Ultrastructure of sheep erythrocyte plasma membranes and cytoskeletons bound to solid supports

Sheep erythrocyte plasma membrane monolayers were formed on positively charged supports by means of controlled lysis and squirting of cells so that the original protoplasmic or inner surfaces (PS) were exposed. The appearance of the surface was studied by transmission electron microscopy of platinum/carbon replicas following freeze-drying of the membranes. After gentle washing with water or dilute buffer, a network of filaments and particles was found to cover the surface. Whole cells bound to positively charged supports were treated with Triton X-100 and hypertonic KCl, which left the protein components of the cytoskeleton on the support. Stereo electron microscopy of Pt/C replicas of these residues showed complex networks of filaments, similar to those seen on the cytoplasmic surfaces of the intact membranes. In both cases the lengths of the filaments correspond to that of the spectrin dimer. Removal of spectrin, actin and other proteins by alkaline treatment led to loss of this network and revealed 15.4-nm particles on the membranes PS. These particles, which were also visible after negative staining, could be removed by treatment with trypsin and were found to correspond to band 4 protein (the equivalent of human erythrocyte band 3 protein). Membranes freeze-fractured following treatment with alkali showed normal intramembranous particles with frequencies similar to those of native membranes. This indicates that band 4 protein spans the sheep erythrocyte membrane and forms a very high proportion of the intramembranous particles. The protoplasmic portions of these particles may be membrane binding sites for the cytoskeleton in sheep erythrocytes.