Changes in Order Parameters Associated with Ceramide-Mediated Membrane Reorganization Measured Using pTIRFM

Abstract

The enzymatic generation of ceramide has significant effects on the biophysical properties of lipid bilayers and can lead to the extensive reorganization of cell membranes. We have synthesized and characterized a headgroup-labeled fluorescent lipid probe (NBD-ceramide, NBD-Cer) and demonstrated that it can be used for polarized total internal reflection fluorescence microscopy experiments to probe changes in membrane order that result from ceramide incorporation. NBD-Cer measures significantly higher order parameters for the liquid-ordered (L o ) domains (⟨P 2 ⟩ = 0.40 ± 0.03) than for the liquid-disordered phase (L d , fluid, ⟨P 2 ⟩ = 0.22 ± 0.02) of phase-separated bilayers prepared from egg sphingomyelin, dioleolyphosphatidylcholine, and cholesterol mixtures. The probe also responds to changes in packing induced by the direct incorporation of ceramide or the variation in the ionic strength of the aqueous medium. Order parameter maps obtained after enzyme treatment of bilayers with coexisting L o and L d phases show two distinct types of behavior. In regions of high enzyme activity, the initial L o /L d domains are replaced by large, dark features that have high membrane order corroborating previous hypotheses that these are ceramide-enriched regions of the membrane. In areas of low enzyme activity, the size and shape of the L o domains are conserved, but there is an increase in the order parameter for the initial L d phase (⟨P 2 ⟩ = 0.30 ± 0.01). This is attributed to the incorporation of ceramide in the L o domains with the concomitant expulsion of cholesterol into the surrounding fluid phase, increasing its order parameter.

Self-Assembly and Visualization of Poly(3-hexyl-thiophene) Chain Alignment along Boron Nitride Nanotubes

“…A similar technique has recently been used in our laboratories to visualize model membrane reorganization and measure order parameters. 19,20 Figure 4 illustrates the fluorescence intensity variation of rra-P3HT/BNNT fragments with the excitation beam polarization indicated by the ⎯ ⇀ E vector. The observation that entire elongated fragments (identified as rra-P3HT-functionalized BNNTs) follow the same intensity variation with excitation polarization indicates a highly anisotropic polymer morphology.…”

Plasma membrane reorganization: A glycolipid gateway for microbes

“…By performing AFM, one could gain data on the nanoscale membrane organisation of cellular and artificial membrane systems [20][21][22]. In the recent past, a combination of AFM with fluorescence microscopy and spectroscopy tools has been established to study ceramide-mediated membrane rafting [23,24]. GUVs with sizes ranging from 10 to 100 μm are one of the most widely exploited models to study lipid rafts by fluorescence microscopy.…”

Sphingomyelinase‐Mediated Multitimescale Clustering of Ganglioside GM1 in Heterogeneous Lipid Membranes

“…In response to various receptor‐mediated or external stimuli, [ 1 , 2 , 3 , 4 , 5 , 6 ] ceramides are generated in the outer leaflet of the plasma membrane through hydrolysis of sphingomyelin (SM) by sphingomyelinases (SMases). Accumulated ceramides are known to modify membrane biophysical properties [ 7 , 8 , 9 ] and to coalesce signaling‐related biomolecules (e.g., membrane receptors, [ 2 , 10 , 11 , 12 ] ion channels, [ 13 , 14 ] caveolin‐1, [ 15 ] and gangliosides [ 3 , 16 , 17 ] ) into specialized microdomains. These ceramide‐rich microdomains are gel phases characterized by high shear viscosity, which effectively slow the lateral diffusion of the captured signaling biomolecules.…”

“…In response to various receptormediated or external stimuli, [1][2][3][4][5][6] ceramides are generated in the outer leaflet of the plasma membrane through hydrolysis of sphingomyelin (SM) by sphingomyelinases (SMases). Accumulated ceramides are known to modify membrane biophysical properties [7][8][9] and to coalesce signaling-related biomolecules (e.g., membrane receptors, [2,[10][11][12] ion channels, [13,14] caveolin-1, [15] and gangliosides [3,16,17] ) into specialized microdomains.…”