Real-Time Measurement of Endosomal Acidification by a Novel Genetically Encoded Biosensor

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. It is well established that Tat protein enters T-cells using clathrin-coated pits and progressively translocates into well-characterized low-pH endosomes. By using E1GFP conjugated to Tat we monitored the changes in vesicle pH, starting from the initial cell-surface value through the intracellular endocytic network, in real time. We demonstrate a progressive acidification within Tat-loaded vesicles that is crucial to the intracellular fate of Tat. The low endosomal pH exploited by Tat-E1GFP after prolonged incubation in live cells, leads to protein degradation in the lysosomal compartment. We also show that treatment with chloroquine raises the vacuolar pH, demonstrating the ability of E1GFP to respond effectively to environmental pH changes. We believe that this implementation illustrates the suitability of E1GFP for use in the surveillance of intracellular pH during dynamic cellular events with high spatio-temporal resolution.