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Vol. 19, Issue 9, 3638-3651, September 2008
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Institute of Developmental Biology and Cancer, Centre National de la Recherche Scientifique Unité Mixte de Recherche 6543, Université de Nice, Faculté des Sciences-Parc Valrose, 06108 Nice Cedex 2, France
Submitted December 26, 2007;
Revised June 11, 2008;
Accepted June 17, 2008
Monitoring Editor: Patrick J. Brennwald
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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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). Rho G proteins are found in eukaryotic cells from yeast to humans, and they are divided into three subfamilies: Cdc42, Rac, and Rho. In humans, >20 Rho G proteins have been identified (Hall, 2005), whereas in yeast such as Saccharomyces cerevisiae, only six Rho G proteins have been described (Park and Bi, 2007). Rho G proteins are highly conserved (Jaffe and Hall, 2005), for example S. cerevisiae Cdc42 has >80% overall sequence identity with its human counterpart. All Rho G proteins cycle between an inactive guanosine diphosphate (GDP)-bound form and an active guanosine triphosphate (GTP)-bound form. These GTPases are temporally and spatially regulated by activators or guanine nucleotide exchange factors (GEFs) and inactivators such as GTPase-activating proteins (GAPs) or guanine nucleotide dissociation inhibitors (DerMardirossian and Bokoch, 2005; Buchsbaum, 2007; Tcherkezian and Lamarche-Vane, 2007).
In humans, there are two major classes of Rho G protein GEFs, which together have >85 members (Hall, 2005). The principal class of GEFs, the Dbl family (Rossman et al., 2005) is made up of several transforming factors or proto-oncogenes, including Dbl, Vav, Tiam1, and LARG, which are involved in a variety of cancers (Karnoub et al., 2004). Cdc24, the sole Cdc42 activator in S. cerevisiae (Gulli and Peter, 2001), is one of the founding members of this class of GEFs, which are characterized by a conserved catalytic domain called a Dbl Homology (DH) domain (Erickson and Cerione, 2004; Rossman and Sondek, 2005). Recently, a new class of GEFs has been identified that has a common catalytic domain, referred to as a DOCKER domain, with no apparent sequence homology to the DH domain (Brugnera et al., 2002; Cote and Vuori, 2002). This class of GEFs, referred to as the CDM zizimin Homologs (CZH) family (Meller et al., 2005) is made up of the CDM (Caenorhabditis elegans Ced-5, Homo sapiens Dock180, and Drosophila melanogaster myoblast city) family (Hasegawa et al., 1996; Erickson et al., 1997; Wu and Horvitz, 1998) and the zizimin family (Meller et al., 2002). These GEFs either activate Rac (typically CDM family) or Cdc42 (typically zizimin family). For example, human Dock180 has been shown to bind nucleotide-free Rac1 (Brugnera et al., 2002; Cote and Vuori, 2002). Homologues of this CZH class of GEFs have also been identified in Dictyostelium discoideum, fungi, and, interestingly, plants such as Arabidopsis thaliana, in which no Dbl family GEF has been identified (Meller et al., 2005). Given their recent identification, much less is known about the CZH GEF family compared with the Dbl family. Nonetheless, the CZH GEFs such as myoblast city have been shown to be involved in myoblast fusion, dorsal closure, cytoskeletal organization, and border cells migration (Erickson et al., 1997; Bianco et al., 2007), whereas Ced-5 plays a critical role in phagocytosis and cell migration (Wu and Horvitz, 1998) and Dock180 is required for cell morphogenesis (Hasegawa et al., 1996) and phagocytosis (Albert et al., 2000; Park et al., 2007). Very recently, Dock180 has been shown to have a critical role in netrin-dependent axon outgrowth and attraction (Li et al., 2008). In S. cerevisiae, where there is no Rac1 homologue, the protein Ylr422wp, which has a region similar to the catalytic DOCKER domain, has been reported to bind human Rac1 (Brugnera et al., 2002), yet its function is unknown. A gene encoding Rac1 is present in virtually all other fungal genomes yet little is known about its activation, in particular the role of CDM family GEFs.
Candida albicans is an opportunistic human fungal pathogen that can cause superficial mucosal infections and systemic infections, in particular when the host immune system is compromised (Odds et al., 1988; Calderone, 2002). The capacity of C. albicans to reversibly switch from "yeast" to filamentous growth is associated with pathogenicity. Several different signals can induce this morphogenesis, including serum, nutrients, or entrapment in an agar matrix (Kumamoto and Vinces, 2005). Both mitogen-activated protein kinase and cAMP-dependent protein kinase A pathways are required for the induction of a range of genes that are critical for this morphological transition (Biswas et al., 2007). This has also been observed for invasive pseudohyphal growth of S. cerevisiae (Park and Bi, 2007). Nonetheless, the cytoskeleton reorganization that is necessary for this morphogenesis in C. albicans can be uncoupled from hyphal specific gene induction (Sinha et al., 2007). Two critical cytoskeleton regulators, Cdc42 and its activator Cdc24 are required for serum-induced invasive filamentous growth and pathogenicity (Michel et al., 2002; Ushinsky et al., 2002; Bassilana et al., 2003; VandenBerg et al., 2004). Recently, we identified an additional Rho G protein in C. albicans, Rac1, which is required for filamentous growth in an agar matrix (Bassilana and Arkowitz, 2006). Despite 
60% sequence identity between C. albicans Rac1 and Cdc42, these two G proteins are required for filamentous growth in response to distinct signals. Furthermore, overexpression of either G protein is unable to complement the function of the other G protein. These results suggest that Cdc42 and Rac1 function in distinct signaling pathways. In contrast to Cdc42, nothing is known about how Rac1 is regulated in C. albicans. Here, we identify Dck1, which contains a region with similarity to the DOCKER catalytic domain, as the Rac1 activator during C. albicans filamentous growth. Our results indicate that CDM family GEF function has been conserved in fungi.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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), Spider medium (Calera et al., 2000) or N-acetyl glucosamine (GlcNAc) (Castilla et al., 1998). For filamentous growth induction on solid media, 2% agar was included. Filamentous growth induction in embedded media was carried out in YEP containing 2% sucrose as described previously (Brown et al., 1999) and for quantification, colonies with five or more filaments were scored as filamentous.
