To study the function behind the molecular arrangement of single integrins in cell adhesion, we designed a hexagonally close-packed rigid template of cell-adhesive gold nanodots coated with cyclic RGDfK peptide by using block-copolymer micelle nanolithography. The diameter of the adhesive dots is < 8 nm, which allows the binding of one integrin per dot. These dots are positioned with high precision at 28, 58, 73, and 85 nm spacing at interfaces. A separation of > or = 73 nm between the adhesive dots results in limited cell attachment and spreading, and dramatically reduces the formation of focal adhesion and actin stress fibers. We attribute these cellular responses to restricted integrin clustering rather than insufficient number of ligand molecules in the cell-matrix interface since "micro-nanopatterned" substrates consisting of alternating fields with dense and no nanodots do support cell adhesion. We propose that the range between 58-73 nm is a universal length scale for integrin clustering and activation, since these properties are shared by a variety of cultured cells.

Cell interactions with adhesive surfaces play a vital role in the regulation of cell proliferation, viability and differentiation, and affect multiple biological processes. Since cell adhesion depends mainly on the nature and density of the adhesive ligand molecules, spatial molecular patterning, which enables the modulation of adhesion receptor clustering, might affect both the structural and signalling activities of the adhesive interaction. We herein show that cells plated on surfaces that present a molecularly defined spacing gradient of an integrin RGD ligand, can sense small but consistent differences in adhesive ligand spacing of about 1 nm across the cell diameter, which is approximately 61 m when the spacing includes 70 nm. Consequently, these positional cues induce cell polarization, and initiate cell migration and signalling. We propose that differential positional clustering of the integrin transmembrane receptors is used by cells for exploring and interpreting their environment, at high spatial sensitivity.Adhesion of tissue cells to the extracellular matrix depends on the activation of specific transmembrane receptors; e.g. integrins, which leads to the assembly of specialized adhesion sites known as focal adhesions (FA). 1 Activation and spatial organization of integrins are mainly controlled by epitopes containing a RGD (R = arginine, G = glycine, D = aspartate) sequence, that are present on a variety of adhesive extracellular matrix (ECM) proteins. Recent studies indicated that beyond the chemical specificity of the adhesive epitope, 2 many physical features of the adhesive surface, including its topography, 3 rigidity, 4 and precise epitope spacing, 5 are critical for guiding receptor-mediated adhesion formation and signalling. Specifically, it was demonstrated that cyclic RGDfK peptides linked to nanogold particles 6 which were arranged in pattern on substrates and interspaced by 58 or 73 nm, influence cell adhesion, 5 spreading, focal adhesion assembly, and migration 7,8 in very different ways. In these studies, control experiments demonstrated that only the specific functionalization of nanogold particles with cyclic RGD induced integrin clustering and such focal adhesion formation upon interaction with cells such as 3T3-fibroblasts or -* Corresponding author: spatz@mf.mpg.de. + Both authors contributed equally to this work. 19 On the molecular length scale, these methods enable quantification of an average molecular concentration, which gradually varies along the adhesive surface; however, they are too "noisy" to quantify the densities themselves, and density variations capable of inducing cell polarization. NIH Public AccessWe herein demonstrate a novel surface patterning technique for the generation of nanoparticle spacing gradients, which is based on controlled modulation of the selfassembly of diblock copolymer micelles. Due to the self-assembly of macromolecules, large-scale surface areas are easily processed by this technique. After successful biofunctionalization of the nano...

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