Analysis of lipid-composition changes in plasma membrane microdomains
Analysis of lipid-composition changes in plasma membrane microdomains
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DOI：
10.1194/jlr.m059972
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发表时间：
2015-08-01
影响因子：
6.5
通讯作者：
Okazaki, Toshiro
中科院分区：
文献类型：
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作者：
Ogiso, Hideo;Taniguchi, Makoto;Okazaki, Toshiro
鞘脂聚集在质膜微区位点，如小窝或脂筏。这种微区被认为是信号转导的重要联系，尽管对信号转导所引起的微区脂质组分的变化知之甚少。在这里，我们应用了阳离子胶体二氧化硅珠的方法来分析质膜脂质从单层细胞培养在一个10厘米的皿。通过LC-MS/MS分析来自二氧化硅珠包被的膜的耐洗涤剂级分以评估微区脂质。该方法揭示了鞘糖脂构成微结构域作为来自SM合酶1/2双KO（DKO）小鼠的小鼠胚胎成纤维细胞（tMEF）中鞘磷脂（SM）的替代物。与WT-tMEFs相比，耐洗涤剂区域的形成速率没有变化。C2-神经酰胺（Cer）的刺激引起更大的二酰基甘油和磷脂酸水平的升高比在WT-tMEFs的微域内的Cer水平。我们还发现，由血清刺激引起的SM缺陷型DKO-tMEFs的微区中的脂质变化与WT-tMEFs的脂质变化以相同的方式发生。这种实用的膜脂分析方法将有助于未来膜微区相关响应的综合分析。
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 micro-domain-associated responses.