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

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 (Lo) domains ([P2] = 0.40 ± 0.03) than for the liquid-disordered phase (Ld, fluid, [P2] = 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 Lo and Ld phases show two distinct types of behavior. In regions of high enzyme activity, the initial Lo/Ld 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 Lo domains are conserved, but there is an increase in the order parameter for the initial Ld phase ([P2] = 0.30 ± 0.01). This is attributed to the incorporation of ceramide in the Lo domains with the concomitant expulsion of cholesterol into the surrounding fluid phase, increasing its order parameter.

Photouncaging of ceramides promotes reorganization of liquid-ordered domains in supported lipid bilayers.

6-Bromo-7-hydroxycoumarin (Bhc)-caged ceramide (Cer) analogs were incorporated into supported lipid bilayers containing a mixture of coexisting liquid-ordered (Lo) and liquid-disordered (Ld) phases. The release of N-palmitoyl and N-butanoyl-D-erythro-sphingosine (C16- and C4-Cer) by the photolysis of caged Cers using long-wavelength UV light was studied using a combination of atomic force microscopy and fluorescence microscopy. This approach demonstrated the ability to generate Cer with spatial and temporal control, providing an alternative method to the enzymatic generation of Cer. The generation of C16-Cer from Bhc-C16-Cer disrupted the Lo domains, with the incorporation of small fluid-phase regions and the disappearance of some smaller domains. Cer-rich gel-phase domains were not observed, in contrast to results reported by either direct Cer incorporation or enzymatic Cer generation. The photorelease of C4-Cer from Bhc-C4-Cer resulted in qualitatively similar changes in bilayer morphology, with the disappearance of some Lo domains and no evidence of Cer-rich gel domains but with a smaller height difference between the ordered and disordered phases.

A new tool to assess ceramide bioactivity: 6-bromo-7-hydroxycoumarinyl-caged ceramide.

The bioactivity of natural, long-chain ceramides has until now been studied after its delivery to cells in organic solvent mixtures containing dodecane. We have synthesized ceramides conjugated to a (6-bromo-7-hydroxycoumarin-4-yl)methyl group. The photocaged ceramide is efficiently released with 350 nm light in aqueous solution at neutral pH, thus providing a promising new tool to study ceramide's properties.

NBD-cholesterol probes to track cholesterol distribution in model membranes.

A series of cholesterol (Chol) probes with NBD and Dansyl fluorophores attached to the 3-hydroxyl position via carbamate linkers has been designed and synthesized and their ability to mimic the behavior of natural cholesterol in bilayer membranes has been examined. Fluorescence spectroscopy data indicate that the NBD-labeled lipids are located in the polar headgroup region of the bilayer with their position varying with the method of fluorophore attachment and the linker length. The partitioning of the Chol probes between liquid-ordered (L(o)) and liquid-disordered (L(o)) phases in supported bilayers prepared from ternary lipid mixtures of DOPC, Chol and either egg sphingomyelin or DPPC was examined by fluorescence microscopy. The carbamate-linked NBD-Chols show a stronger preference for partitioning into L(o) domains than does a structurally similar probe with an ester linkage, indicating the importance of careful optimization of probe and linker to provide the best Chol mimic. Comparison of the partitioning of NBD probes to literature data for native Chol indicates that the probes reproduce well the modest enrichment of Chol in L(o) domains as well as the ceramide-induced displacement of Chol. One NBD probe was used to follow the dynamic redistribution of Chol in phase separated membranes in response to in situ ceramide generation. This provides the first direct optical visualization of Chol redistribution during enzymatic ceramide generation and allows the assignment of new bilayer regions that exclude dye and have high lateral adhesion to ceramide-rich regions.

Enzymatic generation of ceramide induces membrane restructuring: Correlated AFM and fluorescence imaging of supported bilayers.

The effect of enzymatic generation of ceramide on phase separated bilayers with a mixture of co-existing fluid and liquid-ordered phases has been examined using a combination of atomic force microscopy (AFM) and fluorescence imaging. Supported lipid bilayers prepared from a DOPC/sphingomyelin/cholesterol mixture were imaged prior to, during and after incubation with sphingomyelinase by total internal reflection fluorescence (TIRF) microscopy. Enzyme treatment resulted in the growth of large dye-excluded regions. The growth kinetics for these patches are consistent with activity of a variable number of enzyme molecules in different regions of the bilayer. Correlated AFM and fluorescence imaging shows that some of the large dye-excluded patches form around the original liquid-ordered domains, which become heterogeneous in height with many raised ceramide-rich regions around their periphery. However, some of the dye-excluded patches correspond to areas of the bilayer where the initial domains have largely or partially disappeared. The dye-excluded patches observed by fluorescence are shown to be areas of increased adhesion in lateral deflection AFM images and are postulated to form by incorporation of both cholesterol and ceramide in the original fluid phase and to vary in composition throughout the bilayer. This is evident from the observation that the dye-excluded areas are all detected as areas of increased friction, but do not always show a distinct height difference in topographic images. These results highlight the utility of a multi-modal imaging approach for understanding the complex membrane restructuring that occurs upon enzymatic generation of ceramide.