1156 A. Brand, K. Lee, V. Veses and N. A. R. Gow substratum. The response to contact with such obstacles is an immediate re-alignment of the hyphal growth axis (Brand et al., 2007). This form of thigmotropism is calcium-dependent and is attenuated by blockade of calcium signalling pathways, for example by chelation of extracellular calcium or by deletion of the stretch- activated calcium channel, Mid1. This channel is thought to act as a mechanosensor for external, contact-mediated interactions and may also serve to mark the site of new tip expansion via localized calcium inﬂux. Mid1 is a putative regulator of the voltage-gated calcium channel, Cch1, and together they control calcium inﬂux into the cell (Fischer et al., 1997). Calcium ions are an important second mes- senger in developmental and stress signalling pathways, where a rise in cytosolic calcium activates the calcium- dependent signalling pathway via the phosphatase, cal- cineurin (Stathopoulos and Cyert, 1997; Onyewu et al., 2004) and the calcineurin-dependent transcription factor, Crz1 (Karababa et al., 2006). Fungal cells maintain a low cytosolic calcium concentration of ~100 nM by expelling ions from the cytosol into intracellular organelles, such as the Golgi or the vacuole (reviewed by Cunningham and Fink, 1994) and may potentially also expel calcium ions from the cytosol across the plasma membrane to the exterior. This ensures that relatively low calcium ﬂuxes can result in signiﬁcant changes in cytoplasmic Ca2+ con- centration and hence high responsiveness of the signal- ling pathway. In this study, we asked whether the contact- dependent initiation of sinusoidal growth in C. albicans hyphae was dependent on calcium inﬂux and intracellular homeostasis. We ﬁnd that both the initiation and morphol- ogy of sinusoidal curves responded to changes in envi- ronmental conditions and we report that, as with other tropic responses of this organism, calcium signalling and homeostasis are required for normal sinusoidal growth. Results Initiation of sinusoidal growth is dependent on substrate solidity Candida albicans formed primary hyphae that contained two or more consecutive sinusoidal curves when grown on 20% fetal bovine serum (FBS) solidiﬁed with 1%, 2%, 4% or 6% agar (Fig. 1A and B). Helical growth was also observed under these conditions (Fig. 1C). Curve forma- tion on an agar concentration of 1% (w/v) was only half that observed in optimal conditions (P 0.001), demonstrating that hyphae are sensitive to the ﬁrmness of the substratum (Fig. 2A). Preliminary experiments demonstrated that the optimal agar concentration for the formation of sinusoidal hyphae was 4% (w/v). On this surface, zones of sinusoidal growth were observed in 57 4.1% (SD; n = 3) of wild- type hyphae and subsequently this agar concentration was used in all experiments. On 4% (w/v) agar, lowering the serum concentration from 20% to 5% (v/v) only slightly reduced the number of hyphae producing curves, suggest- ing that a certain minimal amount of serum is required as an inducer of morphogenesis but serum concentration did not markedly affect sinusoidal growth initiation (Fig. 2A). There were no differences in the propensity to form sinu- soidal curves between hyphae of the control strain, CAI4/ CIp10, which provides a common genetic background for the deletion mutants used in this study (Brand et al., 2004), its wild-type parent, the clinical isolate SC5314 (Fonzi and Irwin, 1993), and a further wild-type strain, 3153 A (Mack- enzie and Odds, 1991) (data not shown). Carbon source affects sinusoidal growth initiation It has been observed that sinusoidal growth in hyphae occurs in conditions of limited nutrient availability (Sherwood-Higham et al., 1995). To test this further, hyphae were grown in 20% serum supplemented with an additional carbon source in the form of 2% glucose or 2% maltose. To control for the effect of increased osmotic pressure, 2% sorbitol was added as an additional treatment. Sinusoidal initiation decreased signiﬁcantly in the presence of glucose or maltose (P 0.001) but the presence of sorbitol produced no signiﬁcant effect. This suggested that the provision of a utilizable carbon source, but not an increase in osmotic pressure, can reduce the onset of sinusoidal growth (Fig. 2B). Extracellular Ca2+ is required for normal levels sinusoidal growth initiation Thigmotropism, or contact-sensing, on hard surfaces in C. albicans is attenuated in conditions where extracellular [Ca2+] is low (< 5 mM) (Brand et al., 2007). To assess whether Ca2+ ﬂux is involved in the formation of sinusoidal hyphae, the control strain was grown in conditions of varying Ca2+ availability. The [Ca2+] in the 20% FBS used in this study was 0.7 mM, consistent with previous ﬁndings for Ca2+ levels in serum (Blankenship and Heitman, 2005). Sinusoidal growth initiation was not affected by the addition of 1.5 mM Ca2+ to serum but was signiﬁcantly reduced when the Ca2+ in serum was chelated using 1.5 mM 1,2- bis(2-aminophenoxy)ethane-N,N,N′,n′-tetraacetic acid (BAPTA) (Fig. 3A). This effect was reversed by supple- mentation with 1.5 mM Ca2+. An accessible supply of exog- enous Ca2+ is therefore required for maximal initiation of sinusoidal hyphal growth. Deletion of channels involved in Ca2+ homeostasis severely reduces sinusoidal growth formation Ca2+ homeostasis depends on uptake from the environ- ment via plasma-membrane calcium channels, Cch1 and © 2009 The Authors Journal compilation © 2009 Blackwell Publishing Ltd, Molecular Microbiology, 71, 1155–1164