Ceramide-platform formation and -induced biophysical changes in a fluid phospholipid membrane

Abstract

To understand the formation and properties of ceramide-enriched domains in cell membranes, the biophysical properties of the binary system palmitoyloleoylphosphatidylcholine (POPC)/palmitoylceramide were thoroughly characterized. Diverse fluorescent probes and parameters were necessary to unravel the complexity of this apparently simple system. For the first time, a complete phase diagram is reported, characterizing the lamellar phases of these mixtures, and providing a quantitative framework integrating biophysical and biological studies. The diagram suggests that in resting cells no ceramide domains exist, but upon apoptotic stimuli, platforms may form. Moreover, our data show that 2 mol% of Cer strongly affects the POPC fluid matrix, suggesting that a small increase in Cer levels can significantly affect cell membrane properties. In this work, we also show that Cer domains, formed in conditions similar to physiological, are extremely ordered and rigid. The domains composition is estimated from the phase diagram and their large size was concluded from fluorescence resonance energy transfer. Dynamic light scattering and electron microscopy were used to characterize the system morphology, which is highly dependent on ceramide content and includes vesiculation and tubular structure formation.

Changes in membrane biophysical properties induced by sphingomyelinase depend on the sphingolipid N-acyl chain

“…To date, several biophysical studies [e.g., (13)(14)(15)(16)(17)(18)(19)] have focused on the effect of Cer on the properties of model membranes and have demonstrated that Cer increases the global order of the membrane and promotes lateral phase separation in a manner dependent on its acyl chain structure (19)(20)(21). However, no information is currently available on the effect of altering the Cer acyl chain length on the properties of cell membranes.…”

Cholesterol Packing around Lipids with Saturated and Unsaturated Chains: A Simulation Study

“…POPC has also been investigated as part of ternary bilayer mixtures with sphingomeylin and cholesterol 8,20,21 or ceramide. 22 Heerklotz and co-workers used calorimetry to compare the interactions of cholesterol with large unilamellar vesicles of POPC plus 30% cholesterol, sphingomyelin plus 30% cholesterol, and 1:1 sphingomyelin:POPC plus 30% cholesterol.…”

Ceramide-containing membranes: the interface between biophysics and biology

“…Cer also drives lateral phase separation in ‰uid phospholipid bilayers to form regions of Cer-enriched gel domains and Cer-poor ‰uid domains (51,52,55,(57)(58)(59). Only a small amount of Cer (up to 5 molz) is required to drive gel-‰uid phase separation (51,53,56,58) suggesting that these phase changes may occur at physiologically relevant Cer concentrations.…”

“…membranes composed of POPC/SM/Chol, where the ability of Cer to segregate into gel domains is strongly modulated by Chol (61). Use of ‰uorescence techniques, based on multiple probes with diŠerent phase-related properties (53), demonstrated that when the Chol content is low, Cer is able to recruit up to 3 SM molecules to form highly ordered Cer/SM gel domains. Under these conditions, Cer/SM-gel domains are very small (¿4 nm), and are typically surrounded by an lo phase, but are unable to induce raft coalescence (61).…”

“…Cer-gel domains are characterized by an atypical high degree of order (53,54) compared to other lipid gel domains, e.g. SM-gel domains (9).…”