European Journal of Cell Biology 85 (2006) 219–224 Lateral spacing of integrin ligands influences cell spreading and focal adhesion assembly Elisabetta A. Cavalcanti-Adam a,b,Ã , Alexandre Micoulet a,b,c , Jacques Blu¨mmel a,b , Jo¨rg Auernheimer d , Horst Kessler d , Joachim P. Spatz a,b a Department of New Materials and Biosystems, Max-Planck-Institute for Metals Research, Heisenbergstr. 3, D-70 569 Stuttgart, Germany b Department of Biophysical Chemistry, University of Heidelberg, Institute for Physical Chemistry, Heidelberg, Germany c Department of Materials Science and Engineering, Massachusetts Institute of Technology, Cambridge, MA 02139, USA d Technical University of Munich, Institut fu¨r Organische Chemie und Biochemie, Lehrstuhl II, Lichtenbergstrasse 4, D-85747 Garching, Germany Abstract Cell–extracellular matrix (cell–ECM) interactions mediated by integrin receptors are essential for providing positional and environmental information necessary for many cell functions, such as proliferation, differentiation and survival. In vitro studies on cell adhesion to randomly adsorbed molecules on substrates have been limited to sub- micrometer patches, thus preventing the detailed study of structural arrangement of integrins and their ligands. In this article, we illustrate the role of the distance between integrin ligands, namely the RGD (arginine–glycine–aspartate) sequence present in ECM proteins, in the control of cell adhesion. By using substrates, which carry cyclic RGD peptides arranged in highly defined nanopatterns, we investigated the dynamics of cell spreading and the molecular composition of adhesion sites in relation to a fixed spacing between the peptides on the surface. Our novel approach for in vitro studies on cell adhesion indicates that not only the composition, but also the spatial organization of the extracellular environment is important in regulating cell–ECM interactions. r 2005 Elsevier GmbH. All rights reserved. Keywords: Nanotechnology; Focal adhesion; Integrin clustering; RGD peptides Introduction A key event in focal adhesion assembly is the activation and clustering of ligand-occupied integrins. The molecular composition of the extracellular matrix (ECM) determines the recruitment of specific integrins at these adhesion sites: for example, on fibronectin, the major integrin is a 5 b 1 , on vitronectin, the major receptor is a v b 3 (Singer et al., 1988). A common feature of integrin-binding sites present in ECM molecules is a specific tri-peptide sequence, the RGD (arginine–gly- cine–aspartate) motif (Ruoslahti, 1996). The vast majority of in vitro studies on cell–ECM interactions has been focused on the design of bioactive surface coatings by exploring the molecular composition of the ECM (Ito et al., 1991; Dee et al., 1998). Most of the studies on surfaces coated with peptides are based on the assumption that there is an equal distribution of ARTICLE IN PRESS www.elsevier.de/ejcb 0171-9335/$ - see front matter r 2005 Elsevier GmbH. All rights reserved. doi:10.1016/j.ejcb.2005.09.011 Ã Corresponding author. Department of Biophysical Chemistry, University of Heidelberg, Institute for Physical Chemistry, Im Neuenheimer Feld 253, D-69120 Heidelberg, Germany. Tel.: +49 6221 54 4232; fax: +49 6221 54 4950. E-mail address: Ada.Cavalcanti-Adam@urz.uni-heidelberg.de (E.A. Cavalcanti-Adam).