Strains
The strains and primers used in this study are listed in Tables 1 and 2, respectively. The DCK1 and DCK2 Tn7-UAU1 strains (PY284 and PY286) were obtained by transforming an auxotrophic his–ura–arg– strain (BWP17) with the respective Tn7-UAU1 plasmids (CAGE530 and CAGDV28, a gift from A. Mitchell), digested with NotI. ARG+URA+ prototrophs were isolated and verified as described previously (Davis et al., 2002). Dck1
/dck1 mutants were obtained by initially transforming BWP17 with a dck1::HIS1 polymerase chain reaction (PCR) product generated from pGemHis1, by using DCK1.P1 and DCK1.P2 primers, yielding the heterozygote strain PY646. This deletion removed essentially all of the DCK1 open reading frames (ORF), with only 74 base pairs 3' of the ATG and 68 base pairs 5' of the stop codon remaining. Subsequently, a dck1::URA3 PCR product was generated using the same primers and pGemUra3, transformed into PY646 and URA+HIS+ prototrophs were selected, yielding PY704. Correct insertion into the DCK1 locus was confirmed by PCR. Czf1/czf1 mutants were obtained by initially transforming BWP17 with a czf1::HIS1 PCR product generated from pGemHis1, by using CZF1.P1 and CZF1.P2 primers, yielding the heterozygote strain PY523. This deletion removed all of the CZF1 ORF. Subsequently, a czf1::URA3 PCR product was generated using CZF1.P3 and CZF1.P4 primers and pGemUra3, transformed into PY523, yielding the homozygote strain PY897. This deletion removed essentially all of the second CZF1 ORF with 78 base pairs 3' of the ATG and 74 base pairs 5' of the stop codon remaining. Correct insertion into the CZF1 locus was confirmed by PCR. The pExpArg constructs were digested with StuI and targeted to the RP10 locus in the appropriate strains. Similar results were obtained with two independent isolates of each strain.
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7.7 kb) was amplified by PCR from genomic DNA in two fragments and each was cloned into pCR2.1 TA (Invitrogen, Cergy Pontoise, France). The primers DCK1.P3 (with a unique MluI site at the 5' end) and DCK1.P4 were used to amplify a fragment from 987 base pairs 5' of the ATG to 2855 base pairs 3' of the ATG, which was cloned into pCR2.1 TA, yielding pCR-PDCK1DCK1-5'. The primers DCK1.P5 and DCK1.P6 (with a unique XhoI site at the 3' end) were used to amplify a fragment from 2300 base pairs 3' of the ATG to 914 base pairs 3' of the stop codon, which was also cloned into pCR2.1 TA, yielding pCR-DCK1-3'. Subsequently, pCR-PDCK1DCK1-5' was digested with MluI, blunted, and digested with AatII. The released PDCK1DCK1-5' fragment was cloned into pCR-DCK1-3', which was digested with NotI, blunted and digested with AatII, yielding pCR-PDCK1DCK1. A unique NotI site was subsequently introduced by site-directed mutagenesis with the Pfu polymerase (Promega, Charbonnieres Les Bains, France), by using the DpnI method (Weiner et al., 1994), in pCR-PDCK1DCK1, 5' of the DCK1 promoter. The NotI/XhoI PDCK1DCK1 fragment was then cloned into the respective sites in pExpArg (derived from pExp-PADHGFPRAC1) (Bassilana and Arkowitz, 2006). The sequence of all PCR products and mutations were confirmed (ABI [Courtaboeuf, France] PRISM big-dye terminator cycle sequencing kit). Two clones of pCR-DCK1-3' along with DCK1-3' amplified from genomic DNA were sequenced to confirm the difference with the published genome sequence (http://www.candidagenome.org/).
