Ultrastructure of the human erythrocyte cytoskeleton and its attachment to the membrane.
We attached paraformaldehyde-fixed human erythrocyte ghosts to coated coverslips and sheared them to expose the cytoskeleton. Quick-freeze, deep-etch, rotary-replication, or tannic acid/osmium fixation and plastic embedding revealed the cytoskeleton as a dense network of intersecting straight filaments. Previous negative stain studies on spread skeletons found 5-6 spectrin tetramers intersecting at each actin oligomer, with an estimated 250 such intersections/microns 2 of membrane. In contrast, we found 3-4 filaments at each intersection and approximately 400 intersections/microns 2 of membrane. 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. Our results suggest that, in situ, spectrin dimers may associate as hexamers and octamers, rather than tetramers. We present several explanations that can reconcile our observations on intact cytoskeletons with previous reports on spread material. Extracting sheared 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 approximately 3 nm.
Department of Physiology, School of Medicine, University of Maryland, Baltimore 21201.
J A
JA
Ursitti
D W
DW
Pumplin
J B
JB
Wade
R J
RJ
Bloch
D008841:Actin Cytoskeleton / Q000648:ultrastructure; D001457:Anion Exchange Protein 1, Erythrocyte / Q000648:ultrastructure; D003599:Cytoskeleton / Q000648:ultrastructure*; D004910:Erythrocyte Membrane / Q000648:ultrastructure; D004912:Erythrocytes / Q000648:ultrastructure*; D006801:Humans; D007150:Immunohistochemistry; D008854:Microscopy, Electron; D016628:Replica Techniques; D013048:Specimen Handling; D013049:Spectrin / Q000648:ultrastructure
D001457:Anion Exchange Protein 1, Erythrocyte; D013049:Spectrin