Yeast Yeast 2007; 24: 511–522. Published online 13 April 2007 in Wiley InterScience (www.interscience.wiley.com) DOI: 10.1002/yea.1489 Yeast Functional Analysis Report Functional analysis of Candida albicans genes whose Saccharomyces cerevisiae homologues are involved in endocytosis Ronny Martin 1 , Daniela Hellwig 1# , Yvonne Schaub 1# , Janine Bauer 1,2 , Andrea Walther 2 and J¨ urgen Wendland 1,2 * 1 Department of Microbiology, Friedrich-Schiller-University, Jena and Junior Research Group: Fungal Pathogens, Leibniz Institute for Natural Product Research and Infection Biology, Hans-Kn¨ oll Institute, Jena, Germany 2 Carlsberg Laboratory, Yeast Biology, Gamle Carlsberg Vej 10, Valby, Denmark *Correspondence to: J¨ urgen Wendland, Carlsberg Laboratory, Yeast Biology, Gamle Carlsberg Vej 10, DK-2000 Valby, Copenhagen, Denmark. E-mail: [email protected]# Current address: D. Hellwig/Y. Schaub, Fritz-Lipman Institute, Leibniz Institute for Age Research, Beutenbergstrasse 11, D-07745 Jena, Germany. Received: 4 August 2006 Accepted: 26 February 2007 Abstract PCR-based techniques for directed gene alterations have become standard tools in Candida albicans. To help to increase the speed of functional analysis of Candida albicans genes, we previously constructed and updated a modular set of pFA-plasmid vectors for PCR-based gene targeting in C. albicans. Here we report the functional analyses of C. albicans ORFs whose homologues in S. cerevisiae are involved in endocytosis, to explore their potential involvement in polarized cell growth. Three C. albicans genes, ABP1, BZZ1 and EDE1, were found to be non-essential. Yeast and hyphal morphogenesis were not affected by the individual deletions and the mutant strains appeared wild-type-like under the different growth conditions tested. On the other hand, deletion of both alleles of the C. albicans PAN1 homologue was not feasible. Promoter shut-down experiments using a MET3p–PAN1/pan1 strain indicated severe growth defects and abolished endocytosis, indicating that PAN1 is an essential gene. Subcellular distribution of CaAbp1 and CaPan1 was analysed via GFP-tagged proteins. Both proteins were found to localize at the cortex and at hyphal tips in a patch-like manner, supporting their role in endocytosis. Localization patterns of Abp1 and Pan1, however, were distinct from that of the FM4-64 stained Spitzenk¨ orper. Copyright 2007 John Wiley & Sons, Ltd. Keywords: Candida albicans ; human pathogen; polymerase chain reaction; GFP; pFA-plasmids; actin cytoskeleton Introduction Candida albicans is one of the most important human fungal pathogens, which may occur as a commensal, e.g. in the gastrointestinal tract, but can also cause surface infections as well as life- threatening systemic infections in immunocompro- mised patients (Sudbery et al., 2004). The ability to cause disease is promoted by a large set of viru- lence factors that aid in adhesion, colonization and penetration of host tissues, as well as by cell shape changes that induce morphogenetic transitions from yeast to hyphal phases (Sundstrom, 2002; Sudbery et al., 2004; Whiteway and Oberholzer, 2004; Kumamoto and Vinces, 2005; Schaller et al., 2005). The ability to form filaments is regarded as a crucial virulence factor, since afilamentous mutants were shown to be avirulent (Lo et al., 1997). Recent analysis of the C. albicans Wiskott–Aldrich Syndrome Protein (WASP), WAL1, indicated that Wal1 is also required for polarized hyphal growth since wal1 mutants showed delayed endocyto- sis, vacuolar fragmentation, and were defective in forming hyphal filaments (Walther and Wendland, 2004). Copyright 2007 John Wiley & Sons, Ltd.
