A modular biosensor platform for real-time, multiplexed monitoring of receptor tyrosine kinase activity in living cells

Background

Receptor tyrosine kinases (RTKs) are a large family of cell surface receptors that play pivotal roles in regulating cell growth, differentiation, migration, and survival. Aberrant RTK signaling is implicated in a wide range of diseases, including cancer, developmental disorders, and metabolic syndromes. Understanding the precise dynamics of RTK activation in living cells is crucial for unraveling the mechanisms underlying these processes and for developing targeted therapeutics. Traditional biochemical assays, such as Western blotting and immunoprecipitation, offer only static, population-averaged snapshots of RTK activity and lack the spatial and temporal resolution needed to capture the rapid and localized signaling events that occur in single cells. As a result, there is a growing demand for live-cell biosensors that can monitor the activation of specific RTKs in real time, with high sensitivity and specificity.

Current approaches to monitoring RTK activity in live cells, such as FRET-based biosensors or phospho-specific antibodies, face significant limitations. FRET biosensors often require complex engineering for each RTK, can suffer from poor signal-to-noise ratios, and are generally not easily multiplexed to monitor multiple RTKs simultaneously. Phospho-specific antibodies, while useful in fixed-cell or lysate-based assays, are not suitable for dynamic live-cell imaging and may cross-react with multiple phosphorylated proteins, reducing specificity. Moreover, many existing biosensors are not modular, making it difficult to adapt them to different RTKs or to endogenous receptors without extensive re-engineering. These limitations hinder the ability to dissect the nuanced and context-dependent signaling behaviors of individual RTKs, especially in complex multicellular environments or in the presence of closely related receptor subtypes.

Technology

The described technology is a modular biosensor platform called pYtags, designed for real-time monitoring of receptor tyrosine kinase (RTK) activity in living cells. It operates through two main components: a genetically engineered RTK fused at its C-terminus with tandem immunoreceptor tyrosine-based activation motifs (ITAMs), and a cytosolic reporter protein composed of tandem SH2 domains (tSH2) attached to a fluorescent marker. Upon activation of the RTK, the ITAMs are phosphorylated, prompting the tSH2 reporter to bind and translocate from the cytosol to the plasma membrane or receptor-containing compartments. This movement is detected as a decrease in cytosolic fluorescence, providing a sensitive, quantitative, and real-time readout of RTK activation. The system is highly modular, allowing adaptation to various RTKs, supports multiplexed imaging with orthogonal reporter pairs, and can be applied to endogenous receptors via genome editing.

What differentiates this technology is its unique use of immune-derived ITAM/tSH2 pairs, which are orthogonal to endogenous phosphotyrosine signaling in non-immune cells, minimizing background noise and crosstalk. Unlike traditional biosensors, pYtags offer exceptional specificity, enabling the selective monitoring of individual RTKs, even within heterodimeric complexes, and support simultaneous imaging of multiple RTKs in the same cell. The platform achieves high spatiotemporal resolution, capturing rapid and localized signaling events, and is validated for use in both overexpressed and genome-edited endogenous settings. Its versatility extends to drug screening, synthetic biology, and studies of developmental or disease-related signaling, making it a powerful tool for dissecting complex RTK-mediated processes in live cells with unprecedented precision.

Benefits

• Enables specific, real-time monitoring of individual receptor tyrosine kinase (RTK) activity in living cells with high spatial and temporal resolution.
• Modular design allows adaptation to various RTKs and multiplexed imaging of multiple receptors simultaneously using orthogonal biosensor pairs.
• Orthogonality to endogenous signaling minimizes background interference and crosstalk in nonimmune cells.
• Compatible with genome editing for monitoring endogenous RTKs, avoiding artifacts from overexpression.
• Provides quantitative, dynamic readouts of RTK activation, including ligand- and dose-dependent signaling kinetics.
• Supports investigation of RTK signaling in complex multicellular and tissue-like environments.
• Useful for drug screening, synthetic biology, and studying RTK-related processes in development and disease.

Applications

• Live-cell RTK drug screening
• Multiplexed RTK signaling analysis
• Real-time cancer signaling studies
• Endogenous RTK activity monitoring
• Synthetic receptor engineering

Additional info

A modular biosensor system, pYtags, monitors receptor tyrosine kinase activity. It comprises an RTK genetically modified with tandem ITAMs and a cytosolic fluorescent tSH2 reporter. Upon RTK activation, ITAMs phosphorylate, binding the tSH2. This triggers tSH2 translocation from cytosol to membrane, quantified as decreased cytosolic fluorescence, providing a real-time readout.