The possible role of a 140K membrane-associated protein complex (140K) in fibronectin-cytoskeleton associations has been examined. The 140K was identifed by the monoclonal antibody JG22E. Monoclonal and polyclonal antibodies to the 140K showed identical patterns of binding to the cell membranes of fixed and permeabilized chicken embryonic fibroblasts; localization was diffuse, but with marked concentration in cell-toextracellular matrix contact sites. Correlative localization with interference reflection microscopy and double-label or triple-label immunofluorescence showed that 140K co-distributed with extracellular fibronectin fibrils and intracellular a-actinin in microfilament bundles at extracellular matrix contact sites but tended not to co-localize with tropomyosin present in bundles at sites farther from adhesion sites. In addition, binding of antibodies to 140K, aactinin, and fibronectin was excluded from vinculin-rich focal adhesion sites at the cellular periphery. A progressive development of cell surface a-actinin-140K-fibronectin associations was observed in early spreading cells. The anti-140K monoclonal antibody JG22E inhibited the attachment and spreading of both normal and Rous sarcoma virus-transformed chicken embryonic fibroblasts to a fibronectin substratum. However, the anti-140K monoclonal antibody became a positive mediator of cell attachment and spreading if it was adsorbed or cross-linked to the substratum. Our results provide the first description of a membraneassociated protein complex that co-localizes with fibronectin and microfilament bundles, and they suggest that the 140K complex may be part of a cell surface linkage between fibronectin and the cytoskeleton.Interactions of cells with extracellular materials are critically important events during embryonic development and for the maintenance of normal tissue functions. Fibronectin has been shown to promote the adhesion and spreading of cells ~ on a variety of materials including plastic, collagen, gelatin, and fibrin (for reviews, see references 15, 22, 26, 31, 37, and 47). Concomitant with the spreading induced by added fibronectin, cells often acquire highly ordered intracellular microfilament bundles (MFBs) I (1, 45). In highly spread cells, extracellular matrix (ECM) fibers that contain fibronectin are often Abbreviations used in this paper: CEF, chicken embryo fibroblasts; CEL, chicken embryonic lung; ECM, extracellular matrix; IRM, interference reflection microscopy; 140K, a three component, membrane-associated protein complex; MFB, microfilament bundle; RSV, Rous sarcoma virus.observed to correspond in their arrangement with intracellular MFBs (20,23,39). At sites where ECM fibrils appear to attach to the plasma membrane, there is a co-distribution of actin and a-actinin (and sometimes vinculin) inside cells and fibronectin outside cells (4,9,24,40, 41).Immunoelectron microscopy has shown a spatial relationship between fibronectin and a-actinin at membrane attachment sites in spread fibroblasts, which were termed ECM...

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We have characterized a 140-kDa glycoprotein complex purified by a monoclonal antibody and implicated in cell adhesion to the extracellular molecule fibronectin. Three major polypeptide components were purified by monoclonal antibody JG22E, which had apparent molecular weights of 155,000 (band 1), 135,000 (band 2), and 120,000 (band 3). In two-dimensional gel electrophoresis, each subunit migrated as either a broad band or a series of spots at acidic isoelectric points. After treatment with neuraminidase, the spots became focused around pH 6.2 (band 1), pH 5.6 (band 2), and pH 5.3 (band 3). These three major bands were compared by two-dimensional peptide mapping in a series of pairwise combinations and were found to be distinct proteins. In sucrose gradients, these proteins co-migrated as a complex sedimenting at approximately 8.4 S either before or after affinity purification, whereas separated subunits migrated at 4.7 to 5.8 S. Amino acid analysis revealed no detectable hydroxyproline and a composition characterized by a substantial number of cysteine residues compared to the average protein. Our results suggest that a noncovalent complex of structurally distinct glycoproteins is involved in adhesive interactions of fibronectin with cells.

