EXPRESSION OF FUNCTIONAL DOMAINS OF BETA(G)-SPECTRIN DISRUPTS EPITHELIAL MORPHOLOGY IN CULTURED-CELLS

Spectrin is a major structural protein associated with the cytoplasmic surface of plasma membranes of many types of cells. To study the func tions of spectrin, we transfected Caco-2 intestinal epithelial cells w ith a plasmid conferring neomycin resistance and encoding either actin -binding or ankyrin-binding domains of beta(G)-spectrin fused with bet a-galactosidase. These polypeptides, in principle, could interfere wit h the interaction of spectrin with actin or ankyrin, as well as block normal assembly of alpha- and beta-spectrin subunits. Cells expressing the fusion proteins represented only a small fraction of neomycin-res istant cells, but they could be detected based on expression of beta-g alactosidase. Cells expressing spectrin domains exhibited a progressiv e decrease in amounts of endogenous beta(G)-spectrin, although alpha-s pectrin was still present. Beta(G)-spectrin-deficient cells lost epith elial cell morphology, became multinucleated, and eventually disappear ed after 10-14 d in culture. Spectrin-associated membrane proteins, an kyrin and adducin, as well as the Na+, K+-ATPase, which binds to ankyr in, exhibited altered distributions in cells transfected with beta(G)- spectrin domains. E-cadherin and F-actin, in contrast to ankyrin, addu cin, and the Na+, K+-ATPase, were expressed, and they exhibited unalte red distribution in beta(G)-spectrin-deficient cells. Cells transfecte d with the same plasmid encoding beta-galactosidase alone survived in culture as the major population of neomycin-resistant cells, and they exhibited no change in morphology or in the distribution of spectrin-a ssociated membrane proteins. These results establish that beta(G)-spec trin is essential for the normal morphology of epithelial cells, as we ll as for their maintenance in monolayer culture.