Ultrastructure of unit fragments of the skeleton of the human erythrocyte membrane.

Abstract

Fragments of the filamentous network underlying the human erythrocyte membrane were examined by high-resolution EM. Networks were released from ghosts by extraction with Triton X-100, freed of extraneous proteins in 1.5 M NaCl, and collected by centrifugation onto a sucrose cushion. These preparations contained primarily protein bands 1 + 2 (spectrin), band 4.1, and band 5 (actin). The networks were partially disassembled by incubation at 37.degree. C in 2 mM NaPi (pH 7), which caused the preferential dissociation of spectrin tetramers to dimers. The fragments so generated were fractionated by gel filtration chromatography and visualized by negative staining with uranyl acetate on fenestrated carbon films. Unit complexes, which sedimented at .apprx. 40S, contained linear filaments .apprx. 7-8 nm diam from which several slender and convoluted filaments projected. The linear filaments had a mean length of 52 .+-. 17 nm and a serrated profile reminiscent of F-actin. They could be decorated in an arrowhead pattern with S1 fragments of muscle heavy meromyosin which, incidentally, displaced the convoluted filaments. The linear filaments nucleated the polymerization of rabbit muscle G-actin, predominantly but not exclusively from the fast-growing ends. On this basis, the linear filaments were identified as F-actin; inferring that the convoluted filaments are spectrin. Spectrin molecules were usually attached to actin filaments in clusters that showed a preference for the ends of the F-actin. Free globules up to 15 nm diam were observed, usually associated with 3 spectrin molecules, which also nucleated actin polymerization; these may be simple junctional complexes of spectrin, actin and band 4.1. In larger ensembles, spectrin tetramers linked actin filaments and/or globules into irregular arrays. Intact networks were an elaboration of the basic pattern manifested by the fragments. There is ultrastructural evidence that the submembrane skeleton is organized, as widely inferred from less direct information, into short actin filaments linked by multiple tetramers of spectrin clustered at sites of association with band 4.1.