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The leading lamella is a highly dynamic cell compartment of locomoting fibroblasts. Based on its well-characterized internal cytoskeletal architecture, the leading lamella can be divided into three structurally distinct zones. Much less is known about the membrane components of the leading lamella. In this study, we looked at the distribution of spectrin, the major component of the subplasmalemmal membrane skeleton, in the leading lamella and its relation to the subdivision of the lamellar space in cultured fibroblasts. In immunofluorescence microscopy, a general, plasma membrane-associated staining of spectrin was observed especially in the more central regions of detergent-extracted cells. In the leading lamella, spectrin was seen particularly along the lamellar edge and as small protrusions, or nodes, along the lamellar periphery. A weaker staining was observed in the more proximal regions of the lamella. In wet-cleaved cells also, spectrin was observed along the leading edge and in the protrusions of the lamella. In double immunofluorescence microscopy, a close colocalization of spectrin and actin was seen in the lamellar region. In immunoelectron microscopy of whole-mount preparations, spectrin was also found to be in close association with the actin meshwork in the most peripheral zone of the lamella and it was also associated with the actin-containing microspikes. A weaker labeling for spectrin was observed along the filaments in the proximal regions of the lamella. The node-like accumulations of spectrin seen along the lamellar edge were reactive to antibodies raised against talin and paxillin, suggesting that they represent evolving focal adhesions. The results show that spectrin is particularly present along the leading edge of the leading lamella. It is also present in the active protrusion sites of translocating cells, probably representing evolving adhesion sites. The role of spectrin should therefore be considered when studying the mechanisms of events associated with the locomotive behavior of fibroblasts.

The amiloride-sensitive Na+ channel constitutes the rate-limiting step for Na+ transport in epithelia. Immunolocalization and electrophysiological studies have demonstrated that this channel is localized at the apical membrane of polarized epithelial cells. This localization is essential for proper channel function in Na+ transporting epithelia. In addition, the channel has been shown to associate with the cytoskeletal proteins ankyrin and alpha-spectrin in renal epithelia. However, the molecular mechanisms underlying the cytoskeletal interactions and apical membrane localization of this channel are largely unknown. In this study we show that the putative pore forming subunit of the rat epithelial (amiloride-sensitive) Na+ channel (alpha ENaC) binds to alpha-spectrin in vivo, as determined by co-immunoprecipitation. This binding is mediated by the SH3 domain of alpha-spectrin which binds to a unique proline-rich sequence within the C-terminal region of alpha rENaC. Accordingly, the C-terminal region is sufficient to mediate binding to intact alpha-spectrin from alveolar epithelial cell lysate. When microinjected into the cytoplasm of polarized primary rat alveolar epithelial cells, a recombinant fusion protein containing the C-terminal proline-rich region of alpha rENaC localized exclusively to the apical area of the plasma membrane, as determined by confocal microscopy. This localization paralleled that of alpha-spectrin. In contrast, microinjected fusion protein containing the N-terminal (control) protein of alpha rENaC remained diffuse within the cytoplasm. These results suggest that an SH3 binding region in alpha rENaC mediates the apical localization of the Na+ channel. Thus, cytoskeletal interactions via SH3 domains may provide a novel mechanism for retaining proteins in specific membranes of polarized epithelial cells.

