IntroductionThroughout the fungal kingdom, there exists a broad range ofcellular growth forms. Filamentous fungi form long, tube-likehyphae, which show extreme polarised growth from the tip. Incontrast, cells of unicellular budding yeast are ovoid. Inpseudohyphae, cellular compartments are more elongated andcells fail to separate after mitosis, forming branching chains oflong cells. However, pseudohyphal cellular compartments aredistinguished from those of hyphae by virtue of constrictionsat the septal junctions, compared to the uninterrupted, parallel-sided walls of true hyphae (Sudbery et al., 2004). Although themechanisms that drive polarised growth in both filamentousfungi and budding yeast have been studied extensively, it is notclear whether the mechanisms elaborated in yeast areapplicable to filamentous fungi and vice versa. The humanfungal pathogen C. albicans can grow in yeast, pseudohyphaland hyphal growth forms: a property that is thought to beimportant for its virulence. It thus provides the opportunity to

compare, in the same organism, the mechanisms of polarisedgrowth operating in these different growth forms.

S. cerevisiae has provided a model for polarised growth inbudding yeast and pseudohyphal cells (Nelson, 2003; Pruyneand Bretscher, 2000; Pruyne, 2002; Rua et al., 2001). In yeastcells, growth is confined to the daughter buds, which initiallygrow in a polarised fashion and then, in G2, switch to isotropicgrowth (Kron and Gow, 1995). In pseudohyphal cells,polarised growth persists, resulting in longer buds (Kron et al.,1994). Polarised growth depends on the actin cytoskeleton,which consists of cortical actin patches and actin cables(Pruyne and Bretscher, 2000). Actin cables are oriented towardthe site of polarised growth and are thought to form tracksalong which the class V myosin, Myo2, and its regulatory lightchain, Mlc1, transport secretory vesicles that contain the rawmaterials and enzymes for the synthesis of new cell walls andcell membranes in the growing bud (Johnston et al., 1991;Karpova et al., 2000; Schott et al., 1999). Cortical actin patches

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Fungi grow with a variety of morphologies: oval yeast cells,chains of elongated cells called pseudohyphae and long,narrow, tube-like filaments called hyphae. In filamentousfungi, hyphal growth is strongly polarised to the tip and ismediated by the Spitzenkörper, which acts as a supplycentre to concentrate the delivery of secretory vesicles tothe tip. In the budding yeast Saccharomyces cerevisiae,polarised growth is mediated by the polarisome, a surfacecap of proteins that nucleates the formation of actin cablesdelivering secretory vesicles to the growing tip. The humanfungal pathogen, Candida albicans, can grow in all threemorphological forms. Here we show the presence of aSpitzenkörper at the tip of C. albicans hyphae as a ball-likelocalisation of secretory vesicles, together with the forminBni1 and Mlc1, an ortholog of an S. cerevisiae myosinregulatory light chain. In contrast, in C. albicans yeast cells,pseudohyphae and hyphae Spa2 and Bud6, orthologs of S.cerevisiae polarisome components, as well as the mastermorphology regulator Cdc42, localise predominantly, but

not exclusively, to a surface cap resembling the polarisomeof S. cerevisiae yeast cells. A small amount of Cdc42 alsolocalises to the Spitzenkörper. Furthermore, we showdifferences in the genetic and cytoskeletal requirements,and cell cycle dynamics of polarity determinants in yeast,pseudohyphae and hyphae. These results, together with thecytological differences between the cell types, suggest thatthe Spitzenkörper and polarisome are distinct structures,that the polarisome and Spitzenkörper coexist in hyphae,and that polarised growth in hyphae is driven by afundamentally different mechanism to that in yeast andpseudohyphae.

Supplementary material available online athttp://jcs.biologists.org/cgi/content/full/118/13/2935/DC1

Key words: Hyphae, Actin cables, Microtubules, BUD6, MLC1,FM4-64, Pseudohyphae, SPA2

Summary

Candida albicans hyphae have a Spitzenkörper that isdistinct from the polarisome found in yeast andpseudohyphaeHelen Crampin1, Kenneth Finley2, Maryam Gerami-Nejad2, Helen Court1, Cheryl Gale2,3, Judith Berman2,4

and Peter Sudbery1,*1Department of Molecular Biology and Biotechnology, Sheffield University, Western Bank, Sheffield, S10 2TN, UK2University of Minnesota, Department of Genetics, Cell Biology and Development, 6-160 Jackson Hall, 321 Church Street SE, Minneapolis,MN 55455, USA3Department of Pediatrics, University of Minnesota, MMC 39, 420 Delaware Street SE, Minneapolis, MN 55455, USA4Department of Microbiology, University of Minnesota, 6-170 MCB Building, 420 Washington Avenue SE, Minneapolis, MN 55455, USA*Author for correspondence (e-mail: p.sudbery@shef.ac.uk)

Accepted 1 April 2005Journal of Cell Science 118, 2935-2947 Published by The Company of Biologists 2005doi:10.1242/jcs.02414

Research Article

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mediate endocytosis, which is also associated with polarisedgrowth. Polarisation of the actin cytoskeleton is ultimatelycontrolled by the Cdc42 GTPase, which localises to theincipient bud site and the bud tip.

Nucleation of actin cables is mediated by the polarisome, aprotein complex that forms a cap covering the growing bud tip(Lew and Reed, 1995; Sheu et al., 1998; Lew and Reed, 1995).Components of the polarisome include Bud6, Spa2 and theformin Bni1 (Evangelista, 1997; Sheu et al., 1998). After theswitch to isotropic growth, the polarisome disperses, laterrelocalising to the bud neck, to direct the secretory vesicles tothe site of cytokinesis, when a contractile actomyosin ringguides the formation of the primary septum (Lippincott and Li,1998). Like the polarisome proteins, Mlc1 localises to the budtip where it interacts with Myo2 (Boyne et al., 2000). Later,Mlc1 interacts with the class II myosin, Myo1, in thecytokinetic ring (Boyne et al., 2000; Shannon and Li, 2000).Thus, the behaviour of S. cerevisiae Mlc1 is similar to that ofthe polarisome proteins, in that it localises to the bud tip duringpolarised growth and to the contractile ring during cytokinesis.

Polarised growth in filamentous fungi is much more extremethan in budding yeast: on an open Petri dish, Neurosporacrassa hyphae extend at a rate of 38 µm/minute (Read andHickey, 2001). A special structure, called the Spitzenkörper(apical body), which is located at or just behind the hyphal tip,is responsible for this dramatic polarisation of growth(Girbardt, 1957; Harris et al., 2005). The Spitzenkörper wasoriginally recognised as a dark region in phase-contrastmicroscopy at the tip of actively growing hyphae (Girbardt,1957). Subsequently, freeze-substitution electron microscopyrevealed that it is rich in secretory vesicles (Grove and Bracker,1970; Howard, 1981). More recently, it was shown that theamphiphilic styryl dye, FM4-64, labels the Spitzenkörper innumerous fungi (Fischer-Parton et al., 2000). This dye isincorporated into the plasma membrane, internalised byendocytosis and distributed to internal membranes. It is widelyused as a marker of endocytosis and to visualise vacuolarmembranes. However, when added to the growth medium itaccumulates most rapidly at the Spitzenkörper, presumablythrough vesicle trafficking (Read and Hickey, 2001), so thatafter short exposures to FM4-64, the Spitzenkörper ispreferentially labelled.

