Hyphal Orientation of Candida albicans Is Regulated by a Calcium-Dependent Mechanism

Summary Eukaryotic cells from fungal hyphae to neurites that grow by polarized extension must coordinate cell growth and cell orientation to enable them to exhibit growth tropisms and to respond to relevant environmental cues. Such cells generally maintain a tip-high Ca2+ cytoplasmic gradient, which is correlated with their ability to exhibit polarized tip growth and to respond to growth-directing extracellular signals 1, 2, 3, 4, 5. In yeast and other fungi, the polarisome, exocyst, Arp2/3, and Spitzenkörper protein complexes collectively orchestrate tip growth and cell polarity, but it is not clear whether these molecular complexes also regulate cell orientation or whether they are influenced by cytoplasmic Ca2+ gradients. Hyphae of the human pathogenic fungus Candida albicans reorient their growth axis in response to underlying surface topography (thigmotropism) [6] and imposed electric fields (galvanotropism) [7]. The establishment and maintenance of directional growth in relation to these environmental cues was Ca2+ dependent. Tropisms were attenuated in media containing low Ca2+, or calcium-channel blockers, and in mutants where calcium channels or elements of the calcium signaling pathway were deleted. Therefore galvanotropism and thigmotropism may both be mediated by localized Ca2+ influx at sites of polarized growth via Ca2+ channels that are activated by appropriate environmental signals.


Supplemental Experimental Procedures
Strains and Growth Media Strains used in this study are listed in Table S1. Strains were maintained on YPD 1% (w/v) yeast extract, 2% (w/v) mycological peptone, 2% (w/v) glucose, and 2% (w/v) technical agar at room temperature. Media were supplemented with 2% (w/v) glucose or 2% (w/v) maltose for repression and induction of MRP1-regulated genes, respectively. For galvanotropism assays, strains were grown in modified Soll's medium (MSM) [S1-S3] containing 5 mM PIPES, where the medium was modified by removal of all chloride salts and by an increase of medium resistivity to 800-1000 U cm by 10-fold dilution of the total salt content. From overnight cultures at pH 4.5 and 25 C, hyphae were induced by transfer of inocula into 500 ml MSM [pH 7.5] for 6 hr. The temperature was maintained at 37 C 6 1 C by circulation through a heat exchanger. The medium was supplemented with organic blockers of L-type calcium channels, the calcium chelator, BAPTA (1,2-bis(o-aminophenoxy)ethane-N,N,N 0 ,N 0tetraacetic acid), CaSO 4 , or 5 mg/ml of the calcineurin inhibitor, FK506, as appropriate. For thigmotropism assays, strains were grown overnight in SC (0.67% [w/v] yeast nitrogen base [YNB] with amino acids, 2% [w/v] glucose) at 30 C, and hyphae were induced by transfer into 20 ml 20% (v/v) newborn-calf serum and 2% (w/v) glucose at 37 C. Concentrations of 1-10 mg/ml FK506 were added for the inhibition of calcineurin in wild-type cells. For growth-rate measurements, Ca 2+ -accumulation assays, and colony morphology determinations, strains were grown in SD-Ca 2+ (2% [w/v] glucose, 0.67% [w/v] YNB with ammonium sulfate but without CaCl 2 ). The Figure S1. Colony Morphology of C. albicans Ca 2+ -Channel and Ca 2+ -Signaling Mutants Yeast cells (5 3 10 4 ) were spotted on to minimal (YNB) solid medium and grown with or without added Ca 2+ (10 mM). After 17 days' incubation on Ca 2+ -depleted solid minimal medium, wild-type C. albicans colonies start to produce abberant lobes, which can be alleviated by the addition of 10 mM Ca 2+ to the medium. In contrast, emerging colonies of the Cacch1D and Camid1D mutants were lobed. This morphology was alleviated by reintegration or induced expression of the gene (right) but not by supplementation with Ca 2+ , suggesting that exogenous Ca 2+ was not available to the mutant cells. The morphology of Cafig1D mutant was not lobed but instead invaded the underlying agar. The Cacrz1D mutant was the same as the wild-type, and the calcineurin mutants (Cacna1D and Cacnb1D) were partially lobed. The aberrant morphologies of the Cafig1D, Cacna1D, and Cacnb1D mutants were partially alleviated on supplementation with exogenous Ca 2+ , supporting the view that these genes are involved in calcium signaling in C. albicans. Panels depict the following: (A) control strain, and (H) Cacnb1D. Asterisks denote growth on maltose for inducing CaCCH1 in conditional strain. The scale bar represents 10 mm.
only source of Ca 2+ ions in this medium was 0.8 mM Ca 2+ -pantothenoate, supplementation with Ca 2+ where stated, or 45 Ca 2+ in uptake assays.
Construction of Cacch1, Camid1, and Cafig1 Mutants Single homologs of ScCCH1, ScMID1, and ScFIG1 were identified in the C. albicans genome sequence (http://genolist.pasteur.fr/ CandidaDB/) as orf19.3298 (a 6765 bp gene on chromosome 1), orf19.3212 (a 1677 bp gene on chromosome 5), and orf19.138 (a 798 bp gene on chromosome 6), respectively. CaMID1 and CaCCH1 were disrupted in strain CAI4 in accordance with the standard Urablaster method [S4, S5]. For CCH1, the downstream region of the gene was amplified with primers 5 0 -GATCCATACGATGTGACGA-3 0 and 5 0 -GGACATACTATGCTCTCC-3 0 and cloned into the Ura-blaster plasmid, pMB-7, to generate plasmid pVC2. The upstream region of the gene was amplified with primers 5 0 -CTAGACCGAATTCAAGCT-3 0 and 5 0 -TCTCGCACACGTGATAGC-3 0 and cloned into pVC2 to generate plasmid pVC3. pVC3 was linearized by digestion with PvuII and transformed into CAI4 to delete 69% of the gene, including all 24 transmembrane domains and the four pore-forming domains. Three rounds of transformation with the Ura-blaster cassette were required for deleting all wild-type copies of CaCCH1. Trisomy of genes located on chromosome 1 of C. albicans has been reported previously [S6, S7]. For the mid1D mutant, an upstream sequence of 593 bp (bp 2-594) was amplified by PCR with a SacI site at the 5 0 end and a BglII site at the 3 0 end, with primers 5 0 -TGATACCACCTTTT ATTCTACTA-3 0 and 5 0 -CGATTAGTATTTTCCGGTGCTC-3 0 , and cloned into the Ura-blaster vector, pMB-7, for the creation of plasmid pMB7-CaMID-UP. A downstream sequence of 695 bp, containing the last 371 bp of MID1, a 5 0 SalI site, and a 3 0 HindIII site was amplified with primers 5 0 -GGTTATGTGCCGTTACTATACCTC-3 0 and 5 0 -GATGATTATTTGTGGGTTTTATAC-3 0 and cloned into pMB7-CaMID-UP to generate pSGS2, which was linearized by digestion with HindIII and SacI and used for transforming C. albicans strain CAI4. A region comprising 58% of the gene was deleted, including transmembrane domain H3 that is essential for Mid1 function in S. cerevisiae [S8]. CaFIG1 was disrupted by amplification of the dp1200 URA3 mini-blaster cassette from plasmid pDDB57 [S9] by the use of primers with 19 bp homology to the cassette (underlined) and 111 bp homology to CaFIG1 (Biomers, Ulm, Germany): 5 0 -GAG ATATAAAATGAATTTACCATTGAAATTCACCATTGTATTCACAATAA TAATTCAATTCATAACTGCTGCGTTATTAAGTTTCTTATTACTTGGA TGTATAGATACTTCTTTTCCCAGTCACGACGTT-3 0 and 5 0 -GCAGAA ATTTGTACACTAAATTTTTGTATCCACCAGTGGTTGTGGTGGGTGT TGCAGTGATTTTCCATTCAATTTACTCATTCTAATTTCCATCCACCA TGATAAACAAGCTGTGGAATTGTGAGCGGATA-3 0 . The resulting product was transformed into CAI4, resulting in the deletion of bp 102-702 (75% of the gene).

