Table 1.
Strains used in this study.
Figure 1.
Conditions inducing white cell filamentation do not induce opaque cell filamentation.
Environmental cues that induce efficient filamentation in C. albicans white cells (RBY717) include serum (A), high temperature (B), Spider medium (C and F), neutral pH medium (D), and Lee's medium (E). None of these environmental cues induce filamentous growth in opaque cells. Cell photographs were taken after 5 hours incubation at 37°C except for YPD supplemented with serum (2 hour incubation). Colonies grown on Spider medium were incubated at 30°C for 4 days.
Figure 2.
Novel environmental cues induce filamentation in C. albicans opaque cells.
Several culture conditions are described that induce filamentous growth in opaque-locked a cells (CAY2903). These include growth on (A) sorbitol (SOR), (B) minimal (MIN), (C) low nitrogen (SLAD), (D) low phosphate (LP) or (E) N-acetyl glucosamine (GlcNAc) medium at 25°C. These culture conditions do not induce filamentation in white a cells (RBY717). Panels show colony morphologies from mixed white/opaque populations (solid arrow, white colony; dashed arrow, opaque colony). Additional panels show white cells, opaque cells, and calcofluor white (CW)-strained opaque cells.
Figure 3.
Scanning electron micrographs of yeast and filamentous forms of C. albicans white and opaque cells.
(A) White cells (RBY717) were induced to form hyphae by growth in YPD supplemented with serum at 37°C. (B) Opaque cells (CAY2903) were induced to undergo filamentous growth by culture on LP or SOR media at 25°C. Scale bar, 5 µm.
Figure 4.
Differential thermal regulation of filamentous growth in white and opaque cells.
White (RBY717) and opaque (CAY2903) cells were cultured on SCD or filamentation-inducing media (SOR, MIN, SLAD, or LP) at 25°, 30°, or 37°C for 4 days. Opaque filamentation was optimal in the order 25°C > 30°C > 37°C. In contrast, white cell filamentation increased as temperatures increased, as evidenced by weak filamentation of white colonies on SCD medium at 37°C (top panels). (A) SCD, (B) SOR, (C), MIN, (D), SLAD, or (E) LP medium.
Figure 5.
Analysis of the role of the cAMP and MAPK signaling pathways in opaque filamentation.
MTLa strains lacking EFG1 or CPH1 transcription factors that mediate the cAMP or MAPK signaling pathway, respectively, were analyzed for their ability to undergo opaque cell filamentation. Loss of EFG1 resulted in a defect in opaque filamentation under all conditions tested (right panels), while loss of CPH1 did not have a significant effect on opaque filamentation (left panels). Strains were cultured on SOR (A), SLAD (B) or LP (C) medium for 4 days at 25°C (colonies) or 22 hours at 25°C (cells). EFG1 mutants were CAY3526 (white) and CAY3292 (opaque), CPH1 mutants were CAY3524 (white) and CAY3298 (opaque), and the EFG1 complemented strain was CAY4384 (opaque).
Figure 6.
Negative transcriptional regulators of filamentation in white and opaque cells.
The roles of the transcription factors Nrg1, Tup1, and Rfg1, which are negative regulators of white cell filamentation, were tested in the opaque program of filamentous growth. Loss of Tup1 (CAY1616, opaque; CAY1550, white) or Nrg1 (CAY1618, opaque; CAY1552, white) resulted in white and opaque cell filamentation even when cells were grown on SCD medium (A and B). In contrast, loss of Rfg1 (CAY3299, opaque; CAY 3528, white) resulted in decreased filamentation on SOR (C), SLAD (D), and LP (E) medium. Loss of both CPH1 and RFG1 genes (CAY3151, opaque) resulted in a similar phenotype to rfg1 mutants (C–E). Loss of both EFG1 and RFG1 (CAY2822, opaque; CAY2701 white) generated phenotypes similar to efg1 and rfg1 single mutants (C–E). Colonies were grown on the different media for 5 days at 25°C and imaged.
Figure 7.
Role of the Ume6-Hgc1 pathway in opaque cell filamentation.
The transcription factor Ume6 and the G1 cyclin-related protein Hgc1 were analyzed for their potential role(s) in filamentation in opaque cells. Mutant or complemented opaque strains were incubated on SOR (A and E), SLAD (B), LP (C and F), or MIN (D) medium for 4 days at 25°C and colony morphologies determined. Cell morphologies were photographed after 25 hours incubation. Loss of Ume6 (CAY1571) or Hgc1 (CAY4193) compromised opaque filamentation under each of the tested conditions, and filamentation was restored in the complemented strains CAY3697 (UME6 addback) and CAY4197 (HGC1 addback), respectively. Strain DK340 was the white ume6 mutant used.
Figure 8.
Transcriptional profiling of opaque filamentation.
MTLa white (RBY717) or opaque (CAY2903) strains were incubated in SCD medium, a non-filament inducing condition, or sorbitol (SOR) and low phosphate (LP) media, conditions that specifically induce filamentation of opaque cells but not white cells. Cells were harvested after 12 hours growth (LP medium) or 16 hours growth (SOR medium) and cDNA prepared and hybridized against custom C. albicans ORF microarrays. (A) Comparative expression of opaque cell filamentation genes and white hyphal genes. Columns 1 and 2 represent opaque filamentation genes in LP and SOR media (relative to SCD controls) that passed SAM filters. Columns 3 and 4 represent hyphal-induced genes in white cells (serum-treated cells at 1 or 2 h compared to untreated cells) using data from Kadosh and Johnson [36]. Top panel; genes induced in both opaque and white filaments. Bottom panel, genes induced in opaque filaments but not white filaments. Grey boxes indicate no data available. (B) Genes expressed in white hyphal cells but not expressed in filamenting opaque cells. Top panel; genes induced in white hyphal cells only. Bottom panel, genes induced in white hyphal cells and either in opaque LP filaments or SOR filaments (but not both). op, opaque cells; wh, white cells. Gene numbers refer to orf19 gene identifiers (e.g. 1774 refers to orf19.1774).
Figure 9.
Regulation of filamentous growth in C. albicans white and opaque cells.
The central programs regulating filamentation in white cells are shown, including the two core pathways of MAPK and cAMP-PKA signaling. White cell filamentation is also negatively regulated by the transcription factors Tup1 and Nrg1, and positively regulated by Ume6 in concert with the cyclin-like protein Hgc1. Filamentation in opaque cells is regulated by many of the same signaling pathways. In particular, Efg1 and Ume6 appear to be master regulators of filamentous growth in both white and opaque cells. However, there was no detectable role for MAPK signaling through Cph1, or for signaling via Cph2/Tec1, under the conditions used for inducing opaque filamentation.