1-2-oleoylphosphatidylcholine and 7-ketocholesterol

Cholesterol- and glycosphingolipid-enriched membrane lipid microdomains, frequently called lipid rafts, are thought to play an important role in the spatial and temporal organization of immunological synapses. Higher ordering of lipid acyl chains was suggested for these entities and imaging of membrane order in living cells during activation can therefore help to understand the mechanisms responsible for the supramolecular organization of molecules involved in the activation of T cells. Here, we employ the phase-sensitive membrane dye di-4-ANEPPDHQ together with a variety of spectrally-resolved microscopy techniques, including 2-channel ratiometric TIRF microscopy and fluorescence lifetime imaging, to characterize membrane order at the T cell immunological synapse at high spatial and temporal resolution in live cells at physiological temperature. We find that higher membrane order resides at the immunological synapse periphery where proximal signalling through the immunoreceptors and accessory proteins in microclusters has previously been shown to take place. The observed spatial patterning of membrane order in the immunological synapse depends on active receptor signalling.

Topics: 1,2-Dipalmitoylphosphatidylcholine; Animals; Antigen-Presenting Cells; Humans; Immunological Synapses; Jurkat Cells; Ketocholesterols; Membrane Lipids; Membrane Microdomains; Microscopy, Fluorescence; Phosphatidylcholines; T-Lymphocytes

7-Ketocholesterol is an oxidized derivative of cholesterol with numerous physiological effects. In model membranes, 7-ketocholesterol and cholesterol were compared by physical measures of bilayer order and polarity, formation of detergent resistant domains (DRM), phase separation, and membrane microsolubilization by apolipoprotein A-I. In binary mixtures of a saturated phosphatidylcholine (PC), dipalmitoyl-PC (DPPC), and cholesterol or 7-ketocholesterol, the sterols modulate bilayer order and polarity and induce DRM formation to a similar extent. Cholesterol induces formation of ordered lipid domains (rafts) in tertiary mixtures with dioleoyl-PC (DOPC) and DPPC, or DOPC and sphingomyelin (SM). In tertiary mixtures, cholesterol increased lipid order and reduces bilayer polarity more than 7-ketocholesterol. This effect was more pronounced when the mixtures were in a miscible liquid-disordered (L(d)) phase. Substitution of 7-ketocholesterol for cholesterol dramatically reduced the extent of DRM formation in DOPC/DPPC and DOPC/SM bilayers and ordered lipid phase separation in mixtures of a spin-labeled PC with DPPC and with SM. Compared to cholesterol, 7-ketocholesterol decreased the rate for the microsolubilization of dimyristoyl-PC multilamellar vesicles by apolipoprotein A-I. The membrane effects of 7-ketocholesterol were dependent on the phospholipid matrix. In L(d) phase phospholipids, a model for 7-ketocholesterol indicates that the proximity of the 7-keto and 3beta-OH groups puts both polar moieties at the lipid-water interface to tilt the sterol nucleus to the plane of the bilayer. 7-Ketocholesterol was less effective in forming ordered lipid domains, in decreasing the level of bilayer hydration, and in forming phase boundary bilayer defects. Compared to cholesterol, 7-ketocholesterol can differentially modulate membrane properties involved in protein-membrane association and function.

Topics: 1,2-Dipalmitoylphosphatidylcholine; Apolipoprotein A-I; Cholesterol, HDL; Dimyristoylphosphatidylcholine; Humans; Ketocholesterols; Kinetics; Lipid Bilayers; Liposomes; Membrane Microdomains; Octoxynol; Phase Transition; Phosphatidylcholines; Solubility; Spectrometry, Fluorescence; Sphingomyelins

The formation and stability of ordered lipid domains (rafts) in model membrane vesicles were studied using a series of sterols and steroids structurally similar to cholesterol. In one assay, insolubility in Triton X-100 was assessed in bilayers composed of sterol/steroid mixed with dipalmitoylphosphatidylcholine (DPPC), dioleoylphosphatidylcholine, or a 1:1 mixture of these phospholipids. In a second assay fluorescence quenching was used to determine the degree of ordered domain formation in bilayers containing sterol/steroid and a 1:1 mixture of DPPC and a quencher-carrying phosphatidylcholine. Both methods showed that several single modifications of the cholesterol structure weaken, but do not fully abolish, the ability of sterols and steroids to promote ordered domain formation when mixed with DPPC. Some of these modifications included a shift of the double bond from the 5-6 carbons (cholesterol) to 4-5 carbons (allocholesterol), derivatization of the 3-OH (cholesterol methyl ether, cholesteryl formate), and alteration of the 3-hydroxy to a keto group (cholestanone). An oxysterol involved in atherosclerosis, 7-ketocholesterol, formed domains with DPPC that were as thermally stable as those with cholesterol although not as tightly packed as judged by fluorescence anisotropy. It was also found that 7-ketocholesterol has fluorescence quenching properties making it a useful spectroscopic probe. Lathosterol, which has a 7-8 carbon double bond in place of the 5-6 double bond of cholesterol, formed rafts with DPPC that were at least as detergent-resistant as, and even more thermally stable than, rafts containing cholesterol. Because lathosterol is an intermediate in cholesterol biosynthesis, we conclude it is unlikely that sterol biosynthesis continues past lathosterol in order to create a raft-favoring lipid.

Topics: 1,2-Dipalmitoylphosphatidylcholine; Cholesterol; Detergents; Ergosterol; Fluorescence Polarization; Ketocholesterols; Lipid Bilayers; Membrane Microdomains; Octoxynol; Phosphatidylcholines; Solubility; Spectrometry, Fluorescence; Spectrophotometry; Steroids; Sterols; Structure-Activity Relationship

The homogenizing effect of cholesterol and its oxidative derivatives, 7-ketocholesterol, cholesterol 5 alpha, 6 alpha-epoxide and 25-hydroxycholesterol, in liquid-crystalline 1,2-dioleoyl-sn-glycero-3-phosphocholine(DOPC) bilayer vesicles was studied using the fluorescence lifetimes of 2-(3-(diphenylhexatrienyl)propanoyl)-1-hexadecanoyl-sn-glycero-3-p hosphocholine (DPH-PC). The phase and modulation data were fitted either to discrete exponential models or to models characterized by continuous distributional lifetimes. Among all the models tested, it was found that the best one to account for the experimental data was the unimodal Lorentzian distribution. Thus, the DPH-PC lifetime was adequately described by a distributional center and a full width at half-maximum, for DOPC vesicles these values being 6.23 and 0.48 ns, respectively. Increasing the concentration of cholesterol, 7-ketocholesterol, or cholesterol 5 alpha, 6 alpha-epoxide from 0 to 30 mol% resulted in an increase of the lifetime center (e.g., 7.16 ns at 30 mol% cholesterol) and a decrease of the distributional width (e.g., 0.05 ns at 30 mol% cholesterol). On the other hand, up to 30 mol% of 25-hydroxycholesterol incorporated into the bilayer vesicles showed little influence on both lifetime parameters. Our results support the use of lifetime distributional width to evaluate membrane heterogeneity and suggest that oxysterols, depending on their molecular structural particulars, may exert cholesterol-like homogenizing effect in membranes.

Topics: Cholesterol; Diphenylhexatriene; Hydroxycholesterols; Ketocholesterols; Lipid Bilayers; Membrane Fluidity; Membrane Lipids; Phosphatidylcholines; Spectrometry, Fluorescence