affected by the addition of 1.5 mM Ca2+ (Fig. 3A), the limiting extracellular [Ca2+] for helix formation must lie between 1.5 and 3.5 mM. In summary, the reduction of Ca2+ uptake using channel blockers or supplementation of the growth medium with Ca2+ or Mn2+ did not rescue the mutant phenotype of pmr1D but did perturb sinusoidal hypha formation in the control strain. The phenotype of the mid1D/cch1D mutant was similarly unaffected by Ca2+ or Mn2+ supplementation. Sinusoidal growth and the calcium signal transduction pathway In fungi, increased levels of cytosolic calcium activate the calcium signalling pathway via the phosphatase, cal- cineurin, and its transcription factor, Crz1 (Stathopoulos and Cyert, 1997; Onyewu et al., 2004; Karababa et al., 2006). Deletion of either calcineurin subunit, the catalytic Cmp1 or the regulatory Cnb1, in C. albicans results in hypersensitivity to calcium in serum (Blankenship and Heitman, 2005). In accord with these ﬁndings, neither mutant formed hyphae on 20% serum in this study. Dele- tion of CRZ1, which encodes the calcium-dependent tran- scription factor that is activated by calcineurin, reduced sinusoidal hypha formation by 46%, to a similar level as that seen for cch1D (50%) (P 0.001) (Fig. 3A). The tran- scription of genes that are controlled by changes in cyto- solic calcium levels via the calcineurin–Crz1 signalling pathway may therefore contribute to surface sensing or sinusoidal hypha formation. Cell wall composition and sinusoidal hypha initiation In addition to high intracellular calcium levels reported in S. cerevisiae (Halachmi and Eilam, 1996), the phenotype of the pmr1D mutant in C. albicans also results in changes in the composition of the cell wall due to altered surface mannosylation. The outer wall glycoproteins of pmr1D have severely truncated N- and O-linked mannans, accompanied by a compensatory increase in the percent- age of the cell wall that is comprised of glucan (Bates et al., 2005). The pmr1D mutant is also hypersensitive to various cell wall perturbing agents, despite the constitu- tive activation of the cell wall integrity pathway in this strain. To determine whether sinusoidal growth was dependent on normal cell wall structure, initiation was measured in mutants with altered levels of chitin, b-glucan or mannosylation. In addition, control cells were treated with reagents that weaken cross-linking within structural wall polymers, resulting in altered wall integrity. Both the chs3D mutant, which has an 85% reduction in lateral wall chitin (Bulawa et al., 1995), and fks1D, a glucan synthase mutant which has a 50% reduction in cell wall glucan levels and a threefold increase in chitin content (Mio et al., Calcium-regulated sinusoidal hyphal growth 1159 Fig. 4. The effect on curved hypha formation of cell wall-perturbing reagents or deletion of genes involved in cell wall biosynthesis or protein mannosylation. Hyphal growth was induced on solid agar medium at 37°C and the number of primary hyphae forming two or more consecutive sinusoidal waves was determined (error bars are means SD, n = 100 hyphae from three independent experiments). 1997; Walker et al., 2008) (Fig. 4), initiated normal sinu- soidal hyphal curves. Wild-type cells were grown in the presence of 200 mg ml-1 Congo Red or 50 mg ml-1 Cal- coﬂuor White, which disrupt hydrogen bonding between polymeric chains of b-glucan and chitin, respectively. This disruption reduced sinusoidal hypha formation by 30% (P = 0.001) and 25% (P = 0.009), respectively (Fig. 4). Because both N- and O-mannosylation were affected in the pmr1D mutant, we attempted to distinguish whether truncation of one or other glycan correlated with the loss of helix formation. To examine the effect of O-mannan trun- cation, we tested the mnt1D/mnt2D double mutant, where O-linked mannans are truncated at Man2 (Munro et al., 2005). To test N-mannan truncation, we used the mns1D strain, where reduced Golgi a-mannosidase activity results in the truncation of outer-chain N-glycans and loss of phosphomannan (Mora-Montes et al., 2007). Helicity was reduced by 34% when O-mannan was truncated (P 0.001) (Fig. 4) and this phenotype was rescued by the re-integration of MNT1. Although the mns1D mutant was reported to form normal hyphae in liquid 20% serum, the mutant did not form hyphae on solid 20% serum. In the MNS1 re-integrant strain, which has a single copy of MNS1, morphogenesis was normal and helicity was par- tially restored (Fig. 4). Similar results were observed for the mns1D/MNS1 heterozygous strain (data not shown). Taken together, these results suggest that altered manno- sylation status, particularly the lack of outer-chain elabo- ration of N-linked structures, affected helix formation to a greater extent than the disruption of chitin and b-glucan. Regulation of helix morphology Each sinusoidal hypha generally consisted of one cell compartment (Fig. 5). The morphology of hyphal helices © 2009 The Authors Journal compilation © 2009 Blackwell Publishing Ltd, Molecular Microbiology, 71, 1155–1164