For Rac1 and Cdc24 overexpression, pExp-PADHRAC1 and pExp-PADHCDC24GFP plasmids were used (Bassilana et al., 2005; Bassilana and Arkowitz, 2006). Constitutively active [G12V] and [Q61L] and inactive [T17N] Rac1 mutants were generated by site-directed mutagenesis by using pExp-PADHRAC1 as a template. For DCK1 overexpression a pExp-PADH plasmid, which was derived from pExp-PADHGFPRAC1, was used. To generate pExp-PADHDCK1 and pExp-PADHGFPDCK1 plasmids, the primer pairs VECTOR.P1, VECTOR.P2, and VECTOR.P3, VECTOR.P4 were used to introduce an RsrII site into pExp-PADHGFPRAC1, either 3'of the ADH1 promoter or 3' of green fluorescent protein (GFP), respectively, yielding pExp-PADH-R-GFPRAC1 and pExp-PADHGFP-R-RAC1. In parallel, an RsrII site was inserted, two base pairs 5' of the ATG, and an MluI site, 15 base pairs 3' of the stop codon in pCR-PDCK1DCK1, yielding pCR-PDCK1-R-DCK1-M. This plasmid was then digested with RsrII and MluI and the DCK1 ORF was cloned into the respective sites of pExp-PADH-R-GFPRAC1 and pExp-PADHGFP-R-RAC1, yielding pExp-PADHDCK1 and pExp-PADHGFPDCK1. To generate pExp-PDCK1DCK1GFP, site-directed mutagenesis was used to introduce a unique SacI site 5' and a unique MluI site 3' of the stop codon in pExp-PDCK1DCK1, resulting in pExp-PDCK1DCK1-S-M. Subsequently, GFP from pAW6-GFP (Nishikawa et al., 2002) was cloned into pExp-PDCK1DCK1-S-M, resulting in pExp-PDCK1DCK1GFP. To generate DCK1 constructs lacking the DOCKER Homology Region (DHR) domain, the region between base pairs 3998 and 5349 3' of the ATG (encoding amino acids [aa] 1332-1783) was removed by site-directed mutagenesis, using pCR-DCK1-3' as a template and the primers DCK1.P7 and DCK1.P8, yielding pCR-dck1DHR. The AatII/SacI fragment from this plasmid was then cloned into the respective sites in pCR-PDCK1DCK1-5', resulting in pCR-PDCK1dck1DHR. Subsequently, an RsrII site was inserted 5' of the ATG and an MluI site, 3' of the stop codon as described above, resulting in pCR-PDCK1-R-dck1DHR-M. The dck1DHR ORF was then cloned behind either the ADH1 promoter or ADHGFP, as described above.
For overexpression of CZF1, both the MAL2 and ADH1 promoters were used. PMALCZF1 originated from pDB212 (Brown et al., 1999). Initially, a unique XhoI site was introduced 3' of the stop codon by site-directed mutagenesis and subsequently a PMALCZF1 fragment was obtained by PacI digestion, followed by blunting and XhoI digestion. This fragment was then cloned into pExp-PADHGFPRAC1 that was digested with NotI, blunted and digested with XhoI to yield pExp-PMALCZF1. For CZF1 expression behind the ADH1 promoter, a unique BamHI site 5' of the ATG and an MluI site 3' of the stop codon were introduced by site directed mutagenesis, using pDB212. The BamHI/MluI CZF1 ORF was then cloned into compatible sites in plasmid pAW6 resulting in pAW6-PADHCZF1, which was subsequently digested with NotI and XhoI. The NotI-PADHCZF1-XhoI fragment released was finally cloned into respective sites in pExp-PADHGFPRAC1, resulting in pExp-PADHCZF1. The sequence of all site directed mutagenesis products was confirmed (ABI PRISM Big Dye terminator cycle sequencing kit; Applied Biosystems, Foster City, CA).
To generate proteins for in vitro binding experiments, antibody production, and antibody purification, Rac1 from pExp-PADHRAC1 was amplified using the primer pairs RAC1.P1 and RAC1.P2, which introduced a BamHI site 5' of the ATG and a NotI site 3' of the Stop codon. The BamHI/NotI RAC1 ORF was then cloned in pGEX 6P-2 (Amersham, Orsay, France), resulting in pGEX-RAC1. Constitutively active [G12V] and inactive [T17N] GST-Rac1 were generated using the same primers (RAC1.P1 and RAC1.P2), with pExp-PADHrac1[G12V] and pExp-PADHrac1[T17N] as templates. To generate maltose-binding protein (MBP)-Rac1, RAC1 from pGEX-RAC1 was amplified using the primer pairs RAC1.P1 and RAC1.P3, which introduced a BamHI site 5' of the ATG and a SalI site 3' of the Stop codon. The BamHI/SalI RAC1 ORF was then cloned in pMal-c2 (New England Biolabs, Saint Quentin en Yvelines, France), resulting in pMal-RAC1. To generate MBP-DHR, the region encoding aa 1288-1812 of Dck1 was amplified, by using pExp-PADHDCK1 and the primer pairs DCK1.P9 and DCK1.P10. These primers introduced a BamHI site 5' of codon for aa 1288 and a Stop codon, followed by a SalI site, 3' of the codon for aa 1812. The BamHI/SalI DHR fragment was then cloned in pMal-c2 resulting in pMal-DHR. Subsequently, one CTG codon in the DHR domain (residue at position 1773), which codes for a serine in Candida albicans was changed to AGC by site-directed mutagenesis. The sequences of all cloned PCR products and site-directed mutagenesis products were confirmed (ABI PRISM Big Dye terminator cycle sequencing kit).
