Freeze-etch electron tomography for the plasma membrane interface.

To visualize the basal or apical cytoplasmic surface just beneath the plasma membrane, we developed two different methods ("unroof" and "rip-off"). The immunoreplica technique for "unroof" and "rip-off" sample preparation that will be presented in this chapter can determine the distributions of actin, actin-binding proteins, transmembrane proteins, and membrane lipids at the interface of the plasma membrane. We have currently developed freeze-etch electron tomography, which could visualize the 3D molecular architecture of membrane-associated structures (membrane skeleton, clathrin-coated pits, and caveolae) on the cytoplasmic surface of the plasma membrane with 0.85-nm-thick consecutive sections made approximately 100 nm from the cytoplasmic surface using rapidly frozen, deeply etched, platinum-replicated plasma membranes. The membrane skeletons that are closely apposed to the plasma membrane interface are suggested to form the boundaries of the membrane compartments responsible for the temporary confinement of membrane molecules.