Studies on the cytoskeletal and nuclear architecture of Xenopus erythrocytes.

Abstract

A proteinaceous cytoskeletal network is present in nucleated erythrocytes, which is obscured ultrastructurally in whole cells due to the presence of haemoglobin. Lysis of Xenopus erythrocytes in solutions containing Triton X-100 reveals a cytoskeleton that contains a centrally positioned nucleus, which is linked to the cell surface-associated cytoskeleton by intermediate filaments. The marginal band microtubules are also preserved in these structures. In addition, a single or a pair of perinuclear centrioles is frequently observed in thin sections. These structures are surrounded by a mass of intermediate filaments and fibrogranular material. In contrast to the centrioles in invertebrate erythrocytes those in Xenopus erythrocytes are not associated with the marginal band. Cytonuclear skeletons were obtained by DNase I digestion and subsequent high-salt extraction of cytoskeletons. The resulting structures were chromatin-depleted and consisted of a nuclear lamina that was maintained in the same overall shape and position as that of intact nuclei. With the exception of the marginal band, the remaining cytoskeletal elements persisted after these treatments. Although marginal bands were not detectable by electron microscopy, the cytonuclear skeletons contained roughly the same amount of tubulin as cytoskeletons, as indicated by immunoblotting with affinity-purified anti-tubulin antibodies. When intact erythrocytes were exposed to the ionophore A23187 in the presence of calcium, the cell shape and centric nuclear position were altered. Nuclear dislodgement may be attributable to the disruption of intermediate filament associations with the subsurface cytoskeletal shell. Indirect immunofluorescent staining of cytoskeletons lysed in buffers containing either EGTA or calcium indicates that in the absence of calcium, the intermediate filament network extends to the cell periphery. In structures lysed in calcium, however, the filaments are restricted to the vicinity of the nucleus.