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Eukaryotic Cell, May 2009, p. 703-712, Vol. 8, No. 5
1535-9778/09/$08.00+0     doi:10.1128/EC.00333-08
Copyright © 2009, American Society for Microbiology. All Rights Reserved.

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

Tanja Bagar,¹ Kirsten Altenbach,² Nick D. Read,² and Mojca Benina^1*

Department of Biotechnology, National Institute of Chemistry, Hajdrihova 19, SI-1000 Ljubljana, Slovenia,¹ Fungal Cell Biology Group, Institute of Cell Biology, University of Edinburgh, Edinburgh EH9 3JH, United Kingdom²

Received 2 October 2008/ Accepted 10 March 2009

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 µm 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.

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* Corresponding author. Mailing address: Department of Biotechnology, National Institute of Chemistry, Hajdrihova 19, SI-1000 Ljubljana, Slovenia. Phone: 38614760334. Fax: 38614760300. E-mail: mojca.bencina{at}ki.si

Published ahead of print on 13 March 2009.

Supplemental material for this article may be found at http://ec.asm.org/.

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Eukaryotic Cell, May 2009, p. 703-712, Vol. 8, No. 5
1535-9778/09/$08.00+0     doi:10.1128/EC.00333-08
Copyright © 2009, American Society for Microbiology. All Rights Reserved.

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