Cell adhesion and motility depend on nanoscale RGD clustering

Abstract

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 cell functions such as motility, proliferation and differentiation. Two key observations regarding the biophysical nature of integrin-mediated cell-matrix interactions motivated the present study: (1) cell motility can be regulated by modulating the magnitude of cell-substratum adhesion, by varying cell integrin expression level, integrin-ECM binding affinity or substratum ECM surface density; and (2) integrin clustering enables assembly of multiple cytoplasmic regulatory and structural proteins at sites of aggregated integrin cytoplasmic domains, activating certain intracellular signalling pathways. Here, using a minimal integrin adhesion ligand, YGRGD, we test the hypothesis that ligand clustering can affect cell migration in a manner related to its modulation of cell-substratum adhesion. We employ a synthetic polymer-linking method, which allows us to independently and systematically vary both the average surface density and the local (approx. 50 nm scale) spatial 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 average of five ligands per cluster, or in clusters with an average of nine ligands per cluster; for each of these spatial distributions, a range of average ligand densities (1,000-200,000 ligands/micrometer(2)) were examined. Cluster spacing was adjusted in order to present equivalent average ligand densities independently of cluster size. The murine NR6 fibroblast cell line was used as a model because its migration behavior on ECM in the presence and absence of growth factors has been well-characterized and it expresses integrins known to interact with the YGRGD peptide. Using time-lapse videomicroscopy and analysis of individual cell movement paths, we find that NR6 cells can migrate on substrata where adhesion is mediated solely by the YGRGD peptide. As previously observed for migration of NR6 cells on fibronectin, migration speed on YGRGD is a function of the average surface ligand density. Strikingly, clustering of ligand significantly reduced the average ligand density required to support cell migration. In fact, non-clustered integrin ligands support cell attachment but neither full spreading nor haptokinetic or chemokinetic motility. In addition, by quantifying the strength of cell-substratum adhesion, we find that the variation of cell speed with spatial presentation of YGRGD is mediated via its effect on cell adhesion. These effects 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 within the cell, with a greater number of cells exhibiting stress fibers at higher cluster sizes of YGRGD. Our observations demonstrate that cell motility may be regulated by varying ligand spatial presentation at the nanoscale level, and suggest that integrin clustering is required to support cell locomotion.

Soft overcomes the hard: Flexible materials adapt to cell adhesion to promote cell mechanotransduction

“…Traditionally, cell adhesion and proliferation are considered to be inhibited on the soft matrix. However, the matrix stress relaxation successfully compensates for the effect of reduced stiffness on cell adhesion by enabling ligand accumulation after matrix softening [ 44 , 45 ]. This process activates integrin adhesion, actin assembly, and myosin contractility and thereby promotes cell mechanotransduction.…”

Versatile method to generate multiple types of micropatterns

“…The concentrations of proteins adsorbed to the surface can be readily controlled and gradients in the density of surface-bound proteins can be formed. Both parameters are known to influence on cell spreading and migration [18][19][20][21][22] . Since an additional gradient can be set up in the liquid medium bathing the cells with the aid of the chemotaxis chamber, multi-cue situations can be produced, in which the synergy or competition between surface and solution gradients can be studied within the defined geometries provided by a micropattern.…”

“…For instance, it has been found that the size and geometry of the accessible area can alter and direct the axis of cell polarization and division 2,3,5 , the positions and orientation of pseudopodia 4 , or the locations of junctions between adjacent cells 14 . Cell adhesion and migration depends in large part on the protein composition of a cell's surroundings [15][16][17] , but the relative density of adhesion sites [18][19][20] , as well as density gradients of surface-bound proteins, can influence and bias its spreading and motion 21,22 .…”

The advances in nanomedicine for bone and cartilage repair

“…Favourable biocompatibility: Synthetic polymeric materials are widely used in cartilage tissue engineering scaffolds due to their superior controllability over physicochemical properties. That being said, the surface of synthetic polymers lacks certain receptor-specific binding bioactive molecules of natural materials, such as RGD sequences that are important for cell adhesion and growth migration, and functional differentiation [ 289 ]. Therefore, scaffolds derived from synthetic materials such as PLA, PGA, PLGA are mostly modified with natural polymers or functional groups to create a simulated natural biochemical environment suitable for cell survival.…”