The Spitzenkörper is thought to drive hyphal growth becausechanges in the direction of hyphal growth are anticipated bychanges in the position of the Spitzenkörper (Reynaga Pena etal., 1997; Lopez-Franco, 1996). The vesicle supply centre(VSC) model of polarised growth in filamentous fungi positsthat the Spitzenkörper is the repository for secretory vesiclesthat are transported along hyphae towards the tip (Bartnicki-Garcia et al., 1989). Vesicles radiate from the Spitzenkörperand travel to the cell surface, where they fuse with the plasmamembrane and release their cargo. Because of the proximity ofthe Spitzenkörper to the growing tip, a greater concentration ofvesicles per unit area arrives at the tip than in more distant partsof the hypha. Computer modelling shows that a Spitzenkörpergenerates a concentration gradient of vesicles in the form of ahyphoid curve at the cell surface, and this curve closelypredicts the actual shape of the hyphal tip in the 34 differentfungal species examined (Bartnicki-Garcia et al., 1995).Although the behaviour of the Spitzenkörper has been welldocumented, less is known about its molecular composition

and the mechanisms by which it drives polarised growth infilamentous fungi. In Aspergillus nidulans, the formin SepAlocalises to the hyphal tip and to sites of septation.Interestingly, SepA localises to a crescent at the tip surface andto a spot just behind the tip (Sharpless and Harris, 2002). Asthe SepA spot colocalises with FM4-64 (Harris et al., 2005), itis likely to be a component of the Spitzenkörper.

Studies of polarised growth in yeast and filamentous fungihave largely proceeded independently and the relationshipbetween these structures has not been widely addressed,although recently it was suggested that the polarisome complexis a sub-component of the Spitzenkörper (Harris et al., 2005).C. albicans provides the opportunity to use the same organismto compare the molecular mechanisms that drive polarisedgrowth in yeast, pseudohyphae and hyphae. We demonstratehere that C. albicans hyphae contain a Spitzenkörper-likestructure by localisation of Mlc1-YFP and FM4-64 to adiscrete spot at, or just behind, the growing tip. In contrast, wefind that polarisome components and YFP-CDC42 localisepredominantly to a surface crescent or cap. Furthermore, weidentify genetic and cytoskeletal requirements for eachstructure as well as organisational differences during the cellcycle that distinguish the Spitzenkörper from the polarisome.Taken together, our results demonstrate that these structures areindeed distinct and that different molecular mechanisms drivepolarised growth in hyphae as compared to yeast andpseudohyphae. Interestingly, we observed both Spitzenkörperand polarisome structures in hyphal tips, suggesting that bothof these mechanisms may be used, possibly at the same time,in C. albicans hyphae.

Materials and MethodsHeterozygous fusions to green fluorescent protein (GFP), yellowfluorescent protein (YFP) and cyan fluorescent protein (CFP), andgeneration of promoter fusions were constructed as describedpreviously (Gerami-Nejad et al., 2001; Gerami-Nejad et al., 2004).Candida albicans disruption strains were also constructed asdescribed previously (Wilson et al., 1999). Full details of the primersused are presented in supplementary material Table S1. The genotypesof the Candida albicans strains used are listed in Table 1.

Media and growth conditionsYEPD medium consists of 2% Difco peptone, 1% Difco yeast extract(both from BD Diagnostics, Sparks, MD), 2% glucose, and 20 µg ml–1

uridine. SDC medium consists of 0.67% yeast nitrogen base and 2%filter sterilised glucose added after autoclaving. When used, serumwas added after autoclaving.

For all but the time-lapse videos experiments, yeast, pseudohyphaeand hyphae were cultured as follows: cells were grown to saturationovernight in YEPD at 30°C to produce a culture of unbudded yeastcells. For yeast cells, cultures were reinoculated at 106 cells ml–1 intoYEPD, pH 6.0, and incubated at 30°C. For pseudohyphal cells,cultures were reinoculated at 106 cells ml–1 into YEPD, pH 6.0, andincubated at 36°C; in these conditions 10-20% of cells develop ashyphae (Sudbery, 2001). For hyphae, unbudded cells werereinoculated into YEPD, pH 6.0, plus 20% serum and cultured at37°C. For microscopy, cells were washed once in distilled waterbefore examination. Most images were of unfixed cells. However, fortime-course experiments, the sample was removed from the cultureand cells were fixed immediately in 2% formaldehyde for 10 minutes.The GFP-YFP signal was preserved in this procedure.

For time-lapse videos of live cells, 15 µl of the cell suspension was

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placed on a glass slide and the cells were allowed to settle for 10minutes before the liquid was carefully removed by aspiration. Twomicroliters of SDC containing 2% molten SeaPlaque ultra pure lowmelting point agarose (Cambrex, Wokingham, UK) were added tocells on the slide. For induction of hyphae, the SDC/low melting pointagarose mixture was supplemented with 20% serum. A cover slip wasimmediately placed over the cells and sealed with a 1:1:1 mixture ofVaseline®, lanolin, and paraffin wax.

Yeast, pseudohyphae and hyphae were incubated at the appropriatetemperature in an environmental chamber fitted to the microscope(Solent Scientific, Portsmouth, UK). Under these conditions, cellsgrew for several cell cycles, although the mean cell-cycle time wasextended to approximately 140 minutes compared with approximately100 minutes for pseudohyphal cells growing in liquid YEPD.

Expression of YFP-CDC42 from the PCK1 promoter was carriedout as follows. Cells from a freshly streaked plate were grown inYEPD-liquid culture overnight. They were reinoculated into YEPS(YEP plus 2% succinate) and grown to stationary phase at 30°C.These stationary phase cells were reinoculated to a density of 106

cells ml–1 in YEPS plus 5% serum and cultured at 37°C for 90minutes.

Staining with FM4-64, Calcofluor, filipin and DAPICells were stained with Calcofluor and DAPI as described previously(Sudbery, 2001). For filipin staining, cells were grown in YEPD underthe desired inducing conditions, a 1 ml sample of cells was taken and1 µl Filipin (8 µg µl–1 stock, Sigma) was added and incubated at roomtemperature for 20 minutes. The sample was then centrifuged brieflyand resuspended in 1�PBS. For FM4-64 staining, 5 µl of stocksolution (1.64 mM in DMSO) was added directly to 20 ml of cultureand the culture was then incubated for a further 10 minutes. Cells wereharvested by brief centrifugation and washed with 1�PBS.Microscopic imaging was performed immediately, as FM4-64staining of the Spitzenkörper dissipates quickly when hyphae are notactively growing.

MicroscopyFluorescence microscopy was performed with a Delta Vision Spectris4.0 microscope running Softworx™ version 3.2.2 (Applied PrecisionInstruments, Seattle). The standard DAPI filter set was used tovisualise DAPI, Calcofluor and Filipin fluorescence, the standardFITC filter set was used for GFP and YFP, the standard TRITC filterset was used for FM4-64, and a Chroma YFP/CFP filter set (ChromaTechnology Corp., Rockingham, part number 86002) was used for thesimultaneous visualisation of CFP and YFP. Preliminary experimentsconfirmed that there was no bleed-through of either fluorophore intothe inappropriate channel. Z-stack images were collected with stepsizes of 0.2 µm and deconvolved using Softworx™. Wherequantitation of fluorescence was carried out, the exposure time was

kept constant at one second. Except where stated otherwise, imagesare projections of the deconvolved Z-stacks. Where enlargements areshown, the images were depixellated using the interpolated zoomfacility. Images were exported as tif files and image size was adjustedto 300 dpi and cropped using Adobe Photoshop 5.5. Multipart figureswere assembled in Microsoft PowerPoint 9.0 and saved asencapsulated postscript (.eps) files. Where stated, Z-stacks of imagesgenerated with the Delta Vision microscope were further processedby Volocity software (Improvision, Warwick UK). All quantitativemeasurements and model building were carried out using deconvolvedimage files. Quantitation of fluorescence signals was carried out usingthe data inspector in the Softworx suite.

Computer modelling To generate models, we used the modelling module in Softworx™ orthe Volocity software suite (Improvision, Warwick, UK). TheSoftworx™ module operates by identifying polygons in each layerwhere the pixels record fluorescence from each fluorophore that isabove a user-prescribed threshold. It then synthesises a 3D modelfrom the polygons identified in each layer. We set the threshold sothat the polygons corresponded as closely as possible to that visiblein the image presented alongside each model. A limitation of theSoftworx module is that the models show the boundaries of allfluorescence above the threshold, so there is no way of representingvariations in the intensity. However, an advantage is that where onefluorophore is enclosed by a second fluorophore, as was the case withSpa2 and Mlc1 or FM4-64, the outer fluorophore can be representedby a wire frame to allow the boundary of the inner fluorophore to bevisualised. Volocity analyses the Z-stack to identify volumes of equalintensity, called voxels, from which it generates a 3D model. LikeSoftworx, the models represent intensity above a user-prescribedthreshold.

