The polarisome component SPA-2 localizes at the apex of Neurospora crassa and partially colocalizes with the Spitzenkörper

Fungal Genetics and Biology 46 (2009) 551–563

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Fungal Genetics and Biology

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The polarisome component SPA-2 localizes at the apex of Neurospora crassaand partially colocalizes with the Spitzenkörper

Cynthia L. Araujo-Palomares, Meritxell Riquelme, Ernestina Castro-Longoria *

Department of Microbiology, Center for Scientific Research and Higher Education of Ensenada (CICESE), Km. 107 Ctra, Tijuana-Ensenada, 22860 Ensenada, Baja California, Mexico

a r t i c l e i n f o a b s t r a c t

Article history:Received 25 November 2008Accepted 19 February 2009Available online 10 March 2009

Keywords:GermlingsNeurospora crassaPolarisomeSpitzenkörper

1087-1845/$ - see front matter � 2009 Elsevier Inc. Adoi:10.1016/j.fgb.2009.02.009

Abbreviations: Spk, Spitzenkörper; PC-Spk, phase-cSpk; GFP, green fluorescent protein; CHS-1, chitin syn

* Corresponding author. Address: P.O. Box 430222USA. Fax: +52 646 1750589.

E-mail address: [email protected] (E. Castro-Long

In fungal hyphae multiple protein complexes assemble at sites of apical growth to maintain cell polarityand promote nucleation of actin. Polarity allows the directional traffic of vesicles to the Spitzenkörper(Spk) prior to fusing with the plasma membrane to provide precursors and enzymes required for cellextension and nutrition. One of these complexes is the polarisome, which in Saccharomyces cerevisiaecontains Spa2p, Pea2p, Bud6p/Aip3p and Bni1p. To investigate the localization and role of the polarisomeduring Spk establishment in Neurospora crassa we tagged SPA-2 with the green fluorescent protein (GFP)and examined growing cells by laser scanning confocal microscopy in elongating germ tubes and maturehyphae. SPA-2-GFP accumulated gradually at the apex of germ tubes, when a FM4-64 stained Spk was notstill detectable. When the germlings reached about 40 lm in length, a FM4-64 stained Spk started to beapparent and from this point on SPA-2-GFP was observed in the apical region of both germ tubes andmature hyphae, as a hand fan shape with a brighter spot at the base. Fusion of the N. crassa SPA-2-GFPstrain with a N. crassa strain expressing chitin synthase 1 (CHS-1) labeled with mCherryFP indicated onlypartial colocalization of the polarisome and the Spk core. N. crassa SPA-2-GFP was also found at the apexof forming branches but not in septa, suggesting that it participates only in areas of tip growth. A Dspa-2strain displayed hyphae with uneven constrictions, apices with an unstable Spk, reduced growth rate andhigher number of branches than the wild type strain, indicating that SPA-2 is required for the stability,behavior and morphology of the Spk and maintenance of regular apical growth in hyphae of N. crassa,although not for polarity or Spk establishment.

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1. Introduction

Polarization is a fundamental property in all living cells. Deter-mining and maintaining cell polarity are essential features for theappropriate development of any organism (Momany, 2002; Harrisand Momany, 2004). Once the polarization site is determined inthe cell, the machinery that enables polar growth is assembled(Virag and Harris, 2006a). In yeast, the early stages of buddingare described as a number of sequential, coordinated eventsorchestrated by a cascade of small GTPase modules in whichCdc42 is the key player in polarity establishment (Park and Bi,2007). The polarisome was identified in Saccharomyces cerevisiaeas a 12S multiprotein complex that contains Spa2p, Pea2p, Bni1pand Bud6p/Aip3p and it was proposed that this complex promotespolarized morphogenesis through regulation of the actin cytoskel-

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ontrast Spk; FM-Spk, FM4-64thase 1.

, San Ysidro, CA 92143-0222,

oria).

eton and signaling pathways (Sheu et al., 1998). In yeasts, the pola-risome components localize to the tips of growing buds andshmoos during polarized growth, remain at sites of growththroughout the cell cycle and are required for proper morphogen-esis (Gehrung and Snyder, 1990; Pruyne and Bretscher, 2000;Zheng et al., 2003; Shih et al., 2005). Recently, it has been observedthat Msb3p and Msb4p bind to the N-terminal of Spa2p, suggestingthat these proteins are also components of the polarisome (Tche-peregine et al., 2005). Spa2p interacts with numerous proteinssuch as those involved in the protein kinase pathway, actin–inter-acting proteins and with the other polarisome components (Sheuet al., 1998; Van Drogen and Peter, 2002; Shih et al., 2005; Viragand Harris, 2006b; Sudbery and Court, 2007). Deletion of SPA2 inS. cerevisiae revealed a reduced capacity to form shmoos and thus,spa2 mutants were defective in mating (Gehrung and Snyder,1990).

In fungal cells, polarized growth is maintained by several pro-tein complexes and selected components that establish the organi-zation and traffic of vesicles and organelles to the sites ofpolarization (Sheu and Snyder, 2001). A structure that is found onlyat the apex of actively growing hyphae is the Spitzenkörper (Spk).

