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 E(1)GFP which is ideally suited to monitor pH changes in dynamic intracellular structures in real time with high spatio temporal resolution. E(1)GFP 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 E(1)GFP 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 E(1)GFP to the in situ detection of pH changes involved in dynamic biological phenomena.