1158 A. Brand, K. Lee, V. Veses and N. A. R. Gow Fig. 2. The initiation of curved growth in C. albicans hyphae is affected by substrate rigidity (A) or the availability of a carbon source (B). Hyphae were grown on 5% or 20% (v/v) serum and concentrations of agar varied from 1% to 6% (A). Hyphae were grown on 20% (v/v) serum 2% (w/v) glucose, maltose or sorbitol at 37°C for 10 h (B). In both experiments, hyphae were viewed by light microscopy and the number of primary hyphae over 80 mm in length that formed two or more consecutive sinusoidal curves was expressed as a percentage of all hyphae formed (error bars are means SD, n = 100 hyphae from three independent experiments). calcium channels in mammalian cells (Caldwell et al., 1998). No sinusoidal growth was observed in the pmr1D strain, where the primary hypha and the branches grew straight, in a rod-like fashion (Fig. 3B). Re-integration of one copy of PMR1 partially rescued this phenotype, but sinusoidal growth formation was nevertheless 65% lower than the control strain (P 0.001) (Fig. 3A). The pmr1D-like, high cytosolic [Ca2+] phenotype was ameliorated in Ustilago maydis and S. cerevisiae by decreasing or increasing the availability Ca2+ respectively (Halachmi and Eilam, 1996; Adamiková et al., 2004). When hyphae of the C. albicans pmr1D mutant were grown in the presence of 250 mM verapamil together with 500 mM gadolinium in order to reduce Ca2+ entry into the cell, the hypha elongation rate of pmr1D was reduced but the hyphae retained their markedly straight morphology (data not shown). In S. cerevisiae, supplementation of the medium with calcium (10 mM) or manganese (250– 420 mM) ameliorated mid1D or pmr1D mutant phenotypes (Antebi and Fink, 1992; Iida et al., 1994; Dürr et al., 1998). Similarly, supplementation with 10 mM CaCl2 or MnCl2 rescued loss of yeast viability at stationary phase in the C. albicans pmr1D mutant (Bates et al., 2005). Formation of sinusoidal hyphae of the control strain, mid1D/cch1D and pmr1D, was tested in the presence of 10 mM Ca2+ or Mn2+. Morphogenesis was repressed in all three strains grown in 10 mM Mn2+, while in 10 mM Ca2+ morphogen- esis was observed but hypha elongation was inhibited. When [Mn2+] and [Ca2+] were reduced to 100 mM and 3.5 mM respectively, hypha elongation was partially restored in the mutants. In the control strain, hyphal length was fully restored but hyphae meandered (data not shown). As sinusoidal growth in the control strain was not Fig. 3. The effect of varying calcium availability and deletion of calcium-related genes on the initiation of sinusoidal growth (A). Hyphal growth was induced on solid agar medium at 37°C and the number of primary hyphae forming two or more consecutive regular sinusoidal waves was determined (error bars are means SD, n = 100 hyphae from three independent experiments). Light microscopy of hyphae formed by the control strain (B, upper panel) and the pmr1D mutant (B, lower panel) during growth on 20% (v/v) serum solidiﬁed with 4% agar. Bars = 10 mm. © 2009 The Authors Journal compilation © 2009 Blackwell Publishing Ltd, Molecular Microbiology, 71, 1155–1164