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In Vivo Determination of Organellar pH Using a Universal Wavelength-Based Confocal Microscopy Approach

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  • Albert Pineda Rodó
  • Libuše Váchová
  • Zdena Palková

Abstract

Many essential cellular processes are affected by transmembrane H+ gradients and intracellular pH (pHi). The research of such metabolic events calls for a non-invasive method to monitor pHi within individual subcellular compartments. We present a novel confocal microscopy approach for the determination of organellar pHi in living cells expressing pH-dependent ratiometric fluorescent proteins. Unlike conventional intensity-based fluorometry, our method relies on emission wavelength scans at single-organelle resolution to produce wavelength-based pH estimates both accurate and robust to low-signal artifacts. Analyses of Ato1p-pHluorin and Ato1p-mCherry yeast cells revealed previously unreported wavelength shifts in pHluorin emission which, together with ratiometric mCherry, allowed for high-precision quantification of actual physiological pH values and evidenced dynamic pHi changes throughout the different stages of yeast colony development. Additionally, comparative pH quantification of Ato1p-pHluorin and Met17p-pHluorin cells implied the existence of a significant pHi gradient between peripheral and internal cytoplasm of cells from colonies occurring in the ammonia-producing alkali developmental phase. Results represent a step forward in the study of pHi regulation and subcellular metabolic functions beyond the scope of this study.

Suggested Citation

  • Albert Pineda Rodó & Libuše Váchová & Zdena Palková, 2012. "In Vivo Determination of Organellar pH Using a Universal Wavelength-Based Confocal Microscopy Approach," PLOS ONE, Public Library of Science, vol. 7(3), pages 1-12, March.
  • Handle: RePEc:plo:pone00:0033229
    DOI: 10.1371/journal.pone.0033229
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    References listed on IDEAS

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    1. Gero Miesenböck & Dino A. De Angelis & James E. Rothman, 1998. "Visualizing secretion and synaptic transmission with pH-sensitive green fluorescent proteins," Nature, Nature, vol. 394(6689), pages 192-195, July.
    2. Zdena Palková & Blanka Janderová & Jir̂í Gabriel & Blanka Zikánová & Martin Pospíŝek & Jitka Forstová, 1997. "Ammonia mediates communication between yeast colonies," Nature, Nature, vol. 390(6659), pages 532-536, December.
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