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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

View larger version (33K): [in a new window]   Fig. 1. (A) Constructs for multimeric FN7-10 (see Materials and Methods for details). (B) A scale model of the FN7-10 trimer, constructed from the coordinates of FN7-10 (Leahy et al., 1996) (these were also used to model the six FN-III domains in TNfn3-8) with the program RasMol (Sayle and Milner-White, 1995). The FN3 domains are shown as a spacefilling atomic model, and the -helices of CMP are indicated schematically in a ribbon format. For comparison an integrin molecule is drawn to scale (Carrell et al., 1985; Nermut et al., 1988).

View larger version (140K): [in a new window]   Fig. 2. Electron microscopy of rotary shadowed monomeric and oligomeric constructs.

View larger version (114K): [in a new window]   Fig. 3. Distribution of FN7-10 monomers and oligomers bound to fibroblasts. Soluble biotinylated proteins at 3 µM (subunit concentration) were added to fibroblasts grown overnight on a laminin substrate. After 2.5 minutes the cells were fixed and stained with fluorescent avidin to localize bound FN. Two examples are given for each protein. For the tandem dimer and pentamer, the focal plane of the right hand images was set above the substrate to optically section at the level of the cell body, illustrating the diffuse membrane staining for the tandem dimer and the punctate staining for the pentamer. For all other images the focus was on the upper surface of the lamellapodia. Control TN trimer (lacking the FN7-10 segment) does not bind the cell surface (bottom left). A competition experiment is shown in the bottom-right panel, where cells were treated with 3 µM biotinylated FN7-10 trimer in the presence of 30 µM unbiotinylated FN7-10 monomer. Binding of trimer is efficiently competed by excess monomer.

View larger version (113K): [in a new window]   Fig. 4. (A) Binding of FN-TN trimer to fibroblasts at different concentrations. At 0.5 µM and above the trimer localizes to streaks on the cell surface. (B) Competition of trimer binding by FN7-10 monomer. The trimer was at 0.5 µM and FN7-10 monomer was varied. In both A and B 20 µM TN pentamer was included to compete and block binding to FN fibrils. Cells were incubated with the trimer or trimer plus monomer for 4 minutes, then fixed and stained with fluorescent avidin.

View larger version (86K): [in a new window]   Fig. 5. Localization of 5-integrin induced by FN trimer and pentamer. Cells were treated for 5 minutes with different FN-TN constructs and then fixed and labeled simultaneously with avidin to localize the FN-TN construct, and with an antibody against 5-integrin. The monomer and dimer show mostly a diffuse pattern of both FN and integrin. The trimer and pentamer show the strong localization of FN into streaks, as already described. These streaks also stained for 5 integrin, although a substantial pool of diffuse integrin remained in each case. Some plasma FN (pFN) localized over the integrin streaks, but there were also prominent FN patches localized elsewhere.

View larger version (67K): [in a new window]   Fig. 6. Localization of FN oligomers over actin fibers. Cells were treated for 5 minutes with different FN-TN constructs and then fixed and labeled simultaneously with avidin to localize the FN-TN, and with phalloidin to localize actin polymers. The FN-TN streaks produced by the trimer and pentamer are always localized over actin fibers, but only a sub-set of the actin fibers is decorated. The streaks of plasma FN were shorter and more peripheral than those of FN-TN trimer and pentamer, and localized over shorter segments of actin fibers.

View larger version (15K): [in a new window]   Fig. 7. Movement of latex beads coated with the different proteins. (Left traces) X versus Y plots of bead movement in the plane of the membrane. (Right traces) Displacement versus time of beads. At time zero, the beads were placed with the optical trap near the leading edge of the cell and maintained on the cell surface for 4 seconds. The laser was then stopped and the bead released. Unbound beads quickly drifted out of focus while bound beads remained on the cell surface.

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