Atomic force microscopy imaging of lipid rafts of human breast cancer cells
Atomic force microscopy imaging of lipid rafts of human breast cancer cells
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DOI：
10.1016/j.bbamem.2012.07.024
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发表时间：
2012-12-01
影响因子：
3.4
通讯作者：
Rizzo, A. M.
中科院分区：
文献类型：
--
作者：
Orsini, F.;Cremona, A.;Rizzo, A. M.
多项研究表明，质膜由饱和脂质的微域组成，这些微域分离在一起形成脂筏。脂筏在操作上被定义为富含胆固醇和鞘脂的膜微域，在低温下能够抵抗非离子洗涤剂的溶解。在这里，我们报告了一种生物物理方法，旨在通过将原子力显微镜（AFM）研究与生化测定（即蛋白质印迹和高性能薄层色谱）相结合来研究 MDA-MB-231 人乳腺癌细胞的脂筏。使用 Triton X-100 萃取，通过不连续蔗糖梯度超速离心纯化脂筏。生化分析证明，在5%和30%蔗糖界面分离的级分（级分5和6）相对于其他纯化级分具有更高的胆固醇、鞘磷脂和flotillin-1含量。级分 5 的轻敲模式 AFM 成像显示膜斑块的高度与单个脂质双层的高度相对应，并且存在横向尺寸约为数百纳米的微域。此外，使用特定抗体的 AFM 研究表明，在这些微域中存在脂筏的特征标记，即蛋白质 flotillin-1。 (C) 2012 Elsevier B.V. 保留所有权利。
Several studies suggest that the plasma membrane is composed of micro-domains of saturated lipids that segregate together to form lipid rafts. Lipid rafts have been operationally defined as cholesterol- and sphingolipid-enriched membrane micro-domains resistant to solubilization by non-ionic detergents at low temperatures. Here we report a biophysical approach aimed at investigating lipid rafts of MDA-MB-231 human breast cancer cells by coupling an atomic force microscopy (AFM) study to biochemical assays namely Western blotting and high performance thin layer chromatography. Lipid rafts were purified by ultracentrifugation on discontinuous sucrose gradient using extraction with Triton X-100. Biochemical analyses proved that the fractions isolated at the 5% and 30% sucrose interface (fractions 5 and 6) have a higher content of cholesterol, sphingomyelin and flotillin-1 with respect to the other purified fractions. Tapping mode AFM imaging of fraction 5 showed membrane patches whose height corresponds to the one awaited for a single lipid bilayer as well as the presence of micro-domains with lateral dimensions in the order of a few hundreds of nanometers. In addition, an AFM study using specific antibodies suggests the presence, in these micro-domains, of a characteristic marker of lipid rafts, the protein flotillin-1. (C) 2012 Elsevier B.V. All rights reserved.