Position-dependent linkages of fibronectin- integrin-cytoskeleton - PubMed (original) (raw)
Position-dependent linkages of fibronectin- integrin-cytoskeleton
T Nishizaka et al. Proc Natl Acad Sci U S A. 2000.
Abstract
Position-dependent cycling of integrin interactions with both the cytoskeleton and extracellular matrix (ECM) is essential for cell spreading, migration, and wound healing. Whether there are regional changes in integrin concentration, ligand affinity or cytoskeleton crosslinking of liganded integrins has been unclear. Here, we directly demonstrate a position-dependent binding and release cycle of fibronectin-integrin-cytoskeleton interactions with preferential binding at the front of motile 3T3 fibroblasts and release at the endoplasm-ectoplasm boundary. Polystyrene beads coated with low concentrations of an integrin-binding fragment of fibronectin (fibronectin type III domains 7-10) were 3-4 times more likely to bind to integrins when placed within 0.5 microns vs. 0.5-3 microns from the leading edge. Integrins were not concentrated at the leading edge, nor did anti-integrin antibody-coated beads bind preferentially at the leading edge. However, diffusing liganded integrins attached to the cytoskeleton preferentially at the leading edge. Cytochalasin inhibited edge binding, which suggested that cytoskeleton binding to the integrins could alter the avidity for ligand beads. Further, at the ectoplasm-endoplasm boundary, the velocity of bead movement decreased, diffusive motion increased, and approximately one-third of the beads were released into the medium. We suggest that cytoskeleton linkage of liganded integrins stabilizes integrin-ECM bonds at the front whereas release of cytoskeleton-integrin links weakens integrin-ECM bonds at the back of lamellipodia.
Figures
Figure 1
Behavior of a bead coated with a low concentration of FNIII7–10. (a) Video-enhanced differential interference contrast micrographs show binding and release of a FNIII7–10 bead. The bead was carried to the edge of the lamellipodium with an optical tweezers and released from the trap (i). It immediately moved retrogradely across the lamellipodium (ii), and the velocity decreased at the boundary between ectoplasm and endoplasm (iii). The bead then detached from the cell surface (iv) and diffused out of focus into the medium (v and vi). (b) Video micrographs show attachment of diffusing bead and rearward movement after being brought to edge. (c) The trajectory of the bead along the direction of the displacement in a. i–vi Correspond to the micrographs in a. (d) The bead trajectory of b. [Scale bars, 10 μm (a) and 5 μm (b)]. Nuc, nuclear; endo, endoplasm; ecto, ectoplasm; and lamella, lamellipodium.
Figure 2
Histograms are shown of the probability of bead binding. (a) The position dependence of beads coated with a low concentration of FNIII7–10 (total n = 272). At this concentration, we estimate that 300 FNIII7–10 molecules are bound per bead (4–10/bead-membrane contact area). (b) The control experiment for a. Beads were coated with 100% BSA (total n = 75) instead of FNIII7–10. (c) The same experiment as a in the presence of 2 mg/ml RGD peptide, which inhibits fibronectin binding to the β1 integrin (19, 20). This experiment was done within 0.5 μm from the edge (total n = 94). This result coincides with ref. . (d) Edge binding of FN beads is measured as a function of [Mn2+], which is reported to change the affinity of integrin for fibronectin (–22). The binding probability is dependent on the concentration of Mn2+ (total n = 252).
Figure 3
(a, b, and c) Histograms of the bead binding behavior at the front part of migrating fibroblasts. (a) Fibronectin-coated bead. This histogram is based on the same set of data for Fig. 2_a_. (b) ES66 antibody-coated beads. (c) Fibronectin-coated beads in the presence of 100 nM cytochalasin B. For more details, see Materials and Methods.
Figure 4
(a) The position dependence of the unbinding of the beads from cell surfaces. The position where the unbinding of the bead occurred, indicated by the length of bead travel before unbinding (Ltravel), is related to the position of the ectoplasm–endoplasm boundary, indicated by the length of ectoplasm (Lecto). The dashed line is a linear approximation and the slope is 0.87. (b) Video micrographs show the definition of Ltravel and Lecto in a. The bead ceased moving rearward in the third micrograph and diffused away in the fourth micrograph. In micrographs (–3), intervals are 21 s. (Scale bar, 5 μm).
Figure 5
Schematic illustration showing that cytoskeleton binding of integrin-fibronectin complexes at the leading edge could stabilize them. A fibronectin-coated bead (FN-bead) attaches to the dorsal surface of the leading edge and recruits a second integrin, which recruits a second link to the cytoskeleton. Because the two bound integrins are attached to a rigid cytoskeleton, they cannot diffuse away if one should release from the fibronectin. Therefore, bead binding is stabilized until the actin cytoskeleton depolymerizes, which is often seen at the endoplasm–ectoplasm boundary. Upon release from the cytoskeleton, the integrins could diffuse away leading to FN-bead release. On the ventral surface, additional components could stabilize the integrin-cytoskeleton complex perhaps in a force-dependent process (1). Such a position-dependent binding and release cycle could aid cell migration.
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