Molecular Microbiology (2009) 71(5), 1155–1164 doi:10.1111/j.1365-2958.2008.06592.x First published online 20 January 2009 Calcium homeostasis is required for contact-dependent helical and sinusoidal tip growth in Candida albicans hyphae Alexandra Brand, Keunsook Lee, Veronica Veses and Neil A. R. Gow* Aberdeen Fungal Group, School of Medical Sciences, Institute of Medical Sciences, University of Aberdeen, Aberdeen, UK. Summary Hyphae of the dimorphic fungus, Candida albicans, exhibit directional tip responses when grown in contact with surfaces. On hard surfaces or in liquid media, the trajectory of hyphal growth is typically linear, with tip re-orientation events limited to encoun- ters with topographical features (thigmotropism). In contrast, when grown on semisolid surfaces, the tips of C. albicans hyphae grow in an oscillatory manner to form regular two-dimensional sinusoidal curves and three-dimensional helices. We show that, like thigmot- ropism, initiation of directional tip oscillation in C. al- bicans hyphae is severely attenuated when Ca2+ homeostasis is perturbed. Chelation of extracellular Ca2+ or deletion of the Ca2+ transporters that modulate cytosolic [Ca2+] (Mid1, Cch1 or Pmr1) did not affect hyphal length but curve formation was severely reduced in mid1D and cch1D and abolished in pmr1D. Sinusoidal hypha morphology was altered in the mid1D, chs3D and heterozygous pmr1D/PMR1 strains. Treatments that affect cell wall integrity, changes in surface mannosylation or the provision of additional carbon sources had signiﬁcant but less pronounced effects on oscillatory growth. The induction of two- and three-dimensional sinusoidal growth in wild-type C. albicans hyphae is therefore the consequence of mechanisms that involve Ca2+ inﬂux and signalling rather than gross changes in the cell wall architecture. Introduction The growth direction and behaviour of apically growing cells are determined by interactions that occur between the Accepted 20 December, 2008. *For correspondence. E-mail n.gow@ abdn.ac.uk; Tel. (+44) 1224 555879; Fax (+44) 1224 555844. Re-use of this article is permitted in accordance with the Creative Commons Deed, Attribution 2.5, which does not permit commercial exploitation. © 2009 The Authors Journal compilation © 2009 Blackwell Publishing Ltd growing tip and the surrounding environment. While many of the components that establish and maintain cell polarity are highly conserved in apically growing eukaryotic cells, the responses to environmental stimuli are adapted to the life style of the organism, suggesting that sensing and signalling pathways are hard-wired to the intracellular growth machinery in a species-speciﬁc manner. An ob- served feature of anisotropic growth is a tendency to form spiral, sinusoidal or helical curves. Curved growth in tip- growing systems is contact-dependent and observed only during growth on semisolid surfaces. The pronounced clockwise or anticlockwise spiral growth of hyphae ema- nating from a fungal colony is a two-dimensional growth pattern that has been observed in over 22 species of fungi, including Aspergillus nidulans and Sordaria ﬁmicola (Madelin et al., 1978; Lundy et al. 2001) and the oomycete, Achlya bisexualis (Trinci et al., 1979). The formation of three-dimensional helices by hyphae has been described as right-handed for the aerial hyphae of Trichophyton species (Davidson and Gregory, 1937) and the sporangio- phores of Phycomyces blakesleeanus (Trinci et al., 1979). The gravitropic response of primary roots in Arabidopsis thaliana can be subverted to form right-handed helices on contact with hard-agar surfaces (reviewed by Migliaccio and Piconese, 2001). The hyphae of the dimorphic fungal pathogen, Candida albicans, produce regular two- and three-dimensional growth forms on semisolid surfaces. Unlike spiral growth around a colony, where the trajectory of growth is perma- nently offset to one side of the longitudinal growth axis, the trajectory of C. albicans hyphal tips oscillates to produce highly regular, two-dimensional sinusoidal waves. C. albicans hyphae are also observed to grow as helices on agar and Cellophane, where invariant right- handed helices are formed (Sherwood-Higham et al., 1995). Curved growth is thought to involve the displace- ment of the hyphal apex relative to its axis (Madelin et al., 1978; Sherwood-Higham et al., 1995) but the molecular mechanisms are poorly understood. Spiral growth cannot be attributed to electrical or gravitational ﬁelds (Ritchie, 1960), but the requirement for surface contact-sensing, or thigmotropism, is common to all three growth forms. On hard surfaces such as quartz, C. albicans hyphae (grown in 20% v/v serum, 2% w/v glucose) meander slightly but the growing tips are sensitive to obstacles in the