Expression of α-smooth muscle actin (α-SMA) renders fibroblasts highly contractile and hallmarks myofibroblast differentiation. We identify α-SMA as a mechanosensitive protein that is recruited to stress fibers under high tension. Generation of this threshold tension requires the anchoring of stress fibers at sites of 8–30-μm-long “supermature” focal adhesions (suFAs), which exert a stress approximately fourfold higher (∼12 nN/μm2) on micropatterned deformable substrates than 2–6-μm-long classical FAs. Inhibition of suFA formation by growing myofibroblasts on substrates with a compliance of ≤11 kPa and on rigid micropatterns of 6-μm-long classical FA islets confines α-SMA to the cytosol. Reincorporation of α-SMA into stress fibers is established by stretching 6-μm-long classical FAs to 8.1-μm-long suFA islets on extendable membranes; the same stretch producing 5.4-μm-long classical FAs from initially 4-μm-long islets is without effect. We propose that the different molecular composition and higher phosphorylation of FAs on supermature islets, compared with FAs on classical islets, accounts for higher stress resistance.

During cell migration, the physical link between the extracellular substrate and the actin cytoskeleton mediated by receptors of the integrin family is constantly modified. We analyzed the mechanisms that regulate the clustering and incorporation of activated αvβ3 integrins into focal adhesions. Manganese (Mn2+) or mutational activation of integrins induced the formation of de novo F-actin–independent integrin clusters. These clusters recruited talin, but not other focal adhesion adapters, and overexpression of the integrin-binding head domain of talin increased clustering. Integrin clustering required immobilized ligand and was prevented by the sequestration of phosphoinositole-4,5-bisphosphate (PI(4,5)P2). Fluorescence recovery after photobleaching analysis of Mn2+-induced integrin clusters revealed increased integrin turnover compared with mature focal contacts, whereas stabilization of the open conformation of the integrin ectodomain by mutagenesis reduced integrin turnover in focal contacts. Thus, integrin clustering requires the formation of the ternary complex consisting of activated integrins, immobilized ligands, talin, and PI(4,5)P2. The dynamic remodeling of this ternary complex controls cell motility.

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A novel patterning technique based on selective self-assembly of alkane phosphates on metal oxide surfaces is presented. Standard photolithography was used to create patterns of titanium dioxide within a matrix of silicon dioxide. Alkane phosphates were found to self-assemble on TiO2, but not on SiO2, surfaces. Subsequent adsorption of poly(L-lysine)-g-poly(ethylene glycol) (PLL-g-PEG) rendered the exposed SiO2 surface resistant to protein adsorption. X-ray photoelectron spectroscopy and time-of-flight secondary ion mass spectrometry were employed to monitor the assembly processes. Protein-adsorption studies by means of fluorescence microscopy conclusively established that the resulting surfaces displayed proteinadhesive, alkyl phosphate modified TiO2 features, arranged within a protein-resistant PLL-g-PEG-modified SiO2 matrix. Human foreskin fibroblasts, incubated in a serum-containing medium, were found to selectively attach to the protein-adhesive areas, where they developed focal contacts. No interaction of cells with the PLL-g-PEG-coated SiO2 areas was evident for at least 14 days. This patterning approach, termed selective molecular assembly patterning, is considered to be suitable for reproducible and cost-effective fabrication of biologically relevant chemical patterns over large areas.