The focal contact forms beneath F-actin-rich ribs, or cytoplasmic precursors, present in the lamellipodia of fibroblasts. The basal part of the precursor is retained at the contact as the initial adhesion plaque. We have examined the distribution of talin in the lamellipodia and adhesion plaques of chicken embryo fibroblasts relative to the process of focal contact formation. Motility of single cells was recorded with differential interference contrast or interference reflection microscopy before fixation and fluorescent staining for talin, F-actin, and vinculin. Talin is present along the extreme edge of the lamellipodium, where it is further concentrated into a series of nodes. The nodes of talin are present at the tips of both larger and finer F-actin-rich ribs and at small structural nodes at the edge of the lamellipodium. We suggest that the talin in the nodes functions, via a cross-linking activity, in the convergence of actin filaments at the membrane during development of the ribs. Talin accumulates de novo in the adhesion plaque, independent of that at the tip of the precursor, in response to contact with the substrate. This second accumulation of talin at the focal contact starts before vinculin, consistent with a sequential binding of talin at the membrane and of vinculin to talin. The results imply that talin functions independently at two steps during formation of the focal contact: the development of the F-actin-rich precursor of the contact; and development of the contact-associated adhesion plaque, both involving organization of F-actin at the membrane.

The distribution of F-actin and vinculin in chicken embryo fibroblasts has been examined by nitrobenzoxadiazol (NBD)-phallacidin and indirect immunofluorescent staining, respectively, and related to the process of focal contact formation by recording the motility of the cell with differential interference contrast (DIC) or interference reflection microscopy (IRM) before fixation for staining. Linear cytoplasmic precursors of the focal contact, present within unattached lamellipodia, stained intensely with NBD-phallacidin. Without exception new focal contacts, 8 s and older at fixation, were associated with either a longer F-actin rib in the lamellipodium or, in older contacts, an F-actin structure of similar dimensions to the contact. This change in distribution of F-actin over the new contacts was accounted for by the segregation of the structural precursor into an attached part over the focal contact and a separate motile part. These results show that F-actin accumulates in the precursor adjacent to areas of the membrane competent to form the focal contact, and are consistent with the interpretation that this F-actin contributes to the initial adhesion plaque associated with the new contact. Vinculin was essentially absent from motile lamellipodia, showed no preferential association with F-actin rich precursors or very young focal contacts, but accumulated over new contacts during a 90-s period. Therefore, the association of F-actin with the membrane that precedes and persists in the initial focal contact is independent of vinculin, and the role of vinculin in development of the focal contact remains unclear.