Genetically Encoded, pH-Sensitive mTFP1 Biosensor for Probing Lysosomal pH.
ACS Sens
; 6(6): 2168-2180, 2021 06 25.
Article
em En
| MEDLINE
| ID: mdl-34102054
ABSTRACT
Lysosomes are important sites for macromolecular degradation, defined by an acidic lumenal pH of â¼4.5. 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.
Palavras-chave
Texto completo:
1
Coleções:
01-internacional
Base de dados:
MEDLINE
Assunto principal:
Técnicas Biossensoriais
/
Lisossomos
Tipo de estudo:
Diagnostic_studies
Idioma:
En
Ano de publicação:
2021
Tipo de documento:
Article