Novel green fluorescent protein-based ratiometric indicators for monitoring pH in defined intracellular microdomains.
To measure pH in defined intracellular microdomains of living cells, we developed ratiometric indicators based on fusing in tandem two green fluorescent protein (GFP) variants having different pH sensitivities. The indicators function in a single-excitation/dual-emission mode involving fluorescence resonance energy transfer, as well as in a dual-excitation/single-emission mode. The fluorescence ratio from GFpH and YFpH showed pH dependency and pK(a) values were 6.1 and 6.8, respectively. Using these indicators expressed in cultured cells, we measured and visualized pH changes in the cytosol and nucleus. Furthermore, by tethering the indicator to a membrane protein (the alpha(1B) adrenergic receptor), we visualized the pH in the vicinity of the protein during internalization caused by endocytosis after agonist stimulation. These novel probes will serve as a useful tool for monitoring pH in the defined organelle and in the microenvironment of a target protein, to analyze cellular function.
Awaji
T
T
Department of Physiology, Tokyo Women's Medical University School of Medicine, 8-1 Kawada-cho, Shinjuku-ku, Tokyo 162-8666, Japan. tawaji@research.twmu.ac.jp
Hirasawa
A
A
Shirakawa
H
H
Tsujimoto
G
G
Miyazaki
S
S
D000818:Animals; D016466:CHO Cells; D019556:COS Cells; D002462:Cell Membrane / Q000378:metabolism; D002467:Cell Nucleus / Q000378:metabolism; D002627:Chemistry, Physical / Q000379:methods*; D006224:Cricetinae; D003600:Cytosol / Q000378:metabolism; D049452:Green Fluorescent Proteins; D006863:Hydrogen-Ion Concentration; D008164:Luminescent Proteins / Q000378:metabolism*; D008956:Models, Chemical; D010957:Plasmids / Q000378:metabolism; D017434:Protein Structure, Tertiary; D021381:Protein Transport; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D013053:Spectrophotometry
D008164:Luminescent Proteins; D011994:Recombinant Proteins; D049452:Green Fluorescent Proteins