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Single-molecule Super-resolution Imaging of Phosphatidylinositol 4,5-bisphosphate in the Plasma Membrane with Novel Fluorescent Probes
Published on: October 15, 2016
Imaging phosphoinositide dynamics in living cells.
Anne Wuttke1, Olof Idevall-Hagren, Anders Tengholm
1Department of Medical Cell Biology, Uppsala University, Uppsala, Sweden.
Methods in Molecular Biology (Clifton, N.J.)
|July 21, 2010
Summary
This study presents methods for visualizing phosphoinositide dynamics in single cells using fluorescent protein probes. These techniques enable high-resolution imaging of inositol lipid changes crucial for cellular signaling.
Area of Science:
- Cell Biology
- Biochemistry
- Microscopy
Background:
- Inositol lipid derivatives are vital for cellular signaling and processes.
- Measuring phosphoinositide changes in real-time within single cells is essential for understanding their roles.
- Specific protein domains interacting with inositol lipids can be engineered as cellular probes.
Purpose of the Study:
- To develop and present protocols for fluorescence imaging of phosphoinositide dynamics in living cells.
- To enable high spatial and temporal resolution of phosphoinositide concentration changes.
- To visualize various phosphoinositide species in different subcellular compartments.
Main Methods:
- Utilizing protein domains tagged with green fluorescent protein variants as phosphoinositide probes.
- Employing total internal reflection fluorescence microscopy for time-lapse imaging of the plasma membrane.
- Applying these methods to study phospholipase C- and PI3-kinase-induced inositol lipid changes in insulin-secreting cells.
Main Results:
- Demonstrated successful visualization of phosphoinositide dynamics in single living cells.
- Showcased the utility of total internal reflection fluorescence microscopy for capturing rapid inositol lipid fluctuations.
- Quantified changes in inositol lipids induced by specific signaling enzymes.
Conclusions:
- The presented fluorescence imaging protocols offer high spatial and temporal resolution for studying phosphoinositide metabolism.
- These techniques are adaptable for investigating spatio-temporal regulation of phosphoinositides across various cell types.
- This work enhances the understanding of inositol lipid roles in cellular signaling pathways.
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