Calcium-regulated sinusoidal hyphal growth 1157 Fig. 1. Induction of regular sinusoidal and helical hyphae of C. albicans by growth on surfaces in low-nutrient conditions. Sinusoidal curves were induced in hyphae of the control strain by growth on a poly-L-lysine-coated slide in 1% serum. Hyphae were stained with the chitin-speciﬁc brightener, Calcoﬂuor White (A). Other hyphae were imaged using scanning confocal microscopy to view hyphal projections that had been stained with the lipophilic dye, FM4-64. Three-dimensional images were constructed and sequential frames are shown for hyphae rotated through the X-plane by 180° (B and C). When grown on 4% (w/v) agar containing 20% (v/v) serum, most hyphae grew as two-dimensional sinusoidal curves that could be shown to be growing in the plane of the substrate when observed as end-on projections (B). However, two examples are shown in C where hyphae formed three-dimensional helical loops that extended above the plane of the substrate. Scale bars = 10 mm. Mid1, and sequestration in the Golgi compartment via the Ca2+/Mn2+ ATPase, Pmr1 (Antebi and Fink, 1992; Iida et al., 1994; Lapinskas et al., 1995; Sorin et al., 1997; Bates et al., 2005; Brand et al., 2007). Sinusoidal growth initiation was measured in cch1D and mid1D mutants, which have reduced Ca2+ uptake, and in the pmr1D strain, which, in Saccharomyces cerevisiae, accumulates abnor- mally high levels of free cytosolic Ca2+ due to constitutive activation of Mid1-Cch1 (Halachmi and Eilam, 1996; Locke et al., 2000). Deletion of CCH1, which encodes a homologue of the human L-type voltage-gated Ca2+ channel, CaV1.2, reduced sinusoidal growth formation by 50%, a similar level as that observed for Ca2+ chelation (46%) (Fig. 3A). Cch1 is thought to be regulated by the putative stretch-activated Ca2+ channel, Mid1, which acts as a key mechanosensor of topographical changes during the thigmotropic response (Brand et al., 2007). Deletion of MID1 resulted in a more severe phenotype than deletion of CCH1 because sinusoidal growth initiation in mid1D and in the mid1D/cch1D double mutant was reduced by approximately 80% compared with the control strain (P 0.001) (Fig. 3A). In addition, the morphology of the sinusoidal curves formed by mid1D mutants differed to that of control cells (see below). Sinusoidal growth initia- tion was also severely reduced in the presence of vera- pamil, a blocker of voltage-gated calcium channels (Hockerman et al., 1997), and, to a lesser degree, the presence of gadolinium, a blocker of stretch-activated © 2009 The Authors Journal compilation © 2009 Blackwell Publishing Ltd, Molecular Microbiology, 71, 1155–1164