ResultsMlc1 localises to a Spitzenkörper-like structure inhyphaeMlc1-YFP localisation was examined in hyphae,pseudohyphae and yeast using a Delta Vision Spectris 4.0 3Drestoration microscope, which applies iterative deconvolutionto a Z-stack of epifluorescence images (Swedlow et al., 2002).In hyphal germ tubes, Mlc1 appeared as a bright spot offluorescence at the tip of 100% of germ tubes. This spotcolocalised with FM4-64, which has been shown to visualisethe Spitzenkörper at the hyphal tip of filamentous fungi(Fischer-Parton et al., 2000) (Fig. 1A). Close examination ofindividual germ tube tips revealed that, in some cases, theMlc1-YFP spot localised at the tip (Fig. 1B), whereas in others

Table 1. Strains used in this studyStrain Genotype Source or Reference

BWP17 ura3::λimm434/ura3::λimm434 his1::hisG/his1::hisG arg4::hisG/arg4::hisG Wilson et al., 1999YMG7139 BWP17 MLC1/MLC1-YFP:URA3 This studyYMG6709 BWP17 ABP1/ABP1-YFP:URA3 This studyYMG6748 BWP17 SPA2/SPA2-YFP:URA3 This studyYMG8627 BWP17 SPA2/SPA2-YFP:URA3 MLC1/MLC1-CFP:HIS1 This studyYJB7985 BWP17 BUD6/BUD6-GFP:URA3 This studySU601 BWP17 CDC42/URA3:PPCK1-GFP-CDC42 This studyYJB5631 BWP17 TUB1/TUB1-YFP:URA3 This studyYMG8619 BWP17 spa2::HIS1/spa2::ARG4 MLC1/MLC1-YFP:URA3 This studyYJB9066 BWP17 bud6::HIS1/bud6::ARG4 MLC1/MLC1-YFP:URA3 This studyYMG8255 BWP17 MLC1/MLC1-YFP:URA3 NOP1/NOP1-CFP:HIS1 This studyYMG9253 BWP17 BNI1/BNI1-YFP:HIS1 This study

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it was located just behind the tip (Fig. 1B,F). In addition to thespot, 60% of the germ tubes exhibited a crescent of Mlc1-YFPat the tip (Fig. 1B,C). Examination of 200 nm optical sectionsthrough these tips revealed that the crescent is in a differentfocal plane from the spot (Fig. 1C). We found that the crescentis always a surface structure, whereas the spot is sometimesinternal to the germ tube tip. The proportion of cells having acrescent as well as a spot showed little variation through thecell cycle (data not shown).

Deconvolved images in a Z-stack contain informationconcerning the distribution of fluorescence in the Z-plane aswell as the X-Y planes. Two-dimensional (2D) projections of

the Z-stack can summarise this information, but failto recapitulate the full three-dimensional (3D)relationship of the fluorescence in different planes.Computer models utilise the information in the Z-planes to represent a 3D structure (see Materialsand Methods for more detail). To determinewhether the spot of Mlc1-YFP was a 3D objectprojected onto a 2D plane, we generated a 3Dmodel of the Mlc1 structure using the Volocityprogram. Fig. 1D shows such a model in a cell co-stained with filipin to define the position of theplasma membrane at the tip. Mlc1-YFP localises toa 3D ball enclosed by the plasma membrane.

To confirm that the localisation of Mlc1-YFP wasunaffected by the YFP moiety, we investigated the

localisation of Mlc1 in germ tubes of a wild-type strain byimmunocytofluorescence using an antibody to S. cerevisiaeMlc1 (kindly provided by J. Boyne and C. Price, Lancaster,University, Lancaster, UK). As with the Mlc1-YFP strains, adiscrete spot at the hyphal tip is clearly visible byimmunofluorescence, despite the higher background observedwith this method (Fig. 1E). Therefore, the localisation of Mlc1-YFP to a spot at the hyphal tip is not an artefact of the YFPfusion. Furthermore, this localisation is evident in the hyphaeof several independently derived MLC1-YFP strains and in anMLC1-CFP strain, so it is not peculiar to the particular strainused.

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Fig. 1. Mlc1 and FM4-64 colocalise to a Spitzenkörper-like structure at hyphal tips. (A) Colocalisation of Mlc1-YFP (green) and FM4-64 (red). Images shown areprojections of the Z-stack of deconvolved images. In themerged panel, there is extensive overlap between Mlc1-YFP and FM4-64. (B) Detail of Mlc1 localisation athyphal tips. Top left, An Mlc1 spot and crescent arevisible. Right panels, the Mlc1-spot is present either atthe tip (top) or just behind the tip (bottom). (C) Opticalsections at 200 nm intervals through an individual germtube tip. The crescent comes into focus at a differentoptical plane compared to the spot (arrow). Hyphal tipsare outlined in panels B and C. (D) Hyphal germ tubesof an Mlc1-YFP-expressing strain were stained withfilipin to reveal the plasma membrane. A 3D model wasgenerated from the deconvolved Z-stack using Volocity®

software. Mlc1-YFP (green) forms a 3D ball locatedbehind the cell membrane (blue). (E) Localisation ofMlc1 (red) and the septin, Cdc11 (green), revealed byimmunocytofluorescence using polyclonal antisera tothe respective S. cerevisiae proteins. Specificity of theCdc11 antibody has been described elsewhere (Sudbery,2001). The anti-Mlc1 antibody recognised a single bandin western blots that was the same size as S. cerevisiaeMlc1 (data not shown). (F) The shape of a hypha can bepredicted by the VSC model according to the distanceof the spot from the hyphal tip. An Mlc1-YFPexpressing strain was induced to form hyphae and cellwalls were stained with Concanavalin A-Texas Red.Measurements were made of external width at variousdistances from the tip. These were compared with thepredicted widths at these points made by the FungusSimulator program version 4 according to the distanceof the centre of the Mlc1-YFP spot to the outside of thehyphal tip. Bar, 5 µm (A); 1 µm (B,C,F); 0.5 µm (D);2 µm (E).

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The colocalisation of Mlc1-YFP and FM4-64 to a spot at thehyphal tip suggests that a Spitzenkörper is present at the tip ofC. albicans hyphae. If this is the case, then hyphal dimensionsshould conform to a hyphoid curve predicted by the VSCmodel and should be predictable from the position of theSpitzenkörper (Bartnicki-Garcia et al., 1989). Thesepredictions were tested using the Fungus Simulator programversion 4 (Bartnicki-Garcia et al., 1989). The shape of hyphaepredicted by this program, according to the distance of theSpitzenkörper core from the hyphal tip, was in good agreementwith the observed dimensions of hyphae. An example is shownin Fig. 1F, where we visualised the Spitzenkörper with Mlc1-YFP and the cell wall with Concanavalin A conjugated toTexas Red (Fig. 1F). Similar agreement was seen in tenadditional hyphae analysed in this manner. The mean widthmeasured in these hyphae 2 µm from the tip was 2.13±0.09 µm

(mean±s.d.) compared to an averagepredicted width at this point of 2.26±0.19µm. Interestingly, in the experiment shown inFig. 1B, the Spitzenkörper was closer to thetip and the hyphae were narrower (1.59±0.27µm) (Fig. 1B), which is consistent with theprediction from the VSC model (1.51±0.27µm). Thus, the distance of the Spitzenkörperfrom the tip correlates with hyphal width, ingood agreement with the VSC model. Thedifference in hyphal widths observed in thetwo experiments is probably due to therelative growth rates: the culture in Fig. 1Bhad been incubated for 70 minutes afterhyphal induction, whereas the ConcanavalinA-treated culture had been cultured for 120minutes. Taken together, these results areconsistent with the presence of aSpitzenkörper at the tip of C. albicans hyphaeand thus we will refer to the spot of Mlc1 andFM4-64 fluorescence as the Spitzenkörper.

