Direct pH measurements by using subcellular targeting of 5(and 6-) carboxyseminaphthorhodafluor in mammalian cells.
As a means of reliably measuring intracellular pH, we have precisely targeted 5(and 6-) carboxyseminaphthorhodafluor to cellular subcompartments. This was accomplished by combining the well-established pH-sensitive dye with a protein-based reporter system. When expressed in cells, the reporter protein is designed to covalently bind ligands composed of a functional group and a reactive linker. In order to make a pH-sensitive ligand, we chemically coupled the pH sensor to a reactive linker. Several ligands of differing linker lengths were made and tested for their pH responsiveness in vitro. The most responsive of these ligands was then evaluated for its efficacy in live cell labeling and its use as an intracellular pH sensor for ratiometric confocal microscopy. Here we show that we could target this pH sensor within mammalian cells exclusively to either the nucleus or cytoplasm. Exhibiting the versatility of this reporter technology, we were also able to specifically limit pH sensor labeling to within the trafficking pathway of integrins and directly measure pH of this environment. Results correspond well with previously published reports. Both the simplicity and flexibility of the technology used in this study make possible the development of diverse targeted microenvironmental sensors or other moieties of interest.
Research and Development Department, Promega Corporation, Madison, WI 53711, USA. helene.benink@promega.com
Hélène
H
Benink
Mark
M
McDougall
Dieter
D
Klaubert
Georgyi
G
Los
D001578:Benzopyrans / Q000032:analysis* / Q000737:chemistry; D001692:Biological Transport; D015374:Biosensing Techniques / Q000379:methods*; D045744:Cell Line, Tumor; D006801:Humans; D006863:Hydrogen-Ion Concentration; D008024:Ligands; D015394:Molecular Structure; D009284:Naphthols / Q000032:analysis* / Q000737:chemistry; D012235:Rhodamines / Q000032:analysis* / Q000737:chemistry; D013347:Subcellular Fractions / Q000737:chemistry*
D001578:Benzopyrans; D008024:Ligands; D009284:Naphthols; D012235:Rhodamines; C068301:carboxy-seminaphthorhodaminefluoride