Abstract. Using a combined in vivo and in vitro approach, we have analyzed the immunofluorescent localization and function of a 140,000-mol-wt glycoprotein complex implicated in cell adhesion to fibronectin (FN), with particular emphasis on neural crest cell adhesion and migration. This putative fibronectin receptor complex (FN-receptor) was detectable in almost all tissues derived from each of the three primary germ layers. It was present in both mesenchymal and epithelial cells, and was particularly enriched at sites close to concentrations of FN, e.g., at the basal surfaces of epithelial cells. It was also present on neural crest cells.The distribution and function of this putative receptor was then analyzed on individual cells in vitro. It was diffusely organized on highly locomotory neural crest cells and somitic fibroblasts. Both motile cell types also displayed relatively low numbers of focal contacts and microfilament bundles and limited amounts of localized vinculin, a-actinin, and endogenous FN. In contrast, the FN-receptor in stationary embryonic cells, i.e., somitic cells after long-term culture or ectodermal cells, existed in characteristic linear patterns generally co-distributed with a-actinin and fibers of endogenous FN. Anti-FN-receptor antibodies inhibited the adhesion to FN of motile embryonic cells, but not of stationary fibroblasts. However, these same antibodies adsorbed to substrata readily mediated adhesion and spreading of cells, but were much less effective for cell migration.Our results demonstrate a widespread occurrence in vivo of the putative FN-receptor, with high concentrations near FN. Embryonic cell migration was associated with a diffuse organization of this putative receptor on the cell surface in presumably labile adhesions, whereas stationary cells were anchored to the substratum at specific sites linked to the cytoskeleton near local concentrations of FN-receptor. D URING embryonic development, interactions of cellswith extracellular matrix are important in regulating cell behavior (27,59,65). The extracellular molecule fibronectin (FN) ~ promotes the adhesion, spreading, and formation of specialized adhesion sites in a variety of cells (25,31,40,49). Besides this role, FN stimulates the in vitro locomotion of several embryonic cell types, including chick heart fibroblasts and avian neural crest cells (17,42,43,48). In vivo, the presence of FN has often been correlated with the migration of cells, e.g., gastrulating cells, primordial germ cells, and neural crest cells (5,18,21,22,28, 39,50,58,64). In addition, the interaction of FN with the cell surface is a prerequisite for cell movement, since blocking the FN cellAbbreviations used in this paper." DME, Dulbecco's modified Eagle's medium; FN, fibronectin; FN-receptor, putative fibronectin receptor complex; NCS, newborn calf serum.binding domain inhibits the migration of cells (6,7,48). These observations imply that the same molecule is involved in both the transient adhesions involved in cell movement and in the firm ancho...

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...