Atomic force microscopy imaging of lipid rafts of human breast cancer cells

• Published in: Biochimica et biophysica acta Vol. 1818; no. 12; pp. 2943 - 2949
• Main Authors: Orsini, F., Cremona, A., Arosio, P., Corsetto, P.A., Montorfano, G., Lascialfari, A., Rizzo, A.M.
• Format: Journal Article
• Language: English
• Published: Netherlands Elsevier B.V 01.12.2012
• Subjects: Atomic force microscopy; Breast Neoplasms - chemistry; Breast Neoplasms - pathology; Cell Line, Tumor; Cell Membrane - chemistry; Cholesterol - analysis; Female; Humans; Lipid Bilayers - chemistry; Lipid rafts; MDA-MB-231 cancer cells; Membrane Microdomains - chemistry; Membrane Proteins - analysis; Microscopy, Atomic Force; Sphingomyelins - analysis; Atomic force microscopy; MDA-MB-231 cancer cells; Lipid rafts
• ISSN: 0005-2736; 0006-3002; 1879-2642; 1878-2434
• DOI: 10.1016/j.bbamem.2012.07.024

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. [Display omitted] ► Lipid rafts of MDA-MB-231 cancer cells were studied by AFM and biochemical assays. ► AFM showed membrane micro-domains with lateral sizes of a few hundreds of nanometers. ► WB and HP-TLC detected a high CHOL, SM and Flot-1 content in the micro-domains. ► AFM study using specific antibodies identified Flot-1 in the micro-domains.