Changes in order parameters associated with ceramide-mediated membrane reorganization measured using pTIRFM

Area of Science:

• Membrane biophysics
• Lipid biochemistry
• Cell biology

Background:

• Ceramide generation significantly alters lipid bilayer properties and cell membrane organization.
• Understanding these changes requires tools to probe membrane order and domain structure.

Purpose of the Study:

• To synthesize and characterize a fluorescent lipid probe, NBD-ceramide (NBD-Cer), for studying ceramide's effects on membrane order.
• To utilize NBD-Cer with polarized total internal reflection fluorescence microscopy to investigate ceramide incorporation and its impact on lipid domains.

Main Methods:

• Synthesis and characterization of NBD-ceramide (NBD-Cer).
• Utilizing polarized total internal reflection fluorescence microscopy.
• Analyzing phase-separated lipid bilayers composed of sphingomyelin, dioleoylphosphatidylcholine, and cholesterol.
• Enzyme treatment of bilayers to induce ceramide generation.

Main Results:

• NBD-Cer accurately measures higher order parameters in liquid-ordered (Lo) domains compared to liquid-disordered (Ld) phases.
• The probe detects changes in membrane order due to direct ceramide incorporation or ionic strength variations.
• Enzyme treatment revealed ceramide-enriched regions with high membrane order and increased order in Ld phases surrounding Lo domains.

Conclusions:

• NBD-Cer is an effective tool for probing membrane order changes induced by ceramide.
• Ceramide incorporation leads to distinct membrane reorganizations, including ceramide-enriched domains and altered lipid packing.
• These findings provide insights into the biophysical mechanisms underlying ceramide's role in cell membrane dynamics.