Genetically Encoded Ratiometric pH Sensors for the Measurement of Intra- and Extracellular pH and Internalization Rates

Karsten L, Gött-Zink L, Schmitz J, Hoffrogge R, Grünberger A, Kottke T, Müller K (2022)
Biosensors 12(5): 271.

Zeitschriftenaufsatz | Veröffentlicht | Englisch
 
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Abstract / Bemerkung
pH-sensitive fluorescent proteins as genetically encoded pH sensors are promising tools for monitoring intra- and extracellular pH. However, there is a lack of ratiometric pH sensors, which offer a good dynamic range and can be purified and applied extracellularly to investigate uptake. In our study, the bright fluorescent protein CoGFP_V0 was C-terminally fused to the ligand epidermal growth factor (EGF) and retained its dual-excitation and dual-emission properties as a purified protein. The tandem fluorescent variants EGF-CoGFP-mTagBFP2 (pK′ = 6.6) and EGF-CoGFP-mCRISPRed (pK′ = 6.1) revealed high dynamic ranges between pH 4.0 and 7.5. Using live-cell fluorescence microscopy, both pH sensor molecules permitted the conversion of fluorescence intensity ratios to detailed intracellular pH maps, which revealed pH gradients within endocytic vesicles. Additionally, extracellular binding of the pH sensors to cells expressing the EGF receptor (EGFR) enabled the tracking of pH shifts inside cultivation chambers of a microfluidic device. Furthermore, the dual-emission properties of EGF-CoGFP-mCRISPRed upon 488 nm excitation make this pH sensor a valuable tool for ratiometric flow cytometry. This high-throughput method allowed for the determination of internalization rates, which represents a promising kinetic parameter for the in vitro characterization of protein–drug conjugates in cancer therapy.
Stichworte
pH sensor; EGF; ratiometric; GFP; FRET; dynamic range; flow cytometry; live-cell imaging; endocytosis; pH-sensitive fluorescent protein
Erscheinungsjahr
2022
Zeitschriftentitel
Biosensors
Band
12
Ausgabe
5
Art.-Nr.
271
eISSN
2079-6374
Finanzierungs-Informationen
Open-Access-Publikationskosten wurden durch die Universität Bielefeld gefördert.
Page URI
https://pub.uni-bielefeld.de/record/2962620

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Karsten L, Gött-Zink L, Schmitz J, et al. Genetically Encoded Ratiometric pH Sensors for the Measurement of Intra- and Extracellular pH and Internalization Rates. Biosensors. 2022;12(5): 271.
Karsten, L., Gött-Zink, L., Schmitz, J., Hoffrogge, R., Grünberger, A., Kottke, T., & Müller, K. (2022). Genetically Encoded Ratiometric pH Sensors for the Measurement of Intra- and Extracellular pH and Internalization Rates. Biosensors, 12(5), 271. https://doi.org/10.3390/bios12050271
Karsten, L., Gött-Zink, L., Schmitz, J., Hoffrogge, R., Grünberger, A., Kottke, T., and Müller, K. (2022). Genetically Encoded Ratiometric pH Sensors for the Measurement of Intra- and Extracellular pH and Internalization Rates. Biosensors 12:271.
Karsten, L., et al., 2022. Genetically Encoded Ratiometric pH Sensors for the Measurement of Intra- and Extracellular pH and Internalization Rates. Biosensors, 12(5): 271.
L. Karsten, et al., “Genetically Encoded Ratiometric pH Sensors for the Measurement of Intra- and Extracellular pH and Internalization Rates”, Biosensors, vol. 12, 2022, : 271.
Karsten, L., Gött-Zink, L., Schmitz, J., Hoffrogge, R., Grünberger, A., Kottke, T., Müller, K.: Genetically Encoded Ratiometric pH Sensors for the Measurement of Intra- and Extracellular pH and Internalization Rates. Biosensors. 12, : 271 (2022).
Karsten, Lennard, Gött-Zink, Lukas, Schmitz, Julian, Hoffrogge, Raimund, Grünberger, Alexander, Kottke, Tilman, and Müller, Kristian. “Genetically Encoded Ratiometric pH Sensors for the Measurement of Intra- and Extracellular pH and Internalization Rates”. Biosensors 12.5 (2022): 271.
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2022-04-28T06:52:33Z
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