General Techniques
Quantitative reverse transcription (qRT)-PCR analyses were carried out as described previously (Bassilana et al., 2005), and the primers used are listed in Table 2.
Microscopy
For colony morphology, after growth on or in agar containing media for 3–6 d, colonies were imaged with an MZ6 dissection scope (Leica, Rueil-Malmaison, France) at a magnification of 10x or 20x, as described previously (Bassilana et al., 2005). For cell morphology, cells were fixed with formaldehyde after incubation in the different inducing media and imaged with a Leica DMR epifluorescence microscope with a numerical aperture 1.35 63x objective by differential interference contrast optics, as described previously (Bassilana et al., 2005).
Immunoblot Analyses
Yeast protein extracts were prepared from logarithmically growing cells. Western blot analyses were carried out as described previously (Bassilana et al., 2003). Equal amounts of cells or fusion proteins (for in vitro binding studies) were used in each experiment, and proteins in each lane were visualized by Ponceau S staining of nitrocellulose membranes. Blots were probed with either a rabbit polyclonal serum against GFP (1:5000; Nern and Arkowitz, 2000), MBP (1:1000; a gift from W. Wickner), S. cerevisiae Pma1p (1:10,000; a gift from R. Kelly; Kelly et al., 2000), S. cerevisiae Cdc11p (sc-7170, 1:200; Santa Cruz Biotechnology, Heidelberg, Germany), or C. albicans Rac1 (1:1000). Immunoblots were then visualized by enhanced chemiluminescence (luminol-coumaric acid) on a Fuji-Las3000 (Fuji, Clichy, France).
To generate polyclonal sera against C. albicans Rac1, rabbits were immunized with glutathione transferase (GST)-Rac1 purified from Escherichia coli BL21 cells, by using pGEX-RAC1. MBP-Rac1 was purified from E. coli BL21 cells, by using pMal-RAC1, and it was bound to amylose resin. This fusion protein was cross-linked to the resin with dimethyl pimelimidate and used to purify sera as described previously (Harlow and Lane, 1988). The resin was extensively washed with buffer A (10 mM Tris-Cl, pH 7.5) followed by buffer A containing 0.5, 1, and 2 M NaCl. Subsequently, the resin was washed with 100 mM glycine, pH 2.5, followed by 10 mM Tris-Cl, pH 8.8, and then antibodies were eluted with 100 mM triethylamine, pH 11.5, and neutralized as described (Harlow and Lane, 1988). These purified antibodies were stored at 4°C in the presence of 0.5 mg/ml gelatin.
Protein Binding Studies
GST-Rac1 (wild-type and mutants), MBP-Rac1, and MBP-DHR fusion proteins were expressed in BL21 E. coli cells grown at 30°C and induced with 0.1 mM isopropyl β-D-thiogalactoside for 3–4 h. After centrifugation, cells were resuspended in 10% sucrose, 50 mM Tris-Cl, pH 8, and frozen in liquid nitrogen. All subsequent steps were carried out at 4°C. Fusion proteins were purified using glutathione (GSH)-Sepharose 4B (Amersham) or amylose resin (New England Biolabs, Ipswich, MA), as described previously (Barale et al., 2006) in buffer B (50 mM Tris-Cl, pH 7.5, 100 mM NaCl, 2.5 mM MgCl2, 1 mM dithiothreitol [DTT], 1 mM phenylmethylsulfonyl fluoride [PMSF], 10% glycerol, and protease inhibitor cocktail [Roche Diagnostics, Mannheim, Germany]) for G protein fusions and buffer C (20 mM Tris-Cl, pH 7.5, 100 mM NaCl, 2.5 mM MgCl2, 1 mM DTT, and 1 mM PMSF) for the DHR fusion. Protein bound to the respective resin was washed extensively in the same buffer (buffer B or C) containing 250 mM potassium acetate, 500 mM potassium acetate and for MBP-DHR fusions, additionally 1 M potassium acetate. MBP and MBP-DHR were then eluted with buffer C containing 10 mM maltose, and GST-Rac1 was eluted in buffer D (50 mM Tris-Cl, pH 8, 100 mM NaCl, 2.5 mM MgCl2, 1 mM DTT, 1 mM PMSF, 10% glycerol, and 10 mM GSH). For binding assays, 5 µg of GST-Rac1 fusion proteins or GST, bound to GSH-Sepharose, was incubated with 2 µg of MBP-DHR (or MBP) in 50 µl of buffer E (50 mM Tris-Cl pH 7.5, 100 mM NaCl, 5 mM MgCl2, 1 mM DTT, 1 mM PMSF, and 10% glycerol) for 1 h at 25°C. GSH-Sepharose samples were then washed eight times with 200 µl of buffer E, and proteins were eluted in SDS-polyacrylamide gel electrophoresis (PAGE) sample buffer, analyzed by SDS-PAGE, followed by immunoblotting.
