Visualization of the protein associations in the erythrocyte membrane skeleton.

Abstract

We have obtained clear images of the erythrocyte membrane skeleton from negatively stained preparations that originate directly from the intact cell but in which the spectrin meshwork is artificially spread to allow close inspection. Our procedure requires less than 2 min at 50C in phosphate buffers. We flnd 200-nm-long spectrin tetramers crosslinked by junctional complexes. Each junction contains a regular 37-nm rod, probably an actin oligomer of approximately 13 monomers. Densities appear at variable places in the meshwork but distinct globules occur with great frequency 78 nm from the spectrin tetramer's junctional insertion end, very close to the known binding site for ankyrin. Most frequently, five or six spectrin tetramers insert into each junction, producing a meshwork that displays remarkably regular long range order.head end of the spectrin molecules but preserve the associations with actin and band 4.1 at the tail end of spectrin have also been performed (11). The resultant electron micrographs showed fragments containing variable-length actin filaments and globules from which several convoluted filaments of spectrin projected.Although some of the earliest microscopic images of the intact erythrocyte membrane skeleton were obtained by negative staining (1, 2), in only one micrograph published by Pinder et al. (12) can one see individual spectrin tetramers and fragmentary hints of the band 4. 1-actin oligomer junctions. We have now obtained clear images of the spectrin meshwork from negatively stained preparations that originate directly from the intact cell but in which the spectrin filaments are artificially spread to allow close inspection.Electron micrographs of the isolated mammalian erythrocyte cytoskeleton have demonstrated the existence of a continuous reticulum underlying the membrane (1, 2) but have not provided high-resolution information about the details of the structure and connections within this membrane skeleton. Although aspects of the skeleton have been visualized in thin sections of the erythrocyte ghost (3, 4), a major problem when examining the intact erythrocyte membrane or its skeleton is that the high density of proteins makes it very difficult to visualize the individual macromolecules and their connections.The erythrocyte membrane skeleton is believed to consist of a cross-linked meshwork consisting of spectrin, actin, band 4.1, and other associated proteins. Two linkages are viewed as essential for the integrity of this meshwork: first, spectrin heterodimers must associate head to head to form tetramers and, second, tail ends of the tetramers must crosslink the meshwork by binding to junctions that include short actin oligomers and band 4.1 protein. This view of the membrane skeleton has come from studying individual purified molecules and their modes of association in vitro (5-9). While these studies have told us much about the shapes of the individual molecules and the affinities and positions of their binding sites, evidence that the assemblies that have been crea...

Single Molecule Kinetic Analysis of Actin Filament Capping

“…This gives the appearance of uncapping by PPIs. Filament breakage and annealing were not considered in the previous bulk uncapping experiments (4), perhaps because they utilized spectrin-actin seeds with filaments of only 13-14 subunits (40). However, we calculate from the actin polymer concentration and exponential capping rate that half of the filaments were at least 4.1 m long, 25% were at least 6.2 m long, and 2% of filaments were uncapped and 16.9 m long at the time that Schafer et al (4) added PIP 2 in their uncapping experiment.…”

Direct Cytoskeleton Forces Cause Membrane Softening in Red Blood Cells

“…In vivo, the characteristic length of the network is a ¼ 46 5 15 nm (46), indicating that on average, the size of spectrin filaments in the native membrane skeleton is a fraction of its contour length (L z 190 nm) observed in membrane patches stretched on TEM grids (39). Spectrin filaments are pinned to the membrane Biophysical Journal 108(12) 2794-2806 via transmembrane-and membrane-associated proteins (47,48). To assemble the network, the distal tails of the spectrin filaments are connected at pinning nodes via junctional complexes consisting of actin, tropomyosin, protein band 4.1, and other regulatory proteins (Fig.…”

Dynamics of bacteriorhodopsin 2D crystal observed by high-speed atomic force microscopy

“…In particular, it has been shown that proteins in native membranes may not diffuse freely but are in fact confined to specific domains. Cells use several confining mechanisms such as anchoring to the cytoskeleton through hetero-bifunctional proteins (Byers and Branton, 1985), diffusion barriers formed by the accumulation of proteins anchored to cytoskeleton meshes (Halenda et al, 1987;Nakada et al, 2003), or self-assembly into large two-dimensional (2D) crystalline patches, as in the case of bacteriorhodopsin (bR), a light-driven proton pump found in the so-called purple membranes (PM) of Halobacterium salinarum (Blaurock, 1971). Despite these advances, an understanding of the membrane dynamics at the nanoscale remains a major challenge due primarily to the lack of measurement techniques allowing simultaneous spatial and temporal observation of single molecules within native membranes.…”