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. 2013 Dec 19;289(6):3365–3382. doi: 10.1074/jbc.M113.528802

FIGURE 4.

FIGURE 4.

GPI2 mutation results in defective filamentation in C. albicans. A, CAI4, GPI2 heterozygote (CAI4-gpi2/GPI2) and conditional null GPI2 mutant (CAI4-gpi2/PMET3-GPI2) were compared for morphologies by spotting on a YEPD plate and incubating at 37 °C for 7 days. The mutants displayed defective filamentous morphology as compared with CAI4. The experiment was repeated twice for confirmation, and a representative image is shown. B, CAI4, GPI2 heterozygote, and conditional null GPI2 mutant were compared for filamentation pattern by spotting on a Spider plate and incubating at 37 °C for 7 days. The mutants showed defective filamentation as compared with CAI4. The experiment was repeated thrice for confirmation. C, CAI4, GPI2 heterozygote, and conditional null GPI2 mutant were grown in Spider medium at 37 °C, and hypha formation was monitored. Panel i, mutants were found to be defective in filamentation as compared with the wild type strain after 90 and 120 min. However, as shown in panel ii, when hypha formation was monitored in the presence of sodium butyrate (3 mm) after 90 min, the mutant displayed filamentation similar to the wild type strain, suggesting the impaired filamentation in the mutant to be an inhibitory effect of Set3/Hos2 histone deacetylase complex on Ras1/cAMP-PKA signaling (26). The experiment was done twice in duplicate, and a representative image is shown. D, CAI4 and GPI2 heterozygote were also compared for cAMP-dependent PKA activity (7). Lane 1 represents negative experimental control without cell lysate, and lane 2 represents positive experimental control with purified cAMP-dependent PKA, supplied by the kit manufacturer. GPI2 heterozygote was found to have lower PKA activity (lanes 5 and 6) as compared with CAI4 (lanes 3 and 4), correlating with lower Ras activity. E, GPI2 heterozygote was found to be resistant to heat shock when compared with CAI4. The plates shown here are after 48 h of incubation at 30 °C. The experiment was done twice for confirmation, and a representative image is shown. The conditional null GPI2 mutant could not be compared because of its inherent growth defect and reduced viability.