Effect of antibodies to membrane skeletal proteins on the shape of erythrocytes and their ability to respond to shape-modulating agents. Important role of 4.1 protein in the determination/maintenance of the discoid shape of erythrocytes.

Monospecific anti-spectrin, anti-4.1 protein, anti-ankyrin, and anti-band 3 cytoplasmic domain antibodies were individually internalized in human erythrocytes by hypotonic lysis in the presence of 2 mM Mg-ATP. The concentrations of the polyclonal antibodies, or their Fab fragments, varied from 0.5 to 4.0 mg/ml. After resealing and glutaraldehyde fixation, the shape of the erythrocytes was examined by phase-contrast or scanning electron microscopy. The antibody-internalized cells were also treated, prior to fixation, with dinitrophenol, chlorpromazine (membrane-penetrable crenation- and cup-forming agents, respectively), or serum albumin (membrane nonpenetrating cup-forming agent). Anti-spectrin antibodies produced a cup shape in the cells at low concentrations (0.5 mg/ml), with a few cells showing multiple cavities. The proportion of the latter increased with increasing concentration of the antibody. The Fab fragments of the antibody were without any effect on the shape. At lower concentrations of anti-4.1 antibodies (0.5 and 1 mg/ml), crenations were observed in antibody-internalized cells. At higher antibody concentrations, the cells underwent sphering. These looked dense under the phase-contrast microscope and often were found to project vesicles from their surface via long stalks. These were generally absent on cells when viewed under the electron microscope, probably due to their breakage during processing. The Fab fragments of the anti-4.1, at equimolar concentrations, were as effective as the intact antibody in altering the cell shape. Anti-ankyrin antibodies or the Fab fragments produced no effect on the shape of the cell. The intact anti-band 3 cytoplasmic domain antibodies, but not their Fab fragments, produced cups at low concentrations and multiple cavities at higher concentrations and multiple cavities at higher concentrations. The anti-spectrin- and anti-band 3 cytoplasmic domain-internalized cells resisted the shape-modulating actions of dinitrophenol, chlorpromazine, and serum albumin. The anti-4.1 antibody-internalized cells also did not respond to dinitrophenol and serum albumin; however, they were converted to contracted cups in the presence of high concentration of chlorpromazine. The cells in which anti-ankyrin antibodies were internalized responded to the actions of the shape-changing agents similarly to normal cells. The results show that the 4.1 protein plays an important role in the determination or maintenance of the normal shape of the erythrocyte and that the cells possessing antibody-modified membrane skeleton do not respond to amphiphilic ions in the manner that normal cells do.