Bni1 localises to the Spitzenkörper butSpa2, Bud6 and Cdc42 localisepredominantly to the polarisomeWe next investigated the pattern of localisationin hyphal tips of Bni1-YFP, Bud6-GFP andSpa2-YFP, orthologs of polarisomecomponents in S. cerevisiae. Like Mlc1, Bni1localised to a bright spot just behind thehyphal tip (Fig. 2A). Apical localisation ofSpa2 has been reported recently (Zheng et al.,2003), but in contrast to Mlc1 and Bni1, weobserved that both Spa2 and Bud6 localisedpredominantly to a crescent or cap at the tip ofall hyphae (Fig. 2B,C). However, closerexamination revealed that areas of moreintense staining were often visible along withthe cap (Fig. 2C, arrow; Fig. 2D). The extentof Spa2-YFP colocalisation with FM4-64 andMlc1-CFP was determined in colocalisationexperiments (Fig. 2D,E). Spa2-YFP formed acrescent or a cap located slightly closer to thetip than the spots of Mlc1-CFP and FM4-64.

The 3D model of Spa2 and FM4-64 localisation (Fig. 2E) showsthat the Spa2 cap covers the ball of FM4-64-stained material,consistent with the idea that both a polarisome and aSpitzenkörper are present at the hyphal tip. Thus, Spa2 and Bud6localise predominantly to the polarisome, whereas Bni1, Mlc1and FM4-64 localise predominantly to the Spitzenkörper. Themore intensely fluorescing patches of Spa2 and Bud6 mayindicate that some of these proteins are present in theSpitzenkörper. However, close examination reveals that theoverlap between Spa2 and FM4-64 or Mlc1 is limited (Fig.2D,E).

Cdc42 plays many roles in coordinating polarised growth inS. cerevisiae, and in C. albicans it has been localised to thehyphal tip (Hazan and Liu, 2002). We visualised Cdc42 with anN-terminal fusion to YFP (YFP-Cdc42). As found by others, nosignal was detected when YFP-Cdc42 was expressed from its

Fig. 2. Localisation of Bni1-YFP, Spa2-YFP and Bud6-GFP. (A) The fluorescence fromBni1-YFP merged with the DIC image of a germ tube. A single spot is observed justbehind the tip (arrow). (B) Z-stack projection of hyphal cells expressing Spa2-YFP,which can be seen in all cells to form crescents or caps, with areas of higher intensitystaining. (C) Localisation of Bud6-GFP, which predominantly forms a crescent, althoughin some cells a spot of higher fluorescence is observed (arrow). (D) Partial colocalisationof Spa2-YFP and Mlc1-CFP in hyphal cells. (E) Partial colocalisation of Spa2-YFP andFM4-64 in hyphal cells. Arrow indicates the tip that is enlarged in the model below. Themodel was constructed using the model-building module in the Softworx™ suite. Spa2-YFP is shown as a green wireframe, FM4-64 is shown in solid red. The Spa2 cap coversthe solid body of FM4-64. Bar, 5 µm (A); 1 µm (B,E); 2 µm (C,D).

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own promoter (Hazan and Liu, 2002). We thereforeoverexpressed YFP-Cdc42 by placing it under the control of thePCK1 promoter, which is induced by growth on succinate

(Leuker et al., 1997), using a cassette designed for the PCR-mediated construction of such alleles (Gerami-Nejad et al.,2004). Expression of the YFP-Int1, the C. albicans homolog of

S. cerevisiae Bud4, was elevated eightfold whenexpressed from the induced PCK1 promotercompared to the native promoter (Gerami-Nejad etal., 2004). Thus, use of the PCK1 promoter results ina moderate degree of overexpression. Strikingly,YFP-Cdc42 localised to two structures: a brightcrescent at the surface of the hyphal tip and a fainterspot just behind the crescent (Fig. 3A,D). In theseexperiments, FM4-64 stained a discrete spot at thetip (Fig. 3B,E) that colocalised with the subapicalspot of Cdc42 (Fig. 3C,F). We conclude that, likeSpa2 and Bud6, but in contrast to Mlc1 and Bni1, themajority of Cdc42 localises to the polarisome,whereas a smaller amount is located within theSpitzenkörper.

The integrity of the Spitzenkörper requirespolarisome componentsDeletion of SPA2 results in germ tubes that arebroader and less polarised than the wild type (Zhenget al., 2003) (Fig. 4C,E). To investigate whether SPA2affects the integrity of the Spitzenkörper, weconstructed an Mlc1-YFP fusion in a spa2∆/spa2∆strain. Loss of SPA2 resulted in polarisome-likelocalisation of Mlc1-YFP rather than aSpitzenkörper-like localisation in 70 out of 74 hyphaltips examined (Fig. 4A). This mislocalisation ofMlc1-YFP and change in morphology is consistentwith the idea that Spa2 is required for Spitzenkörperformation and, consequently, for hyphal growth.

To determine whether the integrity of theSpitzenkörper was specifically dependent upon SPA2or if it required other components of the polarisomeas well, we introduced Mlc1-YFP into abud6∆/bud6∆ strain. The absence of BUD6 resultedin a phenotype similar to that of spa2∆/spa2∆ strains.When grown under hyphal-inducing conditions, thedaughter cells were wider and less polarised (Fig.4D). Consistent with this change in morphology,Mlc1-YFP no longer localised to a Spitzenkörper,but instead was distributed in a surface crescent in all20 hyphal tips examined (Fig. 4B). Thus, theformation of the C. albicans Spitzenkörper requiresat least two polarisome components, Spa2 and Bud6.

Hyphal growth and Spitzenkörper integrity aredependent on microtubulesThe formation of the Spitzenkörper in Fusariumacuminatum is disrupted by methyl benzimidazole-2-ylcarbamate (MBC), an inhibitor of tubulinpolymerisation (Bartnicki-Garcia et al., 1995;Howard, 1981). There are conflicting reports ofwhether hyphal growth in C. albicans is sensitive toMBC (Akashi et al., 1994; Yokoyama et al., 1990).We found that 0.1 mg/ml MBC blocked hyphalextension within 10 minutes (data not shown). To

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Fig. 3. Localisation of YFP-Cdc42. (A-C) Cells expressing YFP-Cdc42 from thePCK1 promoter were grown in hyphal-inducing conditions and stained with FM4-64. The hyphal tip identified by the arrow in panel C is shown at greatermagnification in panels D-F. Arrow in D indicates the subapical spot of YFP-Cdc42, which colocalises with FM4-64 resulting in the yellow spot in the mergedimage (F). Bar, 5 µm (A-C); 1 µm (D-F).

Fig. 4. Mlc1 localisation to the Spitzenkörper depends on Spa2 and Bud6.(A,B) spa2∆/spa2∆ or bud6∆/bud6∆ strains, both expressing Mlc1-YFP, weregrown under hyphal-inducing conditions. In both mutants, no spot of Mlc1-YFP ispresent, but a crescent remains. (C-E) DIC images of mutant hyphal cells (C,D)compared to wild-type hyphal cells (E). In both mutants, the hyphae are broaderand more kinked than the wild-type hyphae. Bar, 10 µm.

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determine whether microtubules would be disrupted by MBC,we treated a Tub1-YFP-expressing strain with MBC. Inuntreated cells, long microtubules extend along the long axis ofgerm tubes, consistent with previous reports (Fig. 5A) (Bartonand Gull, 1988; Hazan et al., 2002). Microtubules were disruptedwithin 10 minutes of exposure to 0.1 mg/ml MBC. In MBC-

treated hyphae, the organisation of FM4-64 (Fig. 5C) and Mlc1(Fig. 5D) was disrupted in 61 out of 62 hyphal tips examined.Thus, the integrity of the C. albicans Spitzenkörper requiresmicrotubules. As we found for spa2∆/spa2∆ and bud6∆/bud6∆strains, the residual organisation of Mlc1-YFP in MBC-treatedhyphae resembled that of a polarisome, although the structurewas more discontinuous than it was in wild-type pseudohyphaeor yeast (see next section). This suggests that localisation ofMlc1 to a polarisome-like structure requires neithermicrotubules nor the polarisome components Spa2 and Bud6.

