Visualization of the hexagonal lattice in the erythrocyte membrane skeleton

Abstract

The isolated membrane skeleton of human erythrocytes was studied by high resolution negative staining electron microscopy. When the skeletal meshwork is spread onto a thin carbon film, clear images of a primarily hexagonal lattice of junctional F-actin complexes crosslinked by spectrin filaments are obtained. The regularly ordered network extends over the entire membrane skeleton. Some of the junctional complexes are arranged in the form of pentagons and septagons, ~3 and 8% respectively. At least five forms of spectrin crosslinks are detected in the spread skeleton including (a) a single spectrin tetramer linking two junctional complexes, (b) three-armed Y-shaped spectrin molecules linking three junctional complexes, (c) three-armed spectrin molecules connecting two junctional complexes with two arms bound to one complex and the third arm bound to the adjacent complex, (d) double spectrin filaments linking two junctional complexes, and (e) four-armed spectrin molecules linking two junctional complexes. Of these, the crosslinks of single spectrin tetramers and three-armed molecules are the most abundant and represent 84 and 11% of the total crosslinks, respectively. These observations are compatible with the presence of spectrin tetramers and oligomers in the erythrocyte membrane skeleton. Globular structures (9-12 nm in diameter) are attached to the majority of the spectrin tetamers or higher order oligomer-like molecules, ~80 nm from the distal ends of the spectrin tetramers. These globular structures are ankyrin- or ankyrin/band 3-containing complexes, since they are absent when ankyrin and residual band 3 are extracted from the skeleton under hypertonic conditions.