Cyclin Cln3p links G1 progression to hyphal and pseudohyphal development in Candida albicans

Abstract

G1 cyclins coordinate environmental conditions with growth and differentiation in many organisms. In the pathogen Candida albicans, differentiation of hyphae is induced by environmental cues but in a cell cycle-independent manner. Intriguingly, repressing the G1 cyclin Cln3p under yeast growth conditions caused yeast cells to arrest in G1, increase in size, and then develop into hyphae and pseudohyphae, which subsequently resumed the cell cycle. Differentiation was dependent on Efg1p, Cph1p, and Ras1p, but absence of Ras1p was also synthetically lethal with repression of CLN3. In contrast, repressing CLN3 in environment-induced hyphae did not inhibit growth or the cell cycle, suggesting that yeast and hyphal cell cycles may be regulated differently. Therefore, absence of a G1 cyclin can activate developmental pathways in C. albicans and uncouple differentiation from the normal environmental controls. The data suggest that the G1 phase of the cell cycle may therefore play a critical role in regulating hyphal and pseudohyphal development in C. albicans.

FIG. 1.

Repression of CLN3 results in hyphal and pseudohyphal growth under yeast growth conditions. (A) Strains CB488 and CB504 grown on solid SD inducing or repressing medium for 24 h at 30°C; (B) strains grown in liquid inducing or repressing medium at 30°C; (C) Northern blot of CLN3 expression in strains CB488 and CB504 grown in the presence (+) or absence (−) of methionine and cysteine (MC) for the indicated times in liquid SD media. ACT1 was used as a loading control. Bar, 10 μm.

FIG. 2.

Nuclear division and septation occur after filament development in CLN3-repressed cells. Cells incubated in repressing liquid medium for the indicated times were fixed and then stained with DAPI and calcofluor. Control cells grown in IMDM at 37°C for 6 h are also shown. Bar, 10 μm. MC, methionine and cysteine.

FIG. 3.

CLN3 repression in the absence of RAS, EFG1/CPH1, and CST20. (A) Northern analysis of CLN3 expression in ras/ras (CB498), cst20/cst20 (CB499), and efg1/efg1 cph1/cph1 (CB501) backgrounds. CLN3 expression in strains CB504 (CLN3/CLN3) and CB488 (cln3/MET3::CLN3) is also indicated for comparison. Cells were grown for 6 h in repressing or inducing liquid SD medium. (B) Twenty-five microliters of 5 × 10⁵ cells of each strain/ml was spread on plates and incubated for 72 h at 30°C. (C, D) Strains grown on solid or in liquid media, respectively, for 24 h at 30°C. Bars, 20 μm. MC, methionine and cysteine; +, present; −, absent.

FIG. 4.

Northern analysis of CCN1 and PCL2 expression in cells repressed for CLN3. Cells from strain CB504 (CLN3/CLN3) and CB488 (cln3/MET3::CLN3) were incubated in repressing or inducing liquid SD medium for the indicated times. ACT1 was used as a loading control. MC, methionine and cysteine.

FIG. 5.

Repression of CLN3 under hypha-inducing conditions delays nuclear migration and influences hyphal morphology and positioning of septa. (A) Cells were incubated in repressing medium for 1 h at 30°C, transferred to repressing medium containing 10% FCS at 37°C for 1.5 h, fixed, and stained with DAPI and calcofluor; (B) cells were incubated in repressing medium for 3 h at 30°C and then inoculated into IMDM supplemented with methionine and cysteine at 37°C for the times indicated. Bars, 10 μm.