DNA tension assays reveal that force-dependent integrin activation regulates neurite outgrowth in primary cortical neurons

Biomechanical inputs are ubiquitously present in biological systems and are known to regulate various cell functions. In particular, neural cell development is sensitive to mechanical regulation, as these cells reside in one of the softest microenvironments in the body. To fully characterize and comprehend how mechanical force modulates early neuronal processes, we prepared substrates functionalized with DNA probes displaying integrin ligands, including cRGD and laminin, to quantify integrin-mediated molecular tension during neurite initiation in primary cortical neurons. Our live-cell imaging analysis reveals that integrin-mediated tension force is highly dynamic and distributed across the cell body, with the overall tension signal gradually increasing during neurite outgrowth. Notably, we detected a consistent level of mechanical force (amplitude = 4.7–12 piconewtons, pN) for cell integrin-ligand interactions. Further quantifications reveal that neurons exhibit faster cell spreading and neurite outgrowth upon interacting with ligands functionalized with 4.7 pN relative to 12 pN probes. These findings indicate that the magnitude of integrin-mediated mechanical feedback regulates neuronal activity during early neuritogenesis. Additionally, we observed that mechanical tension is correlated with calcium signaling, since inhibiting calcium influx substantially reduced mechanical tension. Thus, our findings support that the magnitude of integrin-mediated mechanical feedback regulates neuronal activity during early neuritogenesis and that mechanical force is an essential element complementing well-known biochemical regulatory mechanisms orchestrating the integrin activation machinery and controlled neurite outgrowth in cortical neurons.