Abstract
Genetically encoded fluorescent proteins are optimal reporters when used to monitor cellular processes as they can be targeted to any subcellular region by fusion to a protein of interest. Here, we present the pH-sensitive fluorescent protein E1GFP which is ideally suited to monitor pH changes in dynamic intracellular structures in real time with high spatio temporal resolution. E1GFP is a ratiometric pH indicator by emission with a pK close to 6.0. We describe an application of this novel pH reporter in the measurement of pH changes along the endo-lysosomal pathway. By fusing E1GFP to the HIV-Tat protein which is endowed with cell-penetrating properties, we were able to monitor multi-step endocytosis from the initial cell-surface binding through to the intracellular endocytic network in real time. This represents a framework for the application of E1GFP to the in situ detection of pH changes involved in dynamic biological phenomena.
The green fluorecent protein variant, E1GFP, is a ratiometric pH-indicator by emission with a pK close to 6.0 and is therefore particularly suitable for pH detection below neutrality. Upon excitation of the neutral state of the chromophore (~400-410 nm), E1GFP emission properties are strongly dependent on the environmental pH. We describe an application of this novel pH-reporter in the measurement of pH changes along the endo-lysosomal pathway. By fusing E1GFP to the HIV-Tat protein, which is endowed with cell-penetrating properties, we were able to monitor in real-time multi-step endocytosis from the initial cell-surface binding through to the intracellular endocytic network.
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Acknowledgments
We thank Dr. Stefano Luin (Scuola Normale Superiore) for the expert technical assistance and helpful in discussion. The authors also gratefully acknowledge partial the financial support of the Italian Ministry for University and Research (FIRB No. RBLA03ER38) and of the Fondazione Monte dei Paschi di Siena.
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Serresi, M., Bizzarri, R., Cardarelli, F. et al. Real-time measurement of endosomal acidification by a novel genetically encoded biosensor. Anal Bioanal Chem 393, 1123–1133 (2009). https://doi.org/10.1007/s00216-008-2489-7
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DOI: https://doi.org/10.1007/s00216-008-2489-7