Several studies suggest that the plasma membrane is composed of microdomains of saturated lipids that segregate together to form lipid rafts. Lipid rafts have been operationally defined as cholesterol- and sphingolipid-enriched membrane microdomains resistant to solubilisation by non-ionic detergents at low temperatures. The formation of lipid rafts is presumed to play an important role in various cellular functions, but their nature remains controversial [1, 2].
Here we report on microdomain formation in isolated, detergent-resistant membranes from MDA-MB-231 human breast cancer cells, studied by Atomic Force Microscopy (AFM) and biochemical assays namely Western blotting and high performance thin layer chromatography.
Membrane microdomains 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 have a higher content of cholesterol, sphingomyelin and flotillin-1 with respect to the other purified fractions [3, 4].
Tapping mode AFM imaging of these fractions showed membrane patches whose height corresponds to the one awaited for a single lipid bilayer as well as the presence of microdomains with lateral dimensions in the order of a few hundreds of nanometers. AFM immunolabeling experiments demonstrated that the microdomains contained flotillin-1, a protein associated with lipid rafts.
Interestingly, whereas microdomains were readily observed at room temperature, they shrunk in size and mostly disappeared at higher temperatures. This shrinking in microdomain size was accompanied by a gradual reduction of the height difference between the microdomains and the surrounding membrane, consistent with the behaviour expected for lipids that are laterally segregated in liquid ordered and liquid disordered domains. The microdomains reversibly dissolved and reappeared, respectively, on heating to and cooling below temperatures around 37°C, which is indicative of radical changes in local membrane order close to physiological temperature.
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 F. Orsini, A. Cremona, P. Arosio, P.A. Corsetto, G. Montorfano, A. Lascialfari, A.M. Rizzo, Atomic force microscopy imaging of lipid rafts of human breast cancer cells, Biochimica et Biophysica Acta-Biomembranes, 1818 (2012), 2943-2949.
 P.A. Corsetto, A. Cremona, G. Montorfano, I.E. Jovenitti, F. Orsini, P. Arosio, A.M. Rizzo, Chemical-Physical Changes in Cell Membrane Microdomains of Breast Cancer Cells After Omega-3 PUFA Incorporation, Cell Biochemistry and Biophysics, 64 (2012), 45-59.