Calcium binding to brain and erythrocyte spectrins was studied at physiological ionic strength by a calcium overlay assay and aqueous two-phase partitioning. When the spectrins were immobilized on nylon membranes by slot blotting, the overlay assay showed that even though both spectrins bound 45Ca2+, the brain protein displayed much greater affinity for calcium ions than erythrocyte spectrin did. Since the observed binding was weaker than that displayed by calmodulin under similar conditions, the overlay assay results indicated that the binding must be weaker than 1 microM. The phase partition experiments showed that there are at least two sites for calcium on brain spectrin and that calcium binding to one of these sites is reduced significantly by magnesium ions. From the partition isotherm, the dissociation constants were estimated as 50 microM for the Mg(2+)-independent site and 150 microM for the Mg(2+)-dependent site. The phase partition results also showed that erythrocyte spectrin bound calcium ions at least 1 order of magnitude weaker. By examining calcium binding to slot-blotted synthetic peptides, we identified two binding sites in brain spectrin. One mapped to the second putative calcium binding site (EF-hand) in alpha-spectrin and the other to the 36 amino acid residue long insert in domain 11. In addition, a tryptic fragment derived from the C-terminal of erythrocyte alpha-spectrin, which contained the two postulated EF-hands, also bound calcium. These findings suggest that the calcium signal system may also involve direct binding of calcium to spectrin beside known calcium modulators such as calmodulin and calpain.(ABSTRACT TRUNCATED AT 250 WORDS)

The alpha- and beta-subunits of spectrin are made of repeated homologous units of 106 residues. In the recently reported partial sequence of the chicken non-muscle alpha-actinin, a repetitive sequence homologous to the internal repeat in spectrin occurs several times. Both spectrin and alpha-actinin are components of the cytoskeletal network, the integrity of which is based on multiple and complex interactions. We suggest that the shared domain structure indicates common structural principles or interactions of spectrin and alpha-actinin and reflects their common evolution.

Immunological screening of a chicken gizzard cDNA expression library was used to isolate two clones encoding a part of the non-erythroid spectrin-like protein. Clones were identified by immunoblotting of the polypeptides synthesized in Escherichia coli cells transformed with cDNA cloned in the pUC8 plasmid vector using polyclonal rabbit antibodies raised against bovine non-erythroid spectrin. The sequence of an approximately 1.5-kb cDNA insert of one clone was determined. Analysis of the predicted amino acid sequence reveals that, despite differences in immunological cross-reactivity and peptide maps, the chicken non-erythroid and the human erythrocyte spectrins are highly homologous proteins. Like the human erythrocyte spectrin, the chicken smooth muscle spectrin appears also to be constructed from repeated, homologous structures of 106 amino acid residues. This is probably a universal structure motif of spectrins.

Antibodies to nonerythroid alpha spectrin (p 230) were used to study the distribution of this polypeptide in mouse germ cells, zygote, and early embryonic cells. In the primordial germ cells, fetal oocytes, and spermatogonia, spectrin was found predominantly in the form of a narrow condensed subplasmalemmal band, as in all other somatic cells. During spermatogenesis, spectrin is condensed into the supraacrosomal cytoplasm and is lost during the reduction of the cytoplasm of the maturing spermatozoa. The postnatal growth of the oocyte is accompanied by a loss of the dense cortical band of spectrin and its redistribution in the cytoplasm. Zygotes also contain granular dispersed spectrin. Cortical condensation of spectrin filaments gradually reappears in the blastomeres at the two-cell stage and in the secondary polar body. Cortically condensed filaments represent thereafter the predominant form of spectrin in all preimplantation stage embryonic cells. Trophoblastic cells spreading out from explanted blastocysts are devoid of the cortically condensed spectrin and contain, instead, spectrin arrays in the cytoplasm. Trophoblastic cells, which surround the implanted embryo in vivo, also show diffuse cytoplasmic spectrin which subsequently undergoes subplasmalemmal condensation. These data show that spectrin is present in all stages of gametogenesis and embryogenesis, except in mature spermatozoa; and that it undergoes cytoplasmic redistribution during morphogenesis.

The complete nucleotide sequence coding for the chicken brain alpha-spectrin was determined. It comprises the entire coding frame, 5'- and 3'-untranslated sequences terminating in a poly(A)-tail. The deduced amino acid sequence shows that the alpha-chain contains 22 segments, 20 of which correspond to the typical 106 residue repeat of the human erythrocyte spectrin. Some segments non-homologous to the repeat structure reside in the middle and COOH-terminal regions. Sequence comparisons with other proteins show that these segments evidently harbour some structural and functional features such as: homology to alpha-actinin and dystrophin, two typical EF-hand structures (calcium-binding) and a putative calmodulin-binding site in the COOH-terminus and a sequence homologous to various src-tyrosine kinases and to phospholipase C in the middle of the molecule. Comparison of our sequence with other partial alpha-spectrin sequences shows that alpha-spectrin is well conserved in different species and that the human erythrocyte alpha-spectrin is divergent.