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552 C.L. Araujo-Palomares et al. / Fungal Genetics and Biology 46 (2009) 551–563

The Spk is a highly dynamic and pleomorphic complex composedof vesicles and other cell components (including microtubules, ac-tin microfilaments and polysomes) that are involved in fungal cellwall growth and hyphal morphogenesis (Girbardt, 1957; Grove,1978; Howard, 1981; Bartnicki-García et al., 1989; Bartnicki-Gar-cía et al., 1995; Riquelme et al., 1998; Harris, 2006; Riquelmeet al., 2007). Although in some fungal species it has been reportedthat the Spk coincides temporally and spatially with polarity-re-lated components, it is not clear whether the function of the Spkoverlaps with the function of the polarisome (Harris et al., 2005;Virag and Harris, 2006a). In the human pathogen Candida albicansSpa2p (CaSpa2p) localizes to growth sites during both yeast andhyphal growth suggesting its role in polarized growth (Zhenget al., 2003; Crampin et al., 2005). The polarisome coexists withthe Spk in the tip of a C. albicans hypha, but apparently polarizedgrowth is driven by a different mechanism to that in yeast andpseudohyphae as it was suggested that the Spk and the polarisomeare distinct structures (Crampin et al., 2005). During yeast growthof Ustilago maydis Spa2 localizes to distinct growth sites in a cellcycle-specific manner and during hyphal growth this protein ispersistently localized to hyphal tips, suggesting a role of Spa2 fordetermination of the growth area (Carbó and Pérez-Martín, 2008).

The filamentous fungus Ashbya gossypii displayed AgSpa2p-GFPat hyphal tips, including tip branches and transitorily at sites ofseptum formation (Knechtle et al., 2003). In Aspergillus nidulansSpaA localizes at the apex of hyphae and phialides and althoughit was found that SpaA colocalizes with the Spk stained withFM4-64, it was suggested that SpaA acts partially independentlyof the Spk (Virag and Harris, 2006b). In Aspergillus niger SpaAwas suggested to play a role in ensuring maximal polar growth rate(Meyer et al., 2008). In this study we report the localization in vivoof SPA-2 labeled with GFP in N. crassa during germ tube elongation,hyphal growth and branching and explore its relationship with theSpk during these different morphogenetic processes.

2. Materials and methods

2.1. Strain and culture conditions

The strains used in this study are listed in Table 1. Unless other-wise stated, N. crassa was routinely grown and maintained at 28 �Cin plates containing Vogel’s Minimal Medium (VMM) (Vogel, 1956)supplemented with 2% (w/v) sucrose as the carbon source andsolidified with 1.5% (w/v) agar. To obtain macroconidia for trans-formation, N. crassa his-3� strain SMRP24 (FGSC #9717) was grownfor 10 days on VMM agar supplemented with histidine

Table 1Strains used and generated for this study.

N. crassastrain

Genotype Source orreference

N1 mat a; wt FGSC # 988N150 mat A; wt FGSC #9013N39 mat A; fl� FGSC # 4317N40 mat a; fl� FGSC # 4347N625 mat a; his-3 FGSC # 6525SMRP24 mat A; his-3 Dmus-51::bar+ FGSC # 9717NCLAP-140 mat A; Dspa-2 FGSC # 11140TNCLAP5-1 mat A; his-3+::Pccg-1::spa-2+::gfp+

heterokaryonThis study

NCLAP-2 mat a; his-3+::Pccg-1::spa-2+::gfp+ This studyNCLAP-3 mat a; his-3 Dspa-2 This studyTNCLAP-4 mat a; his-3+::Pccg-1::Dspa-2+::gfp+

heterokaryonThis study

NCLAP17-04 mat a; his-3+::Pccg-1::Dspa-2+::gfp+ This studyNJV12.1.1 mat A; his-3+::Pccg-1::chs-1+::mchfp+ Verdín et al.

(2008)

(0.5 mg ml�1) under white light. Transformed macroconidia werespread onto agar plates containing VMM plus FGS (0.5% fructose,0.5% glucose and 20% sorbose) and incubated at 30 �C for 3 days.To observe germinating macroconidia, N. crassa was grown andmaintained in test tubes. Once the strain produced sufficient mac-roconidia, they were harvested and resuspended in 5 ml of sterileVMM and filtered to remove any mycelial fragments. A constantvolume (10 ll) of conidial suspension (2.7 � 106 ml�1) was inocu-lated and spread uniformly on plates and incubated until observa-tion under the microscope.

2.2. Plasmid constructs and molecular techniques

N. crassa SPA2 homologue NCU03115.3 was identified byBLASTP search at the Broad Institute N. crassa genome database,using the A. nidulans spaA AN3815.2 open reading frame fromNCBI (National Center for Biotechnology Information) database(http://www.ncbi.nlm.nih.gov). The sequence NCU03115.3, re-named spa-2, is 2667 nucleotides long and encodes a predictedprotein of 888 amino acids, with a molecular mass of97.879 kDa. The amino acids sequence exhibited 37% similaritywith the tested sequence.

The spa-2 gene was amplified by PCR from N. crassa strain N1(FGSC #988) genomic DNA using custom designed primers withXbaI and PacI restriction endonuclease sites at the 50- and 30-ter-mini, respectively (Table 2). PCR was performed in a Bio-RadThermal Cycler (Model iCycler) with Platinum� Taq DNA Polymer-ase High Fidelity (Invitrogen). according to the manufacturer’sinstructions, under the following conditions: denaturation at94 �C for 1 min, followed by 30 cycles of 94 �C (30 s), 55 �C(30 s) and 72 �C (1:30 min), and a final extension step at 72 �Cfor 5 min. The amplified and gel-purified PCR products were di-gested with XbaI and PacI and cloned into XbaI- and PacI-digestedplasmid pMF272 (Freitag et al., 2004) (GenBank accession numberAY598428). This yielded pCLAP6 (spa-2). The insert was se-quenced at the Core Instrumentation Facility of the IntegrativeGenome Biology at the University of California, Riverside, withprimers pMF272F and pMF272R-2 (Table 2) designed for the up-stream and downstream flanks of the multiple cloning site ofpMF272.