Cell Fractionation
Logarithmically growing PY838 cells expressing Dck1-GFP were collected by centrifugation and resuspended in GPL buffer lacking NP-40. All subsequent steps were carried out at 4°C. Cells were broken with glass beads in a RiboLyser (MP Biomedicals, Illkirch, France) for 45 s at maximum power. Cell extracts were clarified by centrifugation at 200 x g for 5 min. This supernatant was then centrifuged for 10 min at 10,000 x g, resulting in P10 pellet and S10 supernatant fractions. The P10 fraction was suspended in a volume equivalent to the S10 fraction. The S10 fraction was subsequently centrifuged for 1 h at 100,000 g in a TL-100 (Beckman, Villepinte, France), resulting in P100 pellet and S100 supernatant fractions. The P100 fraction was resuspended in the same volume as the S100 supernatant fraction. Fractions were subsequently analyzed by 7% (for Dck1-GFP and Pma1) and 12% SDS-PAGE (for Rac1) followed by immunoblotting and probing with anti-GFP, anti-Pma1, and anti-Rac1 sera.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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) and the Rho G protein it activates, Cdc42 (Michel et al., 2002; Ushinsky et al., 2002; Bassilana et al., 2003; VandenBerg et al., 2004), are required for viability and serum-induced hyphal growth. Conversely, cdc42 mutants that were defective in serum-induced hyphal growth were able to form filaments under embedded conditions (VandenBerg et al., 2004). The Rho G protein Rac1, which is 57% identical to Cdc42, is required for filamentous growth when cells are embedded in an agar matrix (Bassilana and Arkowitz, 2006). These responses to different filamentous growth stimuli, together with the inability of overexpressed Rac1 to complement cdc42 mutants, suggest that these two similar G proteins function in distinct signaling pathways. Typically, the GTP bound form of a G protein binds effectors; hence, we examined whether overexpression of a Rac1 mutant, which mimics the GTP-bound state (rac1[Q61L]) or the nucleotide-free state (rac1[T17N]), alters embedded filamentous growth in both wild-type or rac1 deletion strains. Overexpression of the nucleotide-free form of a G protein results in a dominant-negative effect due to sequestration of its GEF (Feig, 1999). Overexpression of rac1[T17N] did not substantially affect growth or serum-dependent hyphal growth of either strain (data not shown), indicating that Rac1 does not sequester Cdc24, suggesting the existence of a distinct Rac1 GEF. In contrast, overexpression of the nucleotide-free Rac1 form in a wild-type strain completely blocked filamentous growth under embedded conditions (Figure 1A). Overexpression of the GTP bound Rac1 form resulted in embedded filamentous growth that was similar, albeit somewhat reduced, compared with strains that overexpressed wild-type Rac1. In a rac1/rac1 strain, overexpression of the GTP bound Rac1 form partially complemented the filamentous growth defect compared with overexpression of wild-type Rac1, whereas that of a nucleotide-free form did not (Figure 1C). Similar results were observed in wild-type and rac1 mutant strains by using different GFPRac1 fusions (RAC1, rac1[Q61L], and rac1[T17N]). Quantitative real-time RT-PCR measurements indicated that the mRNA transcript levels of the different Rac1 forms were expressed at 50-fold higher levels than that of endogenous Rac1 in these strains. To confirm that the Rac1 proteins were overexpressed, we generated specific Rac1 polyclonal antibodies. These antibodies revealed a band corresponding to the expected size of Rac1 in a wild-type strain but not in a rac1 deletion strain and did not cross-react with Cdc42 (Figure 1, B and D, and Supplemental Figure S1). The Rac1 proteins (wild-type and mutants) were overexpressed both in the wild-type strain (Figure 1B) and the rac1 deletion strain (Figure 1D), respectively. In both strain backgrounds, wild-type Rac1 protein overexpression was higher than that of Rac1[Q61L] and Rac1[T17N], the latter was overexpressed the least. Expression of rac1[T17N] was nonetheless sufficient to block embedded filamentous growth in a strain containing both copies of endogenous RAC1 (Figure 1A). Together, these results indicate that the GTP-bound form of Rac1 is required for embedded filamentous growth; yet, given that such a Rac1 mutant, which is blocked for GTP hydrolysis, only partially complements a rac1 deletion strain, it is likely that GDP–GTP cycling is important for efficient filamentous growth in embedded media.
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), or CDM family GEFs, such as Dock180 (Brugnera et al., 2002; Cote and Vuori, 2002). To address whether the Dbl family member Cdc24 could activate Rac1, in addition to Cdc42, we examined whether Cdc24 was required for embedded filamentous growth. Because Cdc42 and Cdc24 are essential for viability, we used strains (cdc42/PMETCDC42 and cdc24/PMETCDC24) with reduced levels of these proteins, which are defective in serum-dependent invasive filamentous growth (Bassilana et al., 2003; Bassilana et al., 2005). Figure 2 shows that although the rac1/rac1 strain formed nonfilamentous smooth colonies when embedded in YEPS, the cdc24/PMETCDC24 strain formed filamentous colonies, similar to that of the cdc42/PMETCDC42 strain (Bassilana and Arkowitz, 2006). It should be noted that colonies of both cdc24 and cdc42 mutant strains are morphologically different from the wild-type strain; they seem to have an increased number of shorter filaments that result in a fur ball-like morphology. Although we cannot rule out the possibility that the reduced level of Cdc24 expression in the cdc24/PMETCDC24 strain (Bassilana et al., 2005) is sufficient for embedded filamentous growth, because the cdc24 mutant is filamentous in this media similar to the cdc42 mutant, we consider it unlikely that Cdc24 activates Rac1 in this process.
