The biconcave disc shape of mammalian erythrocytes has been considered to be maintained only with a membrane underlain by a membranous cytoskeleton. Our improved ion-etching/scanning electron microscopy and saponin-ethanol treatment combined with immunocytochemistry in the human red blood cell revealed the three-dimensional structure of this cytoplasmic endoskeleton apart from the classical membranous cytoskeleton. The endoskeletal meshwork images obtained by the saponin-ethanol treatment corresponded to those by the repeated ion-etching method. The actin-rich endoskeleton was divided into two layers, one superficial and the other deep. The superficial filaments were perpendicularly connected to the membranous cytoskeleton, while the deep filaments formed an irregularly directed complicated meshwork. In the transitional hillside region between the convex periphery and concave center, the endoskeletal filaments containing a neurofilament protein ran parallel to the hillside slope toward the concave center. The endoskeleton of the erythrocyte associating with the membranous cytoskeleton may serve to keep its unique biconcave disc shape deformable, pliable, and restorable against external circumstances.
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