Determination of intracellular pH by fluorescence lifetime imaging in living cells using fluorescent sensors

GABRIELA N. BOSIO; SABINE BALFANZ; FELIX BEINLICH

Brujas

Conferencia; MAF Conference 2017; 2017

KU Leuven

In the past 30 years, fluorescence microscopy has developed from a sparsely used technique describing rare fluorescence phenomena in nature to one of the most powerful methods in the life sciences observing non-invasively the physiologically relevant processes in living cells and biological tissue. It comes as no surprise that two recent Nobel Prizes were awarded for the developments in modern fluorescence microscopy (Nobel Prizes in Chemistry in 2008 and 2014 for Green Fluorescent Protein (GFP) and Super-resolution). A major challenge - besides the optical instrumentation and the image formation andanalysis ? is the development, characterization and application of fluorescent molecules that sense a certain cell parameter (e.g. pH, ion concentrations, kinase activities, temperature or redox state) and report variations of that parameter via changes of the fluorescence.The aim of this work was the quantitative characterization of an already known ratiometric FP-based pH sensor (E1GFP, (1)) with respect to its applicability as a pH-sensor with fluorescence lifetime contrast.Fluorescence lifetime imaging (FLIM) allowed us after proper intracelular calibration to determinate the pH reported by the sensor molecule independent of the number of sensor molecules ? a quantity that cannot be controlled nor be determined in living cells.E1GFP is an attractive pH sensor since it can be targeted to different cell compartments and report in this way local pH values. The apparent low pKa as well as the good fluorescence at pH 5 and below make it a promising alternative to some of the established GFP-based pH-indicators for use e.g. in synapticvesicles. Thus, trying to generate a targeted version of it and characterize its usefulness as well, will be the next task.We performed the measurements in HEK293 and tsa201 cells. The DNA of E1GFP was incorporated into the cells so that they produced the E1GFP protein for two to three days (transient transfection). In addition a stable HEK293 cell line was generated and tested, where the DNA for E1GFP was permanently incorporated. We also compared the performances of E1GFP relative to BCECF, the most popular synthesized organic fluorescent molecule used for intracellular pH measurements.Surprisingly, we were observing a so far not described excitation wavelength dependence of the E1GFP fluorescence lifetime. Even more interesting, its pH-titration revealed two largely different (one pH units) pKa values for two excitation wavelength regions. At exc= 800 nm, we determined a pKa of 6.63 ± 0.04 ideal for normal cytosolic pH and mildly-acidic cellular compartments as parts of the endosome or the cytosol of tumor cells, while at 920 nm excitation a pKa of 5.57 ± 0.03, ideal for acidic compartments as the lysosome, synaptic or some endosomal vesicles. In light of these results and the correspondingabsorption and fluorescence spectra, we hypothesize that E1GFP exist intwo different conformations that have the observed distinctively different pKa values as well as their own spectral and fluorescence lifetimes fortheir protonated and deprotonated forms, respectively.In addition, usefulness of E1GFP fluorescence lifetime determination inliving cells was shown observing an intracellular pH recovery applying FLIM-imaging of E1GFP after different treatments, such as H2O2 or drastic changes in extracellular pH.References:(1) ?Real-time measurement of endosomal acidification by a novel genetically encoded biosensor.? M. Serresi, R. Bizzarri, F. Cardarelli and F. Beltram. Anal. Bioanal. Chem. 2009, 393, 1123?1133.