Architecture of the cytoskeleton in red blood cells


red blood cells

Red blood cells have a characteristic biconcave disc shape that provides a surface area to volume ratio that is optimal for gas exchange. Throughout the course of their 120 day lifespan, the cells are exposed to high amounts of shear force as they navigate the narrow capillaries of the microvasculature and hence need to undergo rapid, reversible deformations. To cope with this stress, the red blood cell is equipped with a specialized cytoskeleton that provides the mechanical stability and flexibility necessary to to withstand forces experienced during circulation.

The red cell membrane is composed of a lipid bilayer, transmembrane proteins, and a filamentous meshwork of proteins that forms a membrane skeleton along the entire cytoplasmic surface of the membrane. The most abundant protein in the membrane skeleton is spectrin, which forms long, flexible heterodimers through the lateral association of ? and ? chains that in turn, associate 'head-to-head' to produce heterotetramers (Sp4). The “tails” of the heterotetramer bind a junctional complex (JC) composed of F-actin, protein 4.1, and actin-binding proteins dematin, adducin, tropomyosin, and tropomodulin. This network is tethered to the cell membrane at two sites: one mediated by ankyrin that couples spectrin to Band 3 and the other mediated by protein 4.1 that couples the junctional complex to Glycophorin C. Mutations in various transmembrane or skeletal proteins give rise to several hereditary red blood cell disorders characterized by membrane fragmentation, hemolysis, and ultimately, anemia, demonstrating the importance of this network.

Early negative-stain electron microscopy studies on isolated membrane skeletons revealed a lattice-like network of thin, straight filaments intersecting at globular junctional complexes (see below). However, this is unlikely to represent the network organization in situ since the samples have been artificially expanded on EM grids, stained with heavy-metal salts, and air-dried. We have used cryo-electron tomography to produce the first three-dimensional representation of the red blood cell cytoskeleton in its intact, native state. For this, we isolated skeletons from ghost membranes, plunge-froze them into liquid ethane, and imaged them over holes in the carbon support at cryogenic temperatures. The resulting three-dimensional volumes (right) revealed a densely packed, heterogeneous network of filaments, instead of the lattice-like network previously seen by negative-stain.

negative stain skeleton

cryo-electron tomogram

red cell segmentation

Based on comparisons between intact and expanded states, we propose that the spectrin filaments substantially reorganize as the membrane is stretched during cell deformation.

spectrin networks