Expression of functional domains of beta G-spectrin disrupts epithelial morphology in cultured cells.

Abstract

Abstract. 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~-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 betaspectrin 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 betac-spectrin, although alpha-spectrin was still present. Betac-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 betac-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 betao-spectrin-deficient cells. Cells transfected with the same plasmid encoding betagalactosidase 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 betac-spectrin is essential for the normal morphology of epithelial cells, as well as for their maintenance in monolayer culture. S PV.CTRIN is an elongated actin-binding protein that is the principal component of a system of structural proteins associated with the cytoplasmic surface of plasma membranes of most metazoan cells (reviewed by Bennett and Gilligan, 1993). Spectrin is comprised of two subunits, termed alpha and beta, that are aligned side-to-side to form heterodimers, and the dimers are linked head-to-head to form tetramers. Beta subunits contain most of the recognition sites of spectrin for other proteins including ankyrin, protein 4.1 actin, as well as the site for ankyrin-independent association of spectrin with membranes. Beta~-spectrin is the most common type of beta subunit, and it is expressed in most vertebrate tissues (Hu et al., 1992). The structure and function of spectrin has been best characterized in mammalian erythrocytes from both in vivo and in vitro studies (Palek and Lambert, 1990;Delaunay and Dhermy, 1993;Gallagher and Forget, 1993;Bennett and Gilligan, 1993). Erythrocyte spectrin forms a membrane-associated polygonal network by the associations of spectrin with actin illaments and with integral membrane proteins through linkages with ankyrin and protein 4.1. Defects and deficiencies in components of the spectrin-actin network result in abnormally fr...

The C-terminal domain ofDrosophilaβHeavy-spectrin exhibits autonomous membrane association and modulates membrane area

“…Overexpression of the βHeavy-spectrin segment 33 induces apoptosis in some tissues Transfection of transgenes expressing fragments of β-spectrin in vertebrate Caco-2 cells has been previously reported to cause the progressive loss of expressing cells from the culture (Hu et al, 1995), so we began by expressing βH33 in a variety of tissues to see if cell lethality also occurs in Drosophila. Crossing this construct to various Gal4-expressing driver lines showed that expression of βH33 often has a catastrophic effect on development.…”

Lateral Membrane Biogenesis in Human Bronchial Epithelial Cells Requires 190-kDa Ankyrin-G

“…The antibody was purified on a GFP affinity column. The affinity-purified ␤-2-spectrin antibody directed against spectrin repeats 4 -9 has been described before (21).…”

“…Depletion of the sole ␣-and ␤-spectrin in C. elegans by RNAi (18) or by null mutation (19,20) revealed that spectrin, although essential for embryonic elongation and survival, was not required for the morphogenesis of polarized epithelial cells. Another caveat is that expression of ␤-2-spectrin domains can cause cell death (21). Moreover, spectrin reversibly dissociates from sites at cell-cell contact under certain experimental conditions, whereas ankyrin remains membrane-associated (22).…”

ELF a β-spectrin is a neuronal precursor cell marker in developing mammalian brain; structure and organization of the elf/β-G spectrin gene

“…Direct evaluation of the functions of spectrins in non erythroid cells has been approached by several methods (Hu et al, 1995;Mishra et al, 1999). Microinjections of N-terminal domain specific anti-b-spectrin inhibits neurite extension in neuroblastoma cells (Sihag et al, 1996).…”

“…Expression of functional domains with b-G spectrin in cultured epithelial cells (CACO cells) resulted in loss of endogenous b-spectrin activity and loss of epithelial cell morphology, the cells becoming multinucleated and eventually disappeared after 10 -14 days in culture. Additionally spectrin associated membrane proteins such as ankyrin, adducin, the Na, 9.0 200 Brain, heart, kidney, Olfactory bulb axons; Fodrin and Ankyrin G/B skeletal muscle purkinje cell axons; associates with Ankyrin stellate cell dendrites; B; anti-sense disrupts developing cortex liver development b Spectrin ELF 4 7.5 240 Heart, skeletal muscle (Hayes et al, 2000) M&Z line, neutrites of Associates with fodaxin cerebellar granule cells 7.0 270 cell bodies of cerebellar granule cell neurons, forebrain, heart, skeletal, muscle and z line, postsynaptic dendrites Non-erythroid b spectrin 9.5 240 Lung (Chen et al, 2001) 7.5 225 Brain, lung, kidney, Associates with Schwannomin cerebellar neurons, Purkinje neurons (not dendrites,) trace-liver: K-ATPase are distributed abnormally reflecting a loss of cell polarity (Nelson and Veshnock, 1987b;Nelson and Hammerton, 1989;Hu et al, 1995). In addition, utilizing antisense oligonucleotides to membrane ELF we have found inhibition of biliary epithelial cell formation in explant cultures .…”