When demembranated axonemes of Chlamydomonas were reactivated with Mg-ATP, the proportion of motile axonemes was significantly increased by the presence of either phosphodiesterase (PDE) or protein inhibitor of cAMP-dependent kinase (PKI). The effect of PDE was cancelled by the addition of cAMP. These findings strongly suggest that the axoneme samples have endogenous cAMP, which can reduce the proportion of motile axonemes via phosphorylation. This inhibitory effect of cAMP on Chlamydomonas axonemes is opposite to its stimulatory effect on the axonemal motility in other organisms so far reported. PKI or PDE activated the motility either in the absence of Ca2+, when the axonemes beat with an asymmetric waveform, or in 10(-5) M Ca2+, when the axonemes beat with a symmetric waveform. This cAMP-dependent regulation of motility was observed with the axonemes from which detergent-soluble material had been removed, indicating that the proteins responsible for the regulation still remained in the axonemes. Preliminary in vitro phosphorylation studies have implicated two polypeptides as candidates for the target protein of cAMP-dependent protein kinase: one with a molecular weight of 270 kD and the other with a much larger molecular weight.

Fibronectin and other cell attachment proteins provide molecular models for beginning to unravel the complex interactions of the cell surface with the extracellular matrix. This area has been reviewed in considerable detail previously [I-lo]. Our brief review will therefore be selective rather than comprehensive, and it will focus on some recent generalizations about this class of proteins, as well as on recent advances in the molecular analysis of the functions of these proteins and their receptors. We shall also present various popular or provocative hypotheses and speculations about future work in the field. CLASSES AND SPECIFICITY OF ATTACHMENT PROTEINS of 1)An emerging generalization about this class of proteins is that they are composed separable functional regions, each specialized for specific binding activities (Fig. . Each appears to have one or more regions essential for binding to the cell surface. Fibronectin (Fig. 2) interacts with the fibroblast cell surface primarily through a region termed the cell-binding or cell-recognition site; recent molecular analysis of the function of this site will be discussed below. Although this site appears to be required for cell interactions with fibronectin [ 11,121, interactions at a heparin-binding domain may provide substantial strengthening of this interaction [ 131. In addition, neuronal cells may be capable of interacting with a heparin-binding domain elsewhere in the molecule in the process of axonal elongation; fibronectin-independent interactions with extracellular materials such as heparan-sulfate-containing molecules appear to be important in some aspects of this process [ 14,151.

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During the process of matrix-driven translocation, certain types of cells or polystyrene latex beads are transported between compositionally different regions of a collagen matrix. Under appropriate conditions this translocation depends on an interaction between the cell or particle surface and fibronectin. We now show that this interaction takes place at a site located within the first 31 kDa of the amino-terminal end of the fibronectin molecule. Using defined fibronectin fragments and monoclonal antibodies directed against specific fibronectin domains, this site is established as both necessary and sufficient for the promotion of matrixdriven translocation. Competition experiments using heparin, heparan sulfate, and other sulfated polysaccharides show that this fibronectin site interacts with heparin-like cell or particle surface components in promoting matrix-driven translocation. Treatment of cells with heparinase renders them unresponsive to the translocational effect. An antibody directed against the amino-terminal domain of fibronectin completely inhibits matrix-driven translocation without interfering with heparin binding, suggesting that a post-binding conformational change in fibronectin may be required for promotion of the effect.Matrix-driven translocation (MDT) is a biophysical process in which there is rapid unidirectional movement of certain types of cells or inert particles from one region to another of a compositionally nonuniform collagen-fibronectin matrix (1). Several characteristics of this phenomenon suggest that it may play a role in morphogenesis and related processes in living organisms: (i) MDT selectively translocates different types of cells. For example, chicken limb precartilage mesenchyme cells are translocated by assembling matrices containing chicken cellular fibronectin, but chicken embryo heart cells are not (1). (ii) MDT is selectively promoted by biologically distinct fibronectin molecules. For example, certain cells that are translocated by chicken cellular fibronectin are not translocated by chicken plasma fibronectin under similar conditions (1). (iii) MDT is dependent on physiological conditions. In particular, when the assay is done at nonphysiological pH or ionic strength, translocation of cells or particles does not occur (1).Because MDT is evidently promoted by an interaction between fibronectin and cell or particle surfaces in an appropriate environment, we have now analyzed the specificity of this interaction in relation to current understanding of the domain organization of the fibronectin molecule. Using defined fragments of human plasma fibronectin (2-4) and monoclonal antibodies directed against the corresponding fibronectin domains, we have located a site within the first 31 kDa of the amino terminus of this molecule that is both necessary and sufficient to promote MDT under appropriate conditions. Molecular competition studies, studies using beads coated with defined macromolecules, and studies with heparinase-treated cells indicate that heparin-intera...