Isolation of lipid rafts from B lymphocytes.

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.

Ordered and disordered phases coexist in plasma membrane vesicles of RBL-2H3 mast cells. An ESR study.

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.

Disruption of lipid order by short-chain ceramides correlates with inhibition of phospholipase D and downstream signaling by FcepsilonRI.

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-FcepsilonRI 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.