Macropinoscope: Real-Time Simultaneous Tracking of pH and Cathepsin B Activity in Individual Macropinosomes

A fluorescent sensor that allows simultaneous analysisof environmentalfactors in a limited cellular space is useful for understanding precisemolecular interactions in live cells and their biological responses.Macropinocytosis is a ubiquitous endocytic pathway for massive uptakeof extracellular fluids, resulting in the formation of macropinosomes.Although macropinocytosis may impact intracellular delivery and cancerproliferation, information on the intracellular behaviors of macropinosomesis limited. Here, we aimed to develop a macropinoscope, a sensor thatsimultaneously detects pH and cathepsin B activity in individual macropinosomes.A macropinosome-specific marker, dextran (70 kDa), was employed asa platform, onto which fluorescein, Oregon Green, and tetramethylrhodaminewere loaded for ratiometric pH sensing and imaging. A cathepsin-B-cleavablepeptide sequence bearing sulfo-Cy5 and the quencher BHQ-3 was alsomounted; cleavage of the sequence was detected as an increase in sulfo-Cy5fluorescence. A steep decrease in pH was observed 5-10 minafter macropinosome formation, which was accompanied by an immediateincrease in cathepsin B activity. Our design concept will lead tothe development of other macropinoscopes for the simultaneous detectionof other parameters in individual macropinosomes.

Automated summary

This study developed a fluorescent sensor that can simultaneously analyze environmental factors in a limited cellular space, allowing for precise understanding of molecular interactions and biological responses in live cells. The sensor was able to detect both pH levels and cathepsin B activity in individual macropinosomes, providing new insights into the intracellular behaviors of macropinocytosis.