Selective binding and lateral clustering of α5ß1 and αvß3 integrins: Unraveling the spatial requirements for cell spreading and focal adhesion assembly.

Coordination of the specific functions of α5ß1 and αvß3 integrins is crucial for the precise regulation of cell adhesion, spreading and migration, yet the contribution of differential integrin-specific crosstalk to these processes remains unclear. To determine the specific functions of αvß3 and α5ß1 integrins, we used nanoarrays of gold particles presenting immobilized, integrin-selective peptidomimetic ligands. Integrin binding to the peptidomimetics is highly selective, and cells can spread on both ligands. However, spreading is faster and the projected cell area is greater on α5ß1 ligand; both depend on ligand spacing. Quantitative analysis of adhesion plaques shows that focal adhesion size is increased in cells adhering to αvß3 ligand at 30 and 60 nm spacings. Analysis of αvß3 and α5ß1 integrin clusters indicates that fibrillar adhesions are more prominent in cells adhering to α5ß1 ligand, while clusters are mostly localized at the cell margins in cells adhering to αvß3 ligand. αvß3 integrin clusters are more pronounced on αvß3 ligand, though they can also be detected in cells adhering to α5ß1 ligand. Furthermore, α5ß1 integrin clusters are present in cells adhering to α5ß1 ligand, and often colocalize with αvß3 clusters. Taken together, these findings indicate that the activation of αvß3 integrin by ligand binding is dispensable for initial adhesion and spreading, but essential to formation of stable focal adhesions.

Segregation versus colocalization: orthogonally functionalized binary micropatterned substrates regulate the molecular distribution in focal adhesions.

Orthogonally functionalized binary micropatterned substrates are produced using a novel protocol. The use of adequate peptido-mimetics enables an unprecedented segregation of purified αvß3 and α5ß1 integrins in adjacent microislands and evidences the preference of U2OS cells to colocalize such receptors. Moreover, this tendency can be altered by varying the geometry and composition of the micropatterns.