In the absence of actin cables, the Spitzenkörperdisappears and hyphal growth becomes isotropicPolarised growth in S. cerevisiae yeast cells is not dependent onmicrotubules, but does require actin cables. In C. albicans, actincables are specifically disrupted with Cytochalasin A (CA),which has no effect on actin cortical patches and microtubules(Akashi et al., 1994). When growing hyphae are treated with CA,the hyphal tips swell (Akashi et al., 1994), as they do in other

Fig. 5. Organisation of the Spitzenkörper depends on microtubules.(A) A strain expressing Tub1-YFP was induced to form hyphal germtubes from unbudded stationary phase yeast cells. Long microtubulesare visible along with spots (arrow), which we presume are spindlepole bodies (SPBs). (B) After 60 minutes, 0.1 mg/ml MBC wasadded and the incubation was continued for a further 10 minutes.Microtubules have disappeared but the SPBs remain (arrow). (C) AnMlc1-YFP expressing strain was also treated with MBC in a parallelexperiment. The cells were stained with FM4-64 after 10 minutes oftreatment with MBC. The spot of FM4-64 staining has disappeared.(D) Localisation of Mlc1-YFP in the same cells shown in C. Thespot of Mlc1-YFP has disappeared but a surface crescent remains insome cells. Bar, 5 µm.

Fig. 6. The role of actincables in Spitzenkörperintegrity and polarisedgrowth. Unbudded yeast cellsexpressing Mlc1-YFP wereinduced to form hyphae,stained with Concanavalin A(Con A) conjugated to eitherAlexa 488 or Texas Red andtreated with 10 µmCytochalasin A (CA) and asdescribed in the flow chart.The protocol results in theformation of young germtubes in which the cell wallsfluoresce green. Cell wallsformed after CA treatment arenot stained with Con A-A488(green) but are stained withCon A-TR (red). (A-C) ACA-treated hypha. (D-F) Anuntreated control hypha. CAtreatment results in swollentips that are stained red butnot green. Thus, the swellingarises from new isotropic growth, not from swelling of a pre-existing hyphal tip. Mlc1-YFP, which is also visible in the green channel,disappears from the tip of CA-treated cells, but remains in the typical Spitzenkörper organisation in untreated cells (D, arrow; F, inset). AnMlc1 cytokinetic ring persists in the CA-treated cells (arrow in C). The response of hyphal germ tubes to CA treatment was uniform in theculture and the images shown are typical. Bar, 5 µm.

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filamentous fungi treated with inhibitors of the actincytoskeleton. We used CA to test the dependency of theSpitzenkörper on actin cables in cells expressing Mlc1-YFP. Wealso wanted to investigate whether the tip swelling caused by CAis due to new cell wall material added through isotropic growth,or whether the swelling arises from expansion of a pre-existingwall weakened by the CA treatment. To do this, we exploited

the cell wall-binding lectin Concanavalin A(ConA) conjugated to different fluorophores todistinguish between old and new cell wallmaterial. ConA-Alexa 488 (green fluorescence)was used prior to CA treatment and ConA-Texas Red (red fluorescence) was used to staincell walls after the CA treatment (Fig. 6).Because the fluorescence of YFP is compatiblewith the filter set used to visualise Alexa 488,the green channel shows Mlc1-YFPfluorescence along with cell wall materialpresent prior to CA addition (Fig. 6A,D). Asreported previously, CA treatment resulted inuniform swelling of all hyphal tips (Fig. 6B,C).The swollen tip exhibited no greenfluorescence, but did stain with ConA-TexasRed (Fig. 6A,B). Thus the swollen tip arisesfrom the isotropic deposition of new cell wallmaterial and is not due to expansion of aweakened cell wall. In CA-treated cells, theSpitzenkörper completely disappeared (Fig.6A,C), whereas in untreated controls theSpitzenkörper remained visible (Fig. 6F inset).Thus, when actin cables are disrupted with CA,the Spitzenkörper disappears and the growthmode switches from polarised to isotropic.Interestingly, Mlc1-YFP localisation to thecytokinetic ring is not disrupted after CAtreatment (arrow, Fig. 6C), thus this inhibitor isspecific to actin cables that deliver cargo to thehyphal tip.

Only the polarisome is present in yeastand pseudohyphaeWe also investigated the localisation of Mlc1-YFP in pseudohyphae and yeast. In some newlyevaginated pseudohyphal buds, Mlc1-YFPlocalised to a spot similar to that seen in hyphae(Fig. 7A, cell 1). However, this quickly changedto a crescent or patch (Fig. 7A, cells 2-4; Fig. 7B,cell 1) which, when analysed by 3D modelling,was located around the surface of the tip (Fig.7D). In elongated buds, Mlc1-YFP fluorescenceat the tip was low or undetectable (Fig. 7B, cell2) and other cells with elongated buds containeda ring of Mlc1-YFP at the bud neck (Fig. 7C).Quantification of the different patterns of Mlc1localisation, categorised according to bud lengthconfirms that the Mlc1-YFP crescent or spotpresent at the tip of young buds disappears ascells enter mitosis to be replaced by a ring at thebud neck. Spa2 and FM4-64 also localised to acrescent at the tips of pseudohyphal buds (Fig.

7E,F). Similarly, 3D modelling showed that both Spa2 and FM4-64 were located in a surface crescent (data not shown). As wefound for pseudohyphal buds, newly evaginated yeast buds had aspot of MIc1 fluorescence (Fig. 8A) that quickly changed to acrescent as the bud enlarged. Again, like pseudohyphae, Mlc1 wasreduced at the tip of large yeast buds compared to small buds andeventually a ring of Mlc1 formed at the bud neck (Fig. 8A). This

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Fig. 7. Mlc1, Spa2 and FM4-64 colocalise to a surface crescent structure inpseudohyphal buds. (A-F) Pseudohyphae were induced in strains carrying the indicatedfusions to YFP or stained with FM4-64. Cells were stained with Calcofluor white (A-C)and with filipin (D). The images shown are a projection of the deconvolved Z-stack,except C, which is a single layer. Numbers in panels A and B refer to cells discussed inthe text. (D) A pseudohyphal cell expressing Mlc1-YFP and stained with filipin. Left,deconvolved image; right, model generated with the Softworx™ suite. (E and F)Localisation of Spa2-YFP and FM4-64, respectively, in pseudohyphae. Arrows in Findicate crescents of FM4-64 staining at the tips of pseudohyphal buds. (G) Mlc1-YFP-expressing pseudohyphal cells were sampled at various times after inoculation and fixedand stained with DAPI. Cells were categorised into bins of bud length and characterisedaccording to whether Mlc1-YFP formed a ball or a crescent at the tip or was not presentat all. In addition, cells that contained two nuclei were categorised as having completedmitosis. Note that the categories at each bud length do not add up to 100% as cells canbe in more than one category. For example, a cell can be categorised as having no Mlc1at the tip as well as having a cytokinetic ring. Each bin contains at least 100 cells.

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pattern of localisation was observed in the localisation of Spa2 inyeast (Fig. 8B). Thus, Mlc1, Spa2 and FM4-64 localise to asurface cap or crescent and are not found in a spot at the tips ofpseudohyphal or yeast cells. We conclude that only a polarisomeis present in pseudohyphae and yeast.

The presence of the polarisome in yeast andpseudohyphae depends on the cell cycleThe reduction in signal intensity for polarity determinants atthe tips of large pseudohyphal and yeast buds suggested thatthe polarisome is regulated in a cell cycle-dependent fashion.To investigate this further, we recorded time-lapse videos ofpseudohyphal cells in a strain coexpressing Nop1-CFP andMlc1-YFP. Nop1 is an abundant nucleolar protein, thus thefluorescent fusion protein allows the nucleus to be visualisedin a living cell (Fig. 9A and supplementary material, Movie 1).During or just after mitosis, an Mlc1 ring appears at the budneck and then contracts (Fig. 9A) as it does during cytokinesisin yeast cells (Lippincott and Li, 1998). In 13/13 videos, thepolarisome disappeared late in the cell cycle and did not coexistwith the cytokinetic ring. Quantification of random populationsof cells showed that Mlc1 is not present at the bud tip in 80%of cells displaying a ring of Mlc1 at the septation site. In themajority of the remaining cells, the residual fluorescence ofMlc1 at the bud tip is faint. Therefore, we conclude that, inpseudohyphae, the polarisome disappears at or before mitosisand rarely coexists with the cytokinetic ring.

