Cells adopt distinct signaling pathways to optimize cell locomotion in different physical microenvironments. However, the underlying mechanism that enables cells to sense and respond to physical confinement is unknown. Using microfabricated devices and substrate-printing methods along with FRET-based biosensors, we report that, as cells transition from unconfined to confined spaces, intracellular Ca(2+) level is increased, leading to phosphodiesterase 1 (PDE1)-dependent suppression of PKA activity. This Ca(2+) elevation requires Piezo1, a stretch-activated cation channel. Moreover, differential regulation of PKA and cell stiffness in unconfined versus confined cells is abrogated by dual, but not individual, inhibition of Piezo1 and myosin II, indicating that these proteins can independently mediate confinement sensing. Signals activated by Piezo1 and myosin II in response to confinement both feed into a signaling circuit that optimizes cell motility. This study provides a mechanism by which confinement-induced signaling enables cells to sense and adapt to different physical microenvironments.

The research is supported by awards from the NIH (R01 DK073368 and DP1 CA174423 to J.Z. and R01 CA183804 and R01 GM114675 to K.K.); Kleberg Foundation and American Heart Association (to K.K.); Spanish Ministry of Economy and Competitiveness (SAF2012-38140 and SAF2015-69762-R); Fondo de Investigación Sanitaria (RD12/0042/0014); and FEDER Funds (to M.A.V.).