Litmus-Body: A Molecularly Targeted Sensor for Cell-Surface pH Measurements

Area of Science:

• Biochemistry
• Molecular Biology
• Cell Biology

Background:

• Accurate pH measurements near cell surface receptors are crucial for understanding disease mechanisms.
• Existing pH sensors lack the specificity and targeting capabilities for localized measurements.
• Aberrant receptor function is often linked to local pH alterations in diseased states.

Purpose of the Study:

• To develop a novel, targeted pH sensor for precise measurements at specific cellular receptors.
• To create a tool for investigating the role of local pH in receptor function and disease.
• To validate the sensor's functionality and specificity on live cells.

Main Methods:

• Engineered a recombinant protein-based pH sensor (Litmus-body) fused to an anti-IgG nanobody for targeted delivery via IgG antibodies.
• Utilized a ratiometric approach normalizing a pH-sensitive green fluorescent protein to a red fluorophore for concentration-independent pH reporting.
• Validated the sensor's performance in solution and on live cells, assessing its ability to track pH changes during receptor-mediated endocytosis.

Main Results:

• Litmus-body demonstrated a >50-fold change in fluorescence across physiological pH ranges.
• The sensor exhibited high specificity for the target protein on live cells.
• Cetuximab antibody fused with Litmus-body retained its EGFR binding and inhibitory function, enabling tracking of endocytic pathway pH.

Conclusions:

• Litmus-body provides a powerful, targeted approach for measuring local pH at specific cell surface receptors.
• This technology facilitates the study of pH dynamics in receptor function and disease pathogenesis.
• The sensor's ability to track pH through endocytosis opens new avenues for investigating cellular processes.