In S. cerevisiae, cells switch from polarised to isotropic growthat a specific point in the cell cycle after phosphorylation ofCdc2/Cdc28 (the cyclin-dependent kinase) by the Swe1 kinase(Lew and Reed, 1995). Prior to this switch, cells exhibit polarisedgrowth with length increasing faster than cell width and, as aresult, the axial ratio (length/width) of the growing bud is greaterthan one and continues to increase as the bud length increases.After the switch to isotropic growth, length and width increase ata similar rate and thus the axial ratio no longer increases relativeto bud length. Plotting bud length versus the axial ratio revealsthe average cell length at which polarised growth (curve has apositive slope) switches to isotropic growth (slope of the curvebecomes shallower). In C. albicans yeast cells, the switch toisotropic growth occurs at a cell length of ~1.75 µm and an axialratio of 1.25 (Fig. 9B). In contrast, pseudohyphal cells switch toisotropic growth at a cell length of 7.5 µm and an axial ratio of4.5 (Fig. 9C). In general, cells with Mlc1 staining at the bud tipfall into the polarised growth part of the curve and cells withoutMlc1 staining fall into the isotropic part of the curve. Thus, thepresence of the polarisome is correlated with polarised growthand this polarisome-associated growth persists for a longer periodin pseudohyphal compared to yeast cells.

In contrast to both yeast and pseudohyphae, hyphal cells donot exhibit a cell cycle-dependent localisation of Mlc1. Rather,Mlc1-YFP localises to the Spitzenkörper in all hyphal cells,even when it localises to the cytokinetic ring (Fig. 9D,E). Thus,the Spitzenkörper is continuously present at the hyphal tipduring all stages of the cell cycle, including septation.

The intensity of Mlc1 fluorescence is greater in hyphaecompared to pseudohyphae and yeastTo compare the amount of Mlc1 in the Spitzenkörper and the

polarisome, we quantified the mean of the peak values of Mlc1intensity at the tips of pseudohyphal buds and hyphal germtubes and plotted these values against germ tube orpseudohyphal bud length (Fig. 10A,B). In hyphae, the meanpeak intensity increased from ~1000 units immediately afterevagination to ~3000 units when the germ tube length hadreached 35-50 µm (Fig. 10A). In contrast, the mean peakintensity in pseudohyphae was ~600 units after evagination,and declined thereafter, consistent with its disappearance fromthe bud tip later in the cell cycle (Fig. 10B). Furthermore, the3D graph of Mlc1-YFP intensity in hyphae (Fig. 10A) revealsa ridge, corresponding to the crescent, on either side of themain peak. The intensity of this ridge of fluorescence is similarto the intensity of the polarisome in pseudohyphae, consistentwith the hypothesis that the crescent at hyphal tips representsa polarisome and the ball at hyphal tips represents aSpitzenkörper. Peak fluorescence intensity in yeast was similarto that of pseudohyphae (Fig. 10C).

Like Mlc1, Spa2 is also distributed as a surface ridge inhyphae (Fig. 10D), in contrast to the cone observed in the 3Dgraph of Mlc1 fluorescence (Fig. 10A) and consistent with themodel shown in Fig. 3D. Interestingly, when we compared therelative fluorescence intensity between the growth forms, wefound that the fluorescence of Mlc1-YFP increasedapproximately fourfold in hyphae compared to yeast andpseudohyphae, whereas the fluorescence intensity of Spa2-YFP did not show much difference between the differentmorphologies (Fig. 10C), supporting the idea that Spa2 is

Fig. 8. Mlc1 and Spa2 localisation in yeast cells. Images wererecorded from exponentially growing yeast cells expressing Mlc1-YFP (A) or Spa2-YFP (B). Mlc1 localises to a surface crescent insmall buds, but to a ring at the mother-bud neck in large cells.

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predominantly in the polarisome,whereas Mlc1 is predominantly in theSpitzenkörper.

DiscussionThe primary aim of this work was totest the hypothesis that there aredifferent properties of polarised growththat account for the distinctive shapesof hyphae relative to yeast andpseudohyphae. To this end, we foundmolecular, genetic and temporalevidence that support this hypothesis.First, we found two informativepatterns of protein localisation athyphal tips. The localisation of Mlc1,which is required to transport vesiclesto the growing tip along actin cables,and the formin, Bni1, which nucleatesactin cables, differs from that of Spa2and Bud6, which are components of theS. cerevisiae polarisome, and of Cdc42,which orchestrates many of theprocesses required for polarisedgrowth. Mlc1 and the Spitzenkörpermarker FM4-64 colocalisedpredominantly to a spot (Fig. 1A). Thespot of Mlc1-YFP fluorescence wasrevealed to be a ball when rendered intoa 3D model (Fig. 1D). In some cells,the spot was located a short distancefrom the tip and the plasma membrane(Fig. 1B,F). In contrast, Spa2, Bud6and Cdc42 localised mostly to acrescent that showed limitedcolocalisation with FM4-64 (Fig. 2E,Fig. 3F). When Spa2-YFP and FM4-64were rendered into three dimensions,Spa2 formed a surface cap at the tip andwas more apical than the FM4-64stained material (Fig. 2E). Thus, weconclude that there are two structurespresent at the hyphal tip: a polarisomeand a Spitzenkörper.

Although Mlc1 localised mainly tothe Spitzenkörper, it was also detectedin the polarisome of many hyphae (Fig.1B,C and Fig. 10A). However, whereasthe Mlc1 spot was sometimes observedwithout the crescent (Fig. 1B,D), wenever observed the crescent alone: allhyphae displayed a spot of Mlc1 at thetip (Fig. 1A). The simultaneouslocalisation Mlc1-YFP to a spot and acrescent in the hyphal tip is strikinglysimilar to the localisation of the A.nidulans formin, SepA, which localisesto two clearly separate structures: aspot a small distance away from the tipand a crescent at the tip (Sharpless and

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Fig. 9. Mlc1 localisation is cell cycle-dependent in yeast and pseudohyphae. (A) Time-lapsevideo recorded from a pseudohyphal cell coexpressing Nop1-CFP and Mlc1-YFP. Numbers ineach frame indicate time elapsed in minutes since inoculation of unbudded cells into freshmedium. The video may be viewed in supplementary material. (B,C) The axial ratio(length/width) of daughter buds of Mlc1-YFP-expressing cells was quantified in culturesgrowing either as yeast (B) or pseudohyphae (C) and plotted against bud length. In addition,the presence or absence of Mlc1-YFP fluorescence at the tip was recorded. The switch toisotropic growth occurs at a longer bud length and greater axial ratio in pseudohyphaecompared to yeast. Note differences in scales. (D) Mlc1-YFP-expressing cells were induced toform hyphal germ tubes. After 105 minutes, cells were fixed and stained with DAPI andexamined with a Delta Vision microscope. Mlc1-YFP fluorescence is visible as a spot at thetip and a ring at the site of septation in the germ tube. Nuclei stained with DAPI are visible ascircular areas within the mother cell, or migrating into the germ tube, or dividing across thecytokinetic ring. (E) Hyphal germ tube showing Mlc1-YFP simultaneously at the tip and theseptation ring. Scale bar, 5 µm (A,E); 15 µm (D).

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Harris, 2002). A small amount of Cdc42 was present in theSpitzenkörper of most cells (Fig. 3). This observation is subjectto the caveat that we could only visualise Cdc42 when it wasoverexpressed, thus the localisation of Cdc42 to theSpitzenkörper could be artefactual. Spa2 and Bud6 were alsoobserved to localise to a spot in some cells (Fig. 2C,D). ThusCdc42, as well as Spa2 and Bud6, may also be components ofthe Spitzenkörper. The observation that Bni1 and Mlc1 wereapparently located predominantly in the Spitzenkörper,whereas Cdc42, Spa2 and Bud6 were found predominantly inthe polarisome, may reflect a genuine difference in theproportions of each protein in the Spitzenkörper andpolarisome. However, the apparent differences could also bedue to technical issues such as different degrees of competitionwith the wild-type protein in the two structures. However, notethat the indirect immunofluorescence with anti-Mlc1antibodies (Fig. 1E) shows that the localisation of Mlc1-YFPto a spot is not artefactual.

