Figure 3.

Generation and validation of novel tools to monitor compartment-specific changes in Ca²⁺ and pH. (A) Aequorin was targeted to cytoplasm or mitochondria in S. cerevisiae, by the addition of N-terminal signal sequence for the latter. Dose-dependent elevations of mitochondrial Ca²⁺ are seen to follow cytoplasmic Ca²⁺ fluxes upon amiodarone (AMD) addition (described previously, Gupta et al., 2003). (B) pH-sensitive version of GFP, pHluorin, was targeted to mitochondria where it colocalized with MitoTracker Red as seen in the inset. Addition of amiodarone acidified mitochondrial matrix, relative to control, followed by slow recovery. (C) Cytosolic pH was monitored by expressing pHluorin in C. albicans, as seen in the inset. Addition of amiodarone elicits dose-dependent acidification as was previously reported in S. cerevisiae. (D) C. albicans vacuolar pH is monitored using the acetoxymethyl precursor of the pH-sensitive fluorescent reporter BCECF, which can stably accumulate in vacuoles (inset) through the action of esterases. The reporter was validated by showing alkalinization of vacuolar pH in vma7^−/− mutants, lacking a functional V-ATPase. DIC, differential interference contrast.