Analysis of EGF Receptor Endocytosis Using Fluorogen Activating Protein Tagged Receptor

Abstract

Functional activities of many transmembrane proteins are controlled by their endocytosis. One of the most studied experimental models is the epidermal growth factor (EGF) receptor (EGFR). However, endocytic trafficking of EGFR has been predominantly analyzed using labeled EGF, whereas quantitative analyses of the endocytosis of the receptor itself have been sparse. The fluorescence microscopy methods described here are designed to directly quantify EGFR internalization in living cells without labeled EGFR ligands or antibodies. These methods utilize an engineered EGFR chimera in which the fluorogen activating protein (FAP) is fused to the receptor extracellular domain (FAP-EGFR). Binding of malachite green (MG) based dyes to FAP results in a strong far-red fluorescence of MG, thus efficiently labeling FAP-EGFR. In particular, binding of the cell impermeant MG-Bis-SA dye to FAP produces the pH-sensitive dual-excitation fluorescence, which allows differentiation of the cell-surface and internalized pools of FAP-EGFR. Two modifications of the methodology are described: 1) singlecell three-dimensional confocal imaging; and 2) high-throughput assay in multi-well plates. These methodologies can be adopted to study endocytosis of any other transmembrane protein extracellularly tagged with FAP.

Mechanism of p38 MAPK–induced EGFR endocytosis and its crosstalk with ligand-induced pathways

“…MG dyes become fluorescent upon binding to FAP with excitation at ∼640 nm and emission at ∼670–700 nm ( Perkins and Bruchez, 2020 ). Two cell-impermeant MG derivatives were used to label cell surface FAP-EGFR: (1) pH-insensitive MG-B-Tau; and (2) pH-sensitive MG-Bis-SA to quantify FAP-EGFR endocytosis using a fluorescence excitation ratiometric imaging (FERI) assay developed in our recent studies ( Larsen et al, 2019 ; Perez Verdaguer et al, 2019 ). Briefly, MG-Bis-SA is a tandem dye in which MG is linked to a pH-sensitive Cy3.…”

“…Labeling and imaging of FAP-EGFR in HeLa/FAP-EGFR cells was described previously ( Larsen et al, 2019 ; Perez Verdaguer et al, 2019 ). Briefly, cells were first incubated with 50 nM MG-B-Tau or 100 nM MG-Bis-SA in DMEM for 5 min at 37°C and then experimentally treated.…”

“…Images were acquired with a spinning-disk Marianas system based on a Zeiss Axio Observer Z1 inverted fluorescence microscope, equipped with CSU-W1 Yokogawa spinning disk; 405-, 436-, 488-, 515-, 561-, and 640-nm lasers; a 63× Plan Apo PH NA 1.4 oil immersion objective; an Evolve EM-CCD camera (Photometrics), a piezo stage controller, a spherical aberration correction module, and a temperature- and CO 2 -controlled chamber, all controlled by Slidebook6 software (Intelligent Imaging Innovation) as described previously ( Larsen et al, 2019 ; Perez Verdaguer et al, 2019 ). Typically, a z-stack of 10 or 20 x-y confocal images was acquired for PAE or HeLa/FAP-EGFR cells, respectively, at 400-nm z-steps.…”

“…EGFR is the most studied experimental model of stimulated endocytosis, in large part owing to the availability of fluorescent and radiolabeled EGF. In this study, we wanted to analyze both ligand-dependent and -independent internalization, and therefore took advantage of an experimental approach for a quantitative analysis of EGFR endocytosis we recently developed, which is completely independent of the ligand ( Larsen et al, 2019 ; Perez Verdaguer et al, 2019 ). Central to this approach is the use of CRISPR/Cas9 gene-edited HeLa cells expressing endogenous EGFR that is extracellularly tagged with a fluorogen-activating protein (FAP; hereafter referred as HeLa/FAP-EGFR cells).…”

Fluorogen activating protein toolset for protein trafficking measurements

“…Understanding the EGFR trafficking mechanisms is difficult due to current methods that utilize overexpressed recombinant EGFR and high ligand concentrations that tend to induce a specific mode of receptor internalization over others . A recent study by Larsen et al, developed a dL5** FAP‐EGFR assay for high throughput screening of proteins important in the clathrin mediated endocytosis of EGFR . This assay design allowed bypass of the difficulties involved with labeled EGFR ligands or EGFR overexpression by using CRISPR/Cas9 technology to maintain endogenous expression levels of dL5** tagged EGFR and response to physiological levels of EGF (sub‐ng/mL).…”