Second, we investigated the genetic requirements for theSpitzenkörper and found that the polarisome components Spa2and Bud6 are required for Spitzenkörper formation (Fig. 4). Inmutants lacking either of these proteins, the Spitzenkörper wasdisrupted and Mlc1 localised only to the polarisome. Moreover,there was an accompanying change in the morphology of themutants, such that they resembled pseudohyphae rather thanhyphae. This is consistent with the Spitzenkörper driving the

hyphal growth of C. albicans. Thus, polarisome componentsare required for hyphal growth, but are not required for yeastor pseudohyphal growth. Similarly, we found that theSpitzenkörper was disrupted by treatment with the microtubuleinhibitor MBC (Fig. 5). This dependence on microtubulefunction is consistent with the observation that MBC disruptsthe Spitzenkörper in the filamentous fungus, Fusariumacuminatum (Howard and Aist, 1981), whereas microtubulesare not required for bud growth and secretory vesicle transportin S. cerevisiae (Huffaker et al., 1988).

Third, we tested the prediction that there would be noSpitzenkörper in yeast and pseudohyphae. Indeed, we foundthat Mlc1, Spa2 and FM4-64 localised to a surface crescentrather than a Spitzenkörper-like structure (Fig. 7A-F and Fig.8). Therefore, we conclude that the Spitzenkörper is specific tohyphae and is not involved in yeast or pseudohyphal growth.

Finally, we observed two differences between the regulationof the polarisome in yeast and pseudohyphae relative to theSpitzenkörper in hyphae. First, polarisome componentsdisappear from the tip of yeast and pseudohyphal buds at orbefore mitosis and reappear at the cytokinetic ring aftermitosis. Mlc1 at the tip rarely coexisted with the cytokineticring (Fig. 9A). In contrast, in hyphae, the Spitzenkörper wascontinually present at the tip and coexisted with the contractileMlc1 ring at the site of septation (Fig. 9D,E). Second, theintensity of Mlc1 fluorescence was approximately fourfold

Fig. 10. Quantification of Mlc1fluorescence in hyphae andpseudohyphae. Strains expressing eitherMlc1-YFP or Spa2-YFP were culturedunder the indicated conditions and theaverage peak fluorescence intensity wasdetermined. An example of the 3Dsurface contour map of fluorescence isshown alongside the hyphal (A,D) orpseudohyphal (B) tip from which it wasderived. (A) A ridge of lesserfluorescence surrounds a focused point ofhigh fluorescence. (B) The contour mapreveals only a structure whose maximumfluorescence resembles that of the ridgedetected in A and no focal point offluorescence. The histograms in A and Bshow the mean maximum fluorescenceintensity in cells divided into binsaccording to bud length. Each bin consistsof at least 100 cells. The time courseexperiments described in panels A and Bhave been carried out on three separateoccasions with similar results.(C) Average peak fluorescence wascompared between Spa2-YFP (black) andMlc1-YFP (grey) in the differentmorphologic forms. Spa2 shows littlevariation in its peak intensity betweengrowth forms whereas Mlc1 fluorescenceis more than four times greater in hyphae(H) than pseudohyphae (PH) or yeast (Y).(D) The shape of the Spa2 fluorescencesurface contour map in hyphae resemblesthat of Mlc1 in pseudohyphae shown inPanel B. Bar, 1 µm.

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greater in the Spitzenkörper as compared to the polarisome.This was true even in newly evaginated yeast andpseudohyphal buds, where the pattern of Mlc1 localisationresembled that of hyphae in some buds (Fig. 9). These resultssupport the existence of two complexes that are subject todifferent modes of regulation.

The role of actin cables and microtubulesMaintenance of the Spitzenkörper requires both actin cablesand microtubules: the Spitzenkörper disappeared on treatmentwith cytochalasin A and was disrupted upon treatment withMBC. However, there were interesting differences between theeffects of these inhibitors on growth patterns. Growthcompletely ceased upon MBC treatment. In contrast, growthcontinued upon Cytochalisin A treatment, but switched from apolarised to an isotropic mode, resulting in a swelling at thehyphal tip (Fig. 6). These observations are consistent with theidea that microtubules mediate long-distance transport ofvesicles and that actin cables mediate short distancedistribution of vesicles from the Spitzenkörper to the growingtip. According to this scenario, growth ceases upon MBCtreatment because secretory vesicles generated in the body ofthe hypha cannot be transported to the tip region. In the absenceof the continued arrival of vesicles, the Spitzenkörperdisperses. The remaining Mlc1-YFP in a crescent at the surfacemay reflect the continued operation of the polarisome-mediated transport of vesicles along actin cables. Upondisruption of actin cables, vesicles would continue to arrive atthe tip region, transported along microtubules. Without aSpitzenkörper to focus these vesicles to the tip, they woulddisperse randomly in all directions, resulting in isotropicgrowth.

Are the polarisome and the Spitzenkörper differentstructures?As the same components are present in both the Spitzenkörperand the polarisome, albeit in different proportions, it may beargued that the structure described here as a Spitzenkörper isa hyperactive polarisome. Three lines of evidence suggest thatthe difference is not simply semantic. First, in hyphae, thepresence of the Spitzenkörper is independent of the cell cycle,whereas in yeast and pseudohyphae it is cell cycle dependent,disappearing before mitosis. Second, the Spitzenkörper isclearly a 3D object, which in some hyphae is clearly separatedfrom the hyphal tip, whereas the polarisome consistently formsa surface cap at the growing tip. Third, loss of Spa2 or Bud6or disruption of the microtubules with MBC results in loss ofthe Spitzenkörper, whereas a polarisome-like structure appearsto persist and growth assumes the pseudohyphal morphology.In S. cerevisiae, microtubules are not required for polarisedgrowth, and so presumably are also not required for theintegrity of the polarisome. Thus, not only is the Spitzenkörperregulated differently from the polarisome, it has differentgenetic and cytoskeletal requirements to the polarisome.

Taken together, our data show that there are considerabledifferences in the properties of the Spitzenkörper, present onlyin hyphae, and the polarisome that is present in hyphae,pseudohyphae and yeast. Importantly, although pseudohyphaemay superficially resemble hyphae, the underlying mechanism

of polarised growth in pseudohyphae is more similar to that inyeast, supporting the view that hyphae and pseudohyphae in C.albicans are qualitatively different states (Sudbery et al., 2004).It will be of great interest to understand the molecularmechanisms responsible for organising these different modesof polarised growth and to determine how signal transductionpathways regulate the structures that give rise to them andthereby mediate morphologic changes.

This work was supported by the Wellcome Trust (Grant No:060862/Z/00/Z) and BBSRC grant to P. E.S., the National Institutesof Health (R01 AI/DE 14666) to J.B. and a University of MNGraduate School grant to C.G. Helen Crampin was in receipt of aBBSRC Research Training Studentship, Helen Court is in receipt ofan MRC Research Training Studentship and K.F. was supported byNIH Biotechnology training grant GM08347. We thank Yue Wang forproviding a spa2∆/spa2∆ strain, Alan Tilley of Improvision forgenerating the 3D models using the Volocity Software suite and JimBoyne and Clive Price for antisera to S. cerevisiae Mlc1.

ReferencesAkashi, T., Kanbe, T. and Tanaka, K. (1994). The role of the cytoskeleton

in the polarized growth of the germ tube in Candida albicans. Microbiology140, 271-280.

Bartnicki-Garcia, S., Hergert, F. and Gierz, G. (1989). Computer-simulation of fungal morphogenesis and the mathematical basis for hyphal(tip) growth. Protoplasma 153, 46-57.

Bartnicki-Garcia, S., Bartnicki, D. D., Gierz, G., Lopez-Franco, R. andBracker, C. E. (1995). Evidence that Spitzenkörper behavior determines theshape of a fungal hypha–a test of the hyphoid model. Exp. Mycol. 19, 153-159.

