Related Experiment Video
Updated: Jun 15, 2025
09:45
Live Imaging of Dense-core Vesicles in Primary Cultured Hippocampal Neurons
Published on: May 29, 2009
12.6K
PI(4,5)P2 Imaging Using a GFP Reporter in Living Cells
Mariam Alkandari1, Christopher R McMaster1, Mahtab Tavasoli1
1Department of Pharmacology, Dalhousie University, Halifax, NS, Canada.
Bio-Protocol
|June 13, 2025
Summary
This study introduces a new method to visualize and quantify phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2) in live cells. The protocol uses a specific fluorescent probe to track PI(4,5)P2 dynamics, aiding in understanding cellular processes and diseases.
Area of Science:
- Cell Biology
- Biochemistry
- Molecular Biology
Background:
- Phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2) is a critical plasma membrane phospholipid.
- PI(4,5)P2 regulates membrane trafficking and cytoskeletal dynamics.
- Dysregulation of PI(4,5)P2 is implicated in various disease states.
Purpose of the Study:
- To present a protocol for visualizing and quantifying PI(4,5)P2 localization in live cells.
- To enable temporal studies of PI(4,5)P2 dynamics under physiological and pathological conditions.
Main Methods:
- Utilizes a specific PI(4,5)P2 probe, PH-PLCD1-GFP.
- Employs fluorescence imaging and confocal microscopy for localization and quantification.
- Includes detailed instructions for reagent preparation and image analysis.
Main Results:
- Successfully demonstrates a method for quantifying cytosol-facing PI(4,5)P2 localization.
- The PH-PLCD1-GFP probe allows for accurate tracking of PI(4,5)P2.
- The protocol is adaptable to various cell types and experimental setups.
Conclusions:
- The developed protocol provides a valuable tool for studying PI(4,5)P2 dynamics in live cells.
- This method can advance research into cellular functions and disease mechanisms involving PI(4,5)P2.
Related Concept Videos
02:11Reporter Genes
11.3K
Reporter genes are a type of protein-coding gene that are often tagged to a gene of interest. Once inside a target cell, reporter genes usually produce visually identifiable characteristics like fluorescence and luminescence when expressed along with the gene of interest. Thus, reporter genes “report” the presence or absence of genes of interest in an organism, determine the gene expression pattern, or track the physical location of a DNA segment or protein in the cell.
11.3K
01:19Protein Dynamics in Living Cells
2.1K
Different fluorescence-based techniques are used to study the protein dynamics in living cells. These techniques include FRAP, FRET, and PET.
Fluorescent recovery after photobleaching (FRAP) is a fluorescent-protein-based detection technique used to quantify protein movement rates within the cell. This method exposes a small portion of the cell to an intense laser beam. The laser beam causes permanent photobleaching of the fluorophore-tagged proteins in the exposed region. As the bleached...
Fluorescent recovery after photobleaching (FRAP) is a fluorescent-protein-based detection technique used to quantify protein movement rates within the cell. This method exposes a small portion of the cell to an intense laser beam. The laser beam causes permanent photobleaching of the fluorophore-tagged proteins in the exposed region. As the bleached...
2.1K