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). Given that members of the CDM family can activate Rac1 in vitro, we used the BLAST algorithm to search the C. albicans genome for proteins with such a domain and found two ORFs (19.815 and 19.816), which will be hereafter referred to as DCK1 and DCK2, respectively. These two ORFs, located on chromosome 2 <1 kb apart, are 5745 base pairs and 5307 base pairs, respectively. Dck1 and Dck2 are 1915 and 1769 aa, respectively, with a region of 452 and 422 aa with similarity to the DOCKER domains (DHR) of HsDock180, CeCed5, DmMyoblast city, and ScYlr422w (Figure 3, A and B). The DHR of Dck1 and Dck2 are similar (26 and 23% identity, respectively) with that of HsDock180. C. albicans Dck1 and Dck2 share 35% overall sequence identity, with 42% identity between their DHRs. HsDock180, CeCed5, DmMyoblast City, have in addition to the DOCKER domain a second homology region, DHR1 and an amino-terminal Src homology 3 (SH3) domain, which are not apparent in Dck1 or Dck2. Dck1 homologues are found in all other fungal species; however, only in Candida species closest to C. albicans, i.e., C. dubliniensis and C. tropicalis is a second Dck protein found, suggesting that DCK2 was generated by a gene duplication (Fitzpatrick and Butler, personal communication).
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. We generated strains in which the Tn7-UAU1 cassette (dck1::Tn7-UAU1 and dck2::Tn7-UAU1) was inserted into both copies of either DCK1 or DCK2, respectively, suggesting that neither of these genes is required for viability. Quantitative real-time RT-PCR confirmed that neither DCK1 nor DCK2 mRNA transcripts were detectable in such dck1 and dck2 mutants, in contrast to the wild-type strain, in which both DCK1 and DCK2 transcripts were present (0.2 ± 0.07 for DCK1 and 0.05 ± 0.02 for DCK2, relative to actin). Figure 4A shows that colonies of the dck2 mutant are filamentous in agar, similar to wild-type colonies. Strikingly, in the same conditions, the dck1 mutant was unable to filament, instead forming smooth colonies, identical to that of the rac1 deletion mutant. These results suggest that DCK1, but not DCK2, is important for matrix-embedded-dependent filamentous growth.
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/dck1 strain was viable and its doubling time in rich media comparable with that of the wild-type strain. DCK1 mRNA transcripts were not detectable in this deletion strain by qRT-PCR. However, neither deletion nor overexpression of DCK1 affected Rac1 protein levels (Supplemental Figure S1). Figure 4B (top) shows that colonies of the dck1/dck1 strain are nonfilamentous when grown in YEPS compared with wild-type colonies. Reintroduction of a copy of DCK1 behind its own promoter, at the RP10 locus restored filamentous growth in these conditions, demonstrating that the dck1/dck1 filamentous growth defect is due to deletion of the DCK1 gene. In contrast, dck1/dck1 colonies are filamentous when grown on serum containing media (Figure 4B, bottom), indicating that Dck1, like Rac1, is specifically required for matrix-induced filamentous growth.
Given that the transcription factor Czf1 is also specifically required for matrix-induced filamentous growth (Brown et al., 1999), we investigated whether overexpression of CZF1 could suppress the defect of the rac1 and dck1 deletion strains. We generated strains in which CZF1 was overexpressed via either the MAL2 or ADH1 promoters, resulting in an increase in CZF1 transcript levels by 9- and 30-fold (compared with the wild-type level), respectively. Such CZF1 overexpression did not suppress the embedded filamentous growth defect of either the rac1 deletion strain (Figure 5A) or the dck1 deletion strain (data not shown), suggesting that this transcription factor does not function downstream of Rac1 and Dck1. To investigate whether CZF1 could function upstream of Rac1, we generated a czf1 deletion mutant, isogenic to the rac1 and dck1 deletion mutants. This czf1 deletion strain was nonfilamentous in embedded media, similar to the czf1 deletion strain generated by Brown et al. (1999), and reintroduction of CZF1 under the control of the ADH1 promoter (Figure 5B) complemented this filamentous growth defect. We examined whether overexpression of a constitutively active Rac1 mutant could restore such filamentous growth in this czf1 mutant (because overexpression of this mutant restores embedded filamentous growth in a dck1 mutant; Figure 10). Overexpression of constitutively active Rac1, rac1[Q61L] (Figure 5B), did not restore filamentous growth in the czf1 deletion mutant. Overexpression of rac1[Q61L] was confirmed both at the mRNA transcript level by qRT-PCR (data not shown) and at the protein level (Figure 5C). Altogether, these results suggest that Rac1 and Dck1 function in a novel signaling pathway, different from that in which Czf1 functions, necessary for matrix-induced filamentous growth.
