Quantification of Genetically Encoded Lipid Biosensors.

Area of Science:

• Cell biology
• Biochemistry
• Microscopy

Background:

• Lipids play crucial roles in cellular functions.
• Visualizing lipid dynamics in living cells is essential for understanding cellular processes.
• Current methods for lipid quantification can be limited.

Purpose of the Study:

• To provide a detailed protocol for visualizing and quantifying relative lipid levels in real time.
• To enable the study of lipid changes in response to genetic or enzymatic alterations.
• To facilitate the investigation of lipid metabolism and signaling pathways.

Main Methods:

• Utilizing genetically encoded biosensors for specific lipid detection.
• Employing fluorescence microscopy for real-time visualization.
• Quantifying sensor localization via fluorescence intensity measurements.
• Applying organelle-specific binary masks to determine lipid enrichment at membranes.

Main Results:

• Demonstrated a method to visualize and quantify lipid levels, including phosphoinositides, in living cells.
• Established a workflow for assessing lipid enrichment at cellular membranes.
• Showcased the ability to detect changes in lipid levels under altered enzyme activity.

Conclusions:

• The described methods offer a robust approach for real-time lipid analysis in cell biology.
• This technique is valuable for studying lipid metabolism and the impact of enzyme mutations.
• The protocol facilitates a deeper understanding of lipid dynamics and cellular signaling.