc42⌬/PMETCDC42 mutant cells resulted in cells that were similar in size to wild-type cells and restored serum-induced hyphal growth, indicating that GFP-Cdc42 is functional. Overexpression of Rac1 or GFP-Rac1 in the cdc42⌬/PMETCDC42 mutant strain resulted in cells which appeared larger than cdc42⌬/PMETCDC42 mutant cells. Wildtype cells were not affected by the overexpression of Rac1 or GFP-Rac1 in rich medium; however, we observed some heterogeneity in cell size (data not shown). Nonetheless, these cells formed hyphae in the presence of serum. Rac1 or GFP-Rac1 overexpression did not restore serum-induced hyphal growth in cdc42⌬/PMETCDC42 mutant cells, and instead, these cells were substantially larger than those not overexpress- ing Rac1 (Fig. 6B). These results suggest that Rac1 can interfere with polarized growth, in particular during hyphal growth, in cells with reduced levels of Cdc42. Perhaps Rac1 titrates out a Cdc42 effector, for example, a CRIB domain-containing protein. Conversely, overexpression of CDC42 did not restore embedded filamentous growth in rac1⌬/rac1⌬ mutant cells (data not shown). Taken together, these results indicate that Cdc42 and Rac1 do not have overlapping functions, but rather that these two G proteins play different roles in C. albicans development.

DISCUSSION
We have identified and characterized Rac1 of C. albicans. This G protein is highly similar to Rac1 proteins in other organisms and to Cdc42. However, in contrast to Cdc42, Rac1 is not necessary for viability in C. albicans. Strikingly, rac1⌬/ rac1⌬ mutant cells do not form filamentous colonies when embedded in an agar matrix, yet these cells form hyphal filaments in response to serum. Rac1 localized to the plasma membrane, albeit with a more uniform distribution than that of Cdc42, which is found clustered at bud and hyphal tips, as previously reported (11). Using FRAP methods, we showed that Rac1 has different dynamics at the plasma membrane than Cdc42, suggesting that these two proteins have different interactions at the membrane. Furthermore, overexpression studies indicated that Cdc42 and Rac1 do not have overlapping functions, as each protein was unable to substitute for the function of the other. Together, our results indicate that these two G proteins have distinct functions in C. albicans.
Surprisingly, Rac1, which is critical for actin cytoskeleton organization in metazoans, is not required for actin organization in C. albicans. The requirement for Rac1 in actin cytoskeleton organization of different fungi varies, as Rac1 in both Y. lipolytica and C. neoformans is not necessary for actin organization (12,22), in contrast to the case of P. marneffei, where Rac1 is important for this process (5). C. albicans Rac1 can bind GTP, as shown by CRIB pull-down experiments. Because Rac1 in the GTP-bound state can bind the S. cerevisiae Ste20 CRIB domain, it is likely that this G protein, similar to Cdc42, binds effector proteins in vivo.
Different environmental signals, such as N-acetylglucosamine, an alkaline extracellular pH, serum, and entrapment in a semisolid matrix, can trigger filamentous growth of the human pathogen C. albicans (26). In response to serum, signaling via a cyclic AMP-protein kinase A pathway results in the induction of Efg1-dependent hypha-specific genes such as HGC1 (28). Cdc42 has been demonstrated to be specifically

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BASSILANA AND ARKOWITZ EUKARYOT. CELL required for the maintenance of Efg1-dependent gene induction (4,23). Both cdc42 mutants and strains expressing reduced levels of this G protein undergo vegetative growth yet do not undergo invasive filamentous growth in response to serum (3,4,17,21,23). However, we showed here that such strains with a reduced level of Cdc42 form filamentous colonies when embedded in a semisolid agar matrix. The phenotype of cells with ectopic Cdc42 expression is similar to that of efg1⌬/efg1⌬ cph1⌬/cph1⌬ mutants, which do not form hyphae in response to serum (16) but, nonetheless, form filamentous colonies in embedded media (9), further supporting the hypothesis that Cdc42 and Efg1 signal via the same pathway. In response to entrapment in a semisolid matrix, the Czf1 transcription factor is required (6). CZF1 was isolated in a screen for genes whose ectopic expression stimulated filamentous colonies when cells were grown under embedded conditions (6). czf1⌬/czf1⌬ mutant cells exhibited a delay in filamentous growth under embedded conditions, and this defect was further enhanced by the deletion of CPH1 from this czf1⌬/ czf1⌬ mutant strain. In contrast, both czf1⌬/czf1⌬ mutant cells and czf1⌬/czf1⌬ cph1⌬/cph1⌬ mutant cells formed filaments similar to those of the wild type in Spider medium, serumcontaining medium, and N-acetylglucosamine-containing medium. rac1⌬/rac1⌬ mutant cells exhibited similar responses to filamentation inducers, i.e., embedded conditions, Spider medium, serum, and N-acetylglucosamine, suggesting that Rac1 may signal via Czf1, raising the possibility that the two very similar G proteins Cdc42 and Rac1 function in different signal transduction pathways. How these two similar G proteins function in different developmental states in C. albicans is currently under investigation.