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.
Highlights
The maintenance of intracellular pH and homeostasis is fundamental in multiple cellular processes, as for instance transmembrane transport, establishment of electrochemical gradients, adaptive responses to environmental pH variations and other metabolic events taking place within particular organelles
Emission spectra of S. cerevisiae BY4742 expressing ATO1 Cterminally tagged with either yEGFP1 or yEGFP3 were recorded with a spectrofluorometer using different pH buffers as well as permeabilizing and invasive treatments to calculate intensity titration curves for the optimization of buffer composition (Figures 1, A and S1)
Ato1p-yEGFP1 fresh cells in P-buffer responded to pH in a Boltzmann sigmoidal pattern comparable to those exhibited by positive permeabilizing and invasive controls (Figure S1)
Summary
The maintenance of intracellular pH (pHi) and homeostasis is fundamental in multiple cellular processes, as for instance transmembrane transport, establishment of electrochemical gradients, adaptive responses to environmental pH variations and other metabolic events taking place within particular organelles. In contrast to more stable parameters, pHi is rapidly altered by almost any manipulation during sample measurement, including common methods for cell harvesting and fluorescent pH probe staining. For all these reasons, the monitoring procedure must be consistent to ensure the reproducibility and reliability of pH estimations. Some of the above requirements are addressed by incorporating genes encoding fluorescent proteins (FP), the fluorescence of which changes according to pH. Metabolic and internal pH changes can be monitored in vivo by targeting FP fusion proteins to subcellular compartments and measuring its fluorescence changes through spectrofluorometry and confocal fluorescence microscopy (CFM) [2]
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