Biophysical mechanisms of single-cell interactions with microtopographical cues.

Biophysical cues encoded in the extracellular matrix (ECM) are increasingly being explored to control cell behavior in tissue engineering applications. Recently, we showed that cell adhesion to microtopographical structures ("micropegs") can suppress proliferation in a manner that may be blunted by inhibiting cellular contractility, suggesting that this effect is related to altered cell-scaffold mechanotransduction. We now directly investigate this possibility at the microscale through a combination of live-cell imaging, single-cell mechanics methods, and analysis of gene expression. Using time-lapse imaging, we show that when cells break adhesive contacts with micropegs, they form F-actin-filled tethers that extend and then rupture at a maximum, critical length that is greater than trailing-edge tethers observed on topographically flat substrates. This critical tether length depends on myosin activation, with inhibition of Rho-associated kinase abolishing topography-dependent differences in tether length. Using cellular de-adhesion and atomic force microscopy indentation measurements, we show that the micropegs enhance cell-scaffold adhesive interactions without changing whole-cell elasticity. Moreover, micropeg adhesion increases expression of specific mechanotransductive genes, including RhoA GTPase and myosin heavy chain II, and, in myoblasts, the functional marker connexin 43. Together, our data support a model in which microtopographical cues alter the local mechanical microenvironment of cells by modulating adhesion and adhesion-dependent mechanotransductive signaling.