Transformation of macroconidia of N. crassa strain SMRP24(FGSC #9717) with plasmid pCLAP6 (linearized with NdeI) was car-ried out by electroporation on a BioRad Gene Pulser (Model Xcell)(capacitance, 25 lF; voltage, 1.5 kV; resistance, 600 X; millisec-onds, 12–14 ms) as previously described (Margolin et al., 1997).Prototrophic His+ transformants were screened for the expressionof SPA-2-GFP by epifluorescence microscopy. A transformantshowing robust consistent fluorescence was selected and namedTNCLAP5-1. To obtain homokaryons TNCLAP5-1 was crossed withN. crassa strain N1 (FGSC #988) on synthetic crossing medium(SCM, Westergaard and Mitchell, 1947). Ascospores were heat-shocked on 1 M sorbitol at 60 �C for 1 h. Once they had germinatedthey were transferred to VMM slants grown for 2 days and ana-lyzed for fluorescence. One strain, NCLAP-2, was selected for fur-ther analyses.

For genomic DNA extraction of N. crassa, we used the DNeasyPlant extraction Kit (Qiagen, Inc.). Mycelium for DNA extractionwas grown for 7 days on VMM liquid medium with no shakingand no light, filtered, submerged in liquid nitrogen and lyophi-lized. Integration of the spa-2-gfp fusion was verified by PCR withtwo sets of primers, MRp10-11 and MRp12-13 (Table 2) to ampli-fy 3.2 and 2.1 kb fragments, respectively. The set of primersRDS478F and RDS482R (Table 2) was used as controls to amplifya 0.6 kb fragment to the small subunit 18S ribosomal DNA in fun-gi. For confirmatory Southern blotting analyses, Neurospora geno-mic DNA from strain NCLAP-2 was digested with EcoRI. The

http://www.ncbi.nlm.nih.gov

Table 2Oligonucleotides and plasmids used or generated in this study. Restriction endonuclease sites for cloning are shown in bold type and start codon is underlined.

Oligonucleotides Sequence Source or reference

Spa2-XbaI-F 50-TGCTCTAGAATGAATGTTCGCAATG-30 This studySpa2-PacI-R 50-CCTTAATTAATGCAAAGTCATCAGCGC-30 This studypMF272F 50-CAAATCAACACAACACTCAAACCA-30 Freitag et al. (2004)pMF272R-2 50-AGATGAACTTCAGGGTCAGCTTG-30 Riquelme et al. (2007)MRp10 50-AGAGACAAGAAAATTACCCCCTTCTT-30 Riquelme et al. (2007)MRp11 50-AACTACAACAGCCACAACGTCTATATC-30 Riquelme et al. (2007)MRp12 50-ATAATGAACGGAAGGTAGTTGTAGAAAG-30 Riquelme et al. 2007MRp13 50-ATGGATATAATGTGGCTGTTGAAAG-30 Riquelme et al. (2007)RDS-478F 50-GTGGTTCTATTTTGTTGGTTTCTA-30 Greene et al. (2000)RDS-482R 50-TAGCGCGCGTGCGGCCCAGA-30 Greene et al. (2000)

Plasmids Description Source or reference

pMF272 Pccg-1::sgfp+ Freitag et al. (2004)pCLAP6 Pccg-1::spa-2+::sgfp+ This study

C.L. Araujo-Palomares et al. / Fungal Genetics and Biology 46 (2009) 551–563 553

hybridization probe was amplified from genomic DNA by PCRusing primers Spa2-XbaI-F and Spa2-PacI-R (Table 2) and labeledwith a nonradioactive digoxigenin-dUTP (DIG high prime DNAlabeling and detection Starter kit II, Roche Applied Science). ForWestern blot analyses, macroconidia from N. crassa NCLAP-2(1 � 106 conidia/ml) were grown in Vogel’s Complete Mediumfor 14 h at 32 �C under agitation at 220 rpm. The mycelium washarvested by filtration through Whatman No. 1 paper, washedwith phosphate buffer (50 mM, pH 8.2), resuspended in phos-phate buffer containing protease inhibitors (Roche’s cocktail,one tablet per 100 ml) and mixed with dry glass beads(0.5 mm). The mycelium was disrupted in a Braun MSK homoge-nizer (Bronwill Scientific, Rochester, NY) for 30 s while the vesselwas cooled with liquid CO2. Crude cell-free homogenates werecentrifuged in a Multifuge 1S-R Heraeus centrifuge at 1000g for5 min at 4 �C. Supernatant was then centrifuged in a L8-70 Multracentrifuge Beckman at 54,000g for 45 min at 4 �C. Seventeenmicrograms of protein sample (Bio-Rad Protein Assay) were re-solved in a 10% polyacrylamide gel and electrotransferred to aPVDF membrane as previously described (Towbin et al., 1979).The membrane was reacted with anti-GFP-antibody conjugatedto HRP (Santa Cruz Biotechnology; 1:3000 dilution), incubatedwith enhanced chemiluminescence detection reagent (Pierce)and, finally, revealed by exposing on an enhanced chemilumines-cence hyperfilm (Amersham).

For complementation, N. crassa Dspa-2 strain NCLAP-140(FGSC #11140) was initially crossed with N. crassa strain N625(FGSC #6525) on SCM containing histidine. Germinated ascosp-ores were grown by duplicate in VMM with and without histi-

Fig. 1. Confirmatory integration of spa-2::gfp in the genome of N. crassa and expressiongenome of strain NCLAP-2 at the his-3 site arrows indicate the oligonucleotides used to chband, indicative of the integration, revealed by Southern blotting analyses; (c) PCR ampliSouthern and (e) Western blotting analyses of DNA and protein extracts from N. crassa

dine. One strain, NCLAP-3 (mat a; Dspa-2 his-3) was selectedand transformed using plasmid pCLAP6 linearized with NdeI. Pro-totrophic His+ transformants were screened for expression of SPA-2-GFP by epifluorescence microscopy. Transformant TNCLAP-4showing robust fluorescence was selected. To obtain homokar-yons, TNCLAP-4 was crossed with N. crassa strain N155 (FGSC#9013) on SCM. One strain NCLAP17-04 was selected for furtheranalyses.

