Organization of the intact human erythrocyte cytoskeleton and its attachment to the membrane: Ultrastructural studies

Abstract

The cytoskeleton of the human erythrocyte is responsible for the remarkable ability of this cell to change shape. In order to study the organization of this structure in the intact human erythrocyte, I attached paraformaldehyde-fixed ghosts to coated coverslips and sheared them to expose the cytoskeleton. Quick-freeze, deep-etch, rotary-replication with Pt/C (QFDERR), or tannic acid/osmium fixation and plastic embedding, revealed the cytoskeleton as a dense network of intersecting straight filaments. I found 3-4 filaments at each intersection and 40% more intersections/{dollar}\mu{dollar}m{dollar}\sp2{dollar} of membrane than predicted on the basis of actin junctions alone. Immunogold labeling verified that the filaments were spectrin, but their lengths (29-37 nm) were approximately one-third that of extended spectrin dimers. The length and diameter of the filaments were sufficient to accommodate spectrin dimers, but not spectrin tetramers. These results suggested that, in situ, spectrin dimers may associate as hexamers and octamers, rather than tetramers. I then isolated the skeleton from the membrane and visualized it by either negative staining or QFDERR. Negatively stained isolated skeletons had 5-7 filaments at an intersection, whereas isolated skeletons visualized by QFDERR had 3-5 filaments at an intersection. This difference appears to arise from filaments unraveling to reveal two strands in negatively stained skeletons. Immunogold labeling revealed that spectrin tetramers and hexamers were the predominant species in the isolated skeletons after QFDERR. Unlabeled skeletons also showed evidence of laterally associated spectrin filaments. Similar associations in the intact cytoskeleton could account for the discrepancy in the number of apparent spectrin hexamers and octamers. Extracting sheared erythrocyte ghosts with solutions of low ionic strength removed the cytoskeleton to reveal projections from the cytoplasmic surface of the membrane. These projections contained band 3, as shown by immunogold labeling, and they aggregated to a similar extent as intramembrane particles (IMP) when the cytoskeleton was removed, suggesting a direct relationship between these structures. Quantification indicated a stoichiometry of 2 IMP for each cytoplasmic projection. Cytoplasmic projections presumably contain other proteins besides band 3 since further treatment with high ionic strength solutions extracts peripheral proteins and reduces the diameter of projections by {dollar}\sp\sim{dollar}3 nm.