Electron microscopic observation of domain movement in reconstituted erythrocyte membranes

The movement of labeled concanavalin A (Con A) receptors in reconstituted human erythrocyte membranes was observed directly in an electron microscope, using an environmental stage that kept the sample fully hydrated at all experimental temperatures. Human erythrocyte membrane ghosts were spread on the air/water interface in a Langmuir trough. The surface monolayer film contained most native proteins and lipids of the erythrocyte membrane. The Con A receptors in the film were labeled with Con A-conjugated, 25-nm-diameter gold microspheres. Unsupported bilayer membranes were reconstituted by dipping a 1000-mesh grid through the labeled surface film. The reconstituted membrane samples were observed under low beam current and photographed by timed exposures with sensitive x-ray films. The total radiation per exposure was kept below the damage threshold of 5 X 10(-4) coulomb/cm2. The Con A-gold labels were observed to move in unison within local areas (domains) of the reconstituted membrane. The size of the domains and the velocity of the labels were measured as functions of temperature. The typical domain size was 10 micron2 and the typical velocity of the labels was 7 nm/sec. The minimum domain size and velocity were found at 17 degrees-28 degrees C. Reduction of the amount of cholesterol in the precursor erythrocyte membrane caused the domain velocity at 7 degrees C to decrease and the domain size to increase; the opposite effect was observed with cholesterol enrichment. The results indicate that the components of the erythrocyte membrane tended to form moving domains and that the motion was related to lipid phase separation in the bilayer.