2.3. Confocal live-cell imaging

Fungal cells were imaged using an inverted Zeiss Laser ScanningConfocal Microscope LSM-510 META. N. crassa cultures were ob-served using the inverted agar block method (Hickey et al.,2005). GFP expression was imaged with Argon-2 laser, Abs/Em488/515–530 nm. An oil immersion objective 100� (PH3)1.3 N.A. Plan Neofluar was used. A photomultiplier module allowedus to combine fluorescence with phase-contrast to provide simul-taneous view of the fluorescently labeled proteins and the entirecell. Confocal images were captured using LSM-510 software (ver-sion 3.2; Carl Zeiss) and evaluated with an LSM-510 Image Exam-iner (version 3.2). Some of the image series were converted intoanimation movies using the same software. Growth rate was mea-sured directly on the computer monitor using the measurementsoption of Image Pro Plus� (version 6.1). The obtained text fileswere then exported into Microsoft Excel spreadsheets and ana-lyzed. For fluorescence recovery after photobleaching (FRAP) anal-ysis, we used the bleach control command of the LSM-510software. Bleaching was applied during a time series sequence

of the SPA-2-GFP fusion. (a) Structure of the spa-2::gfp cassette inserted into theeck the insertion by PCR; (b) Schematic diagram showing the additional EcoRI 6.8 kbcons of 3.2 kb and 2.1 kb using primers MRp10–11 and MRp12–13, respectively; (d)NCLAP-2 strain expressing SPA-2-GFP. (a, b) Not to scale.


acquisition with 125–150 interactions and 95% laser intensity (Ar-gon-2 laser 458, 477, 488 and 514 nm wavelengths). The apicalarea of at least 30 hyphae and the subapical area of at least five hy-phae were photobleached.

Fig. 2. Colocalization of SPA-2-GFP and the Spk in N. crassa NCLAP-2 stained with FM4-6length (early phase III); (e–h) germling of 50 lm in length (late phase III); (i–l) 1 day-oldfluorescence; (c, g and k) SPA-2-GFP fluorescence; (d, h and l) overlapping of SPA-2-GFP anSPA-2-GFP (c, g and k). Scale bar = 10 lm.

Fig. 3. Colocalization of SPA-2-GFP and the Spk in mature hyphae of N. crassa stained wmature hypha of N. crassa NCLAP-2; (e–h) magnification of the apical region of the hyphacontrast microscopy; (b, f and j) FM4-64 fluorescence; (c, g and k) SPA-2-GFP fluorescenindicate accumulation of FM4-64 (a) and SPA-2-GFP (b), black arrow points at the PC-SpScale bar = 10 lm.

2.4. Spk staining

The vital dye FM4-64 (Molecular probes, Invitrogen) was usedas a marker of the Spk in germlings and mature hyphae. A con-

4 and observed by laser scanning confocal microscopy. (a–d) Germling of 29 lm inmature hypha (phase IV); (a, e and i) phase-contrast microscopy; (b, f and j) FM4-64d FM4-64 fluorescence. White arrows indicate accumulation of FM4-64 (f and j) and

ith FM4-64 and observed by laser scanning confocal microscopy. (a–d) one day-oldin a–d; (i–l) one day-old mature hypha of N. crassa NCLAP17-04; (a, e and i) phase-ce; (d, h and l) overlapping of SPA-2-GFP and FM4-64 fluorescence. White arrows

k and arrowheads at the bright spot at the base of the SPA-2-GFP fluorescence area.


centrated stock solution of the dye was diluted in liquid VMM tothe appropriate working solution concentration (25 lM). Thesolution was allowed to warm up to room temperature before

Fig. 4. Colocalization of SPA-2-GFP and CHS-1-mCherryFP in a mature hypha of N. crassCHS-1-mCherryFP fluorescence; (c) overlapping of SPA-2-GFP and CHS-1-mCherryFP fluoCHS-1 (red) distribution in the apical dome. Scale bar = 10 lm.

Figure 5. Time series by laser scanning confocal microscopy showing the retraction of the(d–f) SPA-2-GFP fluorescence; (g–i) overlapping of SPA-2-GFP and FM4-64 fluorescence. Athe hyphoid shape during the retraction (b, e and h). Scale bar = 20 lm (see Supplemen

applying to growing cells. VMM (10 ll) containing FM4-64 wereplaced on coverslips and the sample was placed over it accordingto the inverted agar block method. Coverslips with the sample

a observed by laser scanning confocal microscopy. (a) SPA-2-GFP fluorescence; (b)rescence; (d) Model showing the partial colocalization (yellow) of SPA-2 (green) and

FM-Spk and SPA-2-GFP in a hypha of N. crassa NCLAP-2. (a–c) FM4-64 fluorescence;rrow indicates the retraction of the FM-Spk (b) and SPA-2-GFP (e). Notice the loss of

tary movie 1).


were kept at room temperature for 10 min and then analyzed un-der the confocal microscope using an Argon-2 laser, Abs/Em 514/670 nm.

