Spectrin rearrangement early in erythrocyte ghost endocytosis

Abstract

The endocytic vacuoles induced in white ghosts were found to be depleted of spectrin and therefore it was proposed that they arose from spectrin-free areas in the erythrocyte membrane. To follow changes in spectrin distribution during endocytosis, affinity-purified rabbit antispectrin antibodies were produced. Quantitative techniques were developed for the use of a highly specific 125I-F(ab')2 antispectrin, and these showed that before the appearance of vacuoles, as assessed by phase microscopy, there was a reproducible decrease in immunoreactive spectrin. To determine whether this spectrin decrease represented a local or diffuse spectrin loss or a spectrin rearrangement, morphologic studies were undertaken using transmission electron microscopy on samples treated with rabbit antispectrin and ferritin-conjugated goat anti-rabbit immunoglobulin. These studies showed that endocytosis was preceded by the creation of extensive spectrin-free areas separated by discrete spectrin-containing zones. Pretreatment of ghosts with alkaline phosphatase blocked all forms of endocytosis and prevented the creation of spectrin-free areas. Therefore, it is proposed that under the impetus of endocytosis inducers, phosphorylated spectrin is redistributed so that spectrin-free zones are created, and that endocytic vacuoles form and fuse in spectrin-free areas.