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Updated: Jul 23, 2025
Measuring the pH, Redox Chemistries, and Degradative Capacity of Macropinosomes using Dual-Fluorophore Ratiometric Microscopy
Published on: August 19, 2021
Macropinoscope: Real-Time Simultaneous Tracking of pH and Cathepsin B Activity in Individual Macropinosomes
Hisaaki Hirose1, Eiji Nakata2, Zhengxiao Zhang2
1Institute for Chemical Research, Kyoto University, Uji, Kyoto 611-0011, Japan.
Abstract:
A fluorescent sensor that allows simultaneous analysis of environmental factors in a limited cellular space is useful for understanding precise molecular interactions in live cells and their biological responses. Macropinocytosis is a ubiquitous endocytic pathway for massive uptake of extracellular fluids, resulting in the formation of macropinosomes. Although macropinocytosis may impact intracellular delivery and cancer proliferation, information on the intracellular behaviors of macropinosomes is limited. Here, we aimed to develop a macropinoscope, a sensor that simultaneously detects pH and cathepsin B activity in individual macropinosomes. A macropinosome-specific marker, dextran (70 kDa), was employed as a platform, onto which fluorescein, Oregon Green, and tetramethylrhodamine were loaded for ratiometric pH sensing and imaging. A cathepsin-B-cleavable peptide sequence bearing sulfo-Cy5 and the quencher BHQ-3 was also mounted; cleavage of the sequence was detected as an increase in sulfo-Cy5 fluorescence. A steep decrease in pH was observed 5-10 min after macropinosome formation, which was accompanied by an immediate increase in cathepsin B activity. Our design concept will lead to the development of other macropinoscopes for the simultaneous detection of other parameters in individual macropinosomes.
Insights
Researchers developed a novel sensor, the macropinoscope, to simultaneously track pH and cathepsin B activity within macropinosomes. This tool offers new insights into cellular processes like macropinocytosis and its role in cancer.
Area of Science:
- Cell Biology
- Biochemistry
- Molecular Imaging
Background:
- Macropinocytosis is a cellular process for fluid uptake, crucial for nutrient acquisition and immune surveillance.
- Understanding macropinosome dynamics is vital for insights into intracellular delivery and cancer progression.
- Limited tools exist for simultaneous analysis of multiple parameters within individual macropinosomes.
Purpose of the Study:
- To develop a novel fluorescent sensor, termed a macropinoscope, for simultaneous detection of pH and cathepsin B activity.
- To investigate the dynamic changes in pH and cathepsin B activity within individual macropinosomes.
- To establish a versatile platform for creating sensors to monitor other parameters in macropinosomes.
Main Methods:
- Utilized dextran (70 kDa) as a macropinosome-specific marker and platform.
- Incorporated fluorescein, Oregon Green, and tetramethylrhodamine for ratiometric pH sensing.
- Integrated a cathepsin-B-cleavable peptide sequence with sulfo-Cy5 and BHQ-3 for activity detection.
Main Results:
- Observed a rapid pH decrease (5-10 min) post-macropinosome formation.
- Detected an immediate increase in cathepsin B activity coinciding with the pH drop.
- Successfully demonstrated simultaneous ratiometric pH imaging and cathepsin B activity monitoring in live cells.
Conclusions:
- The developed macropinoscope enables real-time, simultaneous monitoring of pH and cathepsin B activity in macropinosomes.
- This tool provides novel insights into the dynamic intracellular environment of macropinosomes.
- The design concept is adaptable for creating sensors to detect other analytes within macropinosomes, advancing cellular analysis.
