Confinement Sensing and Signal Optimization via Piezo1/PKA and Myosin II Pathways

• Calcium is elevated via Piezo1 as cells transit from unconfined to confined spaces
• PKA activity is suppressed in confined relative to unconfined spaces
• Piezo1-dependent calcium increase negatively regulates PKA via PDE1 in confinement
• Piezo1/PDE1/PKA and myosin II independently mediate confinement sensing

SummaryCells 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 Ca2+ level is increased, leading to phosphodiesterase 1 (PDE1)-dependent suppression of PKA activity. This Ca2+ 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.

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