3. Results

3.1. Selection of positive transformants

N. crassa putative prototrophic transformants that expressedSPA-2-GFP were screened by epifluorescence microscopy. Selectedstrain TNCLAP5-1 was crossed to N. crassa N1. One homokaryoticstrain, NCLAP-2, showing stable fluorescence was selected for fur-ther analyses by laser scanning confocal microscopy. Introductionof spa-2-gfp fusion into N. crassa NCLAP-2 genome at his-3 sitewas confirmed by PCR and Southern blot analyses. Expected 3.2and 2.1 kb PCR products were obtained with primers MRp10-11and MRp12-13, respectively, indicative of the integration (Fig. 1aand c). Confirmation of non-ectopic integrations was conductedby Southern analysis. The host strain revealed a single expected11.3 kb band, while NCLAP-2 displayed an additional 6.8 kb band,

Fig. 6. Time series by laser scanning confocal microscopy showing the retraction and tem(a–g) SPA-2-GFP fluorescence; (h–n) phase-contrast microscopy. White arrow indicatescorresponding PC-Spk. Note the loss of hyphoid shape during the retraction and the bulgi(see Supplementary movie 2).

indicative of the integration (Fig. 1b and d). Western blot analysisof NCLAP-2 extracts revealed a single expected 124.8 kDa band,indicative of the expression of the SPA-2-GFP fusion protein(Fig. 1e).

3.2. SPA-2-GFP localizes at the tips of growing germlings before a Spkcan be detected

To investigate the colocalization of SPA-2-GFP with the Spk, thestrain NCLAP-2 was stained with the vital dye FM4-64. The distri-bution of SPA-2-GFP and FM4-64 stained Spk was determined inliving cells during germ tube elongation (phase III) and mature hy-phal growth (phase IV). A discrete fluorescent accumulation ofSPA-2-GFP was detected in the form of a crescent shape at the apexof germlings from early phase III onwards (in germlings of morethan 24 lm in length; Fig. 2a–h). In mature hyphae the distributionof SPA-2-GFP was clearly concentrated at the apex of the cells,coinciding with the position of the PC-Spk (Fig. 2j and l).

After 10 min of exposure, FM4-64 was internalized into the cellsof N. crassa. SPA-2-GFP appeared at the apices of germ tubes earlier

porary disappearance of the PC-Spk and SPA-2-GFP in a hypha of N. crassa NCLAP-2.the reduced SPA-2-GFP remaining after the retraction (d); black arrow points at theng that remains in the morphology of the hypha (arrowhead in n). Scale bar = 10 lm


than FM-Spk (Fig. 2). In later phases (late III and IV) the dye notonly stained the plasma membrane but also accumulated in the re-gion corresponding to the Spk in both germlings and maturehyphae.

3.3. The Spk colocalizes partially with SPA-2-GFP at the cells tips

In mature hyphae it was more evident that the Spk and SPA-2-GFP displayed a slightly different accumulation pattern. In mostmature cells, SPA-2-GFP fluorescence adopted a hand fan shapewith a brighter spot at the base (Fig. 3c, g and k) while FM4-64staining unveiled a round to amorphous body (Fig. 3b, f and j) thatpartially co-localized with SPA-2-GFP fluorescence (Fig. 3d, h andl). The core of the Spk has been proven to contain different chitinsynthases (CHS) in N. crassa (Riquelme et al., 2007; Sánchez-León et al., 2008). To compare the localization of SPA-2-GFP rela-tive to the Spk core, we fused NCLAP-2 with N. crassa NJV12.1.1,a strain that expresses CHS-1 labeled with mCherryFP (Verdínet al., 2008). It was found that in effect, SPA-2 colocalizes onlypartially with the Spk (Fig. 4). It appears that SPA-2 is highly con-centrated at the core of the Spk, and from that point outward itspreads radially until reaching the apical plasma membrane(Fig. 4c and d).

In addition to the partial colocalization of the Spk and SPA-2-GFP, they showed a close interaction at the cell apex (i.e. changes

Fig. 7. Time series by laser scanning confocal microscopy of SPA-2-GFP localization in thethe apex of the cell; (b) displacement of SPA-2-GFP to the right and detachment of a SPA-2resumes central position; (d–h) gradual accumulation of SPA-2-GFP in the subapex and dthe branch emergence site. Scale bar = 10 lm (see Supplementary movie 3).

in the position of the Spk were accompanied by changes in the po-sition of the SPA-2-GFP). This interaction was more noticeablewhen the PC-Spk suffered a displacement from its typical anteriorposition and the distribution of SPA-2-GFP was clearly observed todisplace along with the PC-Spk (Fig. 5 and Supplementary movie1). Occasionally, the displacement of the PC-Spk and SPA-2-GFPlead to the disassembling of the apical body, which coincided witha complete cessation of further growth (Fig. 6 and Supplementarymovie 2). During this event a spot-like arrangement of SPA-2-GFPremained at the center of the displaced PC-Spk (Fig. 6d and k) andthe formation of a lateral bump coincided with the site of retrac-tion (Fig. 6l and m). After this episode, both the PC-Spk and SPA-2 vanished from the cell apex (Fig. 6e and l) and, shortly after,the hypha resumed elongation rate with both SPA-2 and the PC-Spk resuming their typical position (Fig. 6f–n; Supplementary mo-vie 2). Clearly, the retraction of both the PC-Spk and SPA-2 lead tochanges in hyphal morphology. This suggests that SPA-2 is part ofthe Spk.

