Human erythroleukemic (K562) cells differentiate along the erythroid differentiation pathway in vitro when 0.05 mM hemin is included in the growth medium. In the presence of the inducer the cells continue to proliferate and, after a delay of 24 to 48 h, start to synthesize hemoglobin. However, during differentiation, no changes in the major cell surface proteins were detected using lactoperoxidase-catalyzed iodination, and no change in the synthesis of spectrin, the major cytoskeletal protein of the mature erythrocyte, was detected by specific immune precipitation. Despite this absence of major changes in cell surface proteins, profound changes take place in the organization of the cell membrane. A process similar but not identical to the enucleation observed in erythroid differentiation in vivo occurs in which a smooth-surfaced cell, about 10 jim in diameter, is divided from the nucleus-containing part of the cell. With the exception of ribosomes, these reticulocyte-like cells contain no organelles when examined by transmission electron microscopy, but contain much of the parent cell's hemoglobin, spectrin, and glycophorin.The surface membrane (or ghost) of the human erythrocyte is undoubtedly the best-characterized eucaryotic cell membrane, and a considerable amount of information concerning the structure of its major proteins has accumulated (for a review see reference 23). The major integral membrane glycoproteins of the erythrocyte membrane, glycophorin (which bears the MN antigens [27]) and band 3 protein (which is the major anion transport channel [6]), are located on the external surface of the cell, span the lipid bilayer (3, 28), and interact with peripheral proteins on the cytoplasmic side of the membrane (for a review see reference 23). Of these peripheral proteins the most actively studied have been spectrin (bands 1 and 2 in the nomenclature of Fairbanks and colleagues [10]) and actin (band 5), which together comprise the cytoskeleton lining the inner surface of the membrane (20). It is this cytoskeleton that is responsible for the cells' biconcave shape and spectacular deformability (21 Transmission electron microscopy. Cells were fixed using 2% glutaraldehyde in 0.1 M sodium cacodylate buffer (pH 7.4). They were then incubated with 1% osmium tetroxide in water and processed for electron microscopy using uranyl acetate and lead citrate staining.Scanning electron microscopy. Cells were washed in cold PBS and suspended in 2% glutaraldehyde in PBS at 4°C. After 24 h the cells were gently resuspended, and 1 drop of the suspension was placed on the shiny side of a Nucleopore polycarbonate filter (0.4-um pore size; 13-mm diameter) supported on the grid half of a Gelman Easy-pressure filter holder attached to a 5-ml syringe. The cells were gently drawn down onto the filter and fixed on the filter with 2% glutaraldehyde in PBS. The cells and filter were postfixed in PBS containing 1% osmium tetroxide for 1 h at room temperature and then dehydrated in ethanol, followed by critical-point drying. The filters...