Transcript
YeastYeast 2007; 24: 511–522.Published online 13 April 2007 in Wiley InterScience(www.interscience.wiley.com) DOI: 10.1002/yea.1489
Yeast Functional Analysis Report
Functional analysis of Candida albicans geneswhose Saccharomyces cerevisiae homologuesare involved in endocytosis
Ronny Martin1, Daniela Hellwig1#, Yvonne Schaub1#, Janine Bauer1,2, Andrea Walther2
and Jurgen Wendland1,2*1Department of Microbiology, Friedrich-Schiller-University, Jena and Junior Research Group: Fungal Pathogens, Leibniz Institute for NaturalProduct Research and Infection Biology, Hans-Knoll Institute, Jena, Germany2Carlsberg Laboratory, Yeast Biology, Gamle Carlsberg Vej 10, Valby, Denmark
#Current address: D. Hellwig/Y.Schaub, Fritz-Lipman Institute,Leibniz Institute for AgeResearch, Beutenbergstrasse 11,D-07745 Jena, Germany.
Received: 4 August 2006Accepted: 26 February 2007
AbstractPCR-based techniques for directed gene alterations have become standard tools inCandida albicans. To help to increase the speed of functional analysis of Candidaalbicans genes, we previously constructed and updated a modular set of pFA-plasmidvectors for PCR-based gene targeting in C. albicans. Here we report the functionalanalyses of C. albicans ORFs whose homologues in S. cerevisiae are involved inendocytosis, to explore their potential involvement in polarized cell growth. ThreeC. albicans genes, ABP1, BZZ1 and EDE1, were found to be non-essential. Yeastand hyphal morphogenesis were not affected by the individual deletions and themutant strains appeared wild-type-like under the different growth conditions tested.On the other hand, deletion of both alleles of the C. albicans PAN1 homologue wasnot feasible. Promoter shut-down experiments using a MET3p–PAN1/pan1 strainindicated severe growth defects and abolished endocytosis, indicating that PAN1 isan essential gene. Subcellular distribution of CaAbp1 and CaPan1 was analysedvia GFP-tagged proteins. Both proteins were found to localize at the cortex and athyphal tips in a patch-like manner, supporting their role in endocytosis. Localizationpatterns of Abp1 and Pan1, however, were distinct from that of the FM4-64 stainedSpitzenkorper. Copyright 2007 John Wiley & Sons, Ltd.
IntroductionCandida albicans is one of the most important
human fungal pathogens, which may occur as acommensal, e.g. in the gastrointestinal tract, butcan also cause surface infections as well as life-threatening systemic infections in immunocompro-mised patients (Sudbery et al., 2004). The abilityto cause disease is promoted by a large set of viru-lence factors that aid in adhesion, colonization andpenetration of host tissues, as well as by cell shapechanges that induce morphogenetic transitionsfrom yeast to hyphal phases (Sundstrom, 2002;
Sudbery et al., 2004; Whiteway and Oberholzer,2004; Kumamoto and Vinces, 2005; Schaller et al.,2005). The ability to form filaments is regarded as acrucial virulence factor, since afilamentous mutantswere shown to be avirulent (Lo et al., 1997).Recent analysis of the C. albicans Wiskott–AldrichSyndrome Protein (WASP), WAL1, indicated thatWal1 is also required for polarized hyphal growthsince wal1 mutants showed delayed endocyto-sis, vacuolar fragmentation, and were defective informing hyphal filaments (Walther and Wendland,2004).
Due to its medical importance, C. albicans hasbecome a prime research subject in fungal biol-ogy, which recently resulted in the determinationof its genome sequence (Odds et al., 2004; Braunet al., 2005). Molecular analyses in C. albicans arecomplicated due to its diploidy, the absence ofclassical genetics, and the difficulties in devisingforward genetic screens. Analysis of gene functionthus requires the sequential deletion of both alle-les of a target gene. For consecutive or multiplerounds of gene disruptions ‘URA3-blaster’ or FLP-mediated site-specific recombination strategies andtheir variations are commonly used (Morschhauseret al., 1999; Enloe et al., 2000; Berman and Sud-bery, 2002; Reuss et al., 2004). In conjunctionwith the C. albicans genome project (http://www-sequence.stanford.edu/group/candida/), manynew genes have been discovered that await char-acterization. To increase the speed of functionalanalyses of C. albicans genes, PCR-based methodsof gene disruption were introduced in C. albicans.Using a modular set of pFA-vectors, a limited setof primers allows gene disruptions, promoter swapand GFP-tagging experiments in a single strainbackground without the need of marker recycling(Wilson et al., 1999; Gola et al., 2003; Noble andJohnson, 2005; Schaub et al., 2006).
