Live-Cell Imaging and Measurement of Intracellular pH in Filamentous Fungi Using a Genetically Encoded Ratiometric Probe

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 <1.3 pH units, but it returned to its original value within 20 min following treatment. The weak acid and antifungal food preservative sorbic acid caused prolonged, concentration-dependent intracellular acidification. The inhibition of ATPases with N-ethylmaleimide, dicychlohexylcarbodimide, or sodium azide caused the cytoplasmic pH to decrease by <1 pH unit. Treatment with the protonophore carbonyl cyanide m-chlorophenylhydrazone or cyanide p-(trifluoromethoxy) phenylhydrazone reduced the cytoplasmic pH by <1 pH unit. In older hyphae from 32-h-old cultures, RaVC became sequestered within large vacuoles, which were shown to have pH values between 6.2 and 6.5. Overall, our study demonstrates that RaVC is an excellent probe for visualizing and quantifying intracellular pH in living fungal hyphae.

Low pH regulates the production of deoxynivalenol by Fusarium graminearum

“…2). It is expected that even under such acid extracellular conditions as reported here, F. graminearum would maintain its intracellular pH constant, as has been observed under extreme acidic conditions, including sudden pH changes, in other fungi (Bagar et al, 2009), and it is probable that the GFP produced would remain in the cytoplasm. Therefore, the GFP-based reporter strain used here appears to provide a reliable indicator of TRI5 expression.…”

Ratiometric Imaging of pH Probes

“…In general, cytoplasmic pH in most organisms from bacteria (Martinez et al, 2012;Wilks & Slonczewski, 2007) to yeasts (Bagar, Altenbach, Read, & Bencina, 2009;Grynkiewicz et al, 1985) to mammalian cells (Fig. 23.1) is slightly basic (pH is $7.0-7.8), with the exception of some acidophilic or basophilic bacteria (Paradiso et al, 1984;Slonczewski, Fujisawa, Dopson, & Krulwich, 2009).…”

In Vivo Intracellular pH Measurements in Tobacco andArabidopsisReveal an Unexpected pH Gradient in the Endomembrane System

“…We used pHluorin (Bagar et al, 2009) to generate several genetically encoded pH sensors and target to specific intracellular compartments (see Table 1 for the list and Supplemental Table 1 online). To target the sensor to the ER lumen, the signal peptide from tobacco (Nicotiana tabacum) chitinase (Di Sansebastiano et al, 1998) and the tetrapeptide HDEL (Gomord et al, 1997) was used.…”