3.4. The role of SPA-2-GFP during branching and septum formation inmature hyphae

Branching and septum formation in N. crassa were monitored inmature hyphae by time lapse series. During the formation of newbranches an almost imperceptible accumulation of SPA-2 was de-

emerging site of a new lateral branch in N. crassa. (a) Accumulation of SPA-2-GFP in-GFP fragment; right-lower corner magnification of the apical region; (c) SPA-2-GFP

evelopment of a lateral branch. White arrows indicate accumulation of SPA-2-GFP at


tected at the site of branch emergence and sometimes it was noteven detected due to differences in the focal plane (Fig. 7 and Sup-plementary movie 3). Before a new branch emerged we were ableto detect a detachment of a small fragment of SPA-2-GFP (Fig. 7and Supplementary movie 3). During this episode SPA-2-GFP wasslightly displaced from its typical central position, leaving behinda small fragment (Fig. 7b). Subsequently SPA-2 resumed its typicalcentral position at the apical dome (Fig. 7c). We assume that theSPA-2 detachment was involved in marking the site of branchemergence since a new branch started to form at this site(Fig. 7d–h).

To determine whether the protein SPA-2 was involved in sep-tum formation we used the endocytic marker FM4-64. Duringthe development of septa the distribution of SPA-2-GFP and theSpk-FM4-64 was identical at the tips of main hyphae and branches(Fig. 8a–f) but no accumulation of SPA-2-GFP was detected at theseptation sites (Fig. 8g–l and Supplementary movie 4).

3.5. Analysis of hyphal morphology in Dspa-2 strain

N. crassa Dspa-2 strain (NCLAP-140) displayed some differencesin colony morphology compared with the other strains used in thisstudy. The first obvious difference was a reduced colony diameterat 24 h of incubation. After 48 h the colony developed an aerialmicelial mass distributed in rays emerging from the center of thecolony (Fig. 9). N. crassa Dspa-2 strain was analyzed using thedye FM4-64 to reveal cell and FM-Spk morphology. During earlydevelopmental stages (phases I–III) cells of N. crassa Dspa-2(NCLAP-140) had comparable morphology to the wild type andSPA-2-GFP strains (data not shown). However, mature hyphaeshowed irregular growth generating distorted hyphae with unevenconstrictions (Fig. 10). The Spk had an abnormal behavior with er-ratic movements (Supplementary movie 5). Growth rate of theDspa-2 strain was reduced compared to NCLAP-2 strain(0.39 ± 0.09 lm/s, n = 405 and 0.64 ± 0.18 lm/s, n = 410, respec-tively) (Table 3). Number of branches was higher (3.4 ± 0.7) andcolony diameter was reduced when compared to the other strains

Fig. 8. Septum formation in cells of N. crassa NCLAP-2 stained with FM4-64 by laser scan(red); (d–f) development of septa. White arrowheads indicate the Spk, black arrowhead bSupplementary movie 4).

used in the study (Fig. 9). To determine the functionality of theSPA-2-GFP fusion protein, the Dspa-2 strain of N. crassa (NCLAP-140) was complemented by transformation with the plasmidpCLAP6 that contained the spa-2::gfp fusion under the control ofthe Pccg-1 promoter. This plasmid was integrated into the Dspa-2strain genome at the his-3 site (NCLAP-3). Analysis of the restoredstrain (NCLAP17-04) showed normal growth rate (Table 3) and cellmorphology (Fig. 3i–l), proving a functional SPA-2-GFP fusion andsuggesting that irregular hyphal growth with numerous branching(Fig. 9c, g and k), and abnormal Spk behavior in the Dspa-2 mutantstrain was caused by the lack of the spa-2 gene.

3.6. SPA-2 is possibly synthesized at the cell apex

FRAP analysis was conducted to determine the source of SPA-2-GFP found at the apex. Apical and subapical areas were selected inmature hyphae of the NCLAP-2 strain. In the apical region, the Spkwas included and exposed to a high-intensity laser irradiation.Photobleaching in the subapical area did not produce any changesin the intensity of fluorescence of SPA-2-GFP at the tip (Fig. 11g–l).However, after photobleaching of the GFP at the cell apex, fluores-cence decreased followed by an immediate recovering (Fig. 11a–f).These results suggest that SPA-2-GFP is likely synthesized at the tipof the cell.

4. Discussion

In some fungal species homologues of Spa2p have been identi-fied as one of the components of the polarisome (Knechtle et al.,2003; Crampin et al., 2005; Virag and Harris, 2006b; Carbó andPérez-Martín, 2008; Meyer et al., 2008). The intracellular localiza-tion of this protein has been reported at the apex of fungal hyphaebut there is no information on how this protein relates to theontogeny and behavior of the Spk, a key structure in determininghyphal morphology and growth. The role and distribution ofSpa2 were analyzed in the filamentous fungi A. gossypii, A. nidulans,A. niger and the basidiomycete Ustilago maydis where the protein

ning confocal microscopy. (a–c) Colocalization of SPA-2-GFP (green) and the FM-Spkranch development and white arrows septum development. Scale bar = 20 lm (see

Fig. 9. Radial colony phenotype of N. crassa grown at 28 �C. (a, e and i) SMRP24; (b, f and j) NCLAP-2 strain (SPA-2-GFP); (c, g and k) NCLAP-140 strain (Dspa-2); (d, h and l)NCLAP17-04 strain (Dspa-2 spa-2::gfp). (a–d) 24 h of incubation; (e–h) 48 h of incubation; (i–l) branching pattern at the edge of the colony. Scale bar = 1 mm.


localizes at the tips of hyphae (Knechtle et al., 2003; Virag and Har-ris, 2006b; Carbó and Pérez-Martín, 2008; Meyer et al., 2008). N.crassa is a model filamentous fungus that because of its hyphal sizeand growth rate has proven to be ideal for studying Spk morphol-ogy and behavior by live-cell microscopy. In N. crassa we deter-mined that SPA-2 was also localized exclusively at the apex ofgrowing cells. Our FRAP results suggest that SPA-2 travels as mRNAto the apex where protein synthesis occurs. The cell apex containsa great quantity of polysomes, which are part of the Spk (Girbardt,1969; Howard and Aist, 1979; Howard, 1981; Harris, 2006) sug-gesting that some translation of mRNA during protein synthesis oc-curs at the hyphal apex (Steinberg, 2007).