Growth Curves and Ca 2+ Accumulation
Cells were grown overnight in SD-Ca 2+ at 30 C with shaking. For yeast-growth assays, cells were washed and diluted to OD 600 = 0.1 in a 96-well plate (5 wells/strain). Growth was measured spectrophotometrically over 16 hr at 30 C. The length of hyphae grown over 6 hr were measured with Openlab 2.0 software, and the extension rate was calculated. For Ca 2+ accumulation (adapted from [S14]), cells were washed and diluted to OD 600 = 1 in 8 ml fresh medium and cell concentrations determined with an Improved Neubauer hemocytometer. Cells were incubated for 2 hr at 30 C, pelleted, and resuspended in 8 ml fresh medium supplemented with 0.6 MBq (megabecquerel) 45 Ca 2+ (8 ml of 74 MBq/ml, Amersham Biosciences, Buckinghamshire, UK). The OD 600 was determined at t = 0 and t = 2 hr, when triplicate samples of 2 ml were filtered with Whatman GC/ C filters wetted with medium containing 5 mM calcium and washed with 17 ml of the same. 45 Ca 2+ accumulation was determined by scintillation counting and reported as the mean pmol 45 Ca 2+ accumulated per 10 9 cells 6 SD from three independent experiments.

Scanning Electron Microscopy
Cells were adhered to ridged quartz slides, grown for 6 hr in 20% (v/v) FCS and 2% (w/v) glucose, and fixed with 2.5% glutaraldehyde in 0.1 M phosphate buffer [pH 7.4]. Cells were postfixed with 1% osmium tetroxide, dehydrated in 70%, 90%, 95%, and 100% ethanol, and critical-point-dried in CO 2 . The slide was mounted on a stub and sputter-coated with gold. Cells were viewed in a JEOL35CF scanning electron microscope at a voltage of 10 kV.