Practical labeling methodology for choline-derived lipids and applications in live cell fluorescence imaging

Area of Science:

• Biochemistry
• Cell Biology
• Chemical Biology

Background:

• Plasma membrane lipids are crucial for cellular processes.
• Understanding lipid function requires effective labeling and localization methods.
• Metabolic incorporation of alkyne groups has been used to label phosphocholine lipids.

Purpose of the Study:

• To develop and validate new bioorthogonal metabolic labels for choline-containing lipids and lipid acyl chains.
• To synthesize novel cyanine-based dyes reactive with these labels.
• To apply these labeling strategies for studying lipid dynamics and cell adhesion.

Main Methods:

• Synthesis of alkyne, azide, and ketone derivatives of choline for metabolic labeling.
• Use of 17-octadecynoic acid as a lipid acyl chain label.
• Development of cyanine dyes for bioorthogonal labeling via Cu(I)-catalyzed azide-alkyne cycloaddition (CuAAC).
• Application of labeling in live cells, followed by flow cytometry and fluorescence microscopy.
• Measurement of lipid lateral mobility using fluorescence photobleaching recovery (FPR).

Main Results:

• Successfully metabolically labeled choline-containing lipids and lipid acyl chains using novel derivatives.
• Synthesized and utilized cyanine dyes for efficient fluorophore conjugation.
• Demonstrated the ability to study lipid dynamics and cell adhesion in live cells.
• Validated the robustness of bioorthogonal labeling strategies for native lipids.

Conclusions:

• Developed versatile bioorthogonal labeling strategies for native plasma membrane lipids.
• These methods provide powerful tools for investigating lipid biochemistry and cell membrane functions.
• The described techniques facilitate advanced studies in live cell imaging and biophysics.