title: Analysis of lipid-composition changes in plasma membrane microdomains.
abstract: Sphingolipids accumulate in plasma membrane microdomain sites, such as caveolae or lipid rafts. Such microdomains are considered to be important nexuses for signal transduction, although changes in the microdomain lipid components brought about by signaling are poorly understood. Here, we applied a cationic colloidal silica bead method to analyze plasma membrane lipids from monolayer cells cultured in a 10 cm dish. The detergent-resistant fraction from the silica bead-coated membrane was analyzed by LC-MS/MS to evaluate the microdomain lipids. This method revealed that glycosphingolipids composed the microdomains as a substitute for sphingomyelin (SM) in mouse embryonic fibroblasts (tMEFs) from an SM synthase 1/2 double KO (DKO) mouse. The rate of formation of the detergent-resistant region was unchanged compared with that of WT-tMEFs. C2-ceramide (Cer) stimulation caused greater elevations in diacylglycerol and phosphatidic acid levels than in Cer levels within the microdomains of WT-tMEFs. We also found that lipid changes in the microdomains of SM-deficient DKO-tMEFs caused by serum stimulation occurred in the same manner as that of WT-tMEFs. This practical method for analyzing membrane lipids will facilitate future comprehensive analyses of membrane microdomain-associated responses.
authors.affiliation: Department of Hematology/Immunology Kanazawa Medical University, 1-1 Daigaku, Uchinada, Ishikawa 920-0293, Japan.
authors.forename: Hideo
authors.initials: H
authors.lastname: Ogiso
authors.affiliation: Medical Research Institute, Kanazawa Medical University, 1-1 Daigaku, Uchinada, Ishikawa 920-0293, Japan.
authors.forename: Makoto
authors.initials: M
authors.lastname: Taniguchi
authors.affiliation: Department of Hematology/Immunology Kanazawa Medical University, 1-1 Daigaku, Uchinada, Ishikawa 920-0293, Japan Medical Research Institute, Kanazawa Medical University, 1-1 Daigaku, Uchinada, Ishikawa 920-0293, Japan.
authors.forename: Toshiro
authors.initials: T
authors.lastname: Okazaki
mesh_terms: D000818:Animals; D002460:Cell Line; D055786:Gene Knockout Techniques; D050356:Lipid Metabolism / Q000187:drug effects; D008055:Lipids / Q000737:chemistry*; D021962:Membrane Microdomains / Q000187:drug effects / Q000378:metabolism*; D051379:Mice; D012822:Silicon Dioxide / Q000737:chemistry; D013110:Sphingosine / Q000031:analogs & derivatives / Q000494:pharmacology; D017855:Transferases (Other Substituted Phosphate Groups) / Q000172:deficiency / Q000235:genetics
chemical_list: D008055:Lipids; C064769:N-acetylsphingosine; D012822:Silicon Dioxide; C556686:Sgms1 protein, mouse; D017855:Transferases (Other Substituted Phosphate Groups); C543123:Sgms2 protein, mouse; D013110:Sphingosine
keywords: cationic colloidal silica beads; ceramide; diacylglycerol; lipid raft; lipidomics; liquid chromatography-tandem mass spectrometry; phosphatidic acid; sphingomyelin