Specialized plasma membrane domains known as lipid rafts participate in signal transduction and other cellular processes, and their liquid ordered (L(o)) phase appears to be important for their function. To quantify ordered lipids in biological membranes, we investigated steady-state fluorescence anisotropy of two lipid probes, 2-[3-(diphenylhexatrienyl)propanoyl]-1-hexadecanoyl-sn-glycero-3-phosphocholine (DPH-PC) and N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)-1,2-dihexadecanoyl-sn-glycero-3-phosphoethanolamine (NBD-PE). We show using model membranes with varying amounts of cholesterol that steady-state fluorescence anisotropy is a sensitive measure of cholesterol-dependent ordering. The results suggest that DPH-PC is a more sensitive probe than NBD-PE. In the presence of cholesterol, ordering also depends on the degree of saturation of the phospholipid acyl chains. Using DPH-PC, we find that the plasma membrane of RBL-2H3 mast cells is substantially ordered, roughly 40%, as determined by comparison with anisotropy values for model membranes entirely in a liquid ordered (L(o)) phase and in a liquid disordered (L(alpha)) phase. This result is consistent with the finding that approximately 30% of plasma membrane phospholipids are insoluble in 0.5% Triton X-100. Furthermore, detergent-resistant membranes isolated by sucrose gradient fractionation of Triton X-100 cell lysates are more ordered than plasma membrane vesicles, suggesting that they represent a more ordered subset of the plasma membrane. Treatment of plasma membrane vesicles with methyl-beta-cyclodextrin resulting in 75% cholesterol depletion leads to commensurate decreases in lipid order as measured by anisotropy of DPH-PC and NBD-PE. These results demonstrate that steady-state fluorescence anisotropy of DPH-PC is a useful way to measure the amount of lipid order in biological membranes.

Four chain spin labels and a spin-labeled cholestane were used to study the dynamic structure of plasma membrane vesicles (PMV) prepared from RBL-2H3 mast cells at temperatures ranging from 22 degrees C to 45 degrees C. Analysis shows that the spectra from most labels consist of two components. The abundant spectral components exhibit substantial ordering that is intermediate between that of a liquid-ordered (Lo) phase, and that of a liquid-crystalline (Lc) phase as represented by model membranes. Also, rotational diffusion rates of the spin labels are comparable to those in the Lo phase. In contrast, the ordering for the less abundant components is much lower. These results indicate that a Lo-like region or phase (the abundant component) and an Lc-like region or phase (the less abundant component) coexist in the PMV. In contrast, membranes reconstituted from extracted lipids exhibit the more ordered phase only. This suggests that membrane-associated proteins are important for the coexistence of Lo-like and Lc-like regions in the plasma membrane. In addition, binding of the myristoylated protein, ARF6 to PMV, leads to a new spectral component for a headgroup lipid spin label that indicates the formation of plasma membrane defects by this low molecular weight GTPase.

Specialized plasma membrane domains known as lipid rafts participate in signal transduction and other cellular processes, and their liquid-ordered properties appear to be important for their function. We investigated the possibility of using amphiphiles to disrupt lipid rafts and thereby inhibit IgE-FcϵRI signaling. We find that short-chain ceramides –C2-ceramide and C6-ceramide – decrease plasma membrane lipid order and reduce the extent of fluorescence resonance energy transfer between lipid-raft-associated molecules on intact cells; by contrast,biologically inactive C2-dihydroceramide does neither. Structural perturbations by these ceramides parallel their inhibitory effects on antigen-stimulated Ca2+ mobilization in RBL mast cells in the presence and absence of extracellular Ca2+. Similar inhibition of Ca2+ mobilization is caused by n-butanol, which prevents phosphatidic acid production by phospholipase D, but not by t-butanol, which does not prevent phosphatidic acid production. These results and previously reported effects of short-chain ceramides on phospholipase D activity prompted us to compare the effects of C2-ceramide,C2-dihydroceramide and C16-ceramide on phospholipase D1 and phospholipase D2 activities in vitro. We find that the effects of these ceramides on phospholipase D1 activity strongly correlate with their effects on antigen-stimulated Ca2+ mobilization and with their disruption of lipid order. Our results indicate that phospholipase D activity is upstream of antigen-stimulated Ca2+ mobilization in these cells, and they demonstrate that ceramides can serve as useful probes for investigating roles of plasma membrane structure and phospholipase D activity in cellular signaling.

Recent advances in cell biology have provided evidence that the plasma membrane is not a homogeneous lipid bilayer but rather contains within it sphingolipid- and cholesterol-rich membrane microdomains, termed lipid rafts, which serve as platforms for both receptor signaling and trafficking. In B lymphocytes lipid rafts appear to play a key role in the initiation of B-cell antigen receptor (BCR) signaling. Current methods to isolate lipid rafts rely on the relative detergent insolubility of lipid rafts as compared to the nonraft, glycerophospholipid bilayer. Here a method to isolate and characterize lipid rafts from B lymphocytes is described. Particular emphasis is given to the potential artifacts inherent in current procedures that rely on detergents to isolate lipid rafts and alternative technologies that may circumvent these.

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