Cell adhesion and polarisation on molecularly defined spacing gradient surfaces of cyclic RGDfK peptide patches

Abstract

In vivo cell migration and location are orchestrally guided by soluble and bound chemical gradients. Here, gradients of extracellular matrix molecules are formed synthetically by the combination of a surface nanopatterning technique called block copolymer nanolithography (BCN) and a biofunctionalisation technique. A modified substrate dip-coating process of BCN allows for the formation of precise molecular gradients of cyclic RGDfK peptide patches at interfaces, which are presented to cells for testing cell adhesion and polarisation. Surfaces formed by BCN consist of hexagonally ordered gold dot patterns with a gradient in particle spacing. Each dot serves as a chemical anchor for the binding of cyclic RGDfK peptides, which are specifically recognised by α_vβ₃ integrins. Due to steric hindrance only up to one integrin binds to one functionalised gold dot which forms a peptide patch spacing. We demonstrate how cell morphology, adhesion area, actin and vinculin distribution as well as cell body polarisation are influenced by the peptide patch spacing gradient. As a consequence, these gradients of adhesive ligands induce cell orientation towards smaller particle spacing when the gradient strength is 15 nm/mm at least. This implicates that an adherent cell's sensitivity to differentiate between ligand patch spacing is approximately 1 nm across the cell body.

Introduction

Cell polarisation and directed cell migration plays a crucial role in many physiological processes such as embryonic development, wound healing, tissue remodelling, or angiogenesis, as well as in pathological processes such as inflammatory diseases or cancer metastasis (Lauffenburger and Horwitz, 1996). Cell polarisation and migration is initiated by several external stimuli such as topography, elasticity, mechanic pressure, soluble or bound chemicals. The last is termed haptotaxis (Greek: haptein, to fasten; taxis, arrangement), describing the phenomenon of directed migration along an immobilised extracellular matrix (ECM) gradient (Carter, 1965). Many cells are dependent on contacts with either a substratum like the extracellular environment in a tissue or surrounding cells (Gilmore, 2005). Adhesion-dependent cells which fail to form external contacts initiate apoptosis (Gilmore, 2005). Thus, polarisation and migration of cells is often associated with cells looking for environments, which allow their survival and development. In order to initiate migration, tissue cells such as fibroblasts establish a polarised morphology with spatially differentiated adhesion stability. This allows for the generation of a force which contracts the cytoskeleton and enables it to shift the body forward (Mitchison and Cramer, 1996).

The most common haptotaxis assay is performed with Boyden chambers where cells migrate across a porous membrane which is coated with ECM proteins on its lower side and the number of traversed cells is quantified (Boyden, 1962). However, this system is not suitable for live-cell imaging and completely lacks any information of the molecular composition of the gradient in the porous mesh. Therefore, it is desirable to mimic transparent substrates with a defined gradient of immobilised biomolecules. Although many concentration gradient systems have already been established (DeLong et al., 2005; Kang et al., 2004), none of those can exclude local aggregation effects or possess a precise spatial control of the bound ligands. The latter, however, is crucial for the study of cooperative effects of receptor clustering and spacing in signal transduction and for understanding the mechanism of gradient sensing looking at small membrane protrusions in the micro- and nanometer size range (Maheshwari et al., 2000).

The first approaches to produce immobilised biomolecule gradients were realised by adsorbing proteins to surfaces with gradients of wettability (Fisher et al., 1989). Further progress in the development of more elaborated and better-defined gradients was, for example, achieved with microfluidic systems (Dertinger et al., 2002), photoimmobilisation of peptides on self-assembled monolayers (Herbert et al., 1997) or by coupling electrochemical potential gradients with electrosorption reactions of organothiols (Plummer and Bohn, 2002; Plummer et al., 2003).

So far, all techniques for gradient fabrication consisted only of proteins or bioactive molecules deposited at an average density on a surface where the concentration continuously varied in average as a function of substrate position. However, controlling the average densities of biomolecules and proteins at interfaces is not sufficient to unfold molecular processes involved in cell signalling (Arnold et al., 2004; Cavalcanti-Adam et al., 2006). Therefore, a profound knowledge of local biomolecule or protein density, respectively presentation, with a resolution of single molecules is necessary for understanding the complex cell–ECM interaction at a nanometer level. The importance of designing model surfaces with nanometer accuracy is underlined by the observation that collagen fibres interact at a spatial periodicity of 67 nm with tissue cells in vivo (Poole et al., 2005). Another example is given by the process of integrin clustering which requires a maximum inter-integrin ligand spacing of 58 nm in order to form focal adhesions (Arnold et al., 2004; Cavalcanti-Adam et al., 2006). Different cellular responses can be triggered not only by varying the average bound concentration of bioactive molecules on the surface but also by their mere position relative to each other.

We chose a cyclic RGDfK peptide selective for the α_vβ₃ integrin (Haubner et al., 1996) to perform experiments on chemical gradient sensing of Mc3t3 osteoblasts. The formation of gradients depends on hexagonal arrays of gold nanoparticles which were fabricated on the basis of block copolymer micellar self-assembly (Spatz et al., 2000, Spatz et al., 2002; Glass et al., 2003a, Glass et al., 2003b). By a modified dip-coating procedure we were able to vary the spacing between individual nanoparticles continuously in the range of several mm² on a single substrate with nanometer precision. The gold particles served as anchor points to which cyclic RGDfK peptides provided with a thiol anchor are covalently bound, called cyclic RGDfK peptide patches. Since each cyclic RGDfK patch forms a binding site for up to one integrin per patch (Wolfram et al., 2006), this model platform enabled us to quantify the response of cells to continuously changing nanometer-defined integrin–integrin spacing.