Actin defects contribute to copper sensitivity in C. albicans.
(A) To further analyze the effects of copper on morphogenesis and trafficking mutants, rvs161Δ, rvs167Δ, and arp2Δ arp3Δ were replica-plated onto synthetic medium with 500 μM CuSO4. Only the wild type DIC185 was able to grow, indicating the extreme sensitivity of the deletion mutant strains. Images are representative of two independent experiments performed on different days. (B) Quantification of sensitivity by incubating mutant cells in a dilution series of copper concentrations in synthetic medium demonstrated that the arp2Δ arp3Δ mutant was the most sensitive, being inhibited by concentrations above 3.2 μM. The wild type, DIC185, was able to grow in 400 μM CuSO4. The graph represents the averages of four independent experiments performed on different days. (C) To determine whether an intact actin cytoskeleton is essential for resistance to copper, wild type DIC185 cells were treated with cytochalasin A in synthetic medium for 2 hr to depolarize the actin cytoskeleton, and then viability was assayed following incubation in the indicated concentration of CuSO4 for 2 hr. Relative colony-forming units (CFUs) decreased significantly with depolymerization of actin and exposure to increasing levels of copper. The graph represents averages of four independent experiments performed on different days. Strains used were DIC185, sur7Δ (YJA11), rvs161Δ (YLD14-3), rvs167Δ (YLD16), and arp2Δ arp3Δ (CaEE27).