Although localization of Spa2 has been reported in the apex offilamentous fungi hyphae, some differences have been found inthe possible role that this protein plays in the species analyzed.In A. niger SpaA was localized at the tips of germlings and was de-tected in conidia as an accumulation at certain areas suggestingthat SpaA could possibly mark the site of germ tube emergenceand could play a role in the establishment of a new axis (Meyer

et al., 2008). In germlings of A. gossypii and A. niger SPA-2 was local-ized at the cellular apex during the elongation phase and before aSpk could be detected (Knechtle et al., 2003; Meyer et al., 2008).This could suggest that after polarisome establishment and actinnucleation, more vesicles arrive and accumulate at the polarizationsite until a Spk is visible. In A. nidulans SpaA was present at the tipsof germlings but its localization during the germinating process ofthis fungus was not analyzed (Virag and Harris, 2006b). The anal-ysis of the expression of polarisome components during spore ger-mination is important to determine the role of the polarisome inpolarity establishment. We analyzed the temporal expression ofSPA-2-GFP in N. crassa during phases I and II and we did not ob-serve accumulation of fluorescence in the apical zone (data notshown). Although previous studies have shown that both endoge-nous and ccg-1 driven expression of several GFP tagged proteinsbehave very similarly (Freitag personal communication; unpub-lished results), these results are not conclusive since SPA-2-GFPwas only expressed under the control of the ccg-1 promoter. Theproteins required for bud selection site in yeasts are Rsr1p, Bud5p

Fig. 10. Time series by laser scanning confocal microscopy showing irregular hyphal morphology and erratic Spk position in a mature hypha of N. crassa NCLAP-140 (Dspa-2).(a–e) FM4-64 fluorescence; (f–j) phase-contrast microscopy. Scale bar = 10 lm (see Supplementary movie 5).

Table 3Hyphal extension rate and number of branches of the N. crassa strains used in this study.

No. N. crassa strain Characteristic No. of hyphae analyzed Growth rate (lm s�1) No. of branchesa,b

SMRP24 his-3 Dmus-51::bar+ 415 0.46 ± 0.02 2.5 ± 0.5NCLAP-140 Dspa-2 405 0.39 ± 0.09 3.4 ± 0.7NCLAP-2 spa-2::gfp 410 0.64 ± 0.18 2.0 ± 0.7NCLAP17-04 Dspa-2 spa-2::gfp 420 0.60 ± 0.12 2.0 ± 0.7

a 30 Hyphae analyzed.b Branches/0.5 mm from apex inwards.


and Bud2p (Irazoqui and Lew, 2004). In filamentous fungi,although there is not much information on which proteins areresponsible for tube emergence selection site, different structuresand complexes such as the polarisome, landmark proteins andthe Spk have been implicated in the establishment and mainte-nance of polarity (Harris and Momany, 2004). Cell end marker pro-teins such as TeaA and TeaR were highly concentrated in the Spk ofA. nidulans. Sterol-rich membrane domains localize also at the celltip and are considered to determine destination of those cell endmarker proteins and thereby polarized growth (Takeshita et al.,2008). Rho GTPases have been described in yeasts as essential pro-teins for establishment and maintenance of cell polarity (Benderand Pringle, 1989; Johnson and Pringle, 1990; Matsui and Toh-e,1992; Drgonová et al., 1996; Imai et al., 1996; Kamada et al.,1996; Robinson et al., 1999). One of these proteins is Cdc42 (Adams

et al., 1990; Ayscough et al., 1997), which is a crucial factor in theswitch from isotropic to polarized growth. Also, for Cdc42 activa-tion the guanidine nucleotide exchange factor Cdc24 is required(Ruggieri et al., 1992; Bender, 1993; Zheng et al., 1994, 1995; Parket al., 1999). In A. nidulans Cdc42 is generally required for polarityestablishment events except for the formation of the primary axisof hyphal growth. The localization of SpaA is not altered by the ab-sence of Cdc42, suggesting the possibility that the polarisomecould be a Cdc42 effector in euascomycetes (Virag et al., 2007).However, it has been suggested that Rac1 could substitute Cdc42and recruit the polarisome or that there may be redundant mech-anisms that ensure proper localization of the polarisome indepen-dently of Cdc42 or Rac1.

SPA-2 accumulation in subapical regions of hyphae of filamen-tous fungi has been reported to occur as small patches at initiation

Fig. 11. Time series by laser scanning confocal microscopy showing SPA-2-GFP distribution in N. crassa after photobleaching in apical (a–f) and subapical (g–l) areas. Scalebar = 10 lm.


sites for lateral branches (Knechtle et al., 2003; Bauer et al., 2004).We observed light accumulations of SPA-2-GFP at branch emer-gence sites. Also at times a SPA-2-GFP detachment followed bybranching was noticed. This suggested that SPA-2 might be in-volved in the branching process. However, further analysis of theN. crassa Dspa-2 strain, which branches profusely, suggested thatSPA-2 although localized at emerging branches, is not essentialfor branching. Somehow the absence of SPA-2 may create a mislo-calization of secretory vesicles and as a result there is an increasein the amount of branches.