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/dck1 and rac1/rac1 strains were filamentous, although the length of the filaments was shorter than that of the wild-type strain (Supplemental Figure S2A). At earlier times, the dck1/dck1 and rac1/rac1 strains were less crenelated than the corresponding wild-type strain. When grown in Spider liquid media dck1/dck1 and rac1/rac1 cells formed hyphae similar to wild-type cells, albeit somewhat reduced in length (Supplemental Figure S2B). On GlcNAc containing solid media both dck1/dck1 and rac1/rac1 strains had a strong filamentous growth defect compared with the wild-type strain (Supplemental Figure S3A). Strikingly, in liquid media containing GlcNAc both deletion strains were filamentous similar to the wild-type strain (Supplemental Figure S3B). Together, these data indicate that Dck1 and Rac1 are important for invasive filamentous growth in solid media.
The DOCKER Domain Is Required for Dck1 Function
The DOCKER domain is required for HsDock180 function (Brugnera et al., 2002; Cote and Vuori, 2002). To determine the importance of the homologous DOCKER domain in Dck1, we generated a strain that overexpressed, as a sole DCK1 copy, a version in which the region encoding amino acid residues 1332-1783 (homologous to HsDock180 aa 1152-1607, termed DHR) was removed. This strain overexpressed dck1DHR mRNA 90-fold, similar to the overexpression of DCK1 mRNA, as determined by qRT-PCR. Figure 6A shows that the dck1DHR overexpression strain is unable to form filamentous colonies when embedded in agar, in contrast to the control strain. To verify that the Dck1DHR protein is expressed, we constructed GFP fusions and generated strains containing GFPDck1DHR or GFPDck1 as the sole Dck1 copy. In addition, we confirmed that the expression of these fusion proteins via the ADH1 promoter resulted in a substantial increase in protein level compared with expression via its endogenous promoter (Figure 6B). The immunoblot illustrated in Figure 6C shows that GFPDck1 and GFPDck1DHR migrated consistent with their expected molecular weights (approximately at 250 and 220 kDa, respectively). We did not observe substantial degradation of either fusion. Furthermore, we confirmed that the GFPDck1 fusion, but not GFPDck1DHR, was functional as overexpression of GFPDck1, but not GFPDck1DHR, complemented the filamentous growth defect of the dck1 deletion strain (Figure 6A). These results suggest that the catalytic DOCKER domain is required for Dck1 function in vivo.
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/dck1 strain does not restore filamentous growth in embedded media, whereas overexpression of Dck1 complements the filamentous growth defect of this mutant (Figure 6A). Conversely, we examined whether Dck1 overexpression affected the matrix-dependent filamentous growth of the cdc24/PMETCDC24 strain, and no difference in colony morphology was observed (Figure 7A). We also determined whether Dck1 overexpression could restore serum-dependent filamentous growth of cdc24/PMETCDC24 cells and Figure 7B shows that this strain is still defective. Together, our results indicate that Dck1 and Cdc24 function in response to different inducers of filamentous growth and likely activate the distinct Rho G proteins Rac1 and Cdc42, respectively.
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/rac1 strain was unable to restore filamentous growth, in contrast to Rac1 overexpression. Quantitative RT-PCR revealed that Dck1 was overexpressed 70-fold. Dck1 overexpression complemented the filamentous growth defect of a dck1/dck1 strain, whereas overexpression of Rac1 did not (Figure 10B). Strikingly, overexpression of an activated form of Rac1, Rac1[Q61L], partially restored embedded filamentous growth in the dck1 deletion strain (Figure 10B). These results contrast those observed for rac1 deletion strain (Figure 1C) in which overexpression of activated form of Rac1 results in less extensive filamentous growth compared with Rac1. In contrast, overexpression of the nucleotide-free form of Rac1 ([T17N]) did not restore embedded filamentous growth in all strains examined (Figures 1, A and C, and 10B). Quantification indicated that 80% of the colonies of the dck1 deletion strain overexpressing Rac1[Q61L] were filamentous compared with 10% filamentous colonies in the same strain overexpressing wild-type Rac1 or Rac1[T17N] (Figure 10C). The different Rac1 forms were overexpressed 30- to 50-fold in this dck1 deletion mutant as determined by qRT-PCR; furthermore, Figure 10D shows overexpression of these different forms of Rac1 protein. These results, together with the similar phenotypes of rac1 and dck1 mutants in response to different inducers, indicate that Dck1 activates Rac1 in vivo during invasive filamentous growth.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The Dock180 related GEF subfamily, which is currently made up of 11 mammalian members, has been shown to specifically activate Rac1 in vitro (Cote and Vuori, 2007). Very recent studies suggest that Dock180 is also important for Cdc42 activation in neurons in vivo (Li et al., 2008). In C. albicans, Dck1 does not complement Cdc24 function, suggesting that Dck1 does not activate Cdc42. Proteins with similarity to CDM GEFs are present in a range of fungal species, including Neurospora crassa, Cryptococcus neoformans, Aspergillus nidulans, Magnaporthe grisea, Ustilago maydis, Candida albicans, Ashbya gossypii, and S. cerevisiae (Meller et al., 2005). Interestingly, the latter two fungal species do not possess a discernible Rac1 homologue. In each of these fungal species, there is one protein with similarity to CDM GEFs, except for C. albicans, where two such proteins, Dck1 and Dck2, exist. In a large-scale haploinsufficiency-based transposon mutagenesis for defects in filamentous growth, Uhl et al. (2003) identified insertion mutations in the ORFs corresponding to Dck1 and Dck2 as having reduced filamentous growth on Spider medium. However, we did not detect any defect in dck2 mutants. In contrast to other CDM GEFs, no apparent SH3 domain or conserved DHR1 region is present in Dck1. Nonetheless, we observed weak homology of the amino-terminal half of C. albicans Dck1 to the DHR1 regions of mammalian Dock proteins. DHR1 has been shown to have some similarity to lipid binding modules and in particular binds phosphatidylinositol 3,4,5-triphosphate (Cote et al., 2005). Whether C. albicans Dck1 binds phospholipids and this region plays a role in lipid binding remains to be elucidated. The SH3 domain is critical for Dock180 function in flies and humans (Brugnera et al., 2002; Balagopalan et al., 2006). For example, it has been reported that this domain autoinhibits GEF activity by binding the DOCKER catalytic domain and that it is involved in interactions with ELMO (Lu et al., 2005). ELMO is critical for efficient Rac1 signaling by Dock180 via alteration of the affinity of Dock180 for Rac1 and targeting Dock180 to the plasma membrane (Lu et al., 2004; Park et al., 2007). A protein with similarity to ELMO is present in the C. albicans genome. It remains to be determined whether this protein functions together with Dck1 and is required for Dck1 membrane association.
