title: eGFP-pHsens as a highly sensitive fluorophore for cellular pH determination by fluorescence lifetime imaging microscopy (FLIM).
abstract: The determination of pH in the cell cytoplasm or in intracellular organelles is of high relevance in cell biology. Also in plant cells, organelle-specific pH monitoring with high spatial precision is an important issue, since e.g. ΔpH across thylakoid membranes is the driving force for ATP synthesis critically regulating photoprotective mechanisms like non-photochemical quenching (NPQ) of chlorophyll (Chl) fluorescence or the xanthophyll cycle. In animal cells, pH determination can serve to monitor proton permeation across membranes and, therefore, to assay the efficiency of drugs against proton-selective transporters or ion channels. In this work, we demonstrate the applicability of the pH-sensitive GFP derivative (eGFP-pHsens, originally termed deGFP4 by Hanson et al. [1]) for pH measurements using fluorescence lifetime imaging microscopy (FLIM) with excellent precision. eGFP-pHsens was either expressed in the cytoplasm or targeted to the mitochondria of Chinese hamster ovary (CHO-K1) cells and applied here for monitoring activity of the M2 proton channel from influenza A virus. It is shown that the M2 protein confers high proton permeability of the plasma membrane upon expression in CHO-K1 cells resulting in rapid and strong changes of the intracellular pH upon pH changes of the extracellular medium. These pH changes are abolished in the presence of amantadine, a specific blocker of the M2 proton channel. These results were obtained using a novel multi-parameter FLIM setup that permits the simultaneous imaging of the fluorescence amplitude ratios and lifetimes of eGFP-pHsens enabling the quick and accurate pH determination with spatial resolution of 500 nm in two color channels with time resolution of below 100 ps. With FLIM, we also demonstrate the simultaneous determination of pH in the cytoplasm and mitochondria showing that the pH in the mitochondrial matrix is slightly higher (around 7.8) than that in the cytoplasm (about 7.0). The results obtained for CHO-K1 cells without M2 channels in comparison to M2-expressing cells show that the pH dynamics is determined by the specific H⁺ permeability of the membrane, the buffering of protons in the internal cell lumen and/or an outwardly directed proton pump activity that stabilizes the interior pH at a higher level than the external acidic pH. This article is part of a special issue entitled: photosynthesis research for sustainability: keys to produce clean energy.
authors.affiliation: Technical University of Berlin, Institute of Chemistry, Sekr. PC 14, Straße des 17. Juni 135, D-10623 Berlin, Germany. Electronic address: schmitt@physik.tu-berlin.de.
authors.forename: Franz-Josef
authors.initials: FJ
authors.lastname: Schmitt
authors.affiliation: Free University of Berlin, Department of Veterinary Medicine, Institute of Virology, Robert-Ostertag-Str. 7-13, D-14163 Berlin, Germany.
authors.forename: Bastian
authors.initials: B
authors.lastname: Thaa
authors.affiliation: Technical University of Berlin, Institute of Chemistry, Sekr. PC 14, Straße des 17. Juni 135, D-10623 Berlin, Germany.
authors.forename: Cornelia
authors.initials: C
authors.lastname: Junghans
authors.affiliation: Technical University of Berlin, Institute of Chemistry, Sekr. PC 14, Straße des 17. Juni 135, D-10623 Berlin, Germany.
authors.forename: Marco
authors.initials: M
authors.lastname: Vitali
authors.affiliation: Free University of Berlin, Department of Veterinary Medicine, Institute of Virology, Robert-Ostertag-Str. 7-13, D-14163 Berlin, Germany.
authors.forename: Michael
authors.initials: M
authors.lastname: Veit
authors.affiliation: Technical University of Berlin, Institute of Chemistry, Sekr. PC 14, Straße des 17. Juni 135, D-10623 Berlin, Germany.
authors.forename: Thomas
authors.initials: T
authors.lastname: Friedrich
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D016466:CHO Cells; D006224:Cricetinae; D003412:Cricetulus; D049452:Green Fluorescent Proteins / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D008856:Microscopy, Fluorescence / Q000379:methods*; D008969:Molecular Sequence Data; D010788:Photosynthesis
chemical_list: D049452:Green Fluorescent Proteins
keywords: FLIM; GFP; M2 proton channel; Xanthophyll cycle; pH determination; pH-dependent ROS