Illuminating Transmembrane Proteins Using HiBiT CRISPR Cell Lines

HiBiT technology is a powerful tool for studying transmembrane and extracellular proteins, featuring a small, luminescent tag suitable for sensitive and quantitative detection of protein expression, localization, and dynamics. HiBiT is an 11-amino-acid fragment of the NanoBiT® complex, which, upon binding to its complementary partner LgBiT, reconstitutes a functional luciferase enzyme. Addition of substrate generates a luminescent signal strong enough to detect proteins at their endogenous levels. The minimal size of HiBiT reduces interference with the native function of tagged proteins and facilitates precise CRISPR-based tagging, thus avoiding artifacts common in overexpression models. Additionally, NanoBiT® luciferase activity is compatible with both intra- and extracellular environments, making it particularly effective for analyzing transmembrane proteins. In this article, we demonstrate the utility of HiBiT by tracking Epidermal Growth Factor Receptor (EGFR), a transmembrane receptor tyrosine kinase essential for cell proliferation signaling.

 

We generated endogenous HiBiT-tagged EGFR cells via CRISPR knock-in. Tagging genes at their endogenous locus ensures proteins are expressed at native levels under native regulatory control which is crucial for accurately studying biologically relevant processes such as receptor signaling and trafficking. We selected a knock-in site between the native signal peptide and the N-terminus of the mature EGFR protein (residues 24/25) to preserve proper trafficking and HiBiT integrity up signal peptide removal. When the native signal peptide is unknown or undefined, as is the case for approximately 80% of GPCRs, an alternative, well-characterized signal peptide (e.g., IL-6) can be introduced alongside HiBiT. However, ensuring that only a single signal peptide is present is essential for correct protein trafficking.

When HiBiT is presented extracellularly, live-cell flow cytometry using Anti-HiBiT Monoclonal Antibody, XFD Conjugates enables enrichment of CRISPR-edited pools for HiBiT-positive cells. Here, flow cytometry results revealed a cell pool contained approximately 10% HiBiT-EGFR cells (Figure 1, red trace). A single HiBiT-EGFR clonal cell line was then established following sequencing and allelic counting to verify no additional mutations occurred during the gene editing. Subsequent flow cytometry confirmed robust surface HiBiT-EGFR expression in the clonal line (Figure 1, orange trace), validating that HiBiT tagging did not disrupt EGFR trafficking to the plasma membrane.

Figure 1 Flow cytometry analysis of HiBiT-EGFR CRISPR knock-in cells. A cell pool containing HiBiT-EGFR CRISPR knock-in cells (red trace) was enriched for cells expressing HiBiT on the cell surface using Anti-HiBiT Monoclonal Antibody-FarRed647 relative to parental HeLa cells (blue trace). A clonal HiBiT-EGFR line was established and live-cell flow cytometry confirmed high surface expression (orange trace).

Internalization is a critical mechanism regulating the signaling and degradation of EGFR. After ligand binding, EGFR undergoes endocytosis, followed by recycling or lysosomal degradation, processes that precisely control signaling intensity and duration. Disruption of EGFR internalization can lead to prolonged signaling, contributing to uncontrolled cellular growth and survival commonly observed in cancers (1, 2).

Nano-Glo® HiBiT Extracellular Detection System measures surface expression of HiBiT-tagged EGFR by introducing substrate and purified, cell-impermeable LgBiT directly into live-cell culture media (Figure 2). LgBiT selectively binds to extracellular HiBiT, producing a luminescent signal specific to EGFR localized on the plasma membrane. Internalization removes HiBiT-EGFR from the extracellular environment, decreasing luminescence proportionally to the amount internalized. Nano-Glo® HiBiT Extracellular Detection System facilitates rapid and quantitative assays, either in a plate-based format or through imaging using the GloMax® Galaxy Bioluminescence Imager (For Research Use Only). 

Figure 2 Nano-Glo® HiBiT Extracellular Assay concept. Adding purified LgBiT, the NanoBiT® luciferase fragment with high affinity for HiBiT, to the media of live cells enables selective quantification of extracellular HiBiT as LgBiT cannot cross the cell membrane. 

For a plate-based end-point assay, HiBiT-EGFR cells were treated with various concentrations of EGF, and then Nano-Glo® HiBiT Extracellular Detection Reagent was added. We observed dose-dependent reductions in luminescence corresponding to the expected increased internalization at higher concentrations of EGF. The assay generated a dose-response curve with an EC50 of 3.9 ng/ml and a robust ~7-fold assay window (Figure 3), aligning well with previous studies (3, 4). These findings underscore HiBiT's reliability for quantitatively analyzing receptor internalization at endogenous expression levels.

 

Figure 3 HiBiT-EGFR internalization upon EGF treatment. Increasing doses of EGF resulted in reduced luminescence from HiBiT-EGFR CRISPR knock-in cells, indicating reduced surface expression at those concentrations. Nano-Glo® HiBiT Extracellular Detection System enables robust quantification of receptor internalization at endogenous expression levels within minutes.

Live-cell bioluminescence imaging on GloMax® Galaxy Bioluminescence Imager further validated these findings. HiBiT-EGFR clonal cells were incubated with Nano-Glo® HiBiT Extracellular Detection Reagent, allowing formation of active NanoBiT® luciferase complexes on the cell surface. By preforming the NanoBiT® luciferase complex, internalization can be visualized and quantified kinetically by the pH-dependent decrease in luminescence in the lower pH environment of endosomes. LgBiT-bound HiBiT-EGFR cells were then either left untreated or stimulated with 300 ng/mL EGF to induce internalization and imaged continuously for 30 minutes. Control cells maintained robust membrane localization and luminescent signals, whereas EGF-treated cells showed progressive internalization accompanied by diminished surface luminescence and punctuated intracellular luminescence typical of endosomal localization over time (Figure 4). 

Figure 4 Bioluminescence imaging of HiBiT-EGFR internalization. A) HiBIT-EGFR control cells show steady membrane localization and bioluminescent signal (left) while EGF-treatment induces internalization and a reduction in the bioluminescent signal (right). Images were collected on the GloMax® Galaxy with 2-minute continuous exposures over 30 minutes with the Stagetop Incubator and Controller accessory. Luminescent signal was pseudo colored blue. B) Relative optical density (ROD) of the luminescent signal was quantified by measuring the raw gray values (integrated density) across the image time sequences. See GloMax® Galaxy FIJI Quick Start Guide for more information on image processing and analysis. C) Selected optical zoom images from the 12-minute timepoint highlight the membrane localization in control cells (left) and intracellular puncta in EGF-treated cells (right).

These methods offer powerful, real-time, non-invasive approaches to monitor receptor dynamics at the cell surface, providing insights into receptor trafficking and regulation. The Nano-Glo® HiBiT Extracellular Assay’s high sensitivity, quantitative, and scalable nature make it particularly useful for studying this dynamic process, which plays a critical role in cellular signaling and disease biology.