Figure 1.

Ectopic activation of the RAS/STE pathway via the STE11-4 mutation. (A) The dominant active STE11-4 mutation promotes enhanced filamentous differentiation. Microcolonies formed by the indicated strains (STE11-4 is the STE11:: STE11-4:: LEU2/STE11 strain SKY2183) on rich YPD media and low-nitrogen SLAD media reveal that the requirement for nutrient starvation is bypassed by activation of RAS/STE signaling. A cell deficient for STE7 (ste7/ste7, L5535), an element of the RAS/STE pathway, does not exhibit a filamentous phenotype on YPD or SLAD media. Photographs show representative colonies of each strain after 24 h at 22°C. (B) Diploid cell agar invasion assay. Strains were inoculated on YPD agar and grown for 72 h at 30°C. Photographs were taken after the nonadherent cells were washed away with water. The STE11-4 mutant is hyperinvasive even in the rich media. (C) Cell morphology of mutant cultured in liquid YPD nitrogen-rich media. The STE11-4 mutant exhibits highly elongated cells that grow as adherent cell aggregates reminiscent of filamentous colonies. This phenotype suggests that the agar signal requirement is also bypassed by ectopic RAS/STE signals. Bars, 50 μm. (D) Cell cycle kinetics of the STE11-4/STE11-4 homozygous diploid (SKY2226). Flow cytometry was performed on asynchronous cultures growing on YPD rich media to determine the distribution of nuclear DNA content. The STE11-4 mutant reveals a predominance of cells with G2/M DNA content, which correlates to a large G2/G1 ratio, reflecting the relative percentages of cells falling in the indicated gating intervals of fluorescence intensity.