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

Abstract

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.

Fluorescence Microscopy in Adeno-Associated Virus Research

“…In addition, the present lower resolution techniques benefit from the development of new dyes and fluorescent proteins, which not only report location but also sense the environment or allow pulse-chase settings. Following AAV uptake and endosomal trafficking might be aided by florescent proteins suited for ratiometric pH tracking in endosomes [ 169 ]. Using convertible, switchable or activatable fluorescent proteins may aid in the determination of viral protein dynamics from nuclear protein import to assembly [ 120 ].…”

Progress in pH-Sensitive sensors: essential tools for organelle pH detection, spotlighting mitochondrion and diverse applications

“…Moreover, the ability to bind extracellularly to cells harboring EGF receptors allows for pH monitoring within microfluidic chambers. Notably, the dual-emission feature of EGF-CoGFP-mCRISPRed, which holds potential in ratiometric flow cytometry is a valuable method for assessing internalization rates and characterizing protein-drug conjugates in the context of cancer therapy ( Karsten et al, 2022 ).…”

A Single Fluorescent Protein-Based Indicator with a Time-Resolved Fluorescence Readout for Precise pH Measurements in the Alkaline Range

“…Thus, it must (i) possess a sufficient sensitivity within the relatively narrow pH range of ~5–8, relevant for the cellular organelles; (ii) provide the determination of an absolute pH value with an accuracy of around 0.1 unit; and (iii) be minimally invasive with respect to the live cell physiology. Genetically encoded fluorescent pH indicators (often based on the fluorescent protein (FP) variants) effectively satisfy the third specified demand [ 14 , 15 , 16 , 17 , 18 ]. In most cases, however, the imaging of such probes relies on the fluorescence intensity readout and, thus, enables the monitoring of the relative pH changes but not the determination of the absolute pH value [ 19 , 20 ].…”