Cell adhesion and motility depend on nanoscale RGD clustering

Integrin adhesion receptors play a crucial role in regulating interactions between cells and extracellular matrix (ECM). Integrin activation initiates multiple intracellular signaling pathways and results in regulation of cel l functions such as motility, proliferation and differentiation. Two keg ob servations regarding the biophysical nature of integrin-mediated cell-matri x interactions motivated the present study: (1) cell motility can be regula ted by modulating the magnitude of cell-substratum adhesion, by varying cel l integrin expression level, integrin-ECM binding affinity or substratum EC M surface density; and (2) integrin clustering enables assembly of multiple cytoplasmic regulatory and structural proteins at sites of aggregated inte grin cytoplasmic domains, activating certain intracellular signalling pathw ays. Here, using a minimal integrin adhesion ligand, YGRGD, we test the hyp othesis that ligand clustering can affect cell migration in a manner relate d to its modulation of cell-substratum adhesion. We employ a synthetic poly mer-linking method, which allows us to independently and systematically var y both the average surface density and the local (approx. 50 nm scale) spat ial distribution of the YGRGD peptide, against a background otherwise inert with respect to cell adhesion. In this system, the ligand was presented in three alternative spatial distributions: singly, in clusters with an avera ge of five ligands per cluster, or in clusters with an average of nine liga nds per cluster; for each of these spatial distributions, a range of averag e ligand densities (1,200,000,000 ligands/mu m(2)) were examined. Cluster s pacing was adjusted in order to present equivalent average ligand densities independently of cluster size. The murine NR6 fibroblast cell line was use d as a model because its migration behavior on ECM in the presence and abse nce of growth factors has been well-characterized and it expresses integrin s known to interact with the YGRGD peptide. Using time-lapse videomicroscop y and analysis of individual cell movement paths, we find that NR6 cells ca n migrate on substrata where adhesion is mediated solely by the YGRGD pepti de. As previously observed for migration of NR6 cells on fibronectin, migra tion speed on YGRGD is a function of the average surface ligand density. St rikingly, clustering of ligand significantly reduced the average ligand den sity required to support cell migration. In fact, nonclustered integrin lig ands support cell attachment but neither full spreading nor haptokinetic or chemokinetic motility. In addition, by quantifying the strength of cell-su bstratum adhesion, we find that the variation of cell speed with spatial pr esentation of YGRGD is mediated via its effect on cell adhesion. These effe cts on motility and adhesion are also observed in the presence of epidermal growth factor (EGF), a known motility-regulating growth factor. Variation in YGRGD presentation also affects the organization of actin filaments with in the cell, with a greater number of cells exhibiting stress fibers at hig her cluster sizes of YGRGD. Our observations demonstrate that cell motility may be regulated by varying ligand spatial presentation at the nanoscale l evel, and suggest that integrin clustering is required to support cell loco motion.