Structure and composition of the cytoskeleton of nucleated erythrocytes: III. Organization of the cytoskeleton of Bufo marinus erythrocytes as revealed by freeze-dried platinum-carbon replicas and immunofluorescence microscopy.

Platinum-carbon (Pt-C) replicas of freeze-dried erythrocyte cytoskeletons of the toad, Bufo marinus, were prepared using a modified Balzers 300 system. Examination in stereo of replicas of the microtubule-containing marginal band revealed filaments projecting from the microtubule walls to form links between adjacent microtubules. These cross-bridging proteins may bundle the microtubules into the configuration of the marginal band (MB) and may also serve to stabilize the structure. The MB appears to have linkages to components of the surface-associated cytoskeleton (SAC). The SAC forms a continuous matrix that spreads across the upper and lower surfaces of the cell adjacent to the plasma membrane and extends around the outer perimeter of the MB. Thus, the SAC encapsulates the MB and the central nucleus. After lysis, the elements of the cytoskeleton remain in a configuration similar to that found in the whole cell. Spectrin (fodrin) and actin were identified by immunofluorescence in the region of the SAC. When labeled with antibodies specific for vimentin and synemin, a network of intermediate filaments can be detected in the region between the nucleus and the MB. These vimentin filaments are also enclosed within the SAC and appear in Pt-C replicas to emerge from the area of the nuclear envelope. As the filaments extend toward the periphery of the cell, they form attachments to the SAC. Attachments of intermediate filaments to both the nucleus and the SAC thus appear to anchor the nucleus in its central position within the cytoskeleton.