This methodology was recently employed by ourgroup to analyse the role of C. albicans formingenes in polarized cell growth (Martin et al., 2005).Wal1 proved to be important for polarized morpho-genesis. We went on to analyse C. albicans genesthat are involved with distinct functions duringendocytosis. Endocytosis is a process that generatesintracellular vesicles by plasma membrane invagi-nation and internalization (Wendland and Walther,2005; Moseley and Goode, 2006; Toret and Dru-bin, 2006). In C. albicans, ABP1, BZZ1, EDE1 andPAN1 homologues can be found. ScPAN1 is anessential gene. Pan1 forms a complex with Sla1and End3 and associates with cortical actin patches(Tang and Cai, 1996; Wendland et al., 1996). Par-tial patch co-localization was also observed withthe S. cerevisiae Abp1, Ede1 and Lsb7/Bzz1 pro-teins (Gagny et al., 2000; Soulard et al., 2002;Quintero-Monzon et al., 2005). ScBzz1 was alsofound to directly interact with the S. cerevisiaeWASP homologue Las17 (Soulard et al., 2002).S. cerevisiae Las17 activates the Arp2/3 complexand is thus involved in the organization of theactin cytoskeleton at sites of endocytosis. Other
Arp2/3 activators include Abp1 and Pan1 (Dun-can et al., 2001; Goode et al., 2001). To studythe role and potential involvement of the respec-tive C. albicans homologues in polarized mor-phogenesis, we generated mutant strains bearingdeletions in these genes. The Candida mutantswere subjected to a basic set of phenotypic anal-yses to determine the influence of the targetgenes on C. albicans morphology and growth pat-tern.
Material and methods
Strains and media
C. albicans strains used and generated in this studyare detailed in Table 1. Strains were grown eitherin YPD (1% yeast extract, 2% peptone, 2% dex-trose) or in minimal media complete supplementmixture [CSM; 6.7 g/l yeast nitrogen base (YNB)with ammonium sulphate and without amino acids,0.69 g/l CSM; 20 g/l glucose] or SD (6.7 g/l YNBwith ammonium sulphate and without amino acids,20 g/l glucose) supplemented with the requiredamino acids and uridine. The shut-down of MET3promoter-controlled gene expression was done asfollows: cells were grown overnight in minimalmedium and then diluted in fresh minimal mediumwith or without supplementation of 2.5 mM methio-nine and cysteine. To analyse the hyphal stagesafter a shut-down, cells were incubated for 4 hunder non-permissive conditions. Subsequently,10% serum was added to these cultures, followedby an incubation of 3 h at 37 ◦C. Yeast cells weregenerally grown at 30 ◦C; hyphal induction ofC. albicans cells was done at 37 ◦C in the presenceof 10% serum in the growth medium. Escherichiacoli strain DH5α was used for plasmid propaga-tion.