Previous reports in S. cerevisiae concluded that Spa2p was in-volved in septum formation (Gehrung and Snyder, 1990). In C. albi-cans Spa2p distribution at septation sites was rare (Zheng et al.,2003). In A. gossypii AgSpa2 localizes both at hyphal tips and sep-tation sites (Knechtle et al., 2003). In N. crassa SPA-2 distributionat septation sites was not found as reported for A. nidulans and A.niger (Virag and Harris, 2006b; Meyer et al., 2008).

In mature hyphae of N. crassa it has been demonstrated thatgrowth direction is determined by the Spk (Brunswik, 1924; Gir-bardt, 1957; Bracker et al., 1997; López-Franco and Bracker,1996; Riquelme et al., 1998; Riquelme et al., 2000; Bartnicki-Gar-cía, 2002). The Spk is a structure formed by an accumulation ofmacrovesicles, microvesicles and ribosomes (McClure et al.,1968; Girbardt, 1969; Grove and Bracker, 1970; Howard, 1981;

Riquelme et al., 2007). The use of genetic approaches has allowedus to gain initial insight into the molecular composition of theSpk (Riquelme et al., 2007). In germlings of N. crassa the Spk couldnot be observed by phase-contrast microscopy (Araujo-Palomareset al., 2007). Therefore the vital dye FM4-64 was used to determinethe colocalization of SPA-2-GFP with the Spk. It was observed thatboth the Spk (FM-PC) and SPA-2-GFP partially co-localized at thecellular apex. We analyzed also the relationship of the FM-Spkand SPA-2 in mature hyphae of N. crassa and found that SPA-2-GFP and the FM-Spk partially co-localized at the cells tips. In addi-tion, when the FM-Spk disappeared from the cellular apex SPA-2-GFP also disappeared, or changes in growth direction occurred asreported previously for mature hyphae (Brunswik, 1924; Girbardt,1957; Bracker et al., 1997; López-Franco and Bracker, 1996; Riqu-elme et al., 1998; Riquelme et al., 2000; Bartnicki-García, 2002)and for germlings of N. crassa (Araujo-Palomares et al., 2007).These results differ from those reported by Virag and Harris(2006b), who mentioned that when the FM-Spk disappears, SPA-2-GFP persists at the tips.

These observations suggest that the polarisome forms part ofthe Spk as previously suggested (Harris et al., 2005) and confirmthat SPA-2 is required for Spk integrity (Crampin et al., 2005). Ag-Spa2 is essential for fast radial colony growth in A. gossypii and itwas suggested that the function of AgSpa2p could be in the orga-


nization of incorporating secretory vesicles at the tip (Knechtleet al., 2003). These vesicles surround the core, a region that cor-responds to the central part of the Spk and that is rich in F-actin(Harris et al., 2005; Howard, 1981). The strong accumulation atthe base of the hand fan shape distribution of SPA-2-GFP in N.crassa, might reflect that SPA-2 is participating in the nucleationof actin filaments in the Spk core. The dispersion of SPA-2 fromthe Spk core to the tip of the hypha, seen as a hand fan shape,suggests that the actin filaments are in continuous nucleation toorganize and incorporate secretory vesicles until reaching theplasma membrane at the tip of the cell. The fact that the typicaldistribution of SPA-2-GFP was not clearly seen in young germ-lings of N. crassa, could be due only to the smaller cell diameterof germlings in which SPA-2-GFP was observed as a crescentshape.

It has been suggested that the polarisome mediates the nucle-ation of actin cables (Lew and Read, 1995; Sheu et al., 1998) andparticipates in F-actin polarization, which in turn guides Spk vesi-cles to the cellular apex (Steinberg, 2007). N. crassa hyphae lackingSPA-2 displayed a temporal loss of polarity shown as isotropicgrowth that is reflected by the repetitive enlargements of thehyphae, producing distorted cell morphology. The lack of SPA-2apparently destabilizes the polarisome causing a less efficientassemblage of actin microfilaments and the consequent reducednumber of vesicles arriving to the plasma membrane. The reducednumber of vesicles arriving to the hyphal tip results in a smalleramount of released Ca2+ (Virag and Griffiths, 2004). It has beensuggested that a shallower gradient of Ca2+ results in vesicles moreevenly incorporated at the hyphal tip, creating conditions for addi-tional vesicle accumulation and incorporation that eventually re-sult in dichotomous branching (Virag and Griffiths, 2004). TheDspa-2 strain did not show dichotomous branching, but comparedwith the other strains used in this study a higher number ofbranches were produced. This might reflect as previously men-tioned a lower number of vesicles arriving at the cell apex and inconsequence an altered Spk and gradient of Ca2+. These observa-tions strongly suggest that for N. crassa an intact polarisome isrequired for the integrity, stability, behavior and morphology ofthe Spk which in turn maintains regular hyphal growth. The Spkcould be envisioned as a dynamic structure composed of manyinteracting protein complexes, one of which is the polarisome(Harris et al., 2005). Our findings support this hypothesis; itremains to be determined other constituents of the polarisome,the interaction among them and with other Spk components andits role in cell morphology of N. crassa.

Acknowledgements

This work was supported by a SEP-CONACYT grant (CB-2006-1-61524). We also thank the Consejo Nacional de Ciencia y Tec-nología for a grant to the first author and the Fungal Genetics StockCenter and the Neurospora Genome Project for strains. We wouldlike to thank M. Ferry, M Freitag, J. Verdín and E. Sánchez-Leónfor providing plasmids and strains. We are grateful to G. Amadorand Y.V. Saenz-Aguilar for assistance with Fig. 4d.

Appendix A. Supplementary material

Supplementary data associated with this article can be found, inthe online version, at doi:10.1016/j.fgb.2009.02.009.

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The polarisome component SPA-2 localizes at the apex of Neurospora crassa and partially colocalizes with the Spitzenkörper