A fundamental role of pH as signaling component of cell processes is not yet established. An accurate measurement of intracellular pH in unperturbed cells of filamentous fungi is fraught with difficulties, however, necessary for explaining its function. To study spatial and temporal pH changes in vivo in hyphae of industrially important filamentous fungi Aspergillus niger, a genetically encoded pH probe was modified for expressing in fungi. We showed that a pH sensitive derivative of green fluorescent probe, a ratiometric RaVe_C, which was codon optimized, could be used to image intracellular pH in vivo in fungal cells. RaVe_C has a ratiometric dual excitation and displays reversible emission ratio changes in the range from pH 5.5 to pH 8.0. With a pKa of 7.0 RaVe_C is ideal for monitoring pH changes in the cytosol. In vivo calibration using nigericin proved that the RaVe_C probe is highly pH sensitive in living fungal hyphae, therefore RaVe_C can successfully be used as a noninvasive genetically encoded intracellular sensor. Examination of cytoplasmic pH in growing cells was performed by simultaneous, dual excitation confocal ratio imaging of the ratiometric pH probe RaVe_C. Based on in vivo calibration, estimated value of mean cytoplasmic pH for cells expressing RaVe_C probe was 7.5. No significant cytoplasmic pH gradient was observed along growing hyphae. Sifts of external pH to acidic or alkaline pH values caused a temporal acidification or alkalization in cytoplasmic pH. Stimulation or inhibition of pH homeostasis by pharmacological agents resulted in rapid decrease of pH.
Full conference title:
9th EUROPEAN CONFERENCE ON FUNGAL GENETICS
- ECFG 9th (2008)