Transformation of C. albicans
All PCR-products used in transformation ofC. albicans were amplified from pFA-cassettesusing S1- and S2-primers, as described previously(Gola et al., 2003; Schaub et al., 2006). All primerswere obtained from biomers.net GmbH (Ulm, Ger-many). These S1- and S2-primers contain 100 ntof target homology region at their 5′-ends. Primersequences are shown in Table 2. Transformationwas done either by the lithium acetate procedure
SC5314 C. albicans wild-type Gillum et al., 1984BWP17 ura3::λimm34/ura3::imm34, his1::hisG/his1::hisG, arg4::hisG/arg4::hisG Wilson et al., 1999SN148 arg4/arg4, leu2/leu2, his1/his1 Noble and Johnson, 2005
CJW2 EDE1/ede1::CaHIS1, arg4, ura3, This studyCJW3 EDE1/ede1::CaURA3, arg4, his1, This studyCJW4 ABP1/abp1::CaURA3, arg4, his1, This studyCJW6 ede1::CaHIS1/ede1::CaURA3, arg4, This studyCJW7 ede1::CaURA3/ede1::CaHIS1, arg4, This studyCJW9 abp1::CaURA3/ABP1–GFP::CaHIS1, arg4, This studyGC57 PAN1/pan1::CaURA3, arg4, his1, This studyGC58 pan1::CaURA3/PAN1–GFP::CaHIS1, arg4, This studyGC59 BNI1/BNI1-GFP::CaURA3, his1, arg4 This studyGC129a PAN1/CmLEU2-MET3p-PAN1, arg4, his1, ura3 This studyGC132a CmLEU2-MET3p-PAN1/pan1::CaURA3, arg4, his1 This studyCAJ01 ABP1/abp1::CaHIS1, arg4, ura3, This studyCAJ03 ABP1/abp1::CaURA3, arg4, his1, This studyCAJ05 abp1::CaHIS1/abp1::CaARG4, ura3, This studyCAJ06 abp1::CaURA3/abp1::CaARG4, his1, This studyCAB1 BZZ1/bzz1::CaURA3, arg4, his1, This studyCAB2 BZZ1/bzz1::CaARG4, his1, ura3, This studyCAB4 bzz1::CaARG4/bzz1::CaHIS1, ura3, This studyCAB5 bzz1::CaURA3/bzz1::CaHIS1, arg4, This study
a All strains are derivatives of BWP17, except the strains indicated, which were made in a SN148 background.
or by electroporation (Kohler et al., 1997; Waltherand Wendland, 2003). Varying incubation periods(3–5 days) were used to identify the transformants.For each target gene at least two completely inde-pendent homozygous mutants were generated. Ver-ification of correct integration of the pFA cassettesand the absence of the target ORF in homozygousmutants was done by PCR, as described in detailin Schaub et al. (2006).
Microscopy and staining procedures
Microscopic analyses were performed eitheron an AxioplanII-Imaging or an Axio-Imager
microscope (Zeiss, Jena and Gottingen, Germany)with the aid of Metamorph software tools(Molecular Devices Corp., Downington, PA, USA)and a MicroMax1024 CCD-camera (PrincetonInstruments, Trenton, NJ, USA). Wide-fieldepifluorescence microscopy was performed, usingthe appropriate filter combinations for GFPimaging, chitin staining via Calcofluor white (bydirectly adding 1 µl of a 1 mg/ml stock to a 100 µlcell suspension), and staining of vacuoles or theSpitzenkorper (Spitzenkorper is an apical cluster ofvesicles) using FM4-64 (0.2 µg/ml). Spitzenkorperstaining requires fast sample processing, whereasimaging endocytosis and accumulation of FM4-64
a Abbreviations indicate the origin: C. albicans (Ca) and C. maltosa (Cm).b In long primers, upper case sequences correspond to DNA sequences used as homology regions for recombination, whereas lowercase sequences correspond to 3′-terminal annealing regions for the amplification of transformation cassettes. Short primers were used forverification purposes. All sequences are written from 5′ to 3′ .
a The following domains have been identified in the proteinsequences using searches against the Conserved Domain Databaseat http://www.ncbi.nlm.nih.gov/BLASTADF, actin depolymerisation factor/cofilin-like domain involved inactin binding; SH3, Src-homology 3 domain involved in binding toproline-rich domains; FCH, Fes/CIP4 homology domain implicatedin actin binding; EH, Eps15 homology domain found in proteinsinvolved in endocytosis; coiled-coil domains facilitate protein–proteininteractions; UBA, ubiquitin-associated domains may play a regulatoryrole.
in the vacuoles was done after incubation of cellswith the dye for 1 h. Samples were analysedby generating either single planes or stacks of5–20 images that were processed into single-planeprojections using Metamorph software.
