1160 A. Brand, K. Lee, V. Veses and N. A. R. Gow Fig. 5. Variation in the morphology of sinusoidal curves in gene deletion mutants compared with the control strain. Hyphal growth was induced on 4% solid agar medium at 37°C and the wavelength (l) and amplitude (A) of sinusoidal waves were determined using Openlab software (means SD, n = 100 helices in three independent experiments). Bar = 10 mm. was characterized by measuring the amplitude (A) and wavelength (l) of > 100 sinusoidal compartments per strain for hyphae that were longer than 80 mm. For the control strain, the mean values for wavelength and ampli- tude were 21.9 1.26 mm and 6.8 0.5 mm respectively (mean SD, n = 100). A variety of environmental condi- tions, such as agar concentration, and isogenic mutations affected the propensity to form sinusoidal hyphae, but did not affect the morphology of the curves that were formed. The exceptions were mutants with deletion of MID1 (mid1D, mid1D/cch1D), CHS3 or strains that were het- erozygous for PMR1 (pmr1D/PMR1, pmr1D+PMR1). In the mid1D and chs3D mutants, wavelength (l) was unal- tered compared to control hyphae but the amplitude (A) was lower in mid1D strains (P = 0.008), and higher in the chs3D mutant (P = 0.017). In contrast, the amplitude of the pmr1D+PMR1 re-integrant strain was the same as that of control strain, but the sinusoidal compartments were of shorter wavelength (P = 0.02). To compare the straight pmr1D hyphae further with the control strain, the mean cell length (distance between septa) and hypha length were determined after 8 h growth. In the control strain, there was no difference between sinusoidal and straight hyphae in cell compartment length or total hyphal length after 8 h growth [28.4 0.76 mm and 136 24.2 mm respectively (SD; n = 100)]. In the pmr1D mutant, cell length and hypha length were signiﬁcantly shorter at 14.8 0.89 mm (P = 0.03) and 78 0.11.5 mm (P = 0.33) respectively. Discussion Growing C. albicans hyphae respond to external stimuli such as electric ﬁelds (galvanotropism) and changes in substrate topography (thigmotropism) by re-orienting their growth axis in a calcium-dependent manner (McGillivray and Gow, 1986; Crombie et al., 1990; Lever et al., 1994; Watts et al., 1998; Brand et al., 2007; 2008). Thigmotro- pism is thought to rely on the presence of the putative stretch-activated plasma-membrane calcium channel, Mid1, which may act as a mechanosensor of external obstacles and changes in surface topography. We have proposed that activation of Mid1 allows localized calcium inﬂux to determine a new site for hyphal extension by causing an asymmetry in the tip-high calcium gradient observed in many apically extending polarized eukaryotic cells (Brand et al., 2007). Chelation of extracellular calcium or deletion of MID1 severely curtailed the ability of hyphal tips to respond to contact-dependent stimuli. The formation of regular sinusoidal curves and helices is a further example of a contact-dependent directional response observed in hyphal tips, because it is induced by growth on semisolid surfaces. In this study, we show that the factors that are important for thigmotropism are similarly involved in helix formation. However, unlike gal- vanotropism, which was heightened by the elevation of extracellular Ca2+ concentration, thigmotropism and sinu- soidal hypha formation were not enhanced by this treatment. In contrast, chelation of extracellular calcium reduced the response of hyphae to all three tropic stimuli. Calcium chelation, deletion of CCH1 or the calcium- dependent transcription factor CRZ1, or the addition of gadolinium, a blocker of stretch-activated calcium chan- nels, reduced the formation of helices to a similar extent. The reduction in helicity was more severe in the mid1D mutant than the cch1D mutant. This is consistent with the more pronounced attenuation of thigmotropism of this mutant relative to that in cch1D (Brand et al., 2007) and suggests a key role for this protein in transducing surface- dependent environmental signals. Mutations and treatments that affected the skeletal com- ponents of the cell wall had a relatively small effect on the induction of sinusoidal growth. In contrast, this was com- pletely abolished by deletion of PMR1, which encodes a Golgi-located Ca2+/Mn2+ ATPase that supplies cofactors for glycosidases involved in glycan maturation (Bates et al., 2005). The PMR1 deletion resulted in alterations in intrac- ellular cytoplasmic Ca2+ distribution (Halachmi and Eilam, 1996), and the truncation of N- and O-linked mannans, © 2009 The Authors Journal compilation © 2009 Blackwell Publishing Ltd, Molecular Microbiology, 71, 1155–1164