Development of a novel GFP-based ratiometric excitation and emission pH indicator for intracellular studies.
We report on the development of the F64L/S65T/T203Y/L231H GFP mutant (E2GFP) as an effective ratiometric pH indicator for intracellular studies. E2GFP shows two distinct spectral forms that are convertible upon pH changes both in excitation and in emission with pK close to 7.0. The excitation of the protein at 488 and 458 nm represents the best choice in terms of signal dynamic range and ratiometric deviation from the thermodynamic pK. This makes E2GFP ideally suited for imaging setups equipped with the most widespread light sources and filter settings. We used E2GFP to determine the average intracellular pH (pH(i)) and spatial pH(i) maps in two different cell lines, CHO and U-2 OS, under physiological conditions. In CHO, we monitored the evolution of the pH(i) during mitosis. We also showed the possibility to target specific subcellular compartments such as nucleoli (by fusing E2GFP with the transactivator protein of HIV, (Tat) and nuclear promyelocytic leukemia bodies (by coexpression of promyelocytic leukemia protein).
Bizzarri
Ranieri
R
NEST CNR-INFM, Scuola Normale Superiore, Pisa, Italy. r.bizzari@sns.it
Arcangeli
Caterina
C
Arosio
Daniele
D
Ricci
Fernanda
F
Faraci
Paolo
P
Cardarelli
Francesco
F
Beltram
Fabio
F
D000818:Animals; D001426:Bacterial Proteins / Q000737:chemistry; D016466:CHO Cells; D045744:Cell Line, Tumor; D006224:Cricetinae; D003412:Cricetulus; D049452:Green Fluorescent Proteins / Q000737:chemistry / Q000235:genetics; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007202:Indicators and Reagents; D007424:Intracellular Fluid / Q000737:chemistry* / Q000378:metabolism*; D008164:Luminescent Proteins / Q000737:chemistry; D008856:Microscopy, Fluorescence; D008954:Models, Biological; D013053:Spectrophotometry; D013816:Thermodynamics; D014162:Transfection
D001426:Bacterial Proteins; D007202:Indicators and Reagents; D008164:Luminescent Proteins; C485184:enhanced green fluorescent protein; C054562:yellow fluorescent protein, Bacteria; D049452:Green Fluorescent Proteins