calcium channels have not been identified previously in c. albicans , but high - affinity and low - affinity calcium - uptake systems ( hacs and lacs ) have been described and partially characterized in s. cerevisiae 
 [ 8 , 9 ] .
we identified and deleted in c albicans , homologs of components of these systems  the voltage - gated cch1p channel   and the stretch - activated channel mid1p .
together , these two channels are thought to form a complex that defines the hacs .
cacch1 encodes a putative 2254 amino acid protein with 38.4% identity to its s. cerevisiae homolog . the 24 predicted transmembrane ( tm ) regions in cacch1p are arranged in four repeated units ( i to iv ) of six tm domains , as they are in mammalian calcium channels where they tetramerize to form the core 1-subunit of l - type ca channels .
the tm regions include segments responsible for voltage - dependency , channel - specificity , and association with organic calcium - channel blockers .
the c. albicans cch1p and the human voltage - gated calcium channel cav1.2 are 62.9% similar and 37.7% identical over a 20 amino acid region in the four ca selective , pore - forming p domains . in the voltage - sensitive s4 domains , 13 of the 23 basic residues in  cav1.2
the camid1 gene sequence had 36.9% and 34.4% identity to scmid1 and schizosaccharomyces pombe yam8(+ ) , respectively .
the 559 amino acid protein it encodes contains four putative tm regions ( h1 - 4 ) , potential n - glycosylation sites , a helix - loop - helix domain and 10 conserved cysteines that , in s. cerevisiae , form the c1/c2 domains that are essential for activity and localization [ 11 , 16 ] . unlike in s. cerevisiae , the c1/c2 regions in c. albicans are located between h3 and the c - terminal h4 .
cafig1p shares 48.5% identity with scfig1p , a putative homolog of mammalian pmp-22/emp / mp20/claudins , which are involved in the trafficking and assembly of membrane - associated proteins .
consistent with emp homology , cafig1p has four predicted n - glycosylation sites in the first of its four tm domains .
the role of scfig1p in s. cerevisiae is not well - defined , but it localizes predominantly to the plasma membrane   and is required for low - affinity calcium transport and for the calcium - dependent fusion of mating projections .
control strains were created by the generation of conditional mutants expressing a single remaining wild - type gene from the mrp1 maltose - regulatable promoter ( cch1 or mid1 ) or by reintegration of the gene at a high - expression locus ( fig1 or mid1 ) ( see the experimental procedures in the supplemental data available with this article online ) . in s. cerevisiae , hacs is activated by low ca conditions and is regulated by calcineurin , which controls calcium homeostasis and specific stress responses [ 811 , 18 ] .
scfig1p ( lacs ) activity was only revealed under conditions when hacs was inhibited by rich media .
in contrast to hacs , lacs is insensitive to calcineurin and its affinity for ca is 16-fold lower .
however , both systems are activated on exposure of cells to -pheromone , which stimulates the formation of the polarized mating projection [ 8 , 19 ] .
localization of scmid1p and scfig1p to the mating projection is dependent on scspa2 and scbni1 
 [ 19 , 8 ] . in c. albicans ,
caspa2p and cabni1p are components of the hyphal polarisome and spitzenkrper , respectively , and are required for polarized hyphal extension . mid1p and fig1p may therefore be involved in polarized cell growth in both organisms . in c. albicans , mrna encoding hacs and lacs component proteins
was detected in both yeast and hyphal growth conditions and in c. albicans - infected rabbit kidney ( data not shown ) . also , both hacs and lacs mutants were affected in the thigmotropic response ( see below ) . therefore ,
both hacs and lacs are expressed in c. albicans during the normal in vitro and in vivo growth of this fungus . after prolonged incubation on ca - depleted solid minimal medium ( > 14 days ) , wild - type c. albicans colonies produced aberrant lobed margins that could be alleviated by the addition of 10 mm ca to the medium .
emerging colonies of the cacch1 and camid1 mutants produced lobed colonies at 2 days ( see supplemental data ) .
