Introduction Integrins are transmembrane receptors that mediate cell adhesion by forming links between the extracellular matrix and cytoskeleton. These links are used in cell motility as the actin cytoskeleton generates force pulling the integrin and attached matrix molecules towards the cell center. On the ventral surface this rearward movement of the cytoskeleton contributes to forward motion of the cell relative to the substrate. Similar rearward translocation on the dorsal surface can be demonstrated by attaching beads to integrins. The movement of beads on the dorsal surface has been exploited to follow the pathway, velocity and forces developed by the cytoskeleton. Two types of movement have been observed when beads are attached to integrins on the dorsal surface. Random diffusion is the initial and sometimes only movement, and is thought to indicate lack of attachment of the integrins to the cytoskeleton. When the bead-bound integrins attach to the cytoskeleton they stop diffusive movements and begin a rearward translocation at a constant velocity, typically 1-2 μm/minute (Schmidt et al., 1993). Two factors are important for attachment to the cytoskeleton and rearward translocation – clustering of the integrins and occupancy of the integrin by its matrix ligand. Felsenfeld et al. found that large (1 μm) latex beads coated with a non-interfering antibody against β1 integrin demonstrated rearward translocation, while small (40 nm) gold beads coated with the antibody showed only diffusive movements (Felsenfeld et al., 1996). When the concentration of antibody was reduced, even the large beads showed only diffusive movement, suggesting that integrin clustering was essential for cytoskeletal attachment. The study went on to show that small gold beads coated with FN7-10, a four-domain segment containing the central cell adhesion domain of fibronectin (FN), showed rearward translocation, implying that cytoskeletal attachment was significantly enhanced when the integrin was occupied by its FN ligand. Clustering was still essential, since beads coated with a low concentration of FN, probably one active molecule per bead, bound transiently to the cell but did not attach to the cytoskeleton or translocate. Translocating beads coated with FN7-10 at first form a relatively weak attachment to the cytoskeleton, and they can be pulled off by a modest force using a laser trap (Choquet et al., 1997). However, when beads were subjected to a sustained force for 10 seconds or more, the cell increased the strength of the bond. The cell thus seems to be able to recruit additional cytoskeleton to attach a bead, allowing it to overcome an applied force and continue rearward translocation. This recruitment of cytoskeleton may be related to the findings that when FN-coated beads were placed in contact with a cell they induced formation of an actin cytoskeleton immediately beneath the point of bead binding (Miyamoto et al., 1995a; Miyamoto et al., 1995b). The formation of actin cytoskeleton upon bead contact, and the strengthening of attachment required both ligand binding and integrin clustering. How many integrins are required to form a cluster that will attach to the cytoskeleton? To address this question we have made soluble oligomeric constructs with one, two, three and 2581 Previous studies have shown that small beads coated with FN7-10, a four-domain cell adhesion fragment of fibronectin, bind to cell surfaces and translocate rearward. Here we investigate whether soluble constructs containing two to five FN7-10 units might be sufficient for activity. We have produced a monomer, three forms of dimers, a trimer and a pentamer of FN7-10, on the end of spacer arms. These oligomers could bind small clusters of up to five integrins. Fluorescence microscopy showed that the trimer and pentamer bound strongly to the cell surface, and within 5 minutes were prominently localized to actin fiber bundles. Monomers and dimers showed only diffuse localization. Beads coated with a low concentration (probably one complex per bead) of trimer or pentamer showed prolonged binding and rearward translocation, presumably with the translocating actin cytskeleton. Beads containing monomer or dimer showed only brief binding and diffusive movements. We conclude that clusters of three integrin-binding ligands are necessary and sufficient for coupling to and translocating with the actin cytoskeleton. Key words: Fibronectin, Cell adhesion, Signalling, Multivalent, Dimerization, Oligomer Summary Trimers of the fibronectin cell adhesion domain localize to actin filament bundles and undergo rearward translocation Françoise Coussen*, Daniel Choquet*, Michael P. Sheetz ‡ and Harold P. Erickson ‡,§ Department of Cell Biology, Box 3709, Duke University Medical Center, Durham, NC 27710, USA *Present address UMR CNRS 5091, Université de Bordeaux 2, Institut François Magendie, 33077 Bordeaux, France ‡ Present address: Department of Biological Sciences, Columbia University, New York, NY 10027, USA § Author for correspondence (e-mail: h.erickson@cellbio.duke.edu) Accepted 8 April 2002 Journal of Cell Science 115, 2581-2590 (2002) © The Company of Biologists Ltd Research Article