Staurosporine Induces Filamentation in the Human Fungal Pathogen Candida albicans via Signaling through Cyr1 and Protein Kinase A

Staurosporine-induced filamentation requires Cyr1 and PKA. (A) Farnesol inhibits filamentation induced by staurosporine. SN95 wild-type cells were grown to log phase at 30°C in YPD or YPD plus 0.5 μg/ml staurosporine in the absence or presence of 200 μM farnesol. Cells were imaged by DIC microscopy. The scale bar indicates 10 μm. (B) Cyr1 and PKA are the only components of the cAMP signaling pathway tested that are required for filamentation induced by staurosporine. A CAI4 wild-type strain or mutants lacking components of the cAMP signaling pathway were subcultured to stationary phase in YPD at 30°C in the presence or absence of 0.5 μg/ml staurosporine (STS). Cells were imaged by DIC microscopy. The scale bar indicates 20 μm.

Staurosporine induces a distinct gene expression program from other filament-inducing cues and promotes filamentation independent of Nrg1 degradation. (A) SN95 wild-type cells were subcultured to log phase in YPD at 30°C, YPD plus 10% serum at 37°C, YPD plus 200 mM hydroxyurea at 30°C, or YPD plus 0.5 μg/ml staurosporine at 30°C. cDNA was prepared from total RNA for qRT-PCR. The transcript levels of filament-specific transcripts (ECE1, HGC1, HWP1, and RBT1) and yeast-specific transcripts (YWP1 and NRG1) were monitored by qRT-PCR and normalized to GPD1. The fold change in gene expression under each condition relative to YPD at 30°C is plotted as the mean ± standard deviation from triplicate samples and is representative of two independent experiments. (B) Nrg1 protein persists in filaments induced by staurosporine. SN95 wild-type cells expressing native levels of HA-tagged Nrg1 were subcultured to log phase in YPD plus 10% serum at 37°C, YPD plus 200 mM hydroxyurea at 30°C, or YPD plus 0.5 μg/ml staurosporine at 30°C. Total protein was resolved by SDS-PAGE, and the blot was hybridized with anti-hemagglutinin to detect Nrg1 and anti-PSTAIRE to monitor Cdc28 as a loading control. (C) SN95 wild-type cells were grown to log phase under identical conditions as described for panel B. Cells were imaged by DIC microscopy. The scale bar indicates 10 μm.

Dibutyryl cAMP does not rescue staurosporine-induced filamentation of a cyr1Δ/cyr1Δ mutant. A CAI4 wild-type strain and a cyr1Δ/cyr1Δ mutant were grown to log phase at 37°C in YPD plus 10% serum or at 30°C in YPD plus 0.5 μg/ml staurosporine in the absence or presence of 100 mM dibutyryl cAMP, as indicated. Cells were imaged by DIC microscopy. The scale bar indicates 10 μm.

Mfg1 and Flo8 are not required for polarized growth in response to staurosporine. Strains were grown to log phase at 30°C in YPD, 30°C in YPD plus 0.5 μg/ml staurosporine, or 37°C in YPD plus 10% serum. Cells were imaged by DIC microscopy. The scale bar indicates 20 μm.