The ability of cells to perceive the mechanics and nanostructure of the extracellular matrix (ECM) relies on the activity of a number of intracellular proteins, collectively defined as mechanosome. By modifying the expression, the interaction or the localization of such proteins, the cell dynamically responds to modifications in ECM compliance and nanotopography by activating specific genetic programs. Among the intracellular proteins acting as mechanosensors, those composing the Focal Adhesions (FAs) and the effectors of Hippo pathway YAP/TAZ have been recently described as the main actors. Here, while confirming that both FAs and YAP/TAZ respond to substrate mechanics, we demonstrate that they are sensitive to dynamic modifications of substrate nanotopography by rearranging their structure or their localization. We also show that they are not sensitive to cell polarity when cell area is kept constant. In particular, the assembly of FAs, measured by image analysis through vinculin spike formation at the periphery of the cell, and YAP/TAZ nuclear shuttling to and from the cell nucleus, are proven to be sensitive to the changes in ECM nanostructure as mimicked by thermo-responsive polymers based on crosslinked poly-caprolactone (PCL). By using such a tool, we show that YAP nuclear presence - and thus its transcriptional activity – is promptly impaired by dynamic changes in substrate nanopattern. The same dynamic modifications alter FA size distribution, thus suggesting a change in their molecular composition. In doing so, we show at single cell level that the inhibition of cell spreading and of its ability to perceive ECM mechanics by either micropatterned surfaces or pharmacological inhibitors of RhoA/ROCK or Myosin IIa pathways results in the depletion of YAP/TAZ nuclear activity, the impairment of FAs formation and the regulation of genes involved in cell-matrix interaction. Altogether our results expand the current understanding of cell mechanosensing apparatus and directly connect YAP/TAZ transcriptional activity to the ability of the cell to feel the dynamic modifications of ECM composition.Keywords: mechanobiology, nanotopography, mechanosome, focal adhesion, Hippo pathway
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