In this issue of Blood, An and colleagues demonstrate the interesting finding that depletion of the junctional complex protein tropomyosin from erythrocyte membranes leads to membrane instability.
The erythrocyte membrane is a remarkable structure with unique properties that allow it to withstand the dynamic shear stresses of the circulation, surviving both high arterial flow and reversible deformation in narrow capillary beds and splenic sinusoids. Many of these properties are provided by the membrane skeleton located beneath the cell's lipid bilayer. The membrane skeleton is a 3D structure organized as a hexagonal network. The spokes of the hexagons are composed of extended spectrin tetramers. The junctions of the hexagons are composed of a complex of proteins called the “junctional complex.” At the heart of the junctional complex are short actin filaments approximately 40 nm in length. Other junctional complex proteins include protein 4.1R, adducin, protein 4.9, CapZ, tropomodulin, and tropomyosin.
Tropomyosins are rodlike proteins with a diverse array of cell-type–specific isoforms. In human erythrocytes, the major tropomyosin isoforms are 5 and 5b, which are capable of forming homodimers or heterodimers. Both tropomyosin 5 and 5b exhibit high affinity for actin and erythrocyte tropomodulin, an actin-capping protein. Electron microscopy reveals that tropomyosin is the same length as actin filaments, leading to the suggestion that tropomyosin-tropomodulin complexes function as a “molecular ruler” governing filament length in erythrocytes. In striated muscle, tropomyosin mediates actin-troponin complex interactions, regulating muscle contraction. The function of tropomyosin in erythrocytes is unclear.
An and colleagues hypothesize that tropomyosin contributes to the mechanical stability of the erythrocyte membrane by stabilizing the spectrin-actin junctional complex. To address this, the authors investigated the mechanical properties of erythrocyte membranes depleted of tropomyosin, taking advantage of the observation that washing erythrocytes with magnesium-free buffer depletes the membrane of tropomyosin, apparently without other major membrane effects. As hypothesized, tropomyosin-depleted erythrocytes demonstrated decreased membrane stability. Adding erythrocyte tropomyosin and magnesium back to depleted erythrocytes restored much of the membrane stability. Stability of the spectrin-actin-protein 4.1R ternary complex was examined by introducing a β-spectrin peptide containing its actin-binding and protein 4.1R–binding domains to erythrocyte ghosts. The spectrin peptide destabilized the membrane significantly more when tropomyosin was absent, to a degree comparable with that seen in protein 4.1R–deficient hereditary elliptocytosis.
What are the implications of these studies? Clinically, there are many cases of membrane-linked hereditary hemolytic anemia where the causative mutations are unknown. Mutation of junctional complex proteins may be the answer.
Discovery of numerous attachment points has revealed unexpected complexity in membrane skeleton–plasma membrane interactions. The model of protein 4.1R–glycophorin attaching the junctional complex to the lipid bilayer has recently been called into question, and it is likely additional proteins will be found to participate in these attachments. Other questions include which proteins or interactions direct assembly of the membrane during erythropoiesis, and which are primarily responsible for stabilization of the mature membrane skeleton. Finally, these studies highlight why the erythrocyte continues to be the paradigm for study of membrane structure and function.
The author declares no conflicting financial interests. ▪
