A genetically encoded, pH-sensitive mTFP1 biosensor for probing lysosomal pH

ABSTRACT Lysosomes are important sites for macromolecular degradation, defined by an acidic lumenal pH of ∼4.5. To better understand lysosomal pH, we designed a novel, genetically encoded, fluorescent protein (FP) based pH biosensor called FIRE-pHLy ( F luorescence Indicator RE porting pH in Ly sosomes). This biosensor was targeted to lysosomes with lysosomal-associated membrane protein 1 (LAMP1) and reported lumenal pH between 3.5 and 6.0 with monomeric teal fluorescent protein 1 (mTFP1), a bright cyan pH sensitive FP variant with a pKa of 4.3. Ratiometric quantification was enabled with cytosolically oriented mCherry using high-content quantitative imaging. We expressed FIRE-pHLy in several cellular models and quantified the alkalinizing response to bafilomycin A1, a specific V-ATPase inhibitor. In summary, we have engineered FIRE-pHLy, a specific, robust and versatile lysosomal pH biosensor that has broad applications for investigating pH dynamics in aging and lysosome-related diseases, as well as in lysosome-based drug discovery.