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Updated: Jun 10, 2025
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Radiolabeling and Quantification of Cellular Levels of Phosphoinositides by High Performance Liquid Chromatography-coupled Flow Scintillation
Published on: January 6, 2016
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Acutely Modifying Phosphatidylinositol Phosphates on Endolysosomes Using Chemically Inducible Dimerization Systems.
Wei Sheng Yap1,2, Peter K Kim1,2,3, Maxime Boutry1,4
1Cell Biology Program, Hospital for Sick Children, Peter Gilgan Centre for Research and Learning, Toronto, ON, Canada.
Bio-Protocol
|October 14, 2024
Summary
This study introduces a live imaging method for acute depletion of phosphoinositides, specifically phosphatidylinositol 4-phosphate (PI(4)P) and phosphatidylinositol 3-phosphate (PI(3)P), on endolysosomes. The technique uses inducible dimerization systems for real-time monitoring and modulation of these crucial signaling lipids.
Area of Science:
- Cell Biology
- Molecular Biology
- Biochemistry
Background:
- Phosphoinositides are essential membrane lipids regulating cell signaling and membrane dynamics.
- Phosphatidylinositol 4-phosphate (PI(4)P) and phosphatidylinositol 3-phosphate (PI(3)P) are key phosphoinositides involved in endolysosomal functions.
- Acute, localized modulation of phosphoinositides is a valuable tool for understanding their cellular roles.
Purpose of the Study:
- To develop and describe a live imaging method for the acute depletion of PI(4)P and PI(3)P on endolysosomes.
- To enable real-time monitoring and validation of phosphoinositide depletion using fluorescent biosensors.
- To provide a versatile protocol adaptable for studying other phosphoinositides on various organelle membranes.
Main Methods:
- Utilized chemically inducible dimerization systems (GAI-GID1 or FRB-FKBP) to recruit phosphoinositide phosphatase catalytic domains (Sac1 for PI(4)P, MTM1 for PI(3)P) to endolysosomes.
- Employed fluorescently tagged biosensors (2xP4M for PI(4)P, PX for PI(3)P) for real-time visualization and quantification of phosphoinositide levels.
- Developed a method for normalizing fluorescence measurements to accurately assess relative phosphoinositide abundance during depletion.
Main Results:
- Successfully demonstrated acute, localized depletion of PI(4)P and PI(3)P on endolysosomes in live cells.
- Validated the depletion efficiency and real-time dynamics using specific fluorescent biosensors.
- Established a normalization strategy for reliable monitoring of phosphoinositide levels.
Conclusions:
- The developed method allows for precise, real-time control and monitoring of PI(4)P and PI(3)P levels on endolysosomes.
- This adaptable protocol can be applied to investigate the function of various phosphoinositides in different cellular compartments.
- The technique provides a powerful tool for dissecting the roles of phosphoinositides in cellular signaling and membrane trafficking.
Keywords:
Chemically inducible dimerization systemEndolysosomesHigh-resolution fluorescence microscopyLive-cell imagingPI(4)PPhosphoinositidesMore Related Videos
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