the aberrant morphologies of the colonies of cafig1 , cacna1 , and cacnb1 mutants were partially alleviated on supplementation with exogenous ca , supporting the view that these genes are involved in calcium signaling in c. albicans .
the growth rates of the cacch1 , camid1 , and cacch1-mid1 mutants were reduced by 19% , 23% , and 25% , respectively , when they were grown in the yeast form , compared to the control strain ( p = < 0.037 ) , but the extension rates of hyphae were not affected by the mutations ( data not shown ) .
consistent with the putative roles of camid1p and cch1p as ca channels , ca accumulation in the camid1 and cacch1 mutants was significantly reduced after 2 hr culture in ca - depleted medium supplemented with ca ( p = < 0.001 ; supplemental data ) .
the double cacch1-mid1 mutant had similar ca accumulation and yeast growth rates to the single mutants , consistent with the model that these proteins operate within the same pathway .
this is consistent with reports that , in s. cerevisiae , cch1-mid1p are involved in ca homeostasis under low - ca conditions , where fig1p activity is not detectable .
tropic growth responses to applied external electric fields ( galvanotropism ) have been observed in migratory and tip - growing cells .
growing c. albicans hyphae orient toward the cathode in such fields . to characterize hyphal orientation , we measured the angle at which germ tubes emerged from the mother cell ( emergence angle ) and the angle of the hyphal tip after 6 hr growth ( final angle ) relative to the cathode . to investigate the role of calcium ions and channels in galvanotropism , we measured the emergence and final angles of hyphae exposed to electrical fields in media of varying extracellular [ ca ] or in the presence of pharmacological agents that block the activity of l - type voltage - gated cation channels . in c. albicans ,
site selection of germ tubes is not strictly controlled by the bud proteins that regulate bud - site selection during axial and bipolar budding of s. cerevisiae 
 .
we observed that , in electric fields , germ tubes were formed almost exclusively on the cathode - facing pole of cells , suggesting that imposed electrical fields can override cortical bud evagination markers .
the percentage cathodal germ - tube emergence was positively correlated with extracellular [ ca ] .
cathodal orientation of wild - type cells was attenuated in ca - depleted medium ( figures 1a and 1b ) and was further significantly reduced in the presence of calcium - channel blockers ( p = < 0.05 ) ( figure  2a ) .
therefore , ca influx is important for cathodal evagination of the germ tube in c. albicans . localized ca uptake correlates with sites of germination of other cell types , such as the zygotes of the brown alga silvetia compressa 
 .
even in low ca medium , extending hyphae grew toward the cathode and reoriented their direction of growth when the field polarity was reversed ( figure  1c ) .
deletion of cacch1 resulted in a significant reduction in the cathodal orientation of germ - tube emergence ( p  =  < 0.001 ) ( figure  2b ) .
cathodal orientation was restored in the conditional cacch1 strain when cacch1 expression was induced by growth on maltose but not when camid1 was induced in the cch1/mid1/mrp1-mid1 strain .
we hypothesize that the voltage - gated cacch1p ca channel is activated by membrane depolarization at the cathodal face of yeast cells and that this results in localized ca uptake and subsequent induction of localized polarized growth .
the effects of extracellular [ ca ] deprivation and cacch1 deletion primarily affected the site of germ - tube emergence . in low [ ca ] ,
in the cacch1 mutant and in the presence of l - type ca - channel blockers , cathodal germ - tube emergence was approximately half that of the control strain , but after 6 hr exposure to an electric field , this effect was almost lost ( figure  2a ) .
even hyphae that had emerged from the anodal face of the cacch1 mother cell eventually responded to the electric field .
this suggested that differences exist between the mechanism that establishes cathodal growth and that which maintains it . only the former appears to depend on calcium influx and cch1p .
the ability of fungal hyphae to exhibit tropic growth responses in relation to changes in substratum topography is well - known in plant pathogens and has been demonstrated previously for c. albicans and certain dermatophytes [ 24 , 25 ] .
