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 functions of spectrin, we transfected Caco-2 intestinal epithelial cells with a plasmid conferring neomycin resistance and encoding either actin-binding or ankyrin-binding domains of beta G-spectrin fused with beta-galactosidase. These polypeptides, in principle, could interfere with 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-resistant cells, but they could be detected based on expression of beta-galactosidase. Cells expressing spectrin domains exhibited a progressive decrease in amounts of endogenous beta G-spectrin, although alpha-spectrin was still present. Beta G-spectrin-deficient cells lost epithelial cell morphology, became multinucleated, and eventually disappeared after 10-14 d in culture. Spectrin-associated membrane proteins, ankyrin and adducin, as well as the Na+,K(+)-ATPase, which binds to ankyrin, exhibited altered distributions in cells transfected with beta G-spectrin domains. E-cadherin and F-actin, in contrast to ankyrin, adducin, and the Na+,K(+)-ATPase, were expressed, and they exhibited unaltered distribution in beta G-spectrin-deficient cells. Cells transfected 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-associated membrane proteins. These results establish that beta G-spectrin is essential for the normal morphology of epithelial cells, as well as for their maintenance in monolayer culture.
Hu
R J
RJ
Howard Hughes Medical Institute, Duke University School of Medicine, Durham, North Carolina 27710.
Moorthy
S
S
Bennett
V
V
D017487:Ankyrins / Q000235:genetics / Q000378:metabolism; D016923:Cell Death; D002478:Cells, Cultured; D004847:Epithelial Cells; D004848:Epithelium / Q000378:metabolism; D018014:Gene Transfer Techniques; D015726:Giant Cells / Q000378:metabolism*; D006801:Humans; D007422:Intestines; D008840:Microfilament Proteins / Q000235:genetics / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000378:metabolism; D013049:Spectrin / Q000096:biosynthesis* / Q000235:genetics
D017487:Ankyrins; D008840:Microfilament Proteins; D011993:Recombinant Fusion Proteins; D013049:Spectrin