Integrin clustering, typically nanometers in dimension, is the first and an important step in integrin-mediated cellular signaling processes such as focal adhesion. Engineered nanostructures mimicking extracellular matrices (ECM) provide a new approach for investigation and regulation of this initial step and of downstream cascades of focal adhesion. This work reveals that fibronectin (Fn) nanostructures, even at a small height of 3.2 nm ± 0.5 nm, exhibit high efficacy in guiding cellular orientation and polarization. More interestingly, the Fn nanostructures also impact intracellular structures such as preferential filopodia attachment, and commensurate alignment of intracellular actin stress fibers. The impact can be rationalized by the strong and specific interaction between integrin and Fn, leading to integrin clusters and then focal adhesion assemblies following the underlying nanostructure of Fn. This guided assembly further mediates the downstream behavior, such as actin stress fiber alignment and overall cellular morphology. Our observations collectively demonstrate that engineered nanostructures of Fn provide an alternative and high efficacy biochemical cue for regulation of cellular signaling processes.
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