some plant pathogenic fungi also use topographical features to trigger formation of the appressorium infection structure ( thigmodifferentiation ) .
we tested whether treatments and mutations that attenuated galvanotropism also influenced thigmotropic orientation in c. albicans hyphae . in wild - type cells , 60% of hyphae that contacted  a 0.79 m ridge responded by reorienting their growth axis ( figures 3a and 3b ) .
this is consistent with previous observations that thigmotropism was attenuated by inhibitors of stretch - activated ca channels   and supports a  model whereby changes in the underlying topography induce stresses in the membrane that are  sensed by mid1p ( figure  4b ) .
reorientation of the  cacch1 strain was also significantly reduced ( p = < 0.001 ) compared to the control strain ( figure  3 ) .
thigmotropism was attenuated in the regulatable cacch1/mrp1-cch1 under repressing conditions and restored to normal under mrp1p - inducing conditions .
expression of camid1 in the cacch1/camid1/mrp1-mid1 conditional mutant did not restore the reorientation response , confirming that cacch1p is required for thigmotropism .
we propose that stretch activation of camid1p , and subsequent opening of the cacch1p channel , results in localized ca influx that exerts an influence on the molecular machinery involved in polarized growth of the hyphal tip .
this influence overrides or repositions , or both , the molecular markers that defined the original axis of growth .
however , because hyphal reorientation was observed in almost 30% of ridge interactions in the camid1 mutant , either cacch1p is activated independently of camid1p or other sensing elements also contribute to the regulation of thigmotropism .
the function of fig1p in c. albicans is not known , but in s.  cerevisiae its deletion resulted in defective cell - cell fusion during mating , suggesting it could be involved in the delivery of components to the fusion site . because its deletion in c. albicans reduces hyphal reorientation during contact - sensing , cafig1p may again be involved in targeted delivery of secretory vesicles to the cell surface .
no instances of tip bifurcation were observed when  a hyphal tip contacted a ridge , even when the angle of approach was 90 ( figure  3c ) , suggesting that orientation - determining factors are a nondivisible entity or discrete protein complex .
calcium - dependent gene transcription in eukaryotes involves activation of a transcription factor by the phosphatase , calcineurin .
calcineurin regulates fungal morphogenesis , calcium flux , and homeostasis . in c.  albicans , calcineurin acts on the transcription factor ,
the deletion of cacrz1 affected both cathodal germ - tube emergence ( figure  2b ) and thigmotropism ( figure  3 ) , whereas the inhibition of calcineurin with fk506 or deletion of genes encoding either the catalytic cacna1 or regulatory cacnb1 calcineurin subunits only affected cathodal emergence ( p = 0.006 ) .
our results suggest that calcineurin is required for the establishment of cathodal cell polarity in an electric field but not for the reorientation of already - polarized hyphal tips during contact - sensing .
although cacrz1p influences cacch1 expression , it does not appear to be essential for basal expression or activation of camid1p or cacch1p because the morphology of cacrz1 mutant colonies was the same as the wild - type strain , whereas the camid1 and cacch1 mutants formed aberrant colonies ( see supplemental data ) .
other targets of cacrz1p have been identified in c. albicans and are required for resistance to membrane damage and alkaline stress .
it is not known whether cacrz1p - mediated gene expression regulates events that are upstream or downstream of calcium - influx induced tropisms .
the ability of external cues to influence the orientation of hyphal growth of the human pathogenic fungus , candida albicans , may be relevant to their capacity to infiltrate between human cells during tissue invasion . we have found that reorientation of c. albicans hyphae in relation to electrical fields and topographical signals is ca dependent and is mediated by ca channels and  a ca - dependent transcription factor , cacrz1p .
calcineurin , the primary regulator of cacrz1p , was required for galvanotropism but not thigmotropism .
we observed that the hyphae of mutants lacking the  stretch - activated camid1p or voltage - activated cacch1p proteins grew normally but were attenuated in orientation responses resulting from physical contact or imposed electric fields , respectively .