Barton, R. and Gull, K. (1988). Variation in cytoplasmic microtubularorganisation and spindle length between the two forms of the dimorphicfungus Candida albicans. J. Cell Sci. 91, 211-220.

Boyne, J. R., Yosuf, H. M., Bieganowski, P., Brenner, C. and Price, C.(2000). Yeast myosin light chain, Mlc1p, interacts with both IQGAP andClass II myosin to effect cytokinesis. J. Cell Sci. 113, 4533-4543.

Evangelista, M. (1997). Bni1p, a yeast formin linking Cdc42p and the actincytoskeleton during polarized morphogenesis. Science 276, 118-121.

Fischer-Parton, S., Parton, R. M., Hickey, P. C., Dijksterhuis, J., Atkinson,H. A. and Read, N. D. (2000). Confocal microscopy of FM4-64 as a toolfor analysing endocytosis and vesicle trafficking in living fungal hyphae. J.Microsc. 198, 246-259.

Gerami-Nejad, M., Berman, J. and Gale, C. (2001). Cassettes for PCR-mediated construction of green, yellow and cyan fluorescent protein fusionsin Candida albicans. Yeast 18, 859-880.

Gerami-Nejad, M., Hausauer, D., McClellan, M., Berman, J. and Gale, C.(2004). Cassettes for the PCR-mediated construction of regulatable allelesin Candida albicans. Yeast 21, 429-436.

Girbardt, M. (1957). Der Spitzenkörper von Polystictus versicolor. Planta 50,47-59.

Grove, S. N. and Bracker, C. E. (1970). Protoplasmic organization of hyphaltips amoung fungi: vesicles and Spitzenkörper. J. Bacteriol. 104, 989-1009.

Harris, S. D., Read, N. D., Roberson, R. W., Shaw, B, Seiler, S., Plamann,M. and Momany, M. (2005). Polarisome meets Spitzenkörper: microscopy,genetics, and genomics converge. Eukaryot. Cell 4, 225-229.

Hazan, I. and Liu, H. P. (2002). Hyphal tip-associated localization of Cdc42is F-actin dependent in Candida albicans. Eukaryot. Cell. 1, 856-864.

Hazan, I., Sepulveda-Becerra, M. and Liu, H. P. (2002). Hyphal elongationis regulated independently of cell cycle in Candida albicans. Mol. Biol. Cell13, 134-145.

Howard, R. J. (1981). Ultrastructural analysis of hyphal tip cell-growth infungi–Spitzenkörper, cytoskeleton and endomembranes after freeze-substitution. J. Cell Sci. 48, 89-103.

Howard, R. J. and Aist, J. R. (1981). Cytoplasmic microtubules and fungalmorphogenesis: ultrastructural effects of methyl benzimidazole-2-ylcarbamatedetermined by freeze-substitution of hyphal tip cells. J. Cell Biol. 87, 55-64.

Huffaker, T. C., Thomas, J. H. and Botstein, D. (1988). Diverse effects ofbeta-tubulin mutations on microtubule formation and function. J. Cell Biol.106, 1997-2010.

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Johnston, G. C., Prendergast, J. A. and Singer, R. A. (1991). TheSaccharromyces cerevisiae MYO2 gene encodes an essential myosin forvectorial transport of vesicles. J. Cell Biol. 113, 539-551.

Karpova, T. S., Reck-Peterson, S. L., Elkind, N. B., Mooseker, M. S.,Novick, P. J. and Cooper, J. A. (2000). Role of actin and Myo2p inpolarized secretion and growth of Saccharomyces cerevisiae. Mol. Biol. Cell11, 1727-1737.

Kron, S. J. and Gow, N. A. R. (1995). Budding yeast morphogenesis–signaling,cytoskeleton and cell-cycle. Curr. Opin. Cell Biol. 7, 845-855.

Kron, S. J., Styles, C. A. and Fink, G. R. (1994). Symmetrical cell-divisionin pseudohyphae of the yeast Saccharomyces cerevisiae. Mol. Biol. Cell 5,1003-1022.

Leuker, C. E., Sonneborn, A., Delbruck, S. and Ernst, J. F. (1997).Sequence and promoter regulation of the PCK1 gene encodingphosphoenolpyruvate carboxykinase of the fungal pathogen Candidaalbicans. Gene 192, 235-240.

Lew, D. J. and Reed, S. I. (1995). Cell cycle control of morphogenesis inbudding yeast. Curr. Opin. Genet. Dev. 5, 17-23.

Lippincott, J. and Li, R. (1998). Sequential assembly of myosin II, anIQGAP-like protein, and filamentous actin to a ring structure involved inbudding yeast cytokinesis. J. Cell Biol. 140, 355-366.

Lopez-Franco, R. (1996). Diversity and dynamics of the Spitzenkörper ingrowing hyphal tips of higher fungi. Protoplasma 195, 90-111.

Nelson, W. J. (2003). Adaption of core mechanisms to generate cell polarity.Nature 422, 766-773.

Pruyne, D. (2002). Role of formins in actin assembly: nucleation and barbed-end association. Science 297, 612-615.

Pruyne, D. and Bretscher, A. (2000). Polarization of cell growth in yeast II.The role of the actin cytoskeleton. J. Cell Sci. 113, 571-585.

Read, N. D. and Hickey, P. C. (2001). The Vesicle trafficking network andtip growth in fungal hyphae. In Cell Biology of plant and fungal tip growth(ed. A.Geitman), pp. 137-147. Amsterdam, The Netherlands: IOS Press.

Reynaga Pena, C. G., Gierz, G. and Bartnicki-Garcia, S. (1997). Analysisof the role of the Spitzenkörper in fungal morphogenesis by computer

simulation of apical branching in Aspergillus niger. Proc. Natl. Acad. Sci.USA 94, 9096-9101.

Rua, D., Tobe, B. T. and Kron, S. J. (2001). Cell cycle control of yeastfilamentous growth. Curr. Opin. Microbiol. 4, 720-727.

Schott, D., Ho, J., Pruyne, D. and Bretscher, A. (1999). The COOH-terminaldomain of Myo2p, a yeast myosin V, has a direct role in secretory vesicletargeting. J. Cell Biol. 147, 791-807.

Shannon, K. B. and Li, R. (2000). A myosin light chain mediates thelocalization of the budding yeast IQGAP-like protein during contractile ringformation. Curr. Biol. 10, 727-730.

Sharpless, K. E. and Harris, S. D. (2002). Functional characterization andlocalization of the Aspergillus nidulans formin SEPA. Mol. Biol. Cell 13,469-479.

Sheu, Y. J., Santos, B., Fortin, N., Costigan, C. and Snyder, M. (1998).Spa2p interacts with cell polarity proteins and signaling componentsinvolved in yeast cell morphogenesis. Mol. Cell. Biol. 18, 4053-4069.

Sudbery, P. E. (2001). The germ tubes of Candida albicans hyphae andpseudohyphae show different patterns of septin ring localisation. Mol.Microbiol. 41, 19-31.

Sudbery, P. E., Gow, N. A. R. and Berman, J. (2004). The distinctmorphogenic states of Candida albicans. Trends Microbiol. 12, 317-324.

Swedlow, J. R., Hu, K., Andrews, P. D., Roos, D. S. and Murray, J. M.(2002). Measuring tubulin content in Toxoplasma gondii: A comparison oflaser-scanning confocal and wide-field fluorescence microscopy. Proc. Natl.Acad. Sci. USA 99, 2014-2019.

Wilson, B., Davis, D. and Mitchell, A. P. (1999). Rapid hypothesis testing inCandida albicans through gene disruption with short homology regions. J.Bacteriol. 181, 1868-1874.

Yokoyama, K., Kaji, H., Nishimura, K. and Miyaji, M. (1990). The role ofmicrofilaments and microtubules in apical growth and dimorphism ofCandida albicans. J. Gen. Microbiol. 136, 1067-1075.

Zheng, X. D., Wang, Y. M. and Wang, Y. (2003). CaSPA2 is important forpolarity establishment and maintenance in Candida albicans. Mol.Microbiol. 49, 1391-1405.

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