| Abstract
| - Specialized plasma membrane domains known as lipid rafts participate in signal transductionand other cellular processes, and their liquid ordered (Lo) phase appears to be important for their function.To quantify ordered lipids in biological membranes, we investigated steady-state fluorescence anisotropyof 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-statefluorescence anisotropy is a sensitive measure of cholesterol-dependent ordering. The results suggest thatDPH-PC is a more sensitive probe than NBD-PE. In the presence of cholesterol, ordering also dependson the degree of saturation of the phospholipid acyl chains. Using DPH-PC, we find that the plasmamembrane of RBL-2H3 mast cells is substantially ordered, roughly 40%, as determined by comparisonwith anisotropy values for model membranes entirely in a liquid ordered (Lo) phase and in a liquiddisordered (Lα) phase. This result is consistent with the finding that ∼30% of plasma membranephospholipids are insoluble in 0.5% Triton X-100. Furthermore, detergent-resistant membranes isolatedby sucrose gradient fractionation of Triton X-100 cell lysates are more ordered than plasma membranevesicles, suggesting that they represent a more ordered subset of the plasma membrane. Treatment ofplasma membrane vesicles with methyl-β-cyclodextrin resulting in 75% cholesterol depletion leads tocommensurate decreases in lipid order as measured by anisotropy of DPH-PC and NBD-PE. These resultsdemonstrate that steady-state fluorescence anisotropy of DPH-PC is a useful way to measure the amountof lipid order in biological membranes.
|
