Experimental Cell Research 162 (1986) 449-461 Interaction of Fi bronectin-coated Beads with Attached and Spread Fibroblasts Binding, Phagocytosis, and Cytoskeletal Reorganization FREDERICK GRINNELL’, * and BENJAMIN GEIGER* ‘Uniniversity of Texas Health Science Center, Dallas, TX 75235, and ‘Department of Chemical Immunology, The Weizmann Institute of Science, Rehovot 76100, Israel After 15 min incubations, binding of 0.8-, 6-, and 16ym fibronectin-coated latex beads occurred primarily at the margins of chick embryo tibroblasts that previously were attached and spread on tibronectin-coated glass coverslips. Extensive phagocytosis of the smallest beads and some phagocytosis of the larger beads occurred within 2 h. Following binding of the 16-urn beads, there were no changes in overall cell shape or in the distribution of several cytoskeletal proteins. There was, however, a local accumulation of actin and a-actinin patches adjacent to the sites where the beads were bound. The formation of a-actinin patches could be detected with 6- or 16-urn beads shortly after initial bead binding to the cells, but a similar reorganization of a-actinin in response to the binding of 0.8~urn beads was not detected. The patches of a-actinin appeared to be associated with membrane ruffles, since such structures were observed by scanning electron microscopy @EM) to be sites of cell interaction with 6- but not 0.8~nm beads. Also, two other cytoskeletal proteins normally absent from membrane ruffles, tropomyosin and vinculin, were not detected at the sites of cell-bead interaction. No reorganization of vinculin at the cell-bead interaction sites was observed even when the 16-urn beads remained bound at the cell surfaces for up to 6 h. Nevertheless, prominent vinculin plaques were observed at the marginal attachment sites on the ventral cell surfaces. Consequently, formation of mature focal adhesions may be restricted to linear regions of cell-substratum interac- tion. @ 1986 Academic press, Inc. Cell-substratum adhesion has been found to occur in a series of steps beginning with binding of cell surface receptors to substratum ligands, followed by cyto- skeletal and cytoplasmic reorganization [ 11. While the ligand-receptor interac- tions appear to be specific, a large number of different ligands have been found to be sufficient to initiate the adhesion response [2]. Two types of adhesions have been distinguished based on interference reflec- tion microscopy [3]. Close adhesions, which can be found anywhere on the ventral surface of spread cells, are associated with a microfilament network and a-actinin but lack vinculin [4]. Focal adhesions, on the other hand, which have been located predominantly at the margins of spread cells, are characterized by the insertion of microfilament bundles and the presence of vinculin plaques and a-actinin [4-6]. Close adhesions generally form before focal adhesions [l], are sufficient for cell spreading [7], and are associated with cell motility [8, 91. In * To whom offprint requests should be addressed. Copyright @ 1986 by Academic Press, Inc. All rights of reproduction in any form reserved 0014-4827/86 $03.00