Our results indicate that Cdc42 and its activator Cdc24 are required for filamentous growth in response to serum, whereas Rac1 and its activator Dck1 are required for invasive filamentous growth in solid media. The specificity of these two highly similar G proteins to distinct filamentous growth stimuli could be due to regulation of GEFs, regulation of G protein expression, and/or association of each activated G protein with specific effectors. Even when overexpressed, Cdc24 is unable to replace Dck1 in Rac1 activation and conversely Dck1 is unable to replace Cdc24 in Cdc42 activation. The amino acid at position 56 in Rac1 has been shown to be required for interaction with Dbl family exchange factors (Gao et al., 2001; Karnoub et al., 2001; Hlubek et al., 2008), and mutation of this residue does not substantially impair binding to HsDock180 (Brugnera et al., 2002). We mutated C. albicans Rac1 Trp 56 to Phe, which is present in Cdc42, and this mutant when overexpressed was able to partially complement a rac1 deletion strain for embedded filamentous growth (Arkowitz and Bassilana, unpublished data). In addition, overexpression of the Rac1[W56F] mutant, which has been shown to be activated by other DH family GEFs such as Cdc24, as has been recently shown in U. maydis (Hlubek et al., 2008), did not restore embedded filamentous growth in a dck1/dck1 strain, further consistent with the specific requirement of Dck1 for Rac1 activation during embedded filamentous growth. These results suggest that specificity in part is conferred by G protein–GEF interactions. Although Cdc42 and Rac1 are very similar at the amino acid level, the latter G protein has a 43-amino acid insertion before the carboxy terminus, which may be critical for specific interactions with the Dck1 GEF. Our Cdc42/Rac interactive binding (CRIB) binding experiments indicate that both activated Cdc42 and Rac1 can bind CRIB domain containing effectors (Bassilana and Arkowitz, 2006), consistent with the notion that signaling specificity is achieved by upstream components. The components upstream of Dck1 and Rac1 are unknown; however, recent studies in mammalian systems have identified two membrane proteins that can recruit Dock180, the adhesion-type G protein-coupled receptor brain-specific angiogenesis inhibitor 1 (BAI1), which recruits Dock180 via its association with ELMO (Park et al., 2007) and the transmembrane netrin receptor, deleted in colorectal carcinoma, which binds Dock180 directly (Li et al., 2008). We speculate that Dck1 in C. albicans is recruited to the plasma membrane by a presently unidentified membrane protein, which would be necessary for localized Rac1 activation.
Our results indicate that a GTP bound form of Rac1 partially recovers invasive filamentous growth in both rac1 and dck1 mutants; however, the extent of filamentation in these strains is reduced compared with the wild-type strain. Therefore, it is likely that Rac1 must cycle between GDP-bound and GTP-bound states to fully restore filamentous growth. Ectopic expression of the GTP bound form of Cdc42 was highly detrimental to C. albicans cells, suggesting that cycling of this G protein between GDP and GTP states is also critical (Ushinsky et al., 2002). In addition to Cdc24, the Cdc42 activator, GTPase-activating proteins are also important in filamentous growth (Court and Sudbery, 2007; Zheng et al., 2007). It is unknown whether such Cdc42 GAPs can also stimulate Rac1 GTP hydrolysis or whether specific Rac1 GAPs are required.
We propose that specific stimuli, such as contact with solid agar or presence of serum, result in recruitment and/or activation of Rac1 or Cdc42 GEFs, respectively. It is likely that locally activated Rac1 or Cdc42 recruits effector proteins that are critical for morphological changes. Understanding what determines the specificity for small G protein and GEF interactions will be critical for revealing how these morphological changes occur in response to distinct stimuli. Given that these two classes of exchange factors and the G proteins they activate are conserved from humans to fungi, the specificity determinants are likely to be similar in these organisms.
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ACKNOWLEDGMENTS |
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Footnotes |
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Address correspondence to: Martine Bassilana (mbassila{at}unice.fr)
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4 sequences, respectively. The black line above the sequence represents the DOCKER domain (Dedicator of cytokinesis; pfam06920, 
