Visualizing the localization of sulfoglycolipids in lipid raft domains in model membranes and sperm membrane extracts |
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Authors: | Wattana Weerachatyanukul Ira Probodh Nongnuj Tanphaichitr Linda J Johnston |
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Institution: | a Steacie Institute for Molecular Sciences, National Research Council Canada,100 Sussex Drive, Ottawa, ON Canada K1A 0R6 b Hormones/Growth/Development, Ottawa Health Research Institute, and Departments of Obstetrics/Gynecology and Biochemistry/Microbiology/Immunology, University of Ottawa,725 Parkdale Ave, Ottawa, ON Canada K1Y 4E9 |
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Abstract: | Sulfogalactosylglycerolipid (SGG) is found in detergent-resistant lipid raft fractions isolated from sperm plasma membranes and has been shown to be important in sperm-egg adhesion. In order to provide more direct evidence for the association of sulfoglycolipids with lipid raft domains, we have examined the distribution of two sulfoglycolipids in supported membranes prepared from artificial lipid mixtures and cellular lipid extracts. Atomic force microscopy has been used to visualize the localization of SGG and sulfogalactosylceramide (SGC) in liquid-ordered domains in supported bilayers of ternary lipid mixtures comprised of dipalmitoylphosphatidylcholine, cholesterol and palmitoyldocosahexaenoylphosphatidylcholine. The localization of SGC/SGG in the liquid-ordered raft domains is demonstrated by changes in bilayer morphology in the presence of sulfoglycolipid, by selective antibody labeling of the domains with anti-SGC/SGG and by the effects of the cholesterol-sequestering agent, methyl-β-cyclodextrin, on the supported membranes. In addition, we use a combination of atomic force microscopy and immunofluorescence to show that supported bilayers made from lipids extracted from sperm anterior head plasma membranes (APM) and isolated APM vesicles exhibit small SGG-rich domains that are similar to those observed in bilayers of artificial lipid mixtures. The possible implications of these results for the involvement of SGG-rich lipid rafts in modulating sperm-egg interactions in vivo and the utility of model membranes for studying the behavior of lipid rafts are discussed. |
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Keywords: | AFM atomic force microscopy APM anterior head plasma membrane chol cholesterol DPPC dipalmitoylphosphatidylcholine HPTLC high performance thin layer chromatography lo liquid-ordered MβCD methyl-β-cyclodextrin NCM non-capacitation medium PC phosphatidylcholine PDPC palmitoyldocosahexaenoylphosphatidylcholine PRS preimmune rabbit serum SGC sulfogalactosylceramide SGG sulfogalactosylglycerolipid SM sphingomyelin TLC thin layer chromatography |
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