Atomic force microscopy: a tool to quantitatively perform morpho-dimensional characterization of lipid rafts

BACKGROUND: Lipid rafts are transient liquid-ordered domains that are enriched in sphingolipids, cholesterol and signalling proteins, resistant to solubilization by nonionic detergents at low temperatures. Atomic force microscopy (AFM), providing nanometer spatial resolution and operating in physiological-like conditions without fixation staining, or labeling, is an useful tool to quantitatively perform a morpho-dimensional characterization of lipid rafts (1). AIM: The observation and the characterization of phase separation in purified plasma membranes is important for better understanding the lateral organization of the plasma membrane and lipid rafts. METHODS: Lipid rafts were purified by ultracentrifugation on discontinuous sucrose gradient using extraction with Triton X-100. AFM imaging was performed using a Multimode Nanoscope IIIa. Topography AFM images were collected in tapping mode operating in liquid buffer. Membrane samples were visualized changing the temperature over a large range (25-40°C) using a heating control system (Multimode AFM high temperature heater). RESULTS AND CONCLUSIONS: we have applied tapping mode AFM imaging to investigate MDA-MB-231 human breast cancer cells lipid rafts purified by ultracentrifugation on discontinuous sucrose gradient using Triton X-100. At room temperature, tapping mode AFM imaging showed membrane patches with microdomains protruding from their surface, with lateral dimensions in the range of 100-500nm in agreement with the range expected for lipid rafts as reported in literature. Interestingly, the size of the microdomains depend strongly on the temperature, when the temperature is increased (above 30°C) the smaller microdomains (less than 100nm in diameter) disappear while the larger microdomains (>500nm in diameter) are reduced in size. These data suggest that the dynamics of domain growth, and the distribution of domain sizes strongly depend on the temperature. 1 A Cremona et al. Atomic force microscopy imaging of lipid rafts of human breast cancer cells. Biochim Biophys Acta 2012. 1818(12):2943-9.