We have determined the nucleotide sequence coding for the chicken brain alpha-spectrin. It is derived both from the cDNA and genomic sequences, comprises the entire coding frame, 5' and 3' untranslated sequences, and terminates in the poly(A)-tail. The deduced amino acid sequence was used to map the domain structure of the protein. The alpha-chain of brain spectrin contains 22 segments of which 20 correspond to the repeat of the human erythrocyte spectrin (Speicher, D. W., and V. T. Marchesi. 1984. Nature (Lond.). 311:177-180.), typically made of 106 residues. These homologous segments probably account for the flexible, rod-like structure of spectrin. Secondary structure prediction suggests predominantly alpha-helical structure for the entire chain. Parts of the primary structure are excluded from the repetitive pattern and they reside in the middle part of the sequence and in its COOH terminus. Search for homology in other proteins showed the presence of the following distinct structures in these nonrepetitive regions: (a) the COOH-terminal part of the molecule that shows homology with alpha-actinin, (b) two typical EF-hand (i.e., Ca2+-binding) structures in this region, (c) a sequence close to the EF-hand that fulfills the criteria for a calmodulin-binding site, and (d) a domain in the middle of the sequence that is homologous to a NH2-terminal segment of several src-tyrosine kinases and to a domain of phospholipase C. These regions are good candidates to carry some established as well as some yet unestablished functions of spectrin. Comparative analysis showed that alpha-spectrin is well conserved across the species boundaries from Xenopus to man, and that the human erythrocyte alpha-spectrin is divergent from the other spectrins.

Biochemical and immunofluorescence studies have demonstrated that p36, a major substrate for the tyrosine-specific protein kinases induced by several sarcoma viruses and epidermal growth factor, is associated with plasma membranes and detergent-resistant cytoskeletal structures of cultured cells. We have used here polyclonal antisera and monoclonal antibodies in indirect immunofluorescence microscopy to study the subcellular location of p36 and the p230, which is a subplasmalemmal polypeptide showing immunologic cross-reactivity with erythrocyte alpha-spectrin. Both p36 and p230 showed a diffuse distribution in fixed and permeabilized cells and were localized in a surface lamina-like network in Triton-extracted cells. In double-staining experiments, an extensive co-distribution between these proteins was seen in detergent-treated cultured fibroblasts. These results, together with our previous work, suggest that the p36 protein is an integral part of the detergent-resistant proteinaceous network at the cytoplasmic face of the plasma membrane.

The cytoskeleton of human ventricular myocardium in normal and pathological hearts has been analysed with immunocytochemical techniques. Specific antibodies against the intermediate filament proteins desmin (Mr 55 000) and vimentin (Mr 58 000) and antibodies against two cytoskeleton-associated proteins, a spectrin-like protein (Mr 230 000) and vinculin (Mr 130 000), have been used. We show that desmin is localized in the myocytes as an intermyofibrillar lattice at the Z disk level of the myofibrils, and at the intercalated disks. The spectrin-like protein is localized as a transverse striated pattern interlinked with fine longitudinal strands in the subplasmalemmal region of the myocytes. Vinculin is abundant in the intercalated disks and in myotendinous junctions but occurs also at the peripheral sarcolemma in the form of a regular repeat of dots and of fine bar-like extensions into the cytoplasm from the dots. These patterns were observed both in normal and in abnormal hearts, but a number of altered patterns in pathological myocytes were also seen. It is concluded that the intermediate filament system has important implications in the structural function of normal and abnormal hearts but that further studies are needed to elucidate how the different components are related to each other and how they are influenced by different disease processes.