Real-time measurement of endosomal acidification by a novel genetically encoded biosensor

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. Figure 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. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.