we propose a model whereby localized ca - channel activation , caused by localized changes in membrane potential or membrane stretch , results in calcium influx that directs polarized growth ( figure  4 ) .
stretch - activated ion channel activity has been described in patch - clamp analysis of c. albicans membranes , but the ion selectivity of these channels is not known . in plant cells ,
mechanosensory ca channels produce high cytosolic ca and initiation of localized cell - wall expansion at sites of shear stress .
similarly , in mammalian synapses , localized channel activation produces intracellular ca microdomains where the spatial boundary of the domain correlates with the capacity for efficient vesicle exocytosis .
localized ca influx may result in an asymmetry in the tip - high ca gradient in hyphae of c. albicans and other fungi .
this alters the axis of growth by increasing the rate of vesicle fusion within a local ca - high microdomain or by affecting the activity of calcium - binding proteins that are involved in polarized cell extension .
ca influx may therefore override existing polarity determinants at the cortex of evaginating mother cells and growing hyphae .
thus , both thigmotropic and galvanotropic responses of c. albicans hyphae are dependent on a single ca - regulated orientation mechanism .
for strains used in this study , mutant - strain construction , growth conditions , and determination of ca accumulation , see the supplemental experimental procedures .
yeast cells were adhered to poly - l - lysine - coated microscope slides and placed on the flat bed of a biorad midi - sub cell electrophoresis tank   and cultured in modified soll 's medium at 37c   1c for 6  hr at 10 v / cm and a current of 33  2 ma .
this field strength could be applied without affecting germ - tube formation or hyphal extension rate , yet it is sufficient for inducing cytoplasmic [ ca ] increases in other cell systems [ 3 , 4 ] .
hyphal orientation at the site of germ - tube emergence and at the germ - tube tip relative to the cathode were measured with improvision openlab 2.0 software . the percentage cathodal orientation ( p )
was calculated with p =  ( sin /n )  100 , for n measurements .
a minimum of 100 cells was measured in each of three independent experiments for each treatment .
yeast cells were adhered to poly - l - lysine - coated quartz slides featuring ridges of 0.79 m  40 nm and a pitch of 25 m ( kelvin nanotechnology , glasgow , uk ) .
this ridge height caused maximal hypha reorientation in a preliminary trial of five heights ( data not shown ) .
slides were placed in 20 ml prewarmed 20% ( v / v ) newborn - calf serum and 2% ( w / v ) glucose at 37c for 6 hr for inducing hyphae .
the number of hyphae reorienting on contact with a ridge was expressed as a percentage of the total observed interactions .
a minimum of 100 interactions was observed per strain in each experiment , and results were reported as the mean value from three independent experiments  sd .
 summaryeukaryotic cells from fungal hyphae to neurites that grow by polarized extension must coordinate cell growth and cell orientation to enable them to exhibit growth tropisms and to respond to relevant environmental cues . 
 such cells generally maintain a tip - high ca2 + cytoplasmic gradient , which is correlated with their ability to exhibit polarized tip growth and to respond to growth - directing extracellular signals [ 15 ] . in yeast and other fungi , 
 the polarisome , exocyst , arp2/3 , and spitzenkrper protein complexes collectively orchestrate tip growth and cell polarity , but it is not clear whether these molecular complexes also regulate cell orientation or whether they are influenced by cytoplasmic ca2 + gradients . 
 hyphae of the human pathogenic fungus candida albicans reorient their growth axis in response to underlying surface topography ( thigmotropism ) [ 6 ] and imposed electric fields ( galvanotropism ) [ 7 ] . 
 the establishment and maintenance of directional growth in relation to these environmental cues was ca2 + dependent . 
 tropisms were attenuated in media containing low ca2 + , or calcium - channel blockers , and in mutants where calcium channels or elements of the calcium signaling pathway were deleted 
 . therefore galvanotropism and thigmotropism may both be mediated by localized ca2 + influx at sites of polarized growth via ca2 + channels that are activated by appropriate environmental signals . 