| Title | Development of a novel GFP-based ratiometric excitation and emission pH indicator for intracellular studies |
|---|---|
| Publication Type | Articolo su Rivista peer-reviewed |
| Year of Publication | 2006 |
| Authors | Bizzarri, R., Arcangeli Caterina, Arosio D., Ricci F., Faraci P., Cardarelli F., and Beltram F. |
| Journal | Biophysical Journal |
| Volume | 90 |
| Pagination | 3300-3314 |
| ISSN | 00063495 |
| Keywords | animal cell, Animalia, Animals, article, Bacterial Proteins, Biological, biological marker, cell function, Cell Line, cell pH, cell stimulation, CHO Cells, Cricetinae, Cricetulus, Field emission, filter, fluorescence, green fluorescent protein, Green Fluorescent Proteins, human, human cell, Human immunodeficiency virus, Humans, Hydrogen-Ion Concentration, imaging, Indicators and Reagents, Intracellular Fluid, light, Luminescent Proteins, Microscopy, mitosis, Models, molecular dynamics, molecular evolution, mutant protein, nonhuman, nucleolus, physiology, promyelocytic leukemia, promyelocytic leukemia protein, protein binding, protein expression, Protein Structure, protein synthesis, signal transduction, spectrophotometry, spectrum, target variable, thermodynamics, transactivator protein, Transfection, Tumor |
| Abstract | We report on the development of the F64L/S65T/T203Y/L231H GFP mutant (E 2GFP) as an effective ratiometric pH indicator for intracellular studies. E 2GFP 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 E 2GFP ideally suited for imaging setups equipped with the most widespread light sources and filter settings. We used E 2GFP 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 E 2GFP with the transactivator protein of HIV, (Tat) and nuclear promyelocytic leukemia bodies (by coexpression of promyelocytic leukemia protein). © 2006 by the Biophysical Society. |
| Notes | cited By 76 |
| URL | https://www.scopus.com/inward/record.uri?eid=2-s2.0-33646140570&doi=10.1529%2fbiophysj.105.074708&partnerID=40&md5=9cbfd897d5ceeb983a97cdc9c027dfc9 |
| DOI | 10.1529/biophysj.105.074708 |
| Citation Key | Bizzarri20063300 |
