Figure 1.

Calcineurin pathway is required for thermotolerance in C. glabrata. (A) Pharmacological inhibition of calcineurin phenocopies genetic deletion of calcineurin. Cells were grown overnight in YPD at 24°, 5-fold serially diluted, and spotted onto YPD medium containing FK506 or cyclosporin A (CsA), and incubated at the indicated temperatures for 48 hr. Strains tested were wild-type (CBS138), cna1 mutants (YC67 and YC98), cnb1 mutants (YC191 and YC193), and crz1 mutants (YC267 and YC182). (B) The growth kinetics of C. glabrata wild-type and mutant strains at 24°. Cells were grown overnight at 24°, washed twice with dH₂O, diluted to 0.2 OD/ml in fresh liquid YPD medium, and incubated at 24° with shaking at 250 rpm. The OD₆₀₀ of cultures was measured at 0, 3, 6, 9, 24, and 30 hr. The experiments were performed in triplicate, and data were plotted using Prism 5.03. Strains tested were wild-type (CBS138), cna1 mutant (YC98), cnb1 mutant (YC193), and crz1 mutant (YC182). (C) Calcineurin is required for survival at 40°. Cells were grown overnight in YPD at 24°, 5-fold serially diluted, spotted onto YPD. The plate was incubated at 40° for 48 hr, then transferred to 24° for 48 hr incubation. (D) Calcineurin mutant cells exhibit a shrunken cell morphology at 40°. Cells were grown overnight in YPD medium at 24°, washed twice with dH₂O, diluted to 0.5 OD₆₀₀/ml in fresh liquid YPD medium, and incubated at 24° or 40° with shaking at 250 rpm for 4 hr. Strains tested were wild-type (CBS138), cna1 mutant (YC98), cnb1 mutant (YC193), and crz1 mutant (YC182). The images were taken at 100×. Scale bar = 5 µm. (E) An osmotic stabilizer rescued temperature-sensitive phenotypes of calcineurin mutants from C. glabrata, but not from C. neoformans. Cells were grown overnight in YPD at 24°, 5-fold serially diluted, spotted onto YPD medium containing 1 M sorbitol, and incubated at the temperatures indicated for 48 hr. C. neoformans strains tested were wild-type (H99) and cna1 mutant (KK1).