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Genetically Encoded, pH-Sensitive mTFP1 Biosensor for Probing Lysosomal pH.

  • Author(s): Chin, Marcus Y
  • Patwardhan, Anand R
  • Ang, Kean-Hooi
  • Wang, Austin L
  • Alquezar, Carolina
  • Welch, Mackenzie
  • Nguyen, Phi T
  • Grabe, Michael
  • Molofsky, Anna V
  • Arkin, Michelle R
  • Kao, Aimee W
  • et al.

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 Fluorescence Indicator REporting pH in Lysosomes (FIRE-pHLy). 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.

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