which in turn can result in misfolding or mislocalization of cell surface proteins (Gibson et al., 1981; Machamer and Rose, 1988; Bates et al., 2005). We attempted to distin- guish whether the dramatic loss of sinusoidal growth in pmr1D was due to perturbation of calcium ion homeostasis or alterations to glycosylation by testing mutants with spe- ciﬁc N- or O-mannosylation defects. Disruption of N-linked mannan had a pronounced effect on the propensity to form sinusoidal hyphae. The localization of Mid1 at the plasma membrane is dependent on N-mannosylation in Saccha- romyces cerevisiae (Ozeki-Miyawaki et al., 2005) and the mid1D mutant was markedly compromised in its ability to initiate sinusoidal growth. Therefore, the straight hyphae observed in the pmr1D mutant may be a pleiotropic phe- notype due to alterations in both the cell surface of the hyphae and the calcium signalling required for contact- sensing. In S. cerevisiae, some pmr1D and mid1D pheno- types can be rescued by supplementation with Mn2+ or Ca2+ (Antebi and Fink, 1992; Loukin and Kung, 1995; Dürr et al., 1998). In C. albicans, 10 mM Mn2+ or Ca2+ rescued station- ary phase viability in pmr1D (Bates et al., 2005). However, we were unable to restore sinusoidal growth in pmr1D by supplementation with various concentrations of these cations. Taken together with our observation that perturba- tion of the cell wall had a less signiﬁcant effect on the initiation of curved growth, it is likely that addition of extra- cellular ions further perturbed the intracellular calcium gradient required for normal polarized growth. Similarly, supplementation did not restore the mid1D/cch1D pheno- type, which is consistent with previous ﬁndings (Brand et al., 2007). Initiation of sinusoidal growth was inﬂuenced by the rigidity of the agar substrate and the presence of an additional carbon source but sinusoidal wavelength and amplitude were not affected by these parameters. Changes in sinusoidal hypha morphology were only observed in C. albicans mutant strains. Chs3 is respon- sible for synthesis of short, rod-like chitin ﬁbrils in the lateral wall of C. albicans (Lenardon et al., 2007). The induction rate of sinusoidal hypha formation in chs3D was the same as the control strain but the amplitude of its sinusoidal hyphal compartments was greater. This effect was not observed when hyphae were treated with Calcof- luor White, which destabilizes hydrogen bonding between chitin ﬁbrils (Elorza et al., 1983). Changes in sinusoidal hypha morphology were also observed in the mid1D mutant. In the absence of PMR1, hyphae grew in straight trajectories under conditions that would otherwise induce sinusoidal growth. Re-integration of a single copy of PMR1 partially overcame the block to sinusoidal hypha initiation. Hyphae of the re-integrant strain formed sinu- soidal hyphae with a shorter wavelength than the control strain. The switch to sinusoidal growth therefore appears to involve calcium signalling and to be affected by pro- Calcium-regulated sinusoidal hyphal growth 1161 cesses that may affect the normal distribution and con- centration of intracellular calcium. The mechanisms underlying the formation of regular sinusoidal curves and helices are unknown but, in addi- tion to substrate rigidity, low nutrient availability is a key inducer in fungi that naturally exhibit this behaviour, such as C. albicans, S. ﬁmicola and Saprolegnia ferax (Madelin et al., 1978; Kaminskyj and Heath, 1992; Sherwood- Higham et al., 1995). In other ﬁlamentous fungi, helicity has been observed as a result of mutations that lead to defective delivery of membranous organelles to the hyphal tip, or alter the size, position or mobility of the Spitzenkörper (Wu et al., 1998; Riquelme et al., 2000; Takeshita et al., 2008). The positioning of the Spitzen- körper within the hyphal tip is thought to predict the site of new tip growth (Riquelme et al., 1998). The remarkable regularity of the sinusoidal curves formed by C. albicans hyphae, together with the observed tendency for septa to be localized at alternate apices (Fig. 5), suggest that the Spitzenkörper oscillates in two dimensions across the longitudinal growth axis in a cell cycle-dependent manner. In the green alga, Chara globularis, relocalization of Ca2+ channels and the tip-high calcium gradient are suggested to precede and mediate changes in tropic growth direction by displacing the Spitzenkörper (Braun and Richter, 1999). Our observation that disruption of calcium homeo- stasis by manipulation of Ca2+ availability or by Ca2+ channel deletion severely affects the normal initiation and morphology of sinusoidally curved growth in C. albicans hyphae is consistent with this view. The function and relevance of sinusoidal growth in fungal hyphae is not known. It has been proposed that such directional growth offers greater exploration of the environment than unidirectional growth (Sherwood- Higham et al., 1995; Garrill, 2000). In the pathogenic oomycete, S. ferax, helicity and other changes in hyphal morphology are affected by the presence of bacterial polysaccharides but the relevance of this in vivo is unclear (Kaminskyj and Heath, 1992). C. albicans is an opportu- nistic human pathogen and yeast cells colonize endothe- lial and epithelial surfaces prior to the ensuing process of tissue invasion. To our knowledge, hyphae have not been observed to produce regular sinusoidal or helical growth in tissue sections derived from biopsies or from mouse models of infection, but adhered hyphae have been imaged undergoing directional changes in order to pen- etrate the underlying semisolid tissue in a reconstituted model of oral epithelium (Zakikhany et al., 2007). As uni- directional hyphal growth would not represent the optimal strategy for tissue penetration and ramiﬁcation, it is pos- sible that partial helix formation in the form of hypha tip rotation is a mechanism that enables hyphae to facilitate inﬁltration into a relatively immunologically protected and nutrient-rich host tissue. © 2009 The Authors Journal compilation © 2009 Blackwell Publishing Ltd, Molecular Microbiology, 71, 1155–1164