Live-cell imaging and measurement of intracellular pH in filamentous fungi using a genetically encoded ratiometric probe

Tanja Bagar, Kirsten Altenbach, Nick D. Read, Mojca Benčina

    Research output: Contribution to journalArticlepeer-review

    Abstract

    A novel, genetically encoded, ratiometric pH probe (RaVC) was constructed to image and measure intracellular pH in living hyphae of Aspergillus niger. RaVC is a chimeric protein based on the pH-sensitive probe pHluorin, which was partially codon optimized for expression in Aspergillus. Intracellular pH imaging and measurement was performed by simultaneous, dual-excitation confocal ratio imaging. The mean cytoplasmic pH measured was 7.4 to 7.7 based on calibrating RaVC in situ within nigericin-treated hyphae. Pronounced, longitudinal cytoplasmic pH gradients were not observed in the apical 20 m of actively growing hyphae at the periphery of 18-h-old colonies. The cytoplasmic pH remained unchanged after prolonged growth in buffered medium with pH values between 2.5 or 9.5. Sudden changes in external pH significantly changed cytoplasmic pH by
    Original languageEnglish
    Pages (from-to)703-712
    Number of pages9
    JournalEukaryotic Cell
    Volume8
    Issue number5
    DOIs
    Publication statusPublished - May 2009

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