Abstract
The formation of lipid microdomains (“rafts”) is presumed to play an important role in various cellular functions, but their nature remains controversial. 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). 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. Immunolabeling experiments demonstrated that the microdomains contained flotillin-1, a protein associated with lipid rafts. 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.
Highlights
A typical cell membrane is overwhelmingly complex, as it contains two asymmetric leaflets, proteins, and hundreds of lipid species, such as glycerophospholipids, sphingolipids and cholesterol, that greatly differ in their physico-chemical properties [1]
For low-density fractions at the 5% and 30% sucrose interface, the isolated membranes were enriched in cholesterol and sphingomyelin, as demonstrated by High-Performance Thin Layer Chromatography, and Western Blotting of these membranes (S1 Fig) demonstrated the presence of flotillin-1, a protein associated with lipid rafts [31]
This atomic force microscopy (AFM) study demonstrates the presence of microdomains in detergent-resistant membranes that were isolated from human breast cancer cells [31], similar in size to those observed in isolated erythrocyte membranes at room temperature [32]
Summary
A typical cell membrane is overwhelmingly complex, as it contains two asymmetric leaflets, proteins, and hundreds of lipid species, such as glycerophospholipids, sphingolipids and cholesterol, that greatly differ in their physico-chemical properties [1]. As originally proposed in Singer and Nicolson’s "fluid mosaic" model [2], cellular membranes appear as two-dimensional solutions of integral membrane proteins in a lipid bilayer solvent [3], allowing the lateral movement of membrane components. This fluidity does not preclude the formation of membrane domains that differ in lipid and/or protein composition from that of the surrounding lipid matrix [4].
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