Results and discussion
Sequence comparison
Sequence comparisons showed that the C. albi-cans proteins share approximately 30% iden-tity on the amino acid level with their yeast
Figure 1. Hyphal induction of mutant strains in liquid media.The indicated strains were grown overnight in minimalmedium. Cells were then diluted into fresh media inducinghyphal growth at 37 ◦C. After 4 h incubation the cells wereused for microscopy. Bar = 10 µm
homologues. The proteins are of similar size,with greater sequence variability at the N- andC-termini. Using a domain-searching tool (e.g.
at http://www.expasy.org/tools/), protein domainscould be assigned in these proteins. This indicatedthat the overall organization in homologous pro-teins is highly similar in Bzz1, Ede1 and Pan1homologues. One notable difference is the presenceof two Src homology (SH3)-domains in the C. albi-cans Abp1 protein, whereas only one is found inthe S. cerevisiae Abp1 (Table 3).
Generation of C. albicans mutant strains
PCR-based gene targeting in C. albicans hasbecome a standard tool and the number of genesanalysed in this way, as well as the number offunctional analysis cassettes, is steadily increas-ing (Gerami-Nejad et al., 2001; Gola et al., 2003;Taneja et al., 2004; Schaub et al., 2006). In thisfunctional analysis report we have used two differ-ent approaches to gene function analysis, relyingon different pFA vectors. First, we generated het-erozygous and subsequently, if possible, homozy-gous mutant strains, using different marker genes.Second, for subcellular localization experiments,
we fused CaABP1 and CaPAN1 with the greenfluorescent protein (GFP). All pFA cassettes andtheir efficient usage have recently been describedin detail elsewhere (Schaub et al., 2006).
Analysis of growth morphology of mutantstrains
Previously we were able to show that deletionof the C. albicans WASP homologue not onlyresulted in defects in endocytosis but also abol-ished hyphal growth and mycelium formation(Walther and Wendland, 2004). Thus, we wantedto elucidate whether other genes involved inendocytosis yielded similar morphological defects.ScEde1/Bud15 is a multi-domain protein involvedin endocytosis, and diploid null mutants exhibitrandom budding (Gagny et al., 2000). ScBzz1/Lsb7is an SH3 domain-containing protein that interactswith the yeast WASP homologue Las17 (Soulardet al., 2002). Deletion of CaEDE1 or CaBZZ1 didnot reveal any defect during growth in the yeast orhyphal stages; the distribution of actin patches in
Figure 2. Hyphal growth of mutant strains on solid media. The indicated strains were grown overnight in minimal medium.From each culture, 5 µl were spotted onto different hypha-inducing solid media and grown for 4 days at 37 ◦C prior tophotography
yeast or hyphal cells was similar to that of the wild-type (our unpublished results). Deletion of EDE1impairs the diploid budding pattern in S. cerevisiaebut deletion of the CaEDE1 homologue resultedin only a marginal increase of random budding inC. albicans yeast cells grown at 30 ◦C (5.8%
random budding in Caede1 vs. 0.8% in the C. albi-cans wild-type; n = 500 for each strain).
To extend this analysis, we investigated polarizedhyphal morphogenesis of the mutant strains ondifferent media using either medium containingserum, Spider medium, Lee’s medium or medium
Figure 3. Shut-down of PAN1 expression. Strains were grown overnight in minimal medium at 30 ◦C and then diluted infresh minimal medium, with or without 2.5 mM methionine and cysteine. (A) Prior to microscopy the cultures were grownat 30 ◦C for 4 h. (B) After an incubation of 4 h at 30 ◦C to allow for the shut-down of MET3p–PAN1 expression, 10%serum was added to the cultures and then the cells were grown for an additional period of 3 h at 37 ◦C. Reduced PAN1expression resulted in aberrant and swollen cell morphology. Bars = 10 µm
containing N-acetyl glucosamine, both in liquidculture and on solid medium (Figures 1, 2). Allstrains showed similar abilities to form germ tubesand colonies of wrinkled appearance containinghyphae, suggesting that single mutations in thesegenes did not strongly alter growth behaviour inC. albicans.
Functional analysis of CaPAN1
Three non-myosin activators of the Arp2/3 com-plex and actin assembly are known in yeast (Dun-can et al., 2001; Goode et al., 2001; Winter et al.,1999). In addition to the previously analysed WASPhomologue, we now investigated the function ofthe two other activators in C. albicans encoded byCaABP1 and CaPAN1. Deletion of the C. albicans
homologue of ABP1 did not result in any detectablemorphological phenotype (see Figures 1, 2), whilea homozygous deletion strain of CaPAN1 couldnot be obtained, suggesting that CaPAN1 is anessential gene. Next we went on to study condi-tional PAN1 mutant strains, in which one allelewas deleted and the remaining allele was placedunder the control of the regulatable MET3 pro-moter. Using both yeast and hyphal growth con-ditions, it became apparent that upon shut-down ofMET3-driven expression the cells stopped polar-ized growth, enlarged, and showed a terminalswollen phenotype (Figure 3).
Endocytosis can be monitored in vivo by usingthe membrane selective dye FM4-64. In the wild-type the plasma membrane-bound dye is taken upand delivered to the vacuole, where it accumulates.
Figure 4. Shut-down of PAN1 leads to a block in endocytosis. Strains were grown overnight in minimal medium at 30 ◦Cand then diluted in fresh minimal medium, with or without 2.5 mM methionine and cysteine, followed by 4 h of growthat 30 ◦C. The cells were then incubated with FM4-64 for 1 h prior to fluorescence microscopy. Under these conditions,the wild-type showed uptake of FM4-64 under both regimens, while shut-down of PAN1 expression resulted in defectiveendocytosis and peripheral membrane staining. Bars = 10 µm
Under non-permissive conditions, the swollen cellsgenerated after shut-down of PAN1 expressionshowed a strong defect in the endocytosis of thelipophilic dye FM4-64, which was found to stainthe cellular membrane but was not internalized, incontrast to the rapid uptake found in wild-type cells(Figure 4).
Subcellular localization of CaAbp1 and CaPan1
To analyse the subcellular localization of Abp1and Pan1, we used heterozygous C. albicansABP1/abp1 and PAN1/pan1 strains and fused the
remaining wild-type alleles at their 3′-ends withGFP and monitored the GFP fluorescence of thetagged strains (Figure 5). Both Abp1–GFP andPan1–GFP showed patch-like cortical localizationat sites of polarized growth, e.g. at the hyphal tip,resembling staining of the cortical actin cytoskele-ton. Interestingly, the Abp1–GFP signal was con-centrated at the hyphal tip and also spots in sub-apical parts of hyphae could be detected. In con-trast, Pan1–GFP was found to cluster in brightspots at both the hyphal tips and at subapicalregions (Figure 5). Thus, both proteins localize in
Figure 5. Localization of Abp1–GFP and Pan1–GFP in C. albicans. GFP-tagged strains were grown under yeast- orhypha-inducing conditions. Differential interference contrast (DIC) and fluorescence images (GFP) of yeast cells or hyphalfilaments were acquired sequentially, using appropriate filter sets. Abp1 accumulates at the hyphal tip and is also foundalong the hyphae. Pan1 localization is similar but less concentrated at the hyphal apex. Bars = 10 µm
an overlapping but distinct manner that is also inpart different from the cortical actin cytoskeleton(our unpublished data).
A set of proteins termed the polarisome inS. cerevisiae localize to the tip of the emergingbud. Homologues of polarisome proteins in fila-mentous fungi, e.g. Bni1 and Spa2, were found tolocalize to the tip of C. albicans hyphae (Zhenget al., 2003; Crampin et al., 2005; Li et al., 2005;Martin et al., 2005). Bni1 also co-localizes withthe Spitzenkorper, a structure that can be visualizedby short pulses of FM4-64 staining. To determinethe localization of Abp1 or Pan1 with respect tothe Spitzenkorper, we co-stained the GFP-taggedstrains with FM4-64 (Figure 6). This indicated thatboth Abp1 and Pan1 show a localization that coin-cides with the Spitzenkorper but is not confinedto the Spitzenkorper. Although a clear accumula-tion of Abp1 and Pan1 at the hyphal tips couldbe observed only a minor fraction of the proteinsco-localized with the Spitzenkorper. This may beattributable to the function of the Spitzenkorper inexocytosis while the Abp1 and Pan1 homologuesin yeast are involved in endocytosis.
Conclusion
Using PCR-based methodologies we have gener-ated C. albicans mutant strains in C. albicans genesthat code for proteins involved in endocytosis andperformed initial phenotypic characterizations ofthe mutant strains. CaABP1, CaBZZ1 and CaEDE1were found to be non-essential genes. Single dele-tions in these genes seem not to drastically influ-ence either growth rate or morphogenesis.
On the other hand, our results on CaPAN1indicate that this is an essential gene. Interestingly,the GFP-localization experiments with Abp1 andPan1 showed distinct localization patterns of theseproteins that partially overlap with both the positionof the Spitzenkorper at hyphal tips and with corticalactin patches. Of the mutants generated in thisstudy, only shut-down of PAN1 expression in aconditional mutant revealed a strong defect inpolarized morphogenesis. This defect was muchmore severe than deletion of the C. albicans WAL1gene as was also found in S. cerevisiae. Thewal1 and pan1 mutants suggest that defects inendocytosis may feed back to exocytosis, and thatboth processes are required for directing the vesicle
Figure 6. Localization of Abp1 and Pan1 only partiallyoverlaps with the Spitzenkorper. Cells of the indicatedstrains were induced for filament formation by the additionof 10% serum to CSM medium, followed by incubation at37 ◦C for 4 h. The cells were then stained with FM4-64and immediately analysed by fluorescence microscopy.GFP images (green) and images of the FM4-64-stainedSpitzenkorper (red) were used in an overlay. Bni1 is presentat the hyphal tip, indicating the position of the Spitzenkorper(yellow). In contrast, Abp1 and Pan1 localize to both the tipand subapical parts of the hyphae. Bars = 10 µm
flow at sites of polarized growth. Analysis of thewal1 showed that it slows down endocytosis and
also blocks filamentation, while still allowing yeastcell growth. Analysis of the individual domains ofthis multi-domain protein may yield further insightinto the protein network required for polarizedmorphogenesis in C. albicans.
Large-scale gene deletion and gene functionanalyses as were done in S. cerevisiae are tech-nically feasible with other ascomycetous fungi.PCR-based approaches have been introduced in thefilamentous ascomycete A. gossypii, while othertools based on the use of strains with increasedhomologous recombination efficiencies are avail-able in Neurospora crassa and Aspergillus (Wend-land et al., 2000; Ninomiya et al., 2004; da SilvaFerreira et al., 2006; Takahashi et al., 2006). Dueto the diploidy of C. albicans, gene function anal-yses require more efforts to produce the correctdeletion strains. Furthermore, a variable degree ofectopic integration may yield mutant strains that donot carry the desired genetic alterations. With theuse of PCR-based gene targeting methods, largerscale approaches can also be undertaken in C. albi-cans (Noble and Johnson, 2005). This may help toset the path for establishing a comprehensive genedeletion mutant (or regulated expression mutant)collection similarly as has been established forS. cerevisiae (Winzeler et al., 1999).
Acknowledgements
We would like to thank A. D. Johnson and S. Noblefor generously providing strains used in this study. AnjaMuller provided excellent technical assistance throughoutthe project. This study was funded by the DeutscheForschungsgemeinschaft and by the Leibniz Institute forNatural